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TRANSCRIPT
An Intranasal Delivery Method for Novel Peptide Therapeutics
Designed to treat Major Depressive Disorder
by
Virginia Joan Margaret Brown
A thesis submitted in conformity with the requirements
for the degree of Masters of Science
Department of Physiology
University of Toronto
© Copyright by Virginia Brown, 2013
ii
An Intranasal Delivery Method for Novel Therapeutics designed to treat
Major Depressive Disorder
Virginia Brown
Masters of Science
Department of Physiology
University of Toronto
2013
Abstract
A problem in designing drugs that act upon the central nervous system is developing
effective delivery methods. Major depressive disorder (MDD) affects 12% of men and 20% of
women in the United States, and treatment options are often inadequate. In patients, the
interaction between dopamine D1 and D2 receptors is correlated with major depressive disorder.
A small peptide that disrupts this interaction can be delivered to brain areas using intranasal
delivery. The D1-D2 interfering peptide has an antidepressant effect comparable to imipramine
in the forced swimming test (FST), a test for antidepressant efficacy. At doses greater than 5.75
mg/kg, the D1-D2 interfering peptide has antidepressant action in the FST for 2 hours after
intranasal administration. The D1-D2 interfering peptide disrupts the D1-D2 receptor interaction
in the PFC after intranasal administration. This study provides preclinical support for intranasal
administration of the D1-D2 interfering peptide as a new treatment option for MDD.
iii
Acknowledgments
I would like to thank my supervisor, Dr. Fang Liu, for her guidance and support over the
last 18-20 months. Her encouragement and positive attitude have made working in the lab a
pleasure. Her scientific guidance over the course of this project as well as her personal guidance
has been invaluable.
I would also like to thank my Masters’ committee members, Dr. Paul Fletcher and Dr.
Paul Frankland. Their expertise, positive encouragement and guidance significantly contributed
to my learning throughout this project and to its overall success.
Impel NeuroPharma, the company that developed the POD used throughout my study,
provided training and important input into the development of the protocol we used to administer
substances intranasally. I am grateful to them, especially to John Hokeman, for their patience and
encouragement.
Finally, I would like to thank my wonderful family, friends and roommates for their
encouragement, understanding and support throughout the last two years.
iv
Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
List of Abbreviations ..................................................................................................................... ix
1 Introduction .............................................................................................................................. 1
1.1 Dopamine neurotransmission in the mammalian brain ...................................................... 3
1.1.2 Dopaminergic pathways in the mammalian CNS ................................................. 10
1.1.3 Heterodimerization of Dopamine Receptors ........................................................ 16
1.2 Major Depressive Disorder ............................................................................................... 19
1.2.1 Epidemiology of MDD ......................................................................................... 20
1.2.2 Symptoms and clinical presentation of MDD ....................................................... 21
1.2.3 Treatments for MDD ............................................................................................. 22
1.2.4 Neurobiological changes and pathophysiology of MDD ...................................... 25
1.2.5 Preclinical models of MDD .................................................................................. 31
1.3 Intranasal delivery to the CNS .......................................................................................... 33
1.3.1 Mechanisms of intranasal delivery to the CNS ..................................................... 34
1.3.2 Experimental considerations for successful intranasal delivery to the CNS ........ 36
1.4 Rationale ........................................................................................................................... 36
1.5 Hypothesis ......................................................................................................................... 39
2 Materials and Methods .......................................................................................................... 41
2.1 Animals ............................................................................................................................. 41
2.2 Intranasal administration procedures ................................................................................ 41
2.2.1 Intranasal administration using the POD .............................................................. 41
2.2.2 Verification of POD delivery to the olfactory epithelium .................................... 42
2.2.3 Substances injected intranasally ........................................................................... 42
2.3 Intra-peritoneal injection procedures ................................................................................ 44
2.4 Immunofluorescence and confocal microscopy ................................................................ 44
2.4.1 Tissue fixation and storage ................................................................................... 44
2.4.2 Immunofluorescent staining procedures ............................................................... 45
2.5 The Forced Swimming Test .............................................................................................. 46
2.5.1 FST Procedure ...................................................................................................... 46
2.5.2 FST behavioral scoring method ............................................................................ 48
2.6 FST experiments: experimental design ............................................................................. 50
2.6.1 Effect of the D1-D2 interfering peptide in the FST .............................................. 50
2.6.2 Effect of the D1-D2-FLAG interfering peptide in the FST .................................. 50
2.6.3 Efficacy of the D1-D2 interfering peptide at various intranasal doses ................. 51
2.6.4 Duration of behavioral effect of D1-D2 interfering peptide in the FST ............... 53
2.7 Locomotor activity test ..................................................................................................... 53
2.9 Co-immunoprecipitation and western blots ...................................................................... 55
2.9.1 Tissue Collection .................................................................................................. 55
2.9.2 Co-Immunoprecipitation of D1 receptor by anti-D2DR ....................................... 56
2.9.3 Western Blots ........................................................................................................ 57
3 Results ..................................................................................................................................... 58
v
3.1 Experiment 1: The POD preferentially deposits substances on the olfactory
epithelium within the rat nasal cavity ............................................................................... 58
3.2 Experiment 2: The D1-D2-FLAG interfering peptide can be detected in the prefrontal
cortex after intranasal administration ................................................................................ 58
3.3 Experiment 3: Intranasal administration of the D1-D2 interfering peptide has an
antidepressant effect in the forced swimming test ............................................................ 60
3.3.1 The D1-D2 Interfering Peptide has an Anti-Immobility Effect in the FST .......... 62
3.3.2 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST
after intranasal administration ............................................................................... 64
3.4 Experiment 4: Efficacy of the D1-D2 interfering peptide at various intranasal doses ..... 67
3.4.1 D1-D2 interfering peptide dose: 4.0nmol/g (13.72 mg/kg) .................................. 67
3.4.2 D1-D2 interfering peptide dose: 2.0nmol/g (6.86 mg/kg) .................................... 68
3.4.3 D1-D2 interfering peptide dose: 1.67nmol/g (5.75 mg/kg) .................................. 68
3.4.4 D1-D2 interfering peptide dose: 1.0nmol/g (3.43 mg/kg) .................................... 71
3.5 Experiment 5: Duration of the behavioral effect of the D1-D2 interfering peptide ......... 75
3.5.1 Behavioral Effect in FST 2 hours after intranasal administration ........................ 75
3.5.2 Behavioral Effect in FST 3 hours after intranasal administration ........................ 75
3.5.3 Behavioral effect in the FST 4 hours after intranasal administration ................... 76
3.6 Experiment 6: The D1-D2 interfering peptide does not increase locomotor activity ....... 81
3.6.1 Overall locomotor activity .................................................................................... 81
3.6.2 Effect of time on locomotor activity during 30-minute test ................................. 82
3.7 Experiment 7: Intranasal administration of the D1-D2 interfering peptide disrupts the
interaction between dopamine D1 and D2 receptors in the PFC ...................................... 85
3.8 Experiment 8: The D1-D2 interfering peptide does not change the expression of
dopamine D1 or D2 receptors in the PFC ......................................................................... 85
3.8.1 Expression of Dopamine D1 receptors in the PFC after intranasal
administration of the D1-D2 interfering peptide .................................................. 87
3.8.2 Expression of Dopamine D2 receptors in the PFC after intranasal
administration of the D1-D2 interfering peptide .................................................. 87
4 Discussion ................................................................................................................................ 91
4.1 Overall Findings ................................................................................................................ 91
4.2 The POD delivers biologically active peptides to the CNS .............................................. 92
4.3 Mechanism of transport to the CNS after intranasal administration ................................. 94
4.4 The D1-D2 interfering peptide is effective at intranasal doses ≥ 5.75 mg/kg for up to
2 hours after intranasal administration. ............................................................................. 95
4.5 Possible neurobiological mechanisms of the D1-D2 interfering peptide’s
antidepressant effect .......................................................................................................... 97
4.6 Limitations of the FST as a preclinical test for antidepressant efficacy ........................... 99
4.7 The D1-D2 interfering peptide, TAT-peptide and imipramine significantly decrease
locomotor activity ........................................................................................................... 100
4.8 Future Directions ............................................................................................................ 104
References ................................................................................................................................... 107
Appendix 1: Sufficient intranasal D1-D2 interfering peptide dose to produce antidepressant
effect in the Forced Swimming Test (Calculation) ................................................................ 120
vi
List of Tables
Table 2-1 Efficacy of the D1-D2 interfering peptide at various doses: overall experimental
design and Treatment Groups. ...................................................................................................... 52
Table 2-2 Duration of the anti-immobility effect of the D1-D2 interfering peptide: treatment
groups and overall experimental design ....................................................................................... 54
vii
List of Figures
Figure 1-1 Dopamine receptors: structure and function ............................................................... 9
Figure 1-2 Schematic Representation of D1-D2R receptor interaction and activation of
intracellular signalling pathways. ................................................................................................. 18
Figure 1-3 Previous findings demonstrating the role of the D1-D2R interaction in MDD. ........ 29
Figure 2-1 Pressurized Olfactory Device (POD) for intranasal administration: apparatus ......... 43
Figure 2-2 Overall experimental procedure for FST. .................................................................. 47
Figure 2-3 Representative photographs of behaviors exhibited during the FST. ........................ 49
Figure 3-1 Representative images of deposition of Mark-It Blue tissue marker deposition after
correct POD administration .......................................................................................................... 59
Figure 3-2 Immunofluorescent staining for anti-FLAG antibodies is visible in PFC slices of
animals who were administered TAT-D1-D2-FLAG-IPep (A) but not those who were
administered saline (B). ................................................................................................................ 61
Figure 3-3 The D1-D2 interfering peptide has an antidepressant effect in the FST when
administered intranasally. ............................................................................................................. 63
Figure 3-4 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST. ..... 65
Figure 3-5 The D1-D2 interfering peptide and D1-D2-FLAG tagged interfering peptide have
similar behavioral effects in the FST. ........................................................................................... 66
Figure 3-6 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an
intranasal dose of 4.0nmol/g ......................................................................................................... 69
Figure 3-7 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an
intranasal dose of 2.0nmol/g ......................................................................................................... 70
Figure 3-9 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an
intranasal dose of 1.67 nmol/g. ..................................................................................................... 72
Figure 3-8 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST at
an intranasal dose of 1.0nmol/g .................................................................................................... 73
Figure 3-10 Efficacy of the D1-D2 interfering peptide at various doses in the FST: summary of
findings. ........................................................................................................................................ 74
Figure 3-12 The D1-D2 interfering peptide has an anti-immobility effect in the FST 2 hours
after intranasal administration. ...................................................................................................... 77
viii
Figure 3-11 The D1-D2 Interfering Peptide does not have an anti-immobility effect in the FST 3
hours after intranasal administration. ............................................................................................ 78
Figure 3-13 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST 4
hours after intranasal administration ............................................................................................. 79
Figure 3-14 The D1-D2 interfering peptide no longer has a behavioral effect in the FST 3 hours
after it is administered via intranasal injections. ........................................................................... 80
Figure 3-15 The D1-D2 interfering peptide does not increase locomotor activity during a 30-
minute open field test. ................................................................................................................... 83
Figure 3-16 The D1-D2 interfering peptide decreases overall locomotor activity but does not
change the activity pattern during a 30-minute open field test. .................................................... 84
Figure 3-17 Co-Immunoprecipitation of D1 by anti-D2R is reduced in the PFC of animals who
received intransal injections of TAT-D1-D2-IPep (Dose: 1.67nmol/g) ....................................... 86
Figure 3-18 Intranasal administration of the D1-D2 interfering peptide does not change the
expression of the dopamine D1 receptor in the PFC. ................................................................... 88
Figure 3-19 Intranasal administration of the D1-D2 interfering peptide does not change the
expression of the dopamine D2 Receptor in the PFC ................................................................... 89
Figure 3-20 Representative immunoblot of α-tubulin expression in rat PFC tissue. ................... 90
ix
List of Abbreviations
5-HT serotonin
AC adenyl cyclase
ADHD attention deficit hyperactivity disorder
ANOVA Analysis of Variance
anti α-tubulin immunoglobulin against α-tubulin protein
anti-cy2 immunoglobulin conjugated to cyanine 2 fluorescent dye
anti-D1DR immunoglobulin against dopamine D1 receptor
anti-D2DR immunoglobulin against dopamine D2 receptor
ATP adenosine triphosphate
BDNF brain derived neurotrophic factor
CaMKII calmodulin kinase II
cAMP cyclic adenosine monophosphate
cDNA complementary DNA (deoxyribonucleic acid)
CNS central nervous system
CSF cerebrospinal fluid
C-terminal carboxy-terminal of protein
D1 dopamine D1 receptor
D1-D2 dopamine D1-D2 receptor interaction
D2 dopamine D2 receptor
D2L dopamine D2 receptor - long isoform
D2S dopamine D2 receptor - short isoform
D3 dopamine D3 receptor
D4 dopamine D4 receptor
D5 dopamine D5 receptor
DA dopamine
DAT dopamine transporter protein
DDC DOPA decarboxylase
DSM-IV-TR Diagnostic and Statistical Manual, 4th edition, text revision (2000)
FLAG FLAG octapeptide (protein tag)
FST forced swimming test
GABA gamma-Aminobutyric acid
GABAAR gamma-Aminobutyric acid receptor type A
Gi/o G protein, α subunit type i/o
GPCR G protein coupled receptor
Gq G protein, α subunit type q
Gs
GSK-3β
G protein, α subunit type s
Glycogen Synthase Kinase 3β
GTP guanine triphosphate
Gα G protein, α subunit
x
HIV1 human immunodeficiency virus type 1
IGF-1 insulin-like growth factor 1
IP intraperitoneal injection
IP3 inositol triphosphate
kD kiloDalton
L-DOPA L-3,4-dihydroxyphenylalanine
LH learned helplessness
MAO monomaine oxidase
MAOI monoamine oxidase inhibitors
MCI mild cognitive impairment
MDD major depressive disorder
mg/kg milligrams per kilogram
mRNA messenger RNA (ribonucleic acid)
MSN medium spiny neuron
NAc nucleus accumbens
NE norepinephrine
NGF nerve growth factor
NMDARs n-methyl-D-aspartate receptors
nmol/g nanomoles per gram
OEC olfactory ensheathing cell
ORN olfactory receptor neuron
PBS phosphate buffered saline
PD Parkinson's Disease
PFA 4 % paraformaldehyde
PFC prefrontal cortex
PLC phospholipase C
POD pressurized olfactory device
RGP regulators of G proteins
SNpc substantia nigra pars compacta
SSRI selective serotonin reuptake inhibitor
STAR*D sequenced treatment alternatives to relieve depression clinical trial
TAT membrane permeable protein from HIV1
TAT-D1-D2-FLAG-Ipep TAT-linked membrane permeable D1-D2 interfering peptide with c-
terminal 8-amino acid FLAG tag
TAT-D1-D2-Ipep TAT-linked membrane permeable D1-D2 interfering peptide
TAT-Pep 9-amino acid membrane permeable peptide fragment from HIV1 TAT
protein
TH tyrosine hydroxylase
TrkB tyrosine receptor kinase type B
VMAT2 vesicular monoamine transporter 2
VTA ventral tegmental area
1
1 Introduction
The neurotransmitter dopamine is involved in many processes within the brain, including
motor control, cognition, reward, emotion and pleasure. Dopamine exerts its effects through five
unique dopamine receptors, termed D1 through D5. These receptors are G-protein coupled
receptors (GPCRs) that contain seven trans-membrane domains and initiate intracellular
signaling cascades.4 In addition to existing as unique receptors, dopamine receptors can also
couple with other proteins and receptors to form functional heterodimers that activate signaling
cascades, independent from those activated by each component receptor.5,6
Recently, scientific
evidence has shown that these heterodimers can play a pathological role in the progression of
psychiatric conditions.7 Dopamine D1 and D2 receptors couple in this manner and are thought to
play a role in psychiatric conditions such as Major Depressive Disorder (MDD).3,8
Our laboratory has found a pathophysiological role for the dopamine D1-D2 heterodimer
in MDD.3 MDD is a common, serious psychiatric condition that accounts for 4.4% of total global
disease burden9 and is often left undiagnosed and untreated in patients.
10-12 Furthermore, many
patients do not respond to available pharmacological or psychological treatment for MDD with
over 50% of patients not responding to first-line pharmacological treatment.13,14
Pei et al3
demonstrated that the D1-D2 heterodimer is up-regulated in the striatum of patients with MDD.
Disrupting this interaction using a membrane permeable peptide (the D1-D2 interfering peptide)
had an antidepressant effect in the Forced Swimming Test (FST) and the Learned Helplessness
(LH) task, two strongly validated preclinical tests for antidepressant efficacy.3 These results are
promising, but lack clinical validity since invasive, direct administration methods were used to
deliver the peptide to the prefrontal cortex (PFC). In order for the D1-D2 interfering peptide to
2
become a clinically relevant antidepressant treatment, a less invasive, clinically applicable
method of drug delivery must be developed.
The purpose of this project is to test whether we can effectively administer the D1-D2
interfering peptide to the brain using intranasal delivery. Intranasal delivery is clinically
applicable, offers a direct pathway to the brain and is a non-invasive method to target
therapeutics to the central nervous system (CNS). A number of proteins including insulin and
nerve growth factor have been delivered to the CNS intranasally, both in animals and in
humans.15
The goals of this project are to (1) confirm that the D1-D2 interfering peptide is able
to disrupt the interaction between D1 and D2 when administered intranasally, (2) test whether
the D1-D2 peptide has an antidepressant effect in the FST when administered intranasally, and
(3) further investigate the pharmacological properties of the D1-D2 interfering peptide.
In the introduction, I will briefly review the scientific literature relating the role of
dopamine in cognitive and behavioral processes within the mammalian brain as well as currently
held hypotheses about dopamine receptor heterodimerization and its role in psychiatric
conditions (Section 1.1). I will focus specifically on the Dopamine D1 and D2 receptor-receptor
interaction our laboratory has previously identified (Section 1.1.3). Next, the etiology,
symptoms, and neurobiology of MDD will be reviewed, along with currently available
antidepressant treatment options and their efficacy in treating this disorder (Section 1.2). I will
also discuss preclinical models of depression, and their strengths and weaknesses for identifying
new therapeutics for this complex psychiatric disorder (Section 1.2.5). Finally, I will discuss the
evidence supporting the use of intranasal administration methods to target therapeutic substances
to the CNS (Section 1.3).
3
1.1 Dopamine neurotransmission in the mammalian brain
Dopamine is a catecholamine neurotransmitter synthesized from the amino acid
tyrosine.16
Discovered in the mid-20th
century,17,18
dopamine and its role in the central nervous
system have been the subject of extensive scientific investigation. It is involved in a wide variety
of cognitive and behavioral processes in the brain, such as reward seeking and motivation,
voluntary motor movement, emotional and cognitive processing, attention, and working memory.
It also plays a role in numerous neurological and psychiatric illnesses, including but not limited
to Parkinson’s Disease19
, Huntington’s Disease20
, schizophrenia21
, major depression22
, and
addiction23,24
. For example, Parkinson’s Disease (PD) is a neurodegenerative disorder that
occurs due to a loss of dopaminergic neurons in the substantia nigra (SN) and dopaminergic
innervations to the striatum, a brain area involved in voluntary motor movements.19
Dopamine itself is a small organic compound made up of a benzene ring, an amine group
attached to a 3-carbon chain, and two hydroxyl groups.25
In the central nervous system,
dopamine is synthesized from the amino acid tyrosine in neurons containing the enzymes
necessary for this conversion. These neurons originate in three distinct areas: the substantia nigra
pars compacta (SNpc), the ventral tegmental area (VTA) and the arcuate nucleus of the
hypothalamus.16
Briefly, the amino acid tyrosine is converted into L-DOPA by the enzyme
tyrosine hydroxylase (TH), the rate-limiting enzyme in the production of dopamine.16
L-DOPA
is converted into dopamine by DOPA decarboxylase (DDC).16
Once synthesized, dopamine is
transported from the cytosol into synaptic vesicles by the vesicular monoamine transporter 2
(VMAT2), from where it is released into the synaptic cleft when dopamine neurons fire.4 Once
released into the synaptic cleft, it binds to and activates dopamine receptors on the post-synaptic
(and pre-synaptic) membranes. Subsequently, it can be transported back into the presynaptic
dopaminergic neuron by the dopamine transporter protein (DAT) for re-use or degradation.16
4
Dopamine is broken down into inactive metabolites by a sequence of reactions catalyzed by
monoamine oxidases (MAOs) resulting in the production of homovanillic acid, which is released
into the cerebrospinal fluid (CSF) as metabolic waste. Dopamine also serves as the precursor for
norepinephrine, as the two neurotransmitters differ by the addition of one β-hydroxyl group, a
reaction catalyzed by the enzyme dopamine β-hydroxylase.16
Presynaptic neurons that produce and release dopamine originate from distinct areas in
the basal ganglia and innervate cortical areas, the hippocampus and limbic cortex, and brain
areas related to movement and endocrine function (see Section 1.1.2).4 Like many
neurotransmitters, dopamine exerts its effects in the brain through specific receptors termed D1
through D5, G-protein coupled receptors that each have specific downstream effects in cells that
effect complex intracellular signaling pathways.26,27
Dopamine receptors can form functional
interactions with different types of dopamine receptors3,5,28
while also interacting with other
types of neurotransmitter receptors and a variety of other proteins to facilitate cross-talk between
neurotransmitter systems29-31
(Section 1.1.3). Unlike other neurotransmitters such as glutamate
and GABA, dopamine is not considered an excitatory or an inhibitory neurotransmitter, as the
ultimate effect of dopamine on a given neuronal population depends on the type of dopamine
receptor and the ultimate effect of the intracellular signaling cascade that is activated.
1.1.1 Dopamine receptors and their intracellular effects
As stated above, dopamine exerts its intracellular effects through five distinct receptors,
D1 through D5. These receptors belong to the guanine nucleotide-blinding (G-protein) coupled
receptors superfamily. G-proteins are signal transducers that mediate the transduction of
intracellular signals for a vast number of endocrine, neurotransmitter, autocrine and paracrine
compounds. There are four main types of G proteins, Gs, Gt, Gi and Go, each consisting of three
5
subunits (Gα, Gβ and Gγ).32
There are 20 known Gα, 6 Gβ and 11 Gγ subunits, and G-proteins
are typically named by the identity of their α-subunit.32,33
As a result of their heterogeneity, a
large number of GPCRs can form with resultant activation of vastly different intracellular
signaling pathways.32
Dopamine receptors, like all GPCRs, exert their downstream signaling effects by
activating (or inhibiting) intracellular second messenger cascades.25,34
In the case of dopamine
receptors, the receptor itself contains 7 transmembrane domains, and is coupled to G protein
subunits on the intracellular side (See Figure 1-1).4,25
After dopamine binds to its binding site,
the G-protein becomes activated and causes downstream intracellular effects, which underlie the
cognitive and behavioral changes mediated by dopamine in the brain.35
The existence of dopamine receptors was first proposed in the 1970s when Kebabian and
Greengard published evidence for a dopamine-selective adenyl cyclase36
(AC, an enzyme that
converts ATP to cAMP, a potent second-messenger signaling molecule).4 After the initial
discovery of cAMP-coupled dopamine receptors, Spano et al37
demonstrated that dopamine
receptors exist in two groups, one that is positively coupled to cAMP production and one that is
not.4 Based on their opposing effects on cAMP signaling, these receptors types were named D1
(cAMP-activating) and D2 (cAMP-inhibiting).26
Quickly, the hypothesis that dopaminergic
signaling was mediated by two dopamine receptors with opposing effects was proven to be an
oversimplification,35
as the advent of molecular biology and genetic cloning techniques allowed
for the identification of D1 and D2 cDNA38
and three additional distinct dopamine receptors
activated by dopamine: D339
, D440
and D541
. Currently, dopaminergic pathways in the CNS are
thought to be mediated by these five dopamine receptors, that are separated into two families
based on their effects on cAMP: D1-like and D2-like.4
6
1.1.1.1 D1-like dopamine receptors
The D1-like dopamine receptor family is comprised of dopamine D1 and D5 (formerly
D1B) receptors.41
When agonists bind to these receptors they activate the Gs/α family of GPCRs.
Activation of Gs/α results in activation of AC, and cAMP production.25
D1-like receptors can also
couple to Golf/α (also stimulating AC and cAMP production) in specific brain areas (caudate,
nucleus accumbens (NAc) and olfactory tubercle).42
The genes for both D1 and D5 receptors do
not contain introns in their coding sequences, and thus there are no splice variants of D1 or
D5.34,43
Some pharmacological differences exist between D1 and D5 receptors, as the D5
receptor is more pharmacologically sensitive to dopamine than D1 receptor.34
D1 receptors are the most common dopamine receptors in the CNS, and are highly
expressed in post-synaptic targets of the mesocortical, mesolimbic and nigrostriatal dopamine
pathways (see Section 1.1.2), including the striatum, NAc, amygdala and PFC.25,44,45
D5
receptors are expressed at lower levels in the PFC, the cingulate cortex, substantia nigra,
hypothalamus, hippocampus and dentate gyrus.45-47
D1 and D5 receptors are co-expressed in
pyramidal neurons of the prefrontal, premotor, and cingulate cortices and the dentate gyrus.48,49
Unlike the D2-like dopamine receptors, D1-like receptors are, for the most part,
expressed mostly in the post-synaptic membrane of neurons receiving dopaminergic input4,25
,
although recent evidence suggests that D5 is expressed presynaptically in the basolateral
amygdala and other brain structures.49
Thus, it is probable that dopamine D1-type receptors are
responsible for mediating the diverse effects of dopamine on its post-synaptic cellular targets.
For example, dopaminergic signaling through D1-type receptors in the PFC is critical to working
memory processes50
and to the occurrence of motor movements gated by basal ganglia circuits.51
7
1.1.1.2 D2-like dopamine receptors
The D2-like family of dopamine receptors includes dopamine D2, D3 and D4 receptors.
Originally identified as those receptors that were not coupled to AC activation and cAMP
production26
, they have since been found to play complex roles in various intracellular signaling,
cognitive and behavioral processes. When the dopamine D2, D3 and D4 receptors were cloned in
the early 1990s, it emerged that inhibition of AC was a general property of the D2-like
receptors, although the degree to which AC is inhibited varies by receptor subtype.25
This
property of D2-like dopamine receptors is mediated by coupling to the Gαi/o GPCR subunit,
which inhibits AC function.32,52
Dopamine D2-type receptors also activate intracellular signaling cascades independently
of G-protein activation. For example, the D2 receptor complexes with the regulatory protein β-
arrestin, protein phosphotase 2A and Akt ( a serine/threonine kinase), and this pathway regulates
the function of Glycogen synthase kinase 3β (GSK-3β).53,54
This signaling pathway typically
takes longer to become active, and stays active for a much longer period of time than the G-
protein mediated pathways. GSK-3β is also involved in signaling pathways activated by other
neurotransmitters, such as serotonin. The D2 receptor involvement in GSK-3β function may
represent a point where signaling from numerous neurotransmitters is integrated.4,54
Furthermore,
β-arrestin plays a role in GPCR desensitization and regulation, and thus, dopaminergic signaling
through D2-like receptors may also be involved in dopamine receptor desensitization processes.4
Unlike the D1-type dopamine receptors, the D2-like receptor coding genes contain intron
sequences, allowing for the generation of receptor splice variants.43
The most widely studied of
these splice variants are the D2S (short) and D2L (long) dopamine D2 receptors, generated by
alternative splicing of an 87-base pair exon between introns 4 and 5 of the D2 receptor gene.55,56
As a result, the D2L receptor isoform contains a 29-amino acid sequence in the third intracellular
8
loop that is missing in the D2S receptor isoform.55,56
Interestingly, these isoforms localize
differently within the CNS, with the D2L receptor isoform predominantly located in post-
synaptic targets of dopamine pathways and the D2S isoform located presynaptically, in
dopaminergic neurons.57
D2-like receptors are expressed presynaptically, indicating that they can act as
autoreceptors, providing an important negative feedback mechanism by modulating dopamine
synthesis, neuronal firing rate and dopamine release in response to extracellular dopamine
levels.4,25
The D2S receptor isoform, and not the D2L receptor isoform, of the dopamine D2
receptor is likely at least partially responsible for this autoregulation, as generation of a D2L -/-
transgenic model did not affect the ability of dopaminergic neurons to auto regulate.45,57,58
Dopamine D2 receptors are found in various brain areas, including the striatum, NAc, substantia
nigra, VTA, hypothalamus, cortical areas including the PFC and the hippocampus.39,59,60
D3
dopamine receptors are more limited in their expression than D2s, and are mostly expressed in
the limbic areas.60,61
D4 receptors have the lowest expression of all dopamine receptor subtypes,
but are expressed in the frontal cortex, amygdala, hippocampus, hypothalamus and other brain
areas.62,63
1.1.1.3 Regulation of dopamine receptors
After activation of dopamine receptors and activation of intracellular signaling cascades,
(e.g. cAMP in the case of D1 receptors), the downstream events initiated in the cell occur
regardless of whether dopamine remains bound to its receptor.4 To control these intracellular
signaling events, dopamine receptors are regulated through a number of mechanisms including
G-protein regulatory proteins (RGP family), phosphorylation of intracellular loops, receptor
sensitization and desensitization to agonist binding, and receptor internalization.5,25,32
The
9
D1-type and D2-type Dopamine receptors act on adenyl cyclase (AC) in opposing ways. D1-type receptors activate
AC via coupling with Gs/olf, while D2-type dopamine receptors inhibit AC via coupling with Gi/o G-proteins.
All dopamine receptors contain 7 trans-membrane domains, an extracellular N-terminus and intracellular C-terminal
tail. D2-like receptors have shorter C-terminal cytosolic tails and a larger third intracellular loops. Receptor
function is modulated in part by phosphorylation sites on intracellular loops and C-terminal tails, which can mediate
receptor desensitization and endocytosis. Figure Prepared with help from S.Chen, Liu Lab (2011).
Figure 1-1 Dopamine receptors: structure and function
10
regulators of G-protein family (RGP) typically increase the rate of G-protein GTP hydrolysis,
decreasing the amount of time the proteins spend active, modulating the efficacy of G-protein
mediated signaling.64
For example, activation of protein kinase A by cAMP elevations in
response to D1-type activation will phosphorylate residues on the C-terminal tail of D1
receptors, and initiate the recruitment of adaptor proteins (typically, β-arrestins) that prevent
further G-protein activation.65
Arrestins can also recruit proteins such as clatherin and β-adaptin
that mediate receptor endocytosis, which can reduce signaling in response to high extracellular
levels of dopamine.66
Dopamine receptors are also regulated by their interactions with other
proteins and other transmembrane receptors (see Section 1.1.3), which can change the receptor’s
affinity for agonists and the intracellular pathways activated by each component receptor.5
1.1.2 Dopaminergic pathways in the mammalian CNS
Although neurons producing dopamine are relatively few in number in the brain, they
project extensively to numerous cortical and subcortical structures. There are four main
dopaminergic pathways in the CNS: the nigrostriatal, mesocortical, mesolimbic and
tuberoinfundibular.25
Each pathway plays an important role in the functions of its target areas
and creates a complex system of dopamine-modulated circuits within the brain. The functions of
the mesolimibic (Section 1.1.2.2) and mesocortical (Section 1.1.2.3) dopaminergic pathways
will be the focus of this review, as these are the most relevant to this project and to the
pathogenesis of MDD. The role of the nigrostriatal pathway in motor behavior will be briefly
discussed (Section 1.1.2.1). The tuberoinfundibular pathway, in which dopamine functions as a
neuroendocrine hormone to inhibit prolactin secretion from the anterior pituitary16
, will not be
reviewed here.
11
1.1.2.1 The nigrostriatal dopamine pathway
The nigrostriatal pathway originates in the SNpc and projects to the striatum.16,67
The
striatum plays a major role in the gating of motor movements, and the vast majority of the
neurons originating from the striatum are GABA-ergic (inhibitory) Medium Spiny Neurons
(MSNs).16
Striatal neuronal firing activity has opposing functions: depending on the area that the
neurons project to, it can control both the direct (favoring movement) and indirect (favoring no
movement) basal ganglia circuits.68
D1, D2 and D3 receptors are all involved in the effects of dopamine on motor
movements.4 When dopamine D1 receptors on MSNs are activated and converge with cortical
premotor inputs, the firing of MSN projections “disinhibits” the thalamus, favoring the
behavioral occurrence of that movement.25,51
The role of D2 and D3 receptors in the gating of
locomotor activity is more complex than that of D1 receptors, as they function both as
presynaptic auto-receptors (decrease dopamine release when activated) and post-synaptic
receptors.4,25
In PD, the dopaminergic neurons originating in the SNpc are gradually lost,
gradually reducing dopaminergic input to the striatum, resulting in the symptoms of stiffness and
reduced movement in PD.16,19
1.1.2.2 The mesolimbic dopamine pathway
The mesolimbic dopamine pathway originates in a second brain area containing
dopaminergic neurons, the VTA.69
This pathway projects predominantly the NAc (also known as
the ventral striatum).70
The NAc is highly interconnected with other limbic areas including the
amygdala, cingulate cortex, parahippocampal gyrus, hippocampal formation, anterior thalamic
nuclei and NAc.16
The amygdala, the area most involved with fear and emotional experience,
12
sends neuronal projections to the hypothalamus, which, among other functions, is thought to
regulate the physiological and endocrine changes associated with emotional states.16
Animal models of disrupted mesolimbic dopaminergic input to the limbic areas and
amygdala have demonstrated that dopamine transmission in the amygdala is associated with the
acquisition of Pavlovian-conditioned fear responses.71
Both D1-type and D2-type dopamine
receptors seem to play a role in fear conditioning. Briefly, D1/D5 antagonists diminish
conditioned fear responses69
, and D1 agonists potentiate them72
, indicating that dopamine
signaling via D1 receptors potentiates fear responses in the amygdala. Paradoxically, both D2-
type agonists and antagonists, impair fear conditioning and recall of emotional memory.71
Dopaminergic input to the NAc is highly involved in behavioral reinforcement
mechanisms and reward-dependent learning.16
Most addictive recreational drugs such as
amphetamine, cocaine and nicotine, along with naturally rewarding experiences such as food and
social interactions increase the levels of dopamine in the NAc at the terminals of dopaminergic
projections originating from the VTA.73
Evidence from rodents, non-human primates and human
neuroimaging studies strongly suggest that the mesolimbic system is involved in cue association
to positive rewards, and the reinforcement of behaviors that result in acquisition of a reward.74
The increased levels of dopamine in the NAc are thought to contribute to the strong
reinforcing and addictive properties of recreational drugs and other substances.74-76
Di Chiara et
al75
found that drugs with aversive effects reduced dopamine release in the NAc, implying that
dopamine release is correlated with hedonistic, reinforcing events. In fact, many recreational
drugs enhance dopamine neurotransmission, either by blocking DAT (cocaine and
amphetamine), enhancing release of dopamine through pre-synaptic modulation (nicotine) or
inhibiting inhibitory, GABA-ergic neurons that suppress dopaminergic neurons in the VTA (mu-
opioid agonists).77
Due to the involvement of the dopaminergic system in the CNS response to
13
recreational drugs and other hedonistic experiences, dopaminergic system dysfunction is thought
to play a major role in the pathophysiological changes that accompany addiction.23,24
The reward circuit modulated by dopamine neurotransmission has also been implicated in
the pathogenesis of MDD and other psychiatric conditions. This is not surprising, given the
symptoms of anhedonia, lack of motivation present in MDD and the role of the mesolimibic
pathway in reward-based learning, motivation and emotional processing.22,78
Dopamine signaling
in the VTA – NAc mesolimbic reward circuit modulates motivation for rewards and pleasure,
implying that these common symptoms of MDD could be due to pathological changes in this
circuit.79,80
Interestingly, dopamine does not, as was initially suggested, code for “pleasure” in the
mesolimibic reward pathway.77,81
Studies in mice missing tyrosine hydroxylase (the rate-limiting
enzyme in dopamine synthesis) show that these mice still have hedonic preferences, preferring
sweetened water over unsweetened.77,82
At the same time, dopamine-deficient mice did not seek
rewards during reward-directed tasks, that is, although they enjoyed the reward, they did not seek
it out.83
These studies support the hypothesis that dopamine in the NAc is required for “wanting”
a reward, but not for “liking” it, that is, dopamine signaling encodes incentive salience, leading
to the modification of behavior in order to obtain the reward.77,84,85
The large number of studies
investigating mesolimbic dopaminergic signaling strongly suggest that dopamine is involved in
motivation, reward-based learning and emotional processing in the limbic areas, and that
dysfunction within the mesolimbic system could lead to addiction, MDD and other psychiatric
illnesses.
14
1.1.2.3 The mesocortical dopamine pathway
The mesocortical dopamine pathway consists of dopaminergic neurons originating in the
VTA and projecting to the PFC, insular cortex and cingulate cortex.86
The PFC is highly
involved in higher-order cognitive processing including motivation, planning, attention to salient
stimuli, decision making, behavioral flexibility, and working memory. Working memory is
conceptualized as the manipulation of a number of items in short-term memory storage in order
to effectively plan and organize future thought or actions.86
Early findings by Brozoski et al87
demonstrated that depletion of dopamine in the PFC of monkeys produced cognitive and
working memory deficits comparable to those observed when the frontal lobes were completely
removed. Subsequent research into the role of dopaminergic neurotransmission in working
memory indicated that an optimal range of dopaminergic signaling in the PFC existed, where
“too little” or “too much” signaling through D1 receptors increased errors in the radial arm maze
and other working memory tests mediated by the PFC.50,88,89
The PFC also modulates behavioral flexibility, or the ability to alter behavior in response
to changing environmental conditions. A common test for behavioral flexibility and set-shifting
is the Wisconsin Card Sort Task, which requires the human or animal to disregard a previously
beneficial strategy (e.g. sort cards by shape) and engage in a novel one (e.g. sort cards by color)
to obtain a reward.50,86
Patients with damage to the dorsolateral PFC are unable to alter their
sorting strategy when they are required to organize cards by another dimension, a finding
replicated in non-human primate90,91
and rodent92-94
versions of dimensional set-shifting tasks
and reversal learning (where the animal must discriminate between two or more stimuli, only one
of which is relevant to reinforcement) tasks.50,86
In microdialysis studies that measured PFC
dopamine levels in freely-behaving rats during a set-shifting task, dopamine levels in the PFC
15
increased when the rat had to shift to a different rule in conflict with the first, indicating a role
for DA signaling in behavioral flexibility.50,95
Both attention deficit hyperactivity disorder (ADHD) and schizophrenia patients show
marked impairments in set-shifting, 96,97
and both disorders are associated with various changes
in the mesocortical dopaminergic pathway.98
Pharmacological treatments for ADHD such as
methylphenidate, which increases mesocortical dopamine transmission, are able to decrease
impairments in set shifting seen in patients.95
These clinical findings suggest that dopaminergic
systems are involved in modulating behavioral flexibility, but the exact mechanisms through
which this modulation occurs remain unknown.
The PFC is also very important in decision making processes, specifically when weighing
the advantages and disadvantages of a given choice. Bechara et al99
demonstrated that patients
with damage to the ventromedial PFC were impaired on behavioral tasks designed to simulate
real-life decisions, and the uncertainty and rewards involved.98
In rodents, one can model this
cost/benefit decision making by manipulating the cost (i.e. increasing the delay to reward
delivery, increasing the amount of physical activity required, or making reward delivery
probabilistic) of a reward (typically more, or better, food).98
These different forms of cost/benefit
decision making are regulated by anatomically-distinct regions of the PFC, and all are sensitive
to manipulations in dopamine PFC levels.100,101
Both D1-type and D2-type dopamine receptors
seems to be implicated in cost/benefit decision making paradigms, but they seem to play a
complex role, with their specific function dependent on PFC area and the type of cost/benefit
decision being made.50,102,103
Dopaminergic signaling in the PFC occurs through both D1-type and D2-type dopamine
receptors. In both rodent and monkey PFC, the distribution of D1 receptor messenger RNA
(mRNA) is significantly greater than the other dopamine receptor subtypes.104
Both D1 and D2
16
receptors are found on excitatory, glutamatergic pyramidal neurons and non-pyramidal, GABA-
ergic interneurons in the PFC.86,105,106
In fact, a subset of layer V pyramidal neurons
(approximately 25 %)107
as well as non-pyramidal PFC neurons express both D1 and D2
receptors, indicating that these receptors may co-localize within these cells.8,105,106
Beyond
cellular and sub-cellular expression patterns of dopamine receptors in the CNS, little is currently
understood regarding how intracellular signaling pathways activated by dopamine receptors
eventually modulate the higher-order cognitive processes mediated by the PFC.
1.1.3 Heterodimerization of Dopamine Receptors
After the cloning of the five distinct dopamine receptor subtypes in the early 1990s,
structural, pharmacological and biochemical studies suggested that each receptor had unique
properties, although they fell into the two previously described families of dopamine receptors
(D1-like and D2-like).4 Over the last 20 years, it has become apparent that dopamine receptors
function both as independent entities and form heterodimers with members of the same family
and with structurally divergent families of receptors.5 The pharmacological and functional
profiles of dopamine receptor heterodimers are often very different from that of the component
receptors and these are thought to contribute to the numerous heterogeneous functions of
dopaminergic signaling in the CNS.5,6
Dopamine receptors have been shown to form
heterodimers through direct protein-protein interactions between D1 and D2 receptors,3,108-110
D1 and D3 receptors in the striatum111,112
, D2 and D5 receptors109,113
, D1 receptors and NMDA
receptors (NMDARs) 30,114
and D5 and GABA-A receptors115
, among other transmembrane and
cytoplasmic proteins.
All dopamine receptors subtypes form non-obligatory heterodimers, that is, dimerization
is not necessary in order for the receptor to function.4 However, a large degree of complexity in
17
the signaling effects of dopamine receptors results from their ability to heterodimerize.5 For
example, dopamine receptor heteromers could create novel ligand binding sites, activation of one
or both component receptors could initiate different intracellular signaling pathways than those
initiated by the component receptors, or a synergistic increase in signaling could occur when
both agonists are present.5 The D1-D2 receptor interaction and the activation of independent
intracellular signaling pathways that occurs will be the focus of this section, as this interaction is
implicated in the pathogenesis of MDD3 and is the target of the D1-D2 interfering peptide used
in this project.
1.1.3.1 The Dopamine D1-D2 Receptor Interaction
An interaction between the dopamine D1 and D2 receptors was first investigated because
of the observation that a D1-like receptor could activate Inisitol Phosphate 3 (IP3) production
(leading to increases in intracellular calcium) in various brain regions including striatum,
hippocampus and cortex.116,117
An interaction between dopamine D1 and D2 receptor was
proposed because of the observations that the presence of calcium signaling activated by D1 was
absent in D1-transfected cells, and present in cells transfected with both D1 and D2 receptors.2
Research from our laboratory identified the specific regions through which D1 and D2 receptors
interact as a 15 amino acid sequence within the 30-amino acid insert in the third intracellular
loop of the D2L receptor isoform, and the D1 intracellular C-terminal tail.3 This finding provided
indirect evidence that the D1-D2 receptor interaction occurs in post-synaptic membranes, as both
D1 and D2L dopamine receptors are generally localized to post-synaptic areas.25,57
18
Figure 1-2 Schematic representation of D1-D2R receptor interaction and activation of
intracellular signaling pathways.
(A) Activation of D1 or D2 when those receptors are in complex is thought to activate PLC, resulting
in release of calcium from the endoplasmic reticulum and subsequent activation of CamKII.1,2
(B) The
D1-D2 interfering peptide (TAT-D1-D2-IPep) disrupts the interaction between D1 and D2L receptors,
resulting in disruption in the Gq-mediated downstream signaling pathways.3 (A) and (B) prepared with
help from S.Chen, Liu Lab.
19
Research by George and colleagues2,118
confirmed the interaction between D1 and D2
using co-immunoprecipitation and through fluorescence resonance energy transfer (FRET)
techniques. They also demonstrated that the D1-D2 receptor complex induces intracellular
calcium via a Gq GPCR-dependent pathway in the striatum.119
In the Gq-pathway, Phospholipase
C (PLC) becomes activated by Gq, resulting in an increase in inisitol triphosphate (IP3) which
then causes activation of downstream molecules resulting in increased calcium concentration in
the cytoplasm (Figure 1-2A).32,119
The colocalization of D1 and D2 receptors seems to occur in a
number of brain regions including the dorsal and ventral striatum, and the PFC.105-108
Although the intracellular pathway activated by the D1-D2 receptor heterodimer is
characterized, the physiological relevance of this interaction and its role in neurological and
psychiatric illnesses is not yet clear. Recently, our laboratory demonstrated that the D1-D2
interaction may have a role in the pathogenesis of MDD. Most importantly, the D1-D2 receptor
interaction was up-regulated in post-mortem samples from the striatum of patients with MDD,
implying that this interaction may be disrupted in this illness.3 Uncoupling the D1-D2 receptor
interaction using a small, membrane permeable peptide (TAT-D1-D2-IPep) results in an
antidepressant effect in animal model of depression (Figure 1-2B, Figure 1-3).3 This study
suggests that the D1-D2 receptor interaction may play a role in the pathogenesis of MDD, and
warrants further investigation into possible therapies based on disrupting this interaction.
1.2 Major Depressive Disorder
MDD is the most common psychiatric illness in the world, with the lifetime incidence in
the United States 12% in men and 20% in women.120
Despite its prevalence, many patients who
have MDD are not adequately treated with current antidepressant therapies. Since the discovery
of the first antidepressant compounds over 50 years ago, much progress has been made
20
investigating the neurobiological changes underlying MDD.121
Although numerous hypotheses
attempt to explain the neurobiological and pathological changes underlying the clinical
presentation of MDD, no unitary hypothesis explaining all the pathological changes and the
complex symptoms observed in MDD exists. Here, the epidemiology (Section 1.2.1) and
symptoms of MDD (Section 1.2.2) will be reviewed, along with currently available treatments
and their efficacy in treating MDD (Section 1.2.3). Next, a number of different hypotheses
regarding the pathophysiology underlying MDD will be explored (Section 1.2.4). Since the
treatment in our current investigation targets a protein-protein interaction between D1 and D2
dopamine receptors, the evidence for the involvement of the dopaminergic system in MDD will
be reviewed (Section 1.2.4.2). To conclude, the use of preclinical models to model MDD and
test new antidepressant treatments will be discussed (Section 1.2.5).
1.2.1 Epidemiology of MDD
Depression is a common psychiatric illness characterized by 2 or more weeks of a distinct
change in mood, sadness and/or constant irritability, as well as feelings of hopelessness and loss
of interest in pleasurable activities.122
The lifetime incidence of depression in the United States is
12% in men and 20% in women120
. In fact, women are 70% more likely than men to experience
depression in their lifetimes.123,124
Although depression is closely related to the normal emotions
of sadness, it often does not regress when the external cause of these emotions dissipates, and
can be disproportionate to their cause.78
Often, episodes of depression will recur two or more
times and become classified as MDD.122
At its most severe, MDD can lead to suicide attempts,
which can result in loss of life or significant disability.
MDD is responsible for 4.4 % of the worldwide disease burden and, is the leading cause
of disability worldwide when considering total years lost to disability.125
Depression often goes
21
undiagnosed and untreated because of the prevalent societal stigma associated with psychiatric
conditions and seeking treatment for these conditions. According to statistics from the National
Institute of Mental Health, only 57% of patients with MDD in the United States are receiving
any kind of treatment for the disorder, and only 19% of patients with MDD are receiving
adequate treatment.120,126
Additionally, MDD can often occur in conjunction with other serious illnesses such as
cancer, chronic pain, epilepsy and cardiovascular disease.9,120,127-129
When this is the case, both
MDD and the co-morbid illness are adversely affected, as treatment outcomes in patients who
have diabetes, epilepsy or ischemic heart disease along with MDD have poorer outcomes than
those without MDD.130
Overwhelmingly, epidemiological data regarding the prevalence of MDD
indicates that it is extremely common, often undiagnosed and, in the majority of cases, not
adequately treated.
1.2.2 Symptoms and clinical presentation of MDD
The Diagnostics and Statistical Manual of Mental Disorders IV (DSM IV-TR)122
criteria
for MDD are the most commonly used criteria for MDD diagnosis. A single depressive episode
is categorized by the presence of five or more of the following symptoms during a two-week
period where at least one of the symptoms is either depressed mood most of the day, nearly every
day, or loss of interest or pleasure in almost all activities.122,131
Recurrent MDD occurs when a
patient experiences two or more major depressive episodes, with a symptom-free period of two
or more months separating them. Other symptoms include a change of more than 5% in body
weight over the course of 1 month, insomnia or hypersomnia, psychomotor agitation or
retardation, persistent fatigue and loss of energy, feelings of worthlessness or guilt, diminished
ability to think or concentrate, indecisiveness, and/or recurrent thoughts of death or
22
suicide.8,122,131
These symptoms can cause significant impairment in the patient’s social,
occupation and other functioning. MDD and its symptoms are often variable in both clinical
presentation and severity, which may contribute to the large number of patients left undiagnosed
and untreated.120
1.2.3 Treatments for MDD
Patients diagnosed with MDD are typically treated with pharmaceutical agents that
increase the amount of monoaminergic neurotransmitters at the synapse. Currently, the “first-
line” pharmaceutical therapy for MDD is a class of drugs termed selective serotonin re-uptake
inhibitors (SSRIs).78
When given to patients, these drugs produce an increase in the
neurotransmitter serotonin in the brain by inhibiting its re-uptake into the presynpatic neurons
from where it was released.9 In the CNS, serotonergic neurons project to numerous cortical and
subcortical areas from the brainstem raphe nuclei and are involved in regulation of mood,
appetite, sleeping behavior, learning and memory.78
Serotonin act through serotonin receptors, of
which there are seven families that have diverse effects in cells.16
SSRIs quickly increases the total amount of serotonin available at serotonergic synapses
and, as such, increase the amount of serotonin-mediated neurotransmission in the brain.132
The
side-effects of SSRIs are often apparent almost immediately after the initiation of treatment,
while any therapeutic antidepressant effect from these medications takes approximately three
weeks to become apparent in patients.78
The delay in onset of any therapeutic effect suggests that
the ability to increase serotonin at the synapse may not be the only mechanism through which
SSRIs have an antidepressant effect and that they may be mediating other, longer-term effects in
the CNS responsible for its therapeutic efficacy.8,133
23
In a large clinical trial for antidepressant efficacy, the Sequenced Treatment Alternatives
to Relieve Depression (STAR*D) trial, it was shown that only 27- 33 % of patients with MDD
adequately responded to first line treatment with citalopram, an SSRI medication.134
If, after a
number of weeks of citalopram or other SSRI treatment, little or no improvement on a
standardized rating scale such as the Hamilton Depression Rating Scale135
, is observed, patients
can be treated by increasing the SSRI dose (if side effects are tolerable), switched to a new SSRI,
or started on another antidepressant along with the original SSRI.9 Studies have also indicated
that SSRI efficacy is correlated with the severity of depression when treatment is initiated,
implying that SSRIs are more effective for patients with severe depression and may not be
significantly more effective than placebo for patients with mild or moderate depression.136,137
For patients who do not respond to SSRIs, pharmaceutical treatment alternatives include
SNRIs (Selective norepinephrine reuptake inhibitors), Triple reuptake inhibitors (inhibit re-
uptake of serotonin, dopamine and norepinephrine)138
, tricyclic antidepressants such as
imipramine, and monoamine oxidase inhibitors (MAOIs). MAOIs exert their antidepressant
effect by blocking the breakdown of monoaminergic neurotransmitters (serotonin,
norepinephrine and dopamine) and are effective in the treatment of MDD, but also have strong
and often intolerable side effects.139,140
A final, invasive option in the case of severe, unremitting
MDD is electro-convulsive therapy, which remains the most effective treatment for severe,
unremitting depression.139,141
1.2.3.1 Efficacy of current antidepressant treatments: The STAR*D trial
The STAR*D trial134
is a large clinical trial designed to investigate remission rates after
antidepressant treatment in a large and generalizable sample of patients. In the first level of the
trail, all patients enrolled were treated with citalopram (an SSRI) as a first-line treatment, with
24
their depressive symptoms evaluated every 2 weeks after initiation of treatment.134,142
Between
28 and 33 % of patients treated with citalopram achieved remission within 12 to 14 weeks of
treatment.134
In Level 2 of the STAR*D trial, those patients who did not respond to citalopram
treatment after 14 weeks were given the choice between pharmacotherapy augmentation,
psychotherapy or switching to a different SSRI medication for 12 weeks.14
Of the patients in
Level 2, approximately 30% achieved remission of symptoms within 12 weeks, with no
significant differences in remission rates with any of the treatment strategies employed.14,142
In
Levels 3 and 4 of the trial, patients who had not responded to antidepressant treatments or
psychotherapy in Level 1 or 2 were treated with alternative pharmacotherapies, including
tricyclic antidepressants. In Level 3 and 4 of the STAR*D trial, the remission rats dropped
substantially, ranging from 12 % to 25%, depending on the treatment used.142
In all, only 67% of
patients originally enrolled in the STAR*D achieved remission of their depressive symptoms.142
The STAR*D trial reveals that almost one third of patients with unipolar depression do
not respond to multiple trials of SSRI and other antidepressant treatments. After the first two
levels of the trial, patients were much less likely to respond to further pharmaceutical treatment
trials, indicating the importance of achieving a treatment response with the first few
antidepressants prescribed to patients.142
The STAR*D trial also provides a strong rationale for
further investigation into new antidepressant therapies that could help treat MDD in the subset of
patients who currently do not respond to available antidepressant treatments.
25
1.2.4 Neurobiological changes and pathophysiology of MDD
Currently, there is no unitary hypothesis that explains the various pathological and
neurobiological changes that occur in MDD. What remains clear, however, is that MDD is a
heterogeneous disorder with complex pathological mechanisms that vary considerably between
individuals affected by the disease. In human neuroimaging studies, the brain regions that are
consistently found to be involved in MDD are the PFC, the cingulate cortex (area Cg25), the
hippocampus and the amygdala.79,143,144
These findings are consistent at both the structural level,
where magnetic resonance imaging (MRI) data and other neuroimaging data suggests decreased
hippocampal and PFC volume in depressed patients, and the functional level, where functional
MRI (fMRI) and positron emission tomography studies suggest abnormal connectivity and
decreased functionality of limbic, cingulate and prefrontal areas in depression.143
The monoamine and catecholamine neurotransmitters, serotonin, dopamine and
norepinephrine innervate these areas through extensive axonal projections from the dorsal raphe
nucleus (serotonin), VTA (dopamine) and locus coeruleus (norepinephrine).16,73
Disruptions in
these systems are thought to contribute to the pathogenesis of MDD (Section 1.2.4.1). More
recent investigations into the neurobiological mechanisms behind MDD have also demonstrated
that neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) also play a large
role in MDD (Section 1.2.4.2). The evidence implicating dopamine in the pathogenesis of MDD
will be reviewed, as the D1-D2 interfering peptide used in this project specifically targets the
heterodimerization of two dopamine receptors (Section 1.2.4.3). Although these theories of
MDD all attempt to explain the complex symptoms of the disease, it is currently unclear how
neurobiological, genetic, societal and environmental factors result in the complex and variable
clinical presentation of MDD.
26
1.2.4.1 The Monoaminergic deficiency hypothesis of depression
The most widely accepted hypothesis of the neurobiological basis of depression is the
monoamine deficiency hypothesis. This hypothesis states that depressive symptoms occur due to
a decrease in the amount of monoaminergic neurotransmitters in brain areas implicated in MDD
such as the prefrontal and limbic cortex.78,138
The hypothesis was first proposed in the 1960s
because of the antidepressant actions of two structurally unrelated compounds (tricyclic
compounds and MAOIs). Both tricyclic compounds and MAOIs were found to increase overall
levels of monoamines in the brain, thereby increasing mood in patients being treated with
them.145
The early MAOI – type antidepressants inhibited MAOs, enzymes that break down
serotonin, dopamine or norepinephrine in the presynaptic neuron, rendering these
neurotransmitters inactive.78
Due to their action on MAOs, MAOIs increase the available stores
of monoamines in the CNS, which is the proposed mechanism for their antidepressant effect and
the basis of the monoaminergic hypothesis of MDD.
There is extensive clinical and pre-clinical evidence that suggests that serotonin,
dopamine and norepinephrine are highly involved in the neurobiology of MDD, and that a
sustained deficiency in any one could result in depression.78,138
The clinical efficacy of SSRIs,
SNRIs, and triple reuptake inhibitors, which all increase the availability of monoaminergic
neurotransmitters in the synapse, supports the monoaminergic hypothesis of MDD
pathophysiology. Although these drugs all produce immediate, substantial increases in serotonin
and other monoamines in the brain any antidepressant effect takes a number of weeks to become
apparent.9,134
Thus. it seems probable that the longer-term, antidepressant effects of SSRIs and
other antidepressants are due to adaptive responses in the brain secondary to the effects of
increasing monoaminergic neurotransmission.133
Furthermore, most antidepressant therapies
27
currently in use are based on increasing monoamine levels, and are only effective in
approximately 50% of patients with MDD.121
It is probable that a more complex pathological mechanism underlies MDD than the
monoaminergic deficiency hypothesis. Human dietary studies suggest that depleting tryptophan
stores (rate-limiting for synthesis of serotonin in the brain) or depleting TH (required for
catecholamine synthesis) does not cause depressive symptoms in healthy subjects, but can cause
relapse in patients previously treated for MDD.146
Furthermore, post-mortem studies on human
brain tissue from patients with MDD have not consistently shown decreases in brain monoamine
levels.79,147,148
These findings suggest that monoamines and catecholamine neurotransmitter
levels play an important role in MDD, but depleting these neurotransmitters alone may not be
sufficient to cause MDD.78
Thus, the monoaminergic deficiency hypothesis of MDD may be too
simplistic to explain the pathological changes and clinical presentation of MDD.
1.2.4.2 Role of dopamine in the pathophysiology of MDD
The majority of theories regarding the neurobiology of MDD focus on the role of
disruptions in serotonin and norepinephrine neurotransmitter systems. Disruptions in brain
dopamine levels, as well as changes in dopaminergic neurotransmission have also been identified
as factors contributing to in the neurobiology of MDD, and are the target of a number of
clinically effective antidepressants.8 For example, nomifensine and bupropion block the reuptake
of norepinephrine and dopamine and are both effective antidepressants when used alone or in
conjunction with other antidepressant treatments.149
The involvement of dopamine in MDD is
also supported by clinical studies, as the turnover rate of dopamine, as measured by CSF or
plasma levels of dopamine metabolite homovanillic acid, is decreased in patients with MDD
compared with controls.22,150
28
Many of the common clinical symptoms of MDD, such as anhedonia, loss of ability to
concentrate, flattened affect and motor changes are present in disorders in which dopaminergic
signaling is disrupted, such as PD and schizophrenia.151
Cognitive and behavioral functions
modulated by the mesolimibic and mesocortical pathways such as emotional processing,
planning, motivation and other executive functions, can be impaired in patients with MDD,
implying that dopaminergic signaling in areas such as the cingulate, prefrontal and limbic
cortices may be disrupted in MDD.80,152
Preclinical models of depression have also been used to study the role of dopamine in
MDD. In the LH model of depression, dopamine levels are reduced in the caudate nucleus and
the NAc of animals with MDD, and depressive-like behavior can be prevented by treatment with
a dopamine agonist prior to the behavioral task.22,153,154
In the FST, a commonly used test for
antidepressant efficacy, dopamine agonists and DAT blockers tend to increase mobility,
indicating that these substances have antidepressant-like effects.22,155
Furthermore, dopamine D1
and D2 receptor antagonists can inhibit the effects of antidepressants in the chronic
unconditioned stress model of MDD.156
It is apparent that dopamine is implicated in the
pathology of MDD, but given the complexities of dopaminergic signaling and its role in
prefrontal and limbic processes, we do not have a complete understanding of these mechanisms.
Our laboratory has recently shown that heterodimerization of the dopamine D1 and D2 receptors
is up-regulated in patients with MDD, and disrupting this interaction has an antidepressant effect
in preclinical models of depression (See Figure 1-3 for a summary of these findings) 3,8
Despite
these promising findings, the mechanism by which the D1-D2 receptor interaction is involved in
the neurobiology of depression is unclear. Overall, it is clear that dopamine and dopaminergic
signaling is disrupted in MDD, and may be involved in the pathogenesis of the disorder,
29
Figure 1-3 Previous findings demonstrating the role of the D1-D2R interaction in MDD.
(A) The D1-D2 interaction is significantly increased in post-mortem samples from patients with MDD as assessed
by Co-immunoprecipitation of D1 by anti-D2R. (B) A 15-maino acid, membrane permeable peptide capable of
disrupting the interaction between D1-D2LR (TAT-D2-Il3-29-2) has an anti-immobility effect in the FST when
infused directly into the PFC. (C) Co-imunoprecipitation of D1 by anti-D2R is significantly decreased in the PFC
after infusion with the D1-D2 Interfering peptide (TAT-D2-Il3-29-2). Figures prepared by Pei et al. (2010) and used by
F.Liu in conference presentations. This data was also published, in different figures, in Nature Medicine 16, 1393-
1395 (2010).3
30
although we currently do not have a complete understanding, or a unitary hypothesis, to explain
dopamine’s role in this complex illness.
1.2.4.3 The Neurotrophic Hypothesis of Depression
Neurotrophic factors such as Brain Derived Neurotrophic Factor (BDNF) are expressed
in the brain and act through transmembrane receptors (tropomyosin receptor kinase B receptors
(TrkB)) to promote neuronal survival.157
The neurotrophic hypothesis of MDD postulates that in
MDD, expression of neurotrophic factors such as BDNF is altered in the brain. These alterations
in BDNF expression may be caused by chronic stress often associated with MDD78
, and result in
decreases in BDNF, which may account for its expression in brain areas involved in MDD
pathogenesis.157,158
This hypothesis is supported by the observed decrease in adult neurogenesis
markers in the hippocampus and the PFC of patients with MDD who had committed suicide.159-
161 Antidepressants may cause long-term changes in BDNF expression as post-mortem and
serum BDNF levels increased in the hippocampus and cortex after long-term antidepressant use
compared with patients not taking antidepressants.162
Rodent models of MDD have provided contradictory evidence about the role of BDNF
and other neurotrophic factors in MDD. Infusing BDNF into the hippocampus and surrounding
brain areas has an antidepressant effect in rodent models of depression.163,164
Diverse
antidepressant pharmacological agents increase signaling pathways activated by the BDNF
membrane receptor, TrkB, in a BDNF-mediated manner.165,166
However, male mice who were
knockouts for the BDNF membrane receptor, TrkB, did not exhibit endogenous depressive
behavior in a number of preclinical tests for depression and anxiety,167
and hippocampal BDNF
infusions in male rats did not prevent learned helplessness behaviors.168
Interestingly, BDNF
infusion directly into the VTA and NAc, two areas highly involved in the mesolimibic and
31
mesocortical dopamine pathways, had a pro-depression effect and enhanced social aversion
behaviors in mice.169,170
Since BDNF works to promote neuronal survival (among many other
functions) in areas where it is expressed, it is likely that its role in the neurobiology of MDD
depends on the brain area where it is elevated, and its underlying functions. As such, considering
it as an “antidepressant” in the classical sense oversimplifies its role in the neurobiology of
MDD.
1.2.5 Preclinical models of MDD
Clinical studies investigating the pathological mechanisms in MDD can only provide
correlative, and not causative, clues into this complex disorder. New pharmacological treatment
options for MDD must be validated using preclinical models of MDD to ensure their efficacy
and safety before being tested in the clinical setting. However, accurately modeling a complex,
multi-faceted disease like MDD with unknown neurobiological mechanisms in a laboratory
preclinical animal model is a challenging task. As such, the development of reliable animal
models that model one symptom of MDD (versus attempting to model the spectrum of human
MDD symptoms) has been more useful in the laboratory setting.171
A number of criteria for the validity of animal models of MDD have been proposed.
These include strong predictive validity (i.e. all antidepressants that are clinically effective
produce a similar ‘antidepressant-like’ effect in the behavioral model), that the behavioral output
of the model is reliable within and between laboratories and that a similarity between the
behavioral output in animals and clinical symptoms of depression is present.171-173
In general,
animals must be exposed to some type of stress, either acutely or over a sustained period, in
order to produce depressive-like symptoms.171
In animals, chronic stress can result in
helplessness (inability and unwillingness to escape from a stressful situation), anhedonia (lack of
32
interest in otherwise pleasurable activities), or social aversion (avoidance of other animals).79,171
These behaviors resemble specific human symptoms of depression, albeit in a simplified manner.
Although a number of well-validated models of depression exist including social aversion tests,
and chronic unconditioned stress paradigms,171,172
only the FST and the LH task will be
discussed here, as the D1-D2 interfering peptide used in this study was shown to have an
antidepressant effect using these models.
The FST is a two-day acute test of antidepressant efficacy developed by Porsolt et al.174-
176 The FST is not considered a chronic model of depression, as it is an acute test used to screen
substances that may act as antidepressants.171
Briefly, the animal is placed in an inescapable
plexiglass cylinder for 15 minutes before being given a treatment intervention. The next day, the
animal is replaced in the cylinder for a 5 minute period, and its behavior is scored. An animal
that didn’t receive an antidepressant treatment will display ‘behavioral despair’ or ‘helplessness’
and will assume a floating, immobility posture for the majority of the 5-minute test.176,177
On the
other hand, an animal that receives pharmacological agent with antidepressant properties will
remain active and try to escape for the majority of the 5-minute test (this is referred to as an anti-
immobility effect).175,176
The FST is a useful test because it has high predictive validity, as all
antidepressants currently used in the clinic have an anti-immobility effect in the FST.176,178
On
the other hand, it is not considered an animal model of MDD because it bears little resemblance
to the etiology and symptoms of MDD in patients.
Another widely used depression model is the LH task, a 5-day test in which the animal is
exposed to inescapable shock. Subsequently, its passive response to subsequent shocks (from
which the animal can escape) is measured.179,180
This model is thought to have clinical relevance
and etiological validity since evidence exists indicating that stressful life events perceived as
uncontrollable (such as death of a loved one and romantic breakups) are major predictors of
33
MDD onset and severity.180-182
This clinical finding can be replicated in rodents by controlling
the onset and duration of uncontrollable, aversive events, as is done in the LH paradigm.183,184
The LH model also has pharmacological validity, as administration of substances that act as
antidepressants over the 5-day task results in an increase in the animal’s escape attempts
compared to untreated animals.3,180,185-187
Thus, the LH model is a useful model of both
helplessness in human MDD and has pharmacological validity as a test for novel antidepressant
therapies.
1.3 Intranasal delivery to the CNS
The nasal anatomy, both in humans and rodents, contains a number of features that make
it attractive as a delivery pathway for proteins and peptides targeted to the CNS.15,188
A number
of properties of the vasculature in the CNS limit the entry of molecules, ions, pathogens and
toxins into the CSF, and are together called the Blood Brain Barrier (BBB).189
Tight control of
the extracellular environment in the CNS provided by the BBB is required to maintain proper
neuronal function and prevent injury within the CNS. At the same time, the BBB makes it
difficult to effectively deliver therapeutic substances unable to cross it, such as foreign peptides
and proteins, to the CNS and the brain.189
A number of features of the nasal anatomy make
intranasal administration of peptides and proteins an effective way to bypass the BBB and
specifically deliver these substances to the CNS.15,188
A number of protein and peptide therapies have been effectively delivered to the CNS
using the intranasal pathway. For example, in recent years, intranasal administration of insulin
has been shown to slow memory impairments in rodent models of Alzheimer’s disease (AD).190-
192 A recent meta-analysis suggested an overall beneficial effect of intranasal insulin on cognitive
functions in human trials of intranasal insulin delivery in healthy patients, patients with mild
34
cognitive impairment (MCI) and those with AD, with few detectable side effects and low
systemic levels of insulin.193
Other protein and peptide therapies that have been successfully
delivered to the CNS after intranasal delivery include nerve growth factor (NGF)194,195
other
neurotrophic factors such as BDNF196
, Insulin-like Growth factor 1 (IGF-1)197,198
and numerous
other proteins and drugs (reviewed in Dhuria et al15
).
Furthermore, a TAT-linked membrane permeable peptide similar in size to the D1-D2
interfering peptide was effectively delivered to the CNS using the intranasal approach.196
In fact,
the authors196
found that less than 10% of the intravenous dose administered intranasally resulted
in equivalent brain concentrations of their 22-amino acid TAT-linked peptide. The success of
intranasal insulin delivery to the CNS, along with other proteins and peptides that were delivered
successfully to the CNS after intranasal delivery, suggests that a direct pathway exists between
the nasal olfactory epithelium and the central nervous system.
1.3.1 Mechanisms of intranasal delivery to the CNS
Both the human and the rodent nasal anatomy have several features that make it
conducive to drug transport to the CNS while minimizing systemic exposure to the drug (see
Figure 1-5). The olfactory nerve pathways that connect the olfactory sensing region of the nasal
cavity to the olfactory bulbs and other CNS areas are important for intranasal drug delivery to the
CNS. For example, a fluorescently-labeled 3 kiloDalton (kD) Dextran allowed visualization of
the olfactory pathway after intranasal administration, and demonstrated that the Dextran was
transported to the olfactory bulbs along olfactory nerve pathways in approximately 15
minutes.199
Olfactory receptor neurons (ORNs) are responsible for conveying information about
odors to the CNS.16
ORNs are bipolar cells whose cell bodies are located within the olfactory
35
epithelium, with chemoreceptor-containing dendrites extending into the nasal mucosal layer and
axons travelling via the cribiform plate and olfactory nerve bundle into the CNS and olfactory
bulb.200
The cribiform plate of the ethmoid bone contains many small perforations that allow
ORN axons to extend into the CNS, effectively bypassing the BBB.15,200
Extracellular channels between the olfactory ensheathing cells (OECs) (which protect
the axonal projections of the ORNs) and the ORN axons allow drugs and peptides administered
intranasally to access the CSF and brain directly.15,188
Intracellular transport mechanisms along
the ORN axons may be important for certain substances201,202
but are not currently thought to be
the predominant mode of transportation into the CNS. This is because most intranasal delivery
studies, particularly of proteins and peptides, have demonstrated rapid transport from the
olfactory epithelium and nasal cavity into the CNS, suggesting that these substances are
transported extracellularly via the channels between OECs and ORNs.203-205
In addition,
intracellular transport of intranasally administered substances requires uptake of the substance
into the ORNs, necessitating receptor-mediated transport mechanisms, or the ability of the
substance to cross the phospholipid bilayer, which cannot account for the large variety of drugs,
proteins and peptides that have successfully been delivered to the CNS using the intranasal
route.15
Other potential transport mechanisms from the nasal cavity to the CNS include transport
via the trigeminal nerve pathways and via vascular pathways. The trigeminal nerve innervates
the respiratory and olfactory epithelium of the nose and enters the CNS in the brainstem.200
It is
possible that substances administered to the nasal cavity are also transported to the CNS via this
pathway, as this has been demonstrated for radioactively-labeled IGF-1 and other proteins and
peptides.203,206,207
Secondly, the nasal passages are highly vascularized structures, and
intranasally administered substances can be absorbed into the bloodstream through the
36
endothelial cells making up the capillary wall.15
In order for successful delivery to the CNS to
occur after absorption into the systemic circulation, substances must cross the BBB. This
approach is not thought to mediate the transport of proteins and peptides to the CNS after
intranasal delivery, because they lack the ability to cross the BBB in appreciable amounts.15
1.3.2 Experimental considerations for successful intranasal delivery to the
CNS
For successful delivery of proteins and peptides to the CNS using the olfactory pathway,
experimental considerations including intranasal administration technique, head position and
drug formulation must be taken into account. The vast majority of preclinical studies involving
intranasal administration to rodents have administered substances intranasally in anaesthetized
animals. To optimize delivery to the CNS, the substance being administered must reach the
olfactory epithelium and upper third of the nasal cavity, and different head positions can alter
absorption of substances into the bloodstream and CSF.208
In anaesthetized rodents, a number of
studies have demonstrated that intranasal administration targeted to the CNS can be achieved via
the insertion of flexible tubing into the nostrils, localizing delivery to the olfactory epithelium
and surrounding tissue.208-210
The Pressurized Olfactory Device (POD) used in our present study
combines delivery to the olfactory epithelium using flexible tubing and aerosolized delivery to
deliver substances preferentially to the olfactory epithelium and surrounding tissue, favoring
delivery to the CNS.210,211
1.4 Rationale
After the discovery of the antidepressant effects of MAOIs and tricyclic antidepressants
nearly 60 years ago9, there has been extensive investigation into the pathogenesis of MDD in the
37
human brain and new pharmacological approaches to treating it. Most effective antidepressant
therapies target the monoaminergic (serotonin, norepinephrine and dopamine) neurotransmitter
systems. A disadvantage of these therapies is that they broadly increase monoaminergic
signaling, resulting in a number of unpleasant side effects in patients. These often appear
immediately, a couple of weeks before any appreciable benefit of the antidepressant becomes
apparent.9 As many as 50% of patients do not respond to current antidepressant treatments,
121
necessitating new and more effective therapeutic options for this disorder. New approaches to
treating MDD, and the development of new therapies targeting specific pathological changes
occurring in MDD, are necessary in order to advance the treatment as well as our scientific
understanding of this illness.
Peptides specifically designed to disrupt pathological interactions between
neurotransmitter receptors are promising therapeutics for psychiatric and neurological disorders
because they allow for specific targeting of these pathological interactions.8 They disrupt the
pathological interaction between the two proteins without having an effect on either receptor’s
independent function, minimizing the likelihood of side-effects. Our laboratory has
demonstrated that an interfering peptide specifically designed to disrupt the dopamine D1-D2
receptor heterodimer has a significant antidepressant effect in the FST and the LH task, a
preclinical model of depression, when given directly to the PFC of rats.3 Pei et al
3 also
demonstrated that the interaction between dopamine D1 and D2L receptors is significantly
increased in post-mortem striatal samples from patients with MDD compared with controls. This
finding suggests that the efficacy of the D1-D2 interfering peptide in preclinical models of
depression is relevant to the clinical pathogenesis of this disorder, warranting further
investigation into its pharmacological and biochemical functions.
38
Although the previous findings from our laboratory are promising, they are not yet
clinically applicable, as the administration methods (direct microinjections to the brain and
intracerebral ventricular (ICV) injections) Pei et al3 used are extremely invasive and not feasible
in the clinical setting. For this interfering peptide, and any other peptide that has a therapeutic
effect in animal models of psychiatric or neurological conditions, to translate to the clinical
setting a non-invasive, clinically applicable method of administration must be tested and
developed.
A major challenge in the development of novel therapies for psychiatric or neurological
disorders is successfully and non-invasively delivering them to the central nervous system,
without substantial accumulation of these therapies in the systemic circulation.15
In the last 20
years, intranasal delivery of peptide and proteins targeted to the CNS has been extensively
studied (for review, see Dhuria et al.)15
This relatively non-invasive approach exploits the
weakened blood-brain barrier at the olfactory epithelium to deliver therapeutic substances such
as peptides and proteins to the central nervous system.192,195,196
Many substances, including TAT-
linked membrane permeable peptides212
, insulin190,192
, IGF-1203
and NGF195
, have been
successfully delivered to the CNS using the intranasal route. The POD used to administer the
D1-D2 interfering peptide intranasally is designed to preferentially deposit substances on the
olfactory epithelium within the nasal cavity, favoring absorption of substances into the CNS.
Studies on intranasal delivery to the CNS have also indicated that the intranasal pathway
preferentially delivers substances to anterior brain regions such as the olfactory bulbs, PFC and
adjacent areas.15
Since many patients suffering from MDD do not respond to current antidepressant
therapies, or cannot tolerate these therapies because of aversive side effects, newer and better
therapeutic options must be investigated. For this novel therapeutic approach targeting the D1-
39
D2 interaction in MDD to become a relevant treatment, translational studies in rodents must be
carried out investigating non-invasive methods to deliver the D1-D2 interfering peptide to the
CNS.
This project is designed to test whether the D1-D2 interfering peptide can be successfully
delivered to the central nervous system, and the PFC in particular, using the intranasal approach
and whether it will have an antidepressant effect in the FST after intranasal delivery. We will
also investigate the pharmacological properties of the D1-D2 interfering peptide, including the
intranasal doses required to observe an antidepressant effect in the FST and the amount of time it
remains biologically active in the body. Overall, this project will indicate whether the intranasal
pathway is a viable method to deliver peptides like the D1-D2 interfering peptide to the CNS,
and better inform whether the D1-D2 interfering peptide is suitable for further development as a
novel treatment for depression.
1.5 Hypothesis
Based on our laboratory’s previous findings3 that the D1-D2 interfering peptide, when
infused directly into the PFC, has an antidepressant effect in the FST and the LH task,3 we
hypothesize that it will have this same effect after intranasal delivery. In order for this hypothesis
to be correct, a number of criteria must be met.
First, we hypothesize that the POD used in this study preferentially deposits substances
on the olfactory epithelium, favoring uptake into the CNS via olfactory nerve pathways, and that
after intranasal delivery, we will be able to visualize a FLAG-tagged D1-D2 interfering peptide
in the PFC.
Second, the efficiency of delivery of peptides and protein therapies to the CNS is largely
unknown, but some studies have suggested that between 1 and 5% of the dose delivered
40
intranasally reaches the CNS and anterior brain areas.15
Based on these estimates, we
hypothesize that the D1-D2 interfering peptide will be effective in the FST at a dose 100-fold
larger than that delivered directly to the PFC.
Third, since we currently do not have any information about the efficacy of the D1-D2
interfering peptide after intranasal administration, we will also test whether it is effective at
doses higher or lower than our original 100-fold dose. We predict that at doses 100-fold or
greater than those given directly to the PFC in the previous study, the D1-D2 interfering peptide
will have an antidepressant effect in the FST. Along these same lines, we will investigate the
length of time the D1-D2 interfering peptide remains biologically active in the body, as we are
unsure about how long it remains stable in the CNS once it is administered intranasally.
Finally, the D1-D2 interfering peptide displayed an antidepressant effect when it was
delivered directly to the PFC, but not to other brain areas such as the NAc and hippocampus3.
We hypothesize that if the D1-D2 interfering peptide has an antidepressant effect after intranasal
administration, it will be due, at least in part, to the ability of the D1-D2 interfering peptide to
disrupt the D1-D2 receptor-receptor interaction in the PFC. Taken together, these experiments
will further our understanding of the intranasal delivery route for small interfering peptides as
well as reveal whether the D1-D2 interfering peptide is a promising novel therapeutic for the
treatment of MDD.
41
2 Materials and Methods
2.1 Animals
Adult Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were
used in all experiments. Rats were pair-housed at a constant temperature (20-23°C) on a 12-hour
light/dark cycle (light on at 8:00AM) with unrestricted food and water. After arriving at the
facility, rats were given 1 week to acclimatize before being subjected to behavioral testing and
injections. All rats weighed between 300 and 350g when they underwent behavioral testing. All
experimental procedures were approved by the Animal Care Committee at the Centre for
Addiction and Mental Health (Toronto, ON).
2.2 Intranasal administration procedures
2.2.1 Intranasal administration using the POD
All animals were anaesthetized using 5% isoflurane, an inhalant anesthetic (Benson
Medical Industries, Inc.) for 3-4 minutes. Rats were then placed in a supine position and dosed
with the POD developed by Impel NeuroPharma (Seattle, Washington). When dosing the
animals, the POD tip (with the relevant dose) was inserted approximately 8-10mm into the rat’s
nostril, angled towards the olfactory epithelium (towards the top of the head) and the propellant
can was fired for 1 second. 2 seconds later, the POD tip was slowly removed. The propellant can
and the POD tip were attached by a 30cm-long piece of plastic tubing (see Figure 2-1). This
allowed for maximum maneuverability of the POD tip in the nose. This protocol was adapted
from the POD administration procedure originally developed by Impel NeuroPharma.213
42
After intranasal administration animals were replaced in the anesthetic chamber in the
supine position for 4 minutes at decreasing isoflurane concentrations. This step allows for
maximum absorption of the peptide dose, while decreasing sneezing and other behaviors that
would result in expulsion of the substance from the nose. Animals were then replaced in their
home cages and returned to their housing rooms once they had recovered from the anesthesia and
had regained complete locomotor control. Rats that bled after intranasal administration were
eliminated from the study. The time of injection was recorded as the time at which the animals
woke up from the anesthesia and regained locomotor control.
2.2.2 Verification of POD delivery to the olfactory epithelium
In order to verify that the POD deposits substances preferentially onto the olfactory
epithelium, we gave intranasal injections (IN) of Richard Allen Scientific Mark-It Blue Dye
(5000BL, Thermo Scientific). The Mark-It blue dye allowed us to visualize where within the rat
nose the POD was preferentially depositing substances. Using the POD, we gave 10uL per
nostril using the administration method outlined in Section 2.2a). After POD administration,
animals were immediately sacrificed using trans-cardiac perfusion of 1X Phosphate Buffered
saline (pH=7.4). The nasal anatomy was then dissected and examined for traces of Mark-IT Blue
Dye. After dissection, the nasal anatomy was photographed for qualitative evaluation of the
deposition of substances by the POD.
2.2.3 Substances injected intranasally
In each experiment, animals received the same total number of intranasal injections (IN)
(3-4) in alternating nostrils over a 24-hour period. Animals received either filtered saline (0.9%
9- NaCl, 8-12 L/injection), the D1-D2 interfering peptide (TAT-D1-D2-IPep) (8-12µL, 50mM);
43
Figure 2-1 Pressurized Olfactory Device (POD) for intranasal administration: apparatus
(A) POD apparatus: the Propellant can is attached to the administration tip with a 30cm long piece of plastic
tubing. This allows for maneuverability when inserting the administration tip into the nose and administering the
intranasal dose. (B) Optimal position of the administration tip within the nose for preferential dose deposition
onto the olfactory epithelium.
44
a amino acid membrane permeable TAT peptide (TAT-Pep) (8-12 µL, 50mM) from the Human
Immunodeficiency Virus 1(HIV1) TAT protein214
; or D1-D2-FLAG tagged interfering peptide
(TAT-D1-D2-FLAG-IPep) (8-12µL, 50mM). The 9 amino acid TAT-peptide sequence from the
HIV1 TAT protein (YGRKKQRRR)214
rendered all peptides used in these studies cell-
permeable. All peptides were custom synthesized by Gen Script (New Jersey, USA) and/or
Biomatik, Inc (Cambridge, Ontario) and had purity levels between 95 and 99%. All peptides
were dissolved in filtered saline at a concentration of 50mM and stored at -80°C.
2.3 Intra-peritoneal injection procedures
We administered imipramine hydrochloride (15mg/mL, Sigma-Aldrich) at a dose of
15mg/kg using intra-peritoneal (IP) injections into the abdominal cavity. To control for
anesthetic exposure, these animals were also anaesthetized before administration of IP injections.
Rats were anaesthetized in an induction chamber using 5% isoflurane. Rats were placed in a
supine position and given imipramine via IP injections. After the injection, animals were
replaced in the induction chamber at decreasing isoflurane concentrations for 4 minutes. Animals
were allowed to recover in their home cages and the time of injection was recorded as the time
when the rats regained locomotor control.
2.4 Immunofluorescence and confocal microscopy
2.4.1 Tissue fixation and storage
The purpose of this study was to test whether a biologically active peptide that has an
anti-immobility effect in the FST, can be visualized in the PFC and after intranasal
administration. To do this, we used a peptide with the same sequence as the D1-D2 interfering
45
peptide3, but with an 8-amino acid FLAG-tag (Sequence: DYKDDDDK)
215 fused to the C-
terminal of the peptide. This modification allowed us to use immunofluorescent (see section
2.4.1) methods to detect it in the brain after intranasal administration and completion of the
FST.216
For this experiment, a small number of animals were sacrificed by transcardiac perfusion.
After being anaesthetized for 4 minutes in an induction chamber with 5% isoflurane, animals
were perfused transcardially with 60mL phosphate buffered saline (PBS, pH =7.4) followed by
60mL 4% Paraformaldehyde ( 4% PFA, in PBS). Subsequently, whole brains were dissected and
stored in 4% PFA overnight. The next day, brains were transferred to a 20% sucrose
cryoprotection solution for approximately 48 hours. The tissue was then stored at -80°C for
subsequent use.
2.4.2 Immunofluorescent staining procedures
Rats that had completed the FST and were assigned to the TAT-D1-D2-FLAG-IPep or
saline treatment groups were sacrificed, their tissue fixed and collected as described in Section
2.4.1. The olfactory bulbs and PFC of 2 brains from each condition (TAT-D1-D2-FLAG-IPep
and saline) were cut into 12 M sections using a cryostat. After slicing, we blocked non-specific
antibody interactions using 5% donkey serum for 1 hour, before staining overnight with an anti-
FLAG monoclonal antibody (mouse monoclonal, M2, Sigma-Aldrich). Sections were then
incubated with a secondary immunofluorescent antibody (donkey anti-mouse Cy2-conjugated
antibody, Jackson Immuno Research Laboratories, Inc.) before being counter-stained with
NeuroTrace 530/615 red fluorescent Nissl Stain (Molecular Probes, Invitrogen). Sections were
mounted on slices with PureGold anti-fade mounting reagent (Molecular Probes, Invitrogen.)
and stored at 4°C.
46
Sections were visualized and imaged using a Zeiss LSM 510 confocal microscope.
Images of PFC coronal sections in both conditions were taken under 25X magnification. Cy2
immunofluorescence was imaged using an argon laser with maximum excitation at 488nm. To
detect the NeuroTrace 530/615 stain, we used a helium 1 laser with maximum excitation at 530
nm. Images were overlaid using Image J software.
2.5 The Forced Swimming Test
2.5.1 FST Procedure
The FST is an acute test for antidepressant efficacy originally developed by Porsolt et al.
in 1977.174
On the first day of the test, animals undergo a training session where they are placed
in an inescapable plexiglass cylinder for 15 minutes. The plexiglass cylinders were 60cm high
and 20cm in diameter. The plexiglass cylinders were filled to a height of 40 cm with water at a
temperature of 25 +/- 0.2°C, which was changed between each animal (See Figure 2-3 for a
picture of the FST cylinder).
In accordance with the dosing schedule established in the literature and used
previously3,174-176
subjects were given the treatment intervention three times after the training and
testing sessions: 30 minutes after the 15-minute training session, 5 hours after training and 1 hour
before undergoing the FST. 24 hours after the training session and 1 hour after the last
behavioral intervention, the rat was replaced in the same cylinder for 5 minutes. The session was
video recorded and scored blindly at a later date. After both the training and test FST sessions,
rats were towel-dried and placed in a heated cage for a minimum of 15 minutes. See Figure 2-2
for a detailed schematic of the FST experimental design.
47
Figure 2-2 Overall experimental procedure for FST.
See section 2.5.1 for detailed description of the FST
experimental procedure
48
2.5.2 FST behavioral scoring method
The 5-minute session of the FST was video recorded and animal behavior during the FST
was scored at a later date after the experimenter was blinded to the treatment groups. All FST
videos were scored by the same experimenter who had been blinded to the experimental
conditions of the animals. Behaviors were scored in five-second bins with the predominant
behavior (immobility, swimming, climbing, diving) in each 5-second period recorded for a total
of 60 behavioral counts.
The animal’s activity during the FST was segregated into four behaviors, in keeping with
the literature and previous studies3,174,176
: immobility, which consisted of the animal only making
those movements necessary to keep its nose above the water; swimming, consisting of active
movement of the forepaws and legs and movement around the cylinder; climbing, consisting of
vigorous movement of the forepaws along the sides of the cylinder, as if trying to climb out of it;
and diving, when the animal entered head-first into the water and spent a minimum of 2 seconds
completely submerged. Figure 2-3A-D displays representative photographs of each of these
behaviors.
Mean immobility counts across all groups were analyzed by 1-way independent groups
analysis of variance (ANOVA). Post-hoc Newman-Keuls multiple comparisons tests were used
to evaluate differences across individual groups, as necessary. In some cases, other behaviors
(swimming, diving and climbing) were also analyzed using 1-way independent groups
ANOVAs, followed by post-hoc Newman-Keuls multiple comparisons tests to evaluate
differences between treatment groups. Data were analyzed using Prism (GraphPad Software,
Inc.).
49
Figure 2-3 Representative photographs of behaviors exhibited during the FST.
Top L to Bottom R: immobility, or passive floating with nose out of the water, swimming,
actively moving forepaws and legs to remain afloat, climbing, vigorous movement of
forepaws and legs in tandem, as if to climb up the walls of the FST cylinder, and diving,
head-first submersion of the entire body in the FST cylinder.
50
2.6 FST experiments: experimental design
2.6.1 Effect of the D1-D2 interfering peptide in the FST
We tested whether the D1-D2 interfering peptide would have an anti-immobility effect in
the FST when administered at a dose 100-fold that which was administered directly to the PFC3
(for detailed calculations of the D1-D2 interfering peptide intranasal dose, see Appendix 1.)
Animals were randomly assigned into four treatment groups: TAT-D1-D2-IPep (IN, 1.67nmol/g,
n=7), TAT-Pep (IN, 1.67nmol/g, n=6), saline (IN, 1.67nmol/g, n=6), and imipramine (IP,
15mg/kg, n=6). This group was included in order to confirm that the 9-amino acid, membrane
permeable TAT peptide fragment did not have any behavioral effect in the FST. We followed the
2-day FST protocol and behavioral analysis procedure outlined in Section 2.5. These treatments
were given 30 minutes after the 15-minute FST training session, 5 hours later and 1 hour before
the 5-minute FST test. The 5-minute FST behavioral tests were video-recorded, scored and
analyzed as described in Section 2.5.2.
2.6.2 Effect of the D1-D2-FLAG interfering peptide in the FST
The purpose of this study was to test whether a biologically active peptide that can be
visualized in the PFC areas after intranasal administration also has an anti-immobility effect in
the FST. To do this, we synthesized a peptide with the same sequence as the D1-D2 interfering
peptide3, but with an 8-amino acid FLAG-tag (Sequence: DYKDDDDK)
215 fused to the C-
terminal of the peptide, tested whether it had an anti-immobility effect in the FST and whether
we could visualize it in the PFC.
To test whether the D1-D2-FLAG interfering peptide had an anti-immobility effect in
the FST (for detailed procedure, see Section 2.5), animals were randomly assigned into four
treatment groups: TAT-D1-D2-IPep-FLAG (IN, 1.67nmol/g, n=4), TAT-Pep (IN, 1.67nmol/g,
51
n=4), saline (IN, 1.67nmol/g, n=5), and imipramine (IP, 15mg/kg, n=4). We followed the 2-day
FST protocol and behavioral analysis procedure outlined in Section 2.5. The 5-minute FST
behavioral tests were video-recorded, scored and analyzed as outlined in Section 2.5.2.
2.6.3 Efficacy of the D1-D2 interfering peptide at various intranasal doses
In order to better understand the pharmacological effects of the D1-D2 interfering peptide
upon intranasal administration, information regarding its efficacy at various doses is required. To
address this, we varied the dose of the D1-D2 interfering peptide and analyzed their anti-
immobility effects in the FST. We used saline and imipramine groups as a negative and positive
control, respectively. We followed the general FST design and behavioral analysis procedure
outlined in Section 2.5. Table 2-1 details the treatment groups, doses and the overall
experimental design employed in this study.
Immobility counts during the 5-minute FST in each treatment dose was compared with
saline and imipramine groups using 1-way independent groups ANOVAs, followed by post-hoc
Newman-Keuls multiple comparisons tests. From our results, we identified the minimum dose of
the D1-D2 interfering peptide with the maximal behavioral effect as 1.67nmol/g, which we used
in all subsequent experiments. In order to control for any behavioral effect of the TAT-peptide,
we included two treatment groups that received TAT-pep at a dose of 1.67 nmol/g and 2.0nmol/g
in order to test whether the TAT-peptide’s effect in the FST changes if the dose is increased.
52
Table 2-1 Efficacy of the D1-D2 interfering peptide at various doses: overall experimental design
and Treatment Groups.
Treatment Doses, nmol/g (mg/kg), number of animals per group
saline
Volume-
Controlled
n=4
n=8 (same
group as
1.0nmol/g)
n=6
n=8 (same
group as
2.0nmol/g)
Imipramine
15mg/kg, IP
n=3
n=8 (same
group as
1.0nmol/g)
n=6 n=8 ( same
group as
2.0nmol/g)
TAT-Pep n=7
2.0
nmol/g
n=6
1.67nmol
/g
TAT-D1-D2-
IPep
4.0nmol/g
(13.72mg
/kg)
n=3
2.0nmol/g
(6.86mg/
kg)
n=7
1.67nmol/g
(5.75mg/
kg)
n=7
1 nmol/g
(3.45mg/k
g)
n=7
53
2.6.4 Duration of behavioral effect of D1-D2 interfering peptide in the FST
It is unclear how long the D1-D2 interfering peptide remains active in the CNS after
intranasal administration. To test this, we compared the anti-immobility effect of the D1-D2
interfering peptide in the FST at 2, 3 and 4 hours after intranasal administration. We used the
general FST protocol and analysis procedure outlined in Section 2.5. The overall experimental
design and treatment groups are shown in Table 2-2.
Each animal in this study received intranasal or IP injections 30 min and 5 hrs after the
FST training session while the time point of the last intranasal injection was increased to 2, 3 and
4 hours before the 5-minute FST session. The amount of immobility behavior in treatment
groups was compared using 1-way independent groups ANOVA at each time point (2h, 3h and
4h). Saline and imipramine groups were compared to at all time points (2h, 3h, and 4h).
2.7 Locomotor activity test
We tested whether the increased mobility we observed in the FST after intranasal
administration was due to the antidepressant effect of the D1-D2 interfering peptide or due to an
overall increase in locomotor activity. To do this, we tested whether the D1-D2 interfering
peptide had an effect on activity during a 30-minute locomotor activity test after intranasal
administration. 25 rats that had already been exposed to the FST were used in this experiment.
Rats were given TAT-D1-D2-IPep (IN, 2.0nmol/g, n=5); TAT-D1-D2-IPep (IN, 1.67nmol/g,
n=5); saline (IN, n=5); TAT-Pep (IN 2.0nmol/g, n=5); or 15mg/kg imipramine (IP, n=5) three
times before the open field test: 24 hours before the test, 19 hours before the test, and one hour
before testing. This dosing schedule was identical to that used in the forced swimming
54
Treatment Time point before FST, number of animals/group
Saline 1 hour before FST
Volume-Controlled (10ul/dose)
n=6
Imipramine 1 hour before FST
15mg/kg, IP
n=6
TAT-Pep
(1.67nmol/g)
1 hour before
FST
n=7
2 hours before
FST
n=6
3 hours before
FST
n=6
4 hours before FST
n=6
TAT-D1-D2-
IPep
(1.67nmol/g)
1 hour before
FST
n=6
2 hours before
FST
n=6
3 hours before
FST
n=6
4 hours before FST
n=6
Table 2-2 Duration of the anti-immobility effect of the D1-D2 interfering peptide: treatment groups and
overall experimental design
55
experiments (see Section 2.5.1). Animals were kept in their original treatment groups, for
example if an animal had been assigned to the saline treatment group during the FST, it remained
in the saline group during the locomotor experiments.
To record locomotor activity, rats were placed in a custom-made locomotor activity
recording apparatus. Each animal was placed in a 20cm high, 20 cm wide and 30 cm long cage
(standard housing cage) in the locomotor apparatus for 30 minutes in a dark room. Rats had not
previously been exposed to the testing room or to the locomotor activity boxes. An array of 11
infrared photocells was placed along the long axis of the cages. Interruption of infrared beams
(Beam Breaks) was used as a measure of locomotor activity. The total amount of locomotor
activity was recorded in 5 minute intervals and for the entire 30 minute session. Total activity
data were analyzed via 1-way independent groups ANOVA according to the locomotor measure
followed by Newman-Keuls multiple comparisons tests, using Prism Software (GraphPad
Software, Inc.). The locomotor activity at various intervals during the test was analyzed by 2-
way independent factors ANOVA with treatment group (saline, imipramine, TAT-Pep and TAT-
D1-D2-IPep) and Time Point (5min, 10min, 15min, 20min, 25min, 30min) as main factors.
2.9 Co-immunoprecipitation and western blots
2.9.1 Tissue Collection
Animals were sacrificed the day they completed the FST or, if applicable, the open field
test. Animals were anaesthetized for 3 minutes with 5% isoflurane and were decapitated. Their
brains were quickly removed and relevant tissue areas were dissected on ice. These areas
included the olfactory bulbs, PFC, striatum, hippocampus and VTA. Brain tissue was stored at -
80°C for subsequent use in biochemical assays.
56
2.9.2 Co-Immunoprecipitation of D1 receptor by anti-D2DR
Since the peptide we are testing in the FST is designed to disrupt the interaction between
the D1 and D2 receptors, we investigated the co-immunoprecipitation of the D1 receptor by an
antibody against the D2 receptor in the PFC of animals who had received intranasal injections of
the D1-D2 interfering peptide or saline. We compared animals who had been given intranasal
injections of TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3), or saline (IN, n=3) and had been exposed
to the FST and the open field test.
For the co-immunoprecipitation, solubilized proteins from the PFC and striatum (500 µg)
from each animal were incubated with 1ug goat polyclonal anti-D2DR (N-19, Santa Cruz
Biotechnology) and protein A/G PLUS agarose beads (Santa Cruz Biotechnologies) overnight. A
control sample was incubated with polyclonal Goat IgG (Sigma-Aldrich) to confirm the absence
of non-specific immunoprecipitation.
After incubation, the immunoprecipitated proteins were washed and incubated with SDS
sample buffer (BioRad, Inc.) at 37°C for 40 minutes before being separated from the Protein A/G
PLUS-agarose beads using centrifugation. Immunoprecipitated proteins were then subjected to
separation using 10% SDS-Page gels, transferred onto nitrocellulose membranes and
immunoblotted overnight using anti-D1DR (D187, Sigma-Aldrich). Each immunoblot included
samples from saline and TAT-D1-D2-IPep treatment groups, along with a control sample
incubated with goat IgG (Sigma-Aldrich) and 75µg of tissue-extracted input protein from PFC
tissue. After overnight incubation, secondary antibodies conjugated with horseradish peroxidase
were applied to the blots for approximately two hours. After washing, immunoblots were
developed with ECL reagent (GE Healthcare, Inc.) and imaged using a BioRad ChemiDoc MP
system (BioRad Technologies, Inc.). To quantify the expression of protein, we conducted
57
densitometry analyses using ImageLab software (BioRad Technologies, Inc.). Densitometry data
were analyzed using two-tailed, unpaired Student’s t-tests (Prism Software, GraphPad, Inc).
2.9.3 Western Blots
We compared the expression of the D1 and D2 receptors using western blots in animals
given intranasal injections of TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, n=3). All
groups were given intranasal injections and exposed to the FST and open field tests. 80 µg of
solubilized protein extracts from the PFC, in SDS sample buffer (BioRad Technologies, Inc.)
were denatured by boiling for 5 minutes before being separated by 10% SDS page gel.
After transfer onto nitrocellulose membranes, we immunoblotted for D1 and D2 receptors
with monoclonal anti-D1DR (rat IgG, D187, Sigma-Aldrich) and monoclonal anti-D2DR
(Mouse IgG, B-10, Santa Cruz Biotechnologies). In order to confirm that the total amount of
protein in each sample were equal, we separated 20µg solubilized protein extracts with 10% SDS
page gel and immunoblotted using monoclonal anti-α-tubulin (Mouse IgG, DM1A clone, Sigma-
Aldrich).
After overnight incubation, immunoblots were incubated for 2 hours with species specific
Horseradish Peroxidase conjugated secondary antibodies. After washing, immunoblots were
developed with ECL reagent (GE HealthCare, Ltd.) and visualized using a BioRad ChemiDoc
MP system (BioRad Technologies, Inc). To quantify the expression of protein, we conducted
densitometry analyses using ImageLab software (BioRad Technologies, Inc). Densitometry data
were analyzed with two-tailed, unpaired Student’s T-tests using Prism Software (GraphPad
Software, Inc.).
58
3 Results
3.1 Experiment 1: The POD preferentially deposits substances on the
olfactory epithelium within the rat nasal cavity
In order to verify that the POD deposits substances preferentially onto the olfactory
epithelium, we administered intranasal injections of Richard Allen Scientific Mark-It Blue Dye
using the POD. The Mark-It dye allowed us to visualize where the POD was preferentially
depositing substances within the rat nasal cavity. It also allowed us to optimize our POD
delivery protocol (see Section 2.2.1) in order to maximize deposition of substances on the
olfactory epithelium and surrounding tissue.
We assessed the deposition of the Mark-It dye after delivery using the POD by grossly
dissecting the nasal anatomy. Figure 3-1A and B show representative images of Mark-It dye
deposition after POD delivery on the olfactory epithelium within the rats’ nasal cavity (A) and
visible through the cribiform plate (B), the porous bone through which the olfactory axons travel
to the olfactory bulb and CNS.15
As the images show, the POD preferentially deposited the
Mark-It Dye on the olfactory epithelium, favoring transport mechanisms that deliver substances
to the CNS.
3.2 Experiment 2: The D1-D2-FLAG interfering peptide can be detected in
the prefrontal cortex after intranasal administration
After confirming that the POD preferentially deposits substances on the olfactory
epithelium and olfactory sensory areas of the rat’s nasal anatomy, we next assessed whether a
TAT-linked, membrane-permeable peptide would reach the CNS, and the PFC in particular, after
59
Figure 3-1 Representative images of deposition of Mark-It Blue tissue marker deposition after correct POD
administration
(A) Deposition of Blue Mark-It dye on olfactory epithelium after intranasal administration using the POD
following the protocol outlined in Section 2.2.1. Animal’s nasal anatomy was dissected through the midline in
order to visualize both the left and right nasal cavities. (B) Blue Mark-It Dye is visible through the cribiform plate
(bone separating the nasal olfactory epithelium from the CNS, CSF and olfactory bulbs. The cribiform plate
contains perforations through which the olfactory receptor neuron (ORN) axons travel to the olfactory bulb and
CNS. Representative images shown.
60
administration with the POD. We focused on detecting the peptide in the PFC because Pei et al’s
study3 showed that the D1-D2 interfering peptide only had an antidepressant effect in the FST
when it was administered directly to this brain area.3 After staining selected sections of the PFC
from animals that received TAT-D1-D2-FLAG-IPep (1.67nmol/g, n=2) and saline (n=2) with
anti-FLAG antibodies, followed by a Cy2-conjugated fluorescent secondary antibody, we
visualized the resulting immunofluorescence using a confocal microscope. Figure 3-2A-B shows
representative images of prefrontal cortical slices from individual animals who received
intranasal injections of TAT-D1-D2-IPep (A) or saline (B).
We detected the presence of a TAT-D1-D2-IPep peptide in the PFC, as the Cy-2-
conjugated immunofluorescent signal was visible in slices from animals that had received TAT-
D1-D2-FLAG-IPep intranasal injections and not visible in the PFC of animals that received
saline injections. Immunofluorescence was visible through the anterior PFC coronal sections,
with no extreme variations in staining in dorsal, ventral medial or lateral areas. The results of this
experiment suggest that intranasal administration is a viable method to deliver membrane-
permeable peptides to the PFC.
3.3 Experiment 3: Intranasal administration of the D1-D2 interfering
peptide has an antidepressant effect in the forced swimming test
Although the experiment outlined in Section 3.2 allowed us confirm the presence of the
D1-D2-FLAG interfering peptide in the PFC after intranasal administration, it did not indicate
whether the accumulation of the peptide in the PFC was sufficient to produce a behavioral
antidepressant effect in the FST. Thus, it remained unclear whether intranasal administration
resulted in sufficient accumulation of the D1-D2 interfering peptide in the PFC to produce an
observable behavioral effect in the FST.
61
Figure 3-2 Immunofluorescent staining for anti-FLAG antibodies is visible in PFC slices of animals who
were administered TAT-D1-D2-FLAG-IPep (A) but not those who were administered saline (B). A) 1,2: Representative images of PFC sections of two separate animals who received TAT-D1-D2-FLAG-
Ipep (1.67nmol/g) intranasally. B) 1,2 Representative images of PFC sections from two separate animals who
received intranasal injections of Saline. Representative fluorescent images taken with a Zeiss LM confocal
microscope (25X magnification ) of anti-FLAG (Cy2 secondary antibodies) and NeuroTrace 530/615 neuronal
cell body stain. Representative images shown.
62
3.3.1 The D1-D2 Interfering Peptide has an Anti-Immobility Effect in the
FST
We compared immobility, swimming and diving behaviors during the 5-minute FST
across four treatment groups: animals that received saline (IN, n=6, Volume-controlled)
imipramine (IP, n=6, 15mg/kg), TAT-D1-D2-IPep (IN, n=7, 1.67nmol/g), or TAT-Pep (IN, n=6,
1.67nmol/g).
The D1-D2 interfering peptide had a significant anti-immobility effect in the FST that was
comparable to that of imipramine and significantly greater than that of saline or TAT-Pep. A 1-
way Independent Groups ANOVA revealed a significant difference in immobility behavior
between all groups: TAT-D1-D2-IPep, saline, imipramine and TAT-Pep (n=6-7 per group,
F(3,21)=12.25, p<0.0001). Post-Hoc Newman-Keuls multiple comparisons tests showed
significant differences when comparing TAT-D1-D2-IPep and saline (p<0.01), TAT-D1-D2-
IPep and TAT-Pep (p<0.01), imipramine and saline (p<0.001) and imipramine and TAT-Pep
(p<0.001). No significant differences were present between imipramine and TAT-D1-D2-IPep
groups (p>0.05) and saline and TAT-Pep groups (p>0.05).
For the swimming behavior, a 1-way Independent Groups ANOVA revealed a significant
difference between all treatment groups, (F(3,21)=12.57 p<0.0001). Post-hoc Newman Keuls
multiple comparisons tests revealed significant differences between TAT-D1-D2-IPep and saline
(p<0.01), TAT-D1-D2-IPep and TAT-Pep (p<0.01), imipramine and saline (p<0.001) and
imipramine and TAT-Pep (p<0.001). No significant differences were present between
imipramine and TAT-D1-D2-IPep (p>0.05) and saline and TAT-Pep (p>0.05) treatment groups.
There was no significant difference in diving behavior between saline, imipramine, TAT-D1-D2-
IPep and TAT-Pep groups (F(3,21)=0.99, p>0.05). Data is represented graphically in Figure 3-3.
63
Figure 3-3 The D1-D2 interfering peptide has an antidepressant effect in the FST when administered
intranasally.
After intranasal administration using the POD, the D1-D2 interfering peptide (dose = 1.67nmol/g) significantly
decreases immobility and increases swimming behavior in the FST. Data for each standard behavior (immobility,
swimming, diving) analyzed via 1-way independent groups ANOVA followed by Newman Keuls post-hoc multiple
comparisons tests. ** p<0.01, *** p<0.01 compared to saline; ^^ p<0.01, ^^^ p<0.001 compared to TAT-Pep. No
significant difference was observed between behavior in animals who received TAT-D1-D2-Ipep or imipramine.
Error bars represent SEM.
64
3.3.2 The D1-D2-FLAG interfering peptide has an anti-immobility effect
in the FST after intranasal administration
We tested whether the D1-D2 FLAG peptide produced an anti-immobility effect in the
FST, and whether we could detect it in the PFC of animals who had been exposed to the FST(see
Section 3.2). We hypothesized that, at a sufficient dose, the TAT-linked D1-D2 interfering
peptide with a FLAG tag on the C-terminal end (TAT-D1-D2-FLAG-IPep) would have an anti-
immobility effect in the FST comparable to that of imipramine and the D1-D2 interfering
peptide. The D1-D2-FLAG interfering peptide had a significant anti-immobility effect in the
FST that was comparable to imipramine and significantly greater than that of saline and TAT-
Pep.
A 1-way independent groups ANOVA of immobility behavior across treatment groups
demonstrated a significant difference between all groups (saline, imipramine, TAT-D1-D2-IPep
and TAT-Pep, n=4-5 per group, F(3,14)=7.746, p<0.01). Post-hoc Newman-Keuls multiple
comparisons tests revealed significant differences between imipramine and saline groups (n=4-5,
p<0.01), imipramine and TAT-Pep groups (n=4-5, p<0.05), TAT-D1-D2-FLAG-IPep and saline
groups (n=4-5, p<0.01) and TAT-D1-D2-FLAG-IPep and TAT-Pep groups (n=4-5, p<0.05).
There was no significant differences between TAT-Pep and saline groups (p>0.05) or
imipramine and TAT-D1-D2-IPep groups (p>0.05). Data is represented graphically in Figure 3-
4.
To investigate whether the behavior during the 5-minute FST was significantly different
in animals that received the D1-D2-FLAG peptide and those that received the D1-D2 peptide, we
compared immobility, swimming and diving behavior in these two groups. The D1-D2
interfering peptide and the D1-D2-FLAG tagged interfering peptide had indistinguishable
65
Figure 3-4 The D1-D2-FLAG interfering peptide has an anti-immobility effect in the FST.
The D1-D2-FLAG interfering peptide significantly decreases immobility behaviors in the FST after
intranasal administration. TAT-D1-D2-FLAG-IPep and TAT-Pep administered intranasally at a dose of
1.67nmol/g . Data analyzed via 1-way independent groups ANOVA followed by Newman Keuls Post-Hoc
Multiple Comparisons Tests. ** p<0.01, *** p<0.01 compared to saline; ^^ p<0.01, ^^^ p<0.001 compared
to TAT-Pep. No significant difference was observed between behavior in animals who received saline or
imipramine. Error bars represent SEM. Numbers within bars represent number of animals per group.
66
Figure 3-5 The D1-D2 interfering peptide and D1-D2-FLAG tagged interfering peptide have similar
behavioral effects in the FST. We compared immobility, swimming and diving behavior in treatment groups that that received TAT-D1-D2-
IPep (see Section 3.3.1) and TAT-D1-D2-FLAG-IP (see Section 3.3.2). Two-tailed, unpaired student’s t-tests
revealed no significant differences in immobility (p=0.83), swimming (p=0.73) or diving (p=0.18) behaviors
between these groups, indicating that the D1-D2 interfering peptide and the D1-D2-FLAG interfering peptide’s
effect in the FST are indistinguishable. Error bars represent SEM.
67
behavioral effects during the 5-minute FST. Two-tailed, unpaired Student’s T-tests revealed no
significant differences between immobility, swimming or diving behavior in animals who
received TAT-D1-D2-IPep or TAT-D1-D2-FLAG-IPep (n=4-7 per group, Immobility, t(9)=0.21
, p=0.83; Swimming, t(9)=0.32 , p=0.74 ; Diving t(9)=1.447 , p=0.18).(Figure 3-5).
3.4 Experiment 4: Efficacy of the D1-D2 interfering peptide at various
intranasal doses
The D1-D2 interfering peptide has an antidepressant effect in the FST when given
intranasally at a dose of 1.67nmol/g (5.75mg/kg) (Section 3.3.1., Figure 3-3.). The 1.67nmol/g
dose administered intranasally represents an estimated 100-fold increase over the dose given
directly to the PFC (5nmol/injection).3 Although this particular intranasal dose can produce an
antidepressant effect in the FST, the behavioral effects of the D1-D2 interfering peptide at doses
higher or lower than 1.67nmol/g (5.75 mg/kg) remains unknown.
To investigate the antidepressant effects of the D1-D2 interfering peptide at various
intranasal doses, we varied its dose, exposed animals to the 2-day FST, and analyzed the
resulting immobility behavior in the treatment groups (see Section 2.6.3 and Table 2-1 for
detailed experimental design). We hypothesized that at intranasal doses larger than 1.67nmol/g,
the D1-D2 interfering peptide would have a significant anti-immobility effect in the FST.
3.4.1 D1-D2 interfering peptide dose: 4.0nmol/g (13.72 mg/kg)
At a dose of 4.0nmol/g, the D1-D2 interfering peptide had an anti-immobility effect in
the FST comparable to that of imipramine and significantly greater than that of saline. We
compared immobility behavior during the 5-minute FST in rats that received saline (IN, n=3),
imipramine (IP, 15mg/kg, n=3) and TAT-D1-D2-IPep (IN, n=3, 4.0 nmol/g). A 1-way
68
independent groups ANOVA revealed a significant difference between all groups (F(2,6) =
9.207, p<0.01). Post-hoc Newman-Keuls multiple comparisons tests showed significant
differences between TAT-D1-D2-IPep and saline (p<0.05) and saline and imipramine (p<0.05).
No significant difference was present between TAT-D1-D2-IPep and imipramine groups (Figure
3-6, 3-10).
3.4.2 D1-D2 interfering peptide dose: 2.0nmol/g (6.86 mg/kg)
At an intranasal dose of 2.0nmol/g, the D1-D2 interfering peptide had an anti-immobility
effect in the FST comparable to that of imipramine and significantly greater than that of saline.
We compared immobility behavior during the 5-minute FST in rats that received saline (IN,
n=8), imipramine (IP, 15 mg/kg, n=8), TAT-D1-D2-IPep (IN, 2nmol/g, n=8), and TAT-Pep (IN,
2nmol/g, n=7). A 1-way independent groups ANOVA revealed a significant difference between
immobility behavior in all groups ( F(3,27)=6.836, p<0.01). Post-hoc Newman-Keuls Multiple
comparisons tests revealed significant differences between TAT-D1-D2-IPep and saline
(p<0.01), imipramine and saline (p<0.01) and imipramine and TAT-Pep (p <0.05). There were
no significant differences between saline and TAT-Pep (p>0.05), imipramine and TAT-D1-D2-
IPep (P>0.05) and TAT-Pep and TAT-D1-D2-IPep (p>0.05). Immobility behavioral counts are
represented graphically in Figure 3-7 and summarized in Figure 3-10.
3.4.3 D1-D2 interfering peptide dose: 1.67nmol/g (5.75 mg/kg)
For the purposes of comparison, data relating to immobility behavior in rats that received
our original intranasal dose of 1.67nmol/g (5.57mg/kg) is included here (for complete analysis,
see Section 3.3.1.). Briefly, a 1-way independent groups ANOVA revealed a significant
69
Figure 3-6 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of
4.0nmol/g
Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 4nmol/g) was
compared to those who received saline or IP injections of imipramine. Immobility behavior between groups
compared by 1-way independent groups ANOVA followed by post-hoc Newman Keuls multiple comparisons tests.
* p<0.05 compared to saline. Error bars represent SEM. Numbers within bars represent number of animals per
group.
70
Figure 3-7 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of
2.0nmol/g
Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 2nmol/g) was
compared to those who received saline, TAT-Pep or IP injections of imipramine. Immobility behavior between
groups compared by 1-way Independent Groups ANOVA followed by post-hoc Newman-Keuls multiple
comparisons tests. ** p<0.01 compared to saline, ^ p<0.05 compared to TAT-Pep. Error Bars Represent SEM.
Numbers within bars represent number of animals per group.
71
difference between groups who received saline (IN, n=6), imipramine (IP, 15mg/kg, n=6), TAT-
D1-D2-IPep (IN, 1.67nmol/g, n=7) and TAT-Pep (IN, 1.67nmol/g, n=6), (n=6-7, F(3,21)=12.25,
p<0.001). Post-Hoc Newman-Keuls multiple comparisons tests revealed significant differences
when comparing TAT-D1-D2-IPep and saline (p<0.01), TAT-D1-D2-IPep and TAT-Pep
(p<0.01), imipramine and saline (p<0.001) and imipramine and TAT-Pep (p<0.001). No
significant differences were found between imipramine and TAT-D1-D2-IPep (p>0.05) and
saline and TAT-Pep (p>0.05). Data is represented graphically in Figure 3-3, 3-8 and
summarized in Figure 3-10.
3.4.4 D1-D2 interfering peptide dose: 1.0nmol/g (3.43 mg/kg)
At an intranasal dose of 1.0nmol/g, the D1-D2 interfering peptide did not have an anti-
immobility effect in the FST, as it was not significantly different from that of saline, and
significantly less than that of imipramine. We compared immobility behavior during the 5-
minute FST in rats that received saline (IN, n=8), imipramine (IP, 15 mg/kg, n=8) and TAT-D1-
D2-IPep (IN, n=6, 1.0 nmol/g). A 1-way independent groups ANOVA revealed a significant
difference between all groups (n=6-8 per group, F(2,19)=9.653, p<0.01). Post-hoc Newman-
Keuls multiple comparisons tests revealed significant differences between imipramine and saline
(P <0.001) and imipramine and TAT-D1-D2-IPep (P <0.05). No significant difference was found
between saline and TAT-D1-D2-IPep (p>0.05). Data is represented graphically in Figure 3.9
and summarized in Figure 3.10.
72
Figure 3-8 The D1-D2 interfering peptide has an anti-immobility effect in the FST at an intranasal dose of 1.67
nmol/g.
Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 1.67nmol/g) was
compared to those who received saline, TAT-Pep or IP injections of imipramine. Immobility behavior between groups
compared by 1-way independent groups ANOVA followed by post-hoc Newman-Keuls multiple comparisons tests. ***
p<0.001, ** p<0.01 compared to saline, ^^^ p<0.001, ^^ p<0.01 compared to TAT-Pep. Error bars represent SEM.
Numbers within bars represent number of animals per group.
73
Figure 3-9 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST at an
intranasal dose of 1.0nmol/g
Immobility behavioral of animals who received intranasal injections of TAT-D1-D2-IPep (dose = 1nmol/g) was
compared to those who received saline or IP injections of imipramine. Immobility behavior between groups
compared by 1-way independent groups ANOVA followed by post-hoc Newman-Keuls multiple comparisons
tests *** p<0.001 compared to saline, # p<0.05 compared to imipramine. Error bars represent SEM. Numbers
within bars represent number of animals per group.
74
Figure 3-10 Efficacy of the D1-D2 interfering peptide at various doses in the FST: summary of findings.
Data Points in imipramine and saline group are combined from experiments outlined in Sections 3.4.1 – 3.4.4. For
each dose, complete results represented graphically in Figure 3.6 – 3.9. Data at each dose compared using 1-way
independent groups ANOVA. *** p <0.001, ** p<0.01, * p<0.01 compared to Saline, # p<0.05 compared to
imipramine. Numbers below data points are number of animals per group. Error bars represent SEM. Data from
treatment groups who received TAT-Pep excluded from figure for the purposes of clarity (Figure 3-7, 3-8).
75
3.5 Experiment 5: Duration of the behavioral effect of the D1-D2
interfering peptide
We investigated the length of time that the D1-D2 interfering peptide remains
behaviorally active (has a detectable anti-immobility effect in the FST) after intranasal
administration. To do this, we increased the amount of time between the last intranasal injection
of either the D1-D2 interfering peptide or the 9-amino acid TAT peptide and the beginning of the
FST to 2, 3 or 4 hours. Overall results at all time points are summarized in Figure 3-14.
3.5.1 Behavioral Effect in FST 2 hours after intranasal administration
The D1-D2 interfering peptide had a significant anti-immobility effect in the FST two
hours after the final intranasal injection. We compared immobility behavior during the FST in
rats that had received saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg, IP, 1 hr before
FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN 2 hrs before FST, n=6) or TAT-Pep (1.67nmol/g,
IN, 2 hrs before FST, n=5). A 1-way independent groups ANOVA revealed a significant
difference between all groups (n=5-6 per group, F(3,19)=5.399, p<0.01). Post-Hoc Newman-
Keuls multiple comparisons tests revealed a significant difference between TAT-D1-D2-IPep
and saline (p<0.05), TAT-D1-D2-IPep and TAT-Pep (p<0.05), imipramine and saline (p<0.05)
and imipramine and TAT-Pep (p<0.05). No significant differences exist between imipramine and
TAT-D1-D2-IPep groups (p>0.05) or TAT-Pep and saline groups (p>0.05). Data is represented
graphically in Figure 3-11 and summarized in Figure 3-14.
3.5.2 Behavioral Effect in FST 3 hours after intranasal administration
Three hours after the last intranasal injection, the D1-D2 interfering peptide did not have a
significant anti-immobility effect in the FST. We compared immobility behavior during the 5-
76
minute FST in rats that had received saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg, IP,
1 hr before FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN, 3 hrs before FST, n=7) or TAT- Pep
(1.67nmol/g, IN, 3 hrs before FST, n=5). A 1-way independent groups ANOVA revealed a
significant difference between immobility behaviors across all groups (n=5-6 per group,
F(3,20)=4.669, p<0.01). Post-Hoc Newman-Keuls Multiple Comparisons Tests indicated
significant differences between imipramine and saline groups (p<0.05), imipramine and TAT-
Pep groups (p<0.05) and imipramine and TAT-D1-D2-IPep groups (p<0.05) groups. No
significant differences were present between TAT-D1-D2-IPep and saline groups (p>0.05),
TAT-D1-D2-Ipep and TAT-Pep groups (p>0.05) or TAT-Pep and saline groups (p>0.05). Data is
represented graphically in Figure 3-12 and summarized in Figure 3-14.
3.5.3 Behavioral effect in the FST 4 hours after intranasal administration
Four hours after the last intranasal injection, the D1-D2 interfering peptide did not have a
significant anti-immobility effect in the FST. We compared immobility behavior during the FST
in rats that had received injections of saline (IN, 1 hr before FST, n=6), imipramine (15mg/kg,
IP, 1 hr before FST, n=6), TAT-D1-D2-IPep (1.67nmol/g, IN, 4 hrs before FST, n=6) or TAT-
Pep (1.67nmol/g, IN, 3 hrs before FST, n=6). A 1-way independent groups ANOVA revealed a
significant difference between immobility behaviors across all groups (n=5-6 per group,
F(3,19)=3.727, p<0.05). Post-Hoc Newman-Keuls multiple comparisons tests indicated
significant differences between imipramine and saline groups (p<0.05), imipramine and TAT-
77
Figure 3-11 The D1-D2 interfering peptide has an anti-immobility effect in the FST 2 hours after intranasal
administration.
Saline and imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1-
D2-IPep and TAT-Pep administered via IN injections 2 hours before the FST. Immobility behavioral counts
analyzed by 1 way independent groups ANOVA (p<0.01) followed by post hoc Newman-Keuls multiple
comparisons tests. * p<0.05 compared with saline, ^ p<0.05 compared with TAT-Pep. Error bars represent SEM.
Numbers within bars represent number of animals per group.
78
Figure 3-12 The D1-D2 Interfering Peptide does not have an anti-immobility effect in the FST 3 hours after
intranasal administration.
Saline and imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1-D2-
IPep and TAT-Pep administered via IN injections 3 hours before the FST. Immobility behavioral counts analyzed by 1
way independent groups ANOVA (p<0.01) followed by post hoc Newman-Keuls multiple comparisons tests. * p<0.05
compared with saline, ^ p<0.05 compared with TAT-Pep, # p<0.05 compared with imipramine. Error bars represent
SEM.
79
Figure 3-13 The D1-D2 interfering peptide does not have an anti-immobility effect in the FST 4 hours after
intranasal administration
Saline and Imipramine were administered via IN and IP injections, respectively, 1 hour before the FST. TAT-D1-D2-
IPep and TAT-Pep administered via IN injections 4 hours before the FST. Immobility behavioral counts analyzed by 1
way independent groups ANOVA (p<0.05) followed by post hoc Newman-Keuls multiple comparisons tests. * p<0.05
compared with saline, ^ p<0.05 compared with TAT-Pep, # p<0.05 compared with imipramine. Error bars represent
SEM. Numbers within bars represent number of animals per group.
80
Figure 3-14 The D1-D2 interfering peptide no longer has a behavioral effect in the FST 3 hours after it is
administered via intranasal injections.
Summarized data from Figures 3-11 to 3-13 and Figure 3-3. Immobility data from our original time point of 1 hour after
intranasal administration is included for the purposes of comparison. Imipramine and saline treatment groups received
IP and IN injections, respectively, 1 hour before the FST. Immobility behavioral data from each time point (1,2,3 and 4
hrs) analyzed via 1-way independent groups ANOVA followed by Newman Keuls post hoc tests. *** p<0.001, * p<0.05
compared to saline ^^ p<0.01, ^ p<0.05 compared to TAT-Peptide # p<0.05 compared to imipramine. Numbers below
data points represent number of animals per group. Error bars represent SEM.
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Pep groups (p<0.05) and imipramine and TAT-D1-D2-IPep groups (p<0.05). No significant
differences were present between TAT-D1-D2-IPep and saline (p>0.05), TAT-D1-D2-Ipep and
TAT-Pep (p>0.05) or TAT-Pep and saline (p>0.05). Data is represented graphically in Figure 3-
13 and summarized in Figure 3-14.
3.6 Experiment 6: The D1-D2 interfering peptide does not increase
locomotor activity
In order to investigate whether the anti-immobility effect of the D1-D2 interfering
peptide in the FST was due to its specific anti-depressant effects, we examined the effect of the
D1-D2 interfering peptide on locomotor activity. To test this, we compared the amount of
locomotor activity during a 30-minute test in animals given intranasal injections of saline (n=5),
TAT-Pep (IN, 2nmol/g, n=5), TAT-D1-D2-IPep (IN, 2nmol/g, n=5) or imipramine (IP, 15mg/kg,
n=5).
3.6.1 Overall locomotor activity
Animals in the D1-D2 interfering peptide, TAT-peptide or imipramine treatment groups
had significantly lower overall locomotor activity during the 30-minute test than animals in the
saline group. A 1-way independent groups ANOVA of locomotor activity (as measured by Beam
Breaks) revealed a significant difference between all four treatment groups (n=5 per group,
F(3,16)=9.775 p<0.001). Post-Hoc Newman-Keuls multiple comparisons tests revealed
significant differences between TAT-D1-D2-IPep and saline groups (p<0.001), TAT-Pep and
saline groups (p<0.01), and imipramine and saline groups (p<0.001). No significant difference
was present between imipramine, TAT-Pep or TAT-D1-D2-IPep groups (p>0.05 for all
comparisons). Data is represented graphically in Figure 3-15A.
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to test whether the D1-D2 interfering peptide’s effect on locomotor activity was consistent across
different doses, we compared the locomotor activity of animals in treatment groups that received
the D1-D2 interfering peptide at two doses: 2.0 nmol/g (n=5) and 1.67nmol/g (n=5). A two-
tailed, unpaired student’s t-test revealed no significant differences between overall locomotor
activity in these groups (n=5 per group, t(8)=1.203, p>0.05) (Figure 3-15B).
3.6.2 Effect of time on locomotor activity during 30-minute test
We examined locomotor activity across the treatment groups during the open-field test in
5-minute intervals. There was a significant difference in the amount of locomotor activity across
different treatment groups, as well as the amount at different time points in the test. However, all
treatment groups displayed the same pattern of locomotor activity (highest at the beginning of
the test) (Figure 3-16). Locomotor activity at various time course data was analyzed by 2-way
Independent Groups ANOVA, with treatment groups (saline, imipramine, TAT-D1-D2-IPep or
TAT-Pep) and time point (5min, 10min, 15min, 20min, 25min, 30min) as independent factors.
The analysis revealed a significant main effect of treatment groups (F(3,96)= 20.24, p<0.001)
and time point (F(5,96)= 30.66, p<0.001). The interaction between treatment group and time
point was not significant (F(15,96)=1.06, p>0.05).
83
Figure 3-15 The D1-D2 interfering peptide does not increase locomotor activity during a 30-minute open field
test.
(A) Total locomotor activity during a 30-minute open field test. Locomotor activity was assessed during a 30-
minute open field test in a novel environment, and measured by the amount of movement around the testing
chamber (Beam Breaks). Animals were given intranasal injections of saline, TAT-D1-D2-IPep or TAT Pep
(2.0nmol/g) or IP injections of imipramine 24 hours, 19hours and 1 hour before the open field test. Locomotor
activity data was analyzed by 1-way independent groups ANOVA (p<0.001) followed by post-hoc Newman-Keuls
multiple comparisons tests. *** p<0.001, ** p<0.01, compared to saline. (B) The TAT-D1-D2-IPep has a similar
effect in the open field test at 2.0nmol/g and 1.67nmol/g. Differences in locomotor activity produced by TAT-D1-
D2-IPep dose evaluated by unpaired, two-tailed Student’s t-test (p>0.05).
84
Figure 3-16 The D1-D2 interfering peptide decreases overall locomotor activity but does not change the
activity pattern during a 30-minute open field test.
Each Point on the test represents total number of beam breaks during the previous 5-minutes of the open field
test. Data across treatment group and time points analyzed using 2-way independent groups ANOVA with
time point and treatment group as independent factors. There was a significant main effect for time course
(p<0.001) and treatment group (p<0.001) but no significant interaction between the two factors. n=5 animals
per group. TAT-D1-D2-Ipep and TAT-Pep were given at an intranasal dose of 2.0nmol/g. Error bars represent
SEM.
85
3.7 Experiment 7: Intranasal administration of the D1-D2 interfering
peptide disrupts the interaction between dopamine D1 and D2 receptors
in the PFC
The D1-D2 interfering peptide is designed to disrupt the interaction between the
Dopamine D1 receptor and the Dopamine D2-Long Receptor isoform by interacting with a 15-
amino acid segment in the third intracellular loop of the D2L receptor.3 In previous studies from
our laboratory, administering this peptide directly to the PFC decreased the interaction between
dopamine D1 and D2 receptors in the PFC (as assessed by co-immunoprecipitation) and had an
anti-immobility effect in the FST (see Figure 1-4).3 The D1-D2 interfering peptide also had a
significant antidepressant effect in the FST after intranasal administration (Figure 3-3, 3-10),
thus we examined whether there was a concurrent decrease in the D1-D2 dopamine receptor
interaction in the PFC after intranasal administration of the D1-D2 interfering peptide.
A representative immunoblot of D1R (anti-D1DR) immunoprecipitated by anti-D2DR is
shown in Figure 3-17A. Densitometry analysis of immunoblots revealed that the detectable
interaction between the Dopamine D1 and D2 receptors was significantly reduced in prefrontal
tissue from animals that received TAT-D1-D2-IPep compared with animals who received saline
(Student’s t-test, n=3 per group, t(4)=3.872, p=0.018) (Figure 3-17B).
3.8 Experiment 8: The D1-D2 interfering peptide does not change the
expression of dopamine D1 or D2 receptors in the PFC
After intranasal administration, the D1-D2 interfering peptide can disrupt the interaction
between Dopamine D1 and D2 receptors (Figure 3-17). We tested if this disruption is due to the
D1-D2 interfering peptide’s pharmacological effect or due to a change in the expression of either
86
Figure 3-17 Co-Immunoprecipitation of D1 by anti-D2R is reduced in the PFC of animals who received
intranasal injections of TAT-D1-D2-IPep (Dose: 1.67nmol/g)
(A) Representative immunoblot of anti-D2DR immunoprecipitated tissue from the PFC of rats that received
intranasal injections of saline (n=3) or TAT-D1-D2-Ipep (IN, 1.67nmol/g, n=3). Input lane: 75 µg solubilized PFC
tissue, IgG: tissue incubated with non-specific immunoglobulin antibody. (B) The interaction between D1 and D2R
is significantly reduced in the PFC of animals who received TAT-D1-D2-IPep. The interaction between D1 and
D2R quantified by densitometry analysis of immunoblots. All samples were standardized to control (saline) samples
and analyzed by two-tailed, unpaired student’s T-test (n=3 per group=0.018).
87
the dopamine D1 or D2 receptor proteins. To do this, we evaluated the expression of D1 and D2
receptors in the PFC of animals that were given intranasal injections of TAT-D1-D2-IPep (n=3)
or saline (n=3) using Western Blot analysis.
3.8.1 Expression of Dopamine D1 receptors in the PFC after intranasal
administration of the D1-D2 interfering peptide
We compared prefrontal expression of D1 in the same subsample of animals used in the
co-immunoprecipitation experiment (Section 3.8). The Western Blot analysis showed no change
in the overall expression of the D1 dopamine receptor in the PFC after intranasal administration
of the D1-D2 interfering peptide (IN, 1.67nmol/g, n=3) compared with saline (IN, n=3)
(Student’s t-test, t(4)=0.167, p>0.05), Figure 3-18A-B.
3.8.2 Expression of Dopamine D2 receptors in the PFC after intranasal
administration of the D1-D2 interfering peptide
We compared prefrontal expression of D2 in the same subsample of animals used in the
co-immunoprecipitation analysis described in Section 3.7. We found no change in the overall
expression of D2 dopamine receptors in the PFC after intranasal administration of the D1-D2
interfering peptide (IN, 1.67nmol/g, n=3) compared with saline (IN, n=3) (Student’s t-test,
t(4)=0.009, p>0.05), Figure 3-19A-B. We also performed an immunoblot for α-tubulin (Figure
3-20) to verify that the amount of protein in each sample was equivalent.
88
Figure 3-18 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the
dopamine D1 receptor in the PFC.
(A) Representative immunoblot of D2R in PFC tissue from animals who received intranasal injections of TAT-D1-
D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, n=3). (B) Expression of D2 is unchanged in animals who received
intranasal injections of TAT-D1-D2-IPep compared with those who received Saline (Control). Data quantified using
densitometry and analyzed using unpaired, two-tailed student’s t-test (n=3 per group, p>0.05). Error bars represent
SEM.
89
Figure 3-19 Intranasal administration of the D1-D2 interfering peptide does not change the expression of the
dopamine D2 Receptor in the PFC
(A) Representative Immunoblot of D1 in PFC tissue from animals who received intranasal injections of TAT-D1-
D2-IPep (IN, 1.67nmol/g, n=3) or saline (IN, 1.67nmol/g, n=3). (B) Expression of D2 is unchanged in animals who
received intranasal injections of TAT-D1-D2-IPep compared with those who received saline (control). Data
quantified using densitometry and analyzed using unpaired, two-tailed student’s t-test (n=3 per group, p>0.05). Error
bars represent SEM.
90
Figure 3-20 Representative immunoblot of α-tubulin expression in rat PFC tissue.
Rats received intranasal injections of saline (IN, n=3) or TAT-D1-D2-IPep (IN, 1.67nmol/g, n=3). 20 µg protein
from PFC of each animal resolved using SDS page and immunoblotted with anti- α-tubulin. α-tubulin levels were
used as a loading control for experiments assessing expression of D1 or D2R in the PFC.
91
4 Discussion
4.1 Overall Findings
This project addressed whether we can effectively deliver peptide therapies designed to
disrupt pathological interactions between two cell membrane receptors to the CNS in a non-
invasive manner. We show that the D1-D2 interfering peptide, designed to disrupt the interaction
between the D1 and D2 dopamine receptors3, has an antidepressant effect in the FST after
intranasal delivery (Figure 3-3 and 3-10). These findings, along with experiments investigating
the pharmacological and biological properties of the D1-D2 interfering peptide, provide a solid
basis for further development of this therapy as a novel treatment for MDD.
We demonstrated that the POD used in this study deposits substances preferentially on
the olfactory epithelium and that a FLAG-tagged biologically active peptide can be detected in
the PFC after intranasal administration. Administration of the D1-D2 interfering peptide at doses
greater than 5.75 mg/kg (1.67nmol/g) had a significant anti-immobility effect that was
comparable to that of imipramine in the FST, for two, but not three, hours after intranasal
administration. The D1-D2 interfering peptide, the TAT-peptide and imipramine all significantly
decreased locomotor activity during a 30-minute locomotor activity test, suggesting that these
substances do not increase the overall occurrence of motor movements and that the anti-
immobility effect observed in the FST is specific to its antidepressant effects. Together, these
finding demonstrated the efficacy of the intranasal pathway for peptide delivery to the CNS,
expanded upon our laboratory’s previous findings that the D1-D2 interfering peptide had an
antidepressant effect in animal models of depression, and developed a non-invasive, clinically
applicable method to deliver this peptide to the CNS.3
The D1-D2 interfering peptide significantly disrupted the interaction between D1 and D2
in the PFC without changing the expression of either receptor, indicating that the antidepressant
92
effect of the peptide may be due to its ability to disrupt this protein-protein interaction. I will
discuss the implications and the shortcomings of these findings and propose future experiments
to further understand the pharmacological effects of the D1-D2 interfering peptide.
4.2 The POD delivers biologically active peptides to the CNS
The POD preferentially deposits substances on the olfactory epithelium and surrounding
tissue when using the administration protocol we developed (outlined in Section 2.2.1). After
intranasal delivery with the POD, Mark-It tissue dye was preferentially deposited onto the
olfactory epithelium and surrounding tissue (Figure 3.1). Numerous previous studies regarding
protein and peptide delivery to the CNS have indicated that proteins such as NGF194,195
, BDNF196
and IGF-1197,198
preferentially enter the CNS through the extracellular channels between the
ORN axons and the OECs protecting them from the cribiform plate.16
It is probable that
preferentially depositing substances onto the olfactory epithelium, as the POD does, where ORN
axons originate would increase the amount of the D1-D2 interfering peptide delivered to the
CNS while minimizing loss to the periphery.
Next, we investigated whether we could detect the D1-D2 interfering peptide in the PFC
after intranasal administration. To do this, we added an 8-amino acid, immunoreactive FLAG215
tag to the C-terminal end of the D1-D2 interfering peptide. We detected fluorescent Cy-2-
conjugated antibodies for FLAG both intracellularly and in extracellular areas of PFC coronal
sections (Figure 3-2). As well, the immunofluorescence was visible in most areas of the PFC
coronal section, with no extreme variations in fluorescent signals between dorsal and ventral,
lateral or medial PFC areas. We focused on investigating whether the D1-D2-FLAG peptide
could be detected in the PFC because in our laboratory’s previous study, the D1-D2 interfering
93
peptide only had an antidepressant effect when infused directly to the PFC and not in other brain
areas.3
These experiments were designed to qualitatively assess where substances are deposited
by the POD, and whether they can be transported to the CNS. As a result, we are unable to make
direct conclusions about the quantitative aspects of this process, such as the efficiency of
delivery to the CNS, and the mechanisms through which this occurs. One of the shortcomings of
this method of detecting the D1-D2-FLAG peptide in the PFC is that we were not able to
quantify the amount of D1-D2 interfering peptide effectively delivered to the PFC. Thus, the
proportion of the original intranasal dose of the D1-D2-FLAG peptide present after intranasal
administration is not known. In addition, these experiments did not provide any information
about the mechanism through which the D1-D2 interfering peptide gains access to the central
nervous system.
These results do not provide a quantitative measure of the efficiency of delivery to the
CNS after intranasal administration. However, we observed an anti-immobility effect in the FST
at an intranasal D1-D2 interfering peptide dose equal to or greater than 1.67nmol/g. This dose
was approximately 100-fold larger than that given directly to the PFC (5nmol per injection) in
Pei et al’s previous study.3 We chose this dose based on previous studies of intranasal
administration of small proteins such as BDNF, NGF and insulin to the CNS that indicated that
the efficiency of delivery to the CNS is between 1 and 5%.192,194,196,212,217
Using these studies, we
estimated that approximately 1-5% of the dose originally given intranasally will be delivered to
the CNS in a biologically-active form (see Appendix 1 for detailed calculations) and our
subsequent results (see Figure 3-10) are consistent with this estimate.
94
4.3 Mechanism of transport to the CNS after intranasal administration
The results outlined above show that the POD preferentially deposits substances on the
olfactory epithelium, and, hypothetically, once there, they are transported to the CNS via
extracellular pathways around the olfactory receptor neuron axons (see section 1.3 for review of
the intranasal pathways to the CNS). However, the mechanism by which the D1-D2 interfering
peptide is transported to the CNS and PFC is not known. As stated above, it is likely that the
peptide is being transported to the CNS via extracellular transport pathways. This hypothesis is
supported by our experimental observations that the POD preferentially deposits substances on
the olfactory epithelium, the relatively short (1 hour) amount of time required for transport to the
PFC, and previous studies demonstrating that peptides similar to the one used in this study are
also transported to the CNS via extracellular mechanisms.203,212
Alternately, the peptide could be
transported by the ORNs via intracellular mechanisms, a process that would require diffusion of
the peptide into the ORNs and subsequent transport via axonal transport mechanisms to the axon
terminals in the olfactory bulbs.15
This is unlikely, as previous studies have shown that
intracellular transport of this type takes significantly longer than 1 hour.15,218
An alternate possibility is that the D1-D2 interfering peptide is absorbed into the systemic
circulation through the nasal capillary bed, as the nasal cavity contains extensive capillaries and
vascularization.15
After uptake into the bloodstream, the peptide would be transported throughout
the body via the systemic circulation, resulting in diffuse administration of the peptide and
proportionally less of the intranasal dose transported to the CNS.15
Second, the peptide would
need to cross the BBB, and, although peptides containing a membrane-permeable TAT sequence
are permeable to the BBB214
, they require an extremely high systemic dose to enter the CNS in
appreciable amounts. This is supported by a number of studies from our laboratory suggesting
that TAT-linked peptides need to be administered at a systemic dose of ≥ 3nmol/g in order to be
95
transported to the CNS, and have any detectable CNS-mediated pharmacological effects in vivo
(unpublished data, Liu laboratory, 2008-2013). In the present study, the minimum effective dose
we tested was 1.67nmol/g, 55% that of the typical systemic dose. This suggests that our
intranasal delivery method has some specificity to the CNS, although it is not conclusive as we
have not yet tested the antidepressant effects of the D1-D2 interfering peptide after systemic
administration. In conclusion, although it is likely that after intranasal administration some of the
D1-D2 peptide is taken up into the circulation, it is unlikely that this is the principle mechanism
of transport to the CNS.
A recent study by Yang et al212
showed that for a 22-amino acid, TAT-linked membrane
permeable peptide similar in size to the D1-D2 interfering peptide, an intranasal dose only 7%
that of the IV doses previously administered219
was able to alleviate hypoxia-induced ischemic
brain injury in a rat preclinical model. Yang et al212
also showed that their TAT-linked peptide
was detectable in the olfactory bulbs and anterior brain areas such as the PFC 10-30 minutes
after delivery, a result that suggests that their peptide was being transported to the CNS via the
extracellular pathways between the olfactory epithelium and the CNS. Overall, when the results
from this project are evaluated within the context of the scientific literature regarding intranasal
delivery of peptides of this type, it seems likely that the D1-D2 interfering peptide is being
delivered to the CNS via extracellular transport pathways along olfactory receptor axons.
4.4 The D1-D2 interfering peptide is effective at intranasal doses ≥ 5.75
mg/kg for up to 2 hours after intranasal administration.
The D1-D2 interfering peptide has a significant anti-immobility effect in the FST at doses
greater than or equal to 1.67nmol/g (5.75 mg/kg) (Figure 3-10). At these doses, the
antidepressant effect in the FST was comparable to that of imipramine, a clinically effective
96
tricyclic antidepressant. We tested if the behavioral effect of the D1-D2 interfering peptide in the
FST would differ at doses higher or lower than the original dose (1.67nmol/g) we tested. We did
not observe any appreciable increase or decrease in immobility behavior at D1-D2 interfering
peptide doses greater than 1.67nmol/g. Thus, there may be some threshold dose of the D1-D2
interfering peptide that is required to produce an anti-immobility effect in the FST, beyond
which no additional antidepressant efficacy is conferred. This experiment also allowed us to
identify the minimum intranasal dose of the D1-D2 interfering peptide with maximal behavioral
effect in the FST as 1.67nmol/g.
The lowest intranasal dose of the D1-D2 interfering peptide we tested was 1.0nmol/g. At
this dose, we did not observe a significant difference between the behavior of animals who
received the D1-D2 interfering peptide and those who received saline (Figure 3-9). However,
the mean amount of immobility in the saline group was greater than in the D1-D2 interfering
peptide group. (Figure 3-9, 3-10) It is possible that the D1-D2 interfering peptide has some sub-
threshold antidepressant effect in the FST when administered at this dose. In order to test this
hypothesis and to gain a more complete understanding of the antidepressant efficacy of the D1-
D2 interfering peptide at various intranasal doses, doses of the D1-D2 interfering peptide lower
than 1.0nmol/g should be tested in the FST.
We evaluated the duration of the effect of the D1-D2 interfering peptide in the FST. We
show that at the minimally effective intranasal dose, 1.67nmol/g, the D1-D2 interfering peptide
has an anti-immobility effect in the FST for two, but not three, hours after intranasal
administration. (Figure 3-14) At the 3 and 4 hour time points, we were no longer able to detect a
significant anti-immobility effect in animals that were given intranasal administrations of the D1-
D2 interfering peptide. The absence of a behavioral effect in the FST 3 or 4 hours after
intranasal administration is likely due to degradation of the D1-D2 interfering peptide as it is
97
transported from the olfactory epithelium to the central nervous system. The mucous membranes
in the nasal cavity and covering the olfactory epithelium contain proteases capable of degrading
peptide bonds.15
Furthermore, small peptides like the D1-D2 interfering peptide disrupt the
interaction between two proteins by competitively binding to the interacting regions, and, as
such, must remain properly folded in order to effectively bind to these regions. Likely, the longer
the D1-D2 interfering peptide in the body, the more degradation occurs, resulting in loss of its
pharmacological effects in the CNS. Thus, strategies to improve the stability of the D1-D2
interfering peptide in the CNS will be useful in increasing the duration of its antidepressant
effect.
4.5 Possible neurobiological mechanisms of the D1-D2 interfering peptide’s
antidepressant effect
In Pei et al.’s study,3 they document an increase in D1-D2 interaction in the striatum of
patients with MDD. This observation is correlative in nature and could be due to neurobiological
changes from antidepressant treatments or a result of other confounding factors inherent in
human studies. As such, no causal role for the D1-D2 in the pathogenesis of MDD in the
striatum has been shown. Beyond the D1-D2 interfering peptide’s ability to disrupt the
interaction between D1 and D2 in the PFC, the cellular and neurobiological mechanisms leading
to its antidepressant efficacy remain unknown. It is unclear how disrupting the D1-D2 interaction
translates into a behavioral antidepressant effect in the FST and, in Pei et al’s previous study, the
LH task.3
However, a number of studies have indicated that the D1-D2 heterodimer results in
increases in intracellular calcium levels through activation of the Gq – PLC pathway.2 Increases
in cytoplasmic calcium levels have a myriad of effects within neurons, including changing
98
neuronal excitability, activating intracellular processes such as transcription and translation of
proteins and trophic factors.220
Any of these sub-cellular changes could have effects on
mesocortical dopaminergic circuits and the higher-order cognitive processes, thereby having an
effect on the neurobiology of MDD.70,152
One possible mechanistic explanation for the antidepressant effects of the D1-D2
interfering peptide is that it causes an increase in expression of BDNF in the PFC and other brain
areas. Unpublished data from our laboratory indicates that after ICV delivery of the D1-D2
interfering peptide to animals undergoing the LH task, BDNF protein levels were elevated in
striatal tissue compared to animals who had received saline infusions. (T.Lai, Liu lab,
Unpublished Data, 2011). Presumably, the D1-D2 interfering peptide has this effect because it
blocks intracellular pathways that inhibit BDNF expression. Furthermore, evidence from post-
mortem human studies indicate that BDNF serum levels are decreased in the PFC of patients
who had MDD and committed suicide, and increased in patients on long term antidepressant
treatment.159-161
This hypothesis remains unproven, as a study by Hasbi et al. 118
contradicts these findings
and demonstrates a link between activation of the D1-D2 heterodimer and subsequent increases
in BDNF expression in the NAc. The authors hypothesize that an increase in intracellular
calcium caused by activation of the D1-D2 heteromer results in phosphorylation of calmodulin
Kinase II (CamKII), which then acts as a transcription factor and activates transcription of the
BDNF gene.118
One limitation of Hasbi et al’s118
study is that it was conducted in cultured
neurons from the ventral striatum, and, as such, may not be generalizable to the in vivo
pathogenesis of complex psychiatric conditions such as MDD.
A number of studies have demonstrated that directly infusing BDNF into the NAc and the
VTA results in a ‘pro-depression’ phenotype in mice, increasing social aversion
99
behaviors.121,169,170
Overall, these contradictory results suggest that the effect of BDNF on the
pathogenesis of MDD is complex and may depend on the brain region where it is expressed.
Thus, the specific pathway linking D1-D2 heteromerization and upregulation or down-regulation
of BDNF expression with the pathogenesis of MDD remains unknown.
The intranasal administration method used to deliver the D1-D2 interfering peptide to the
brain is much less anatomically specific than the direct microinjections to the PFC used in Pei et
al’s study.3 Thus, it is likely that, after intranasal administration, the D1-D2 interfering peptide
was transported diffusely to numerous brain areas after intranasal administration. As a result, it is
possible that the anti-immobility effect we observed in the FST may not be solely due to the
effect of the D1-D2 interfering peptide in the PFC, but also to its concurrent effects in other brain
areas.
There are numerous lines of evidence that implicate both the NAc and the mesolimbic
dopaminergic reward system in the pathogenesis of MDD.3,80,152
In the NAc, there is a
population of neurons that express both D1 and D2 receptors where these receptors co-
localize.116,118,119
It is possible that, after intranasal administration, the D1-D2 interfering peptide
interferes with the interaction between D1 and D2 in the PFC and these other areas. To better
understand the regions and mechanisms involved in the antidepressant effect of the D1-D2
interfering peptide, biochemical studies in brain areas other than the PFC along with studies
investigating the mechanisms behind the antidepressant effect of the D1-D2 interfering peptide
should be conducted.
4.6 Limitations of the FST as a preclinical test for antidepressant efficacy
It is important to note that the behavioral test we used throughout this study, the FST, is
not a model of MDD. Instead, it is a pharmacologically valid test for antidepressant efficacy, as
100
all clinically effective therapeutics have the same behavioral effect in the FST, that of decreasing
the time spent immobile during the test.171
One disadvantage of the FST is that it lacks
etiological validity, as it does not accurately model the clinical symptoms and manifestation of
MDD.79,171
Notwithstanding, the FST was an appropriate test for this study, as we were
examining whether intranasal administration of the D1-D2 interfering peptide would result in a
behavioral antidepressant effect in the FST, as well as evaluating pharmacological parameters of
this administration method.
A disadvantage of using the FST is that we were unable to examine whether the D1-D2
interfering peptide, after intranasal administration, would continue to have an antidepressant
effect in a chronic animal model of depression such as chronic mild stress or the LH task. We
hypothesize that this is likely the case as intra-cerebral administration of the D1-D2 interfering
peptide decreased escape failures in the 5-day LH Task.3 Additionally, the acute nature of the
FST also presented limitations in investigating whether intranasal administration of the D1-D2
interfering peptide has any appreciable neurobiological changes other than that of disrupting the
interaction between D1 and D2 receptors in vivo. Future studies investigating the antidepressant
efficacy of the D1-D2 interfering peptide should examine its effects in a chronic model of MDD
such as chronic mild stress paradigms.
4.7 The D1-D2 interfering peptide, TAT-peptide and imipramine
significantly decrease locomotor activity
We tested whether the anti-immobility effect of the D1-D2 interfering peptide and of
imipramine during the FST was due to the specific antidepressant effects of these treatments or
due to an overall increase in locomotor activity. To determine this, we tested the effect of these
treatments in a 30-minute locomotor activity test (Figure 3-15). Surprisingly, we found that the
101
D1-D2 interfering peptide, TAT-peptide and imipramine all significantly decreased locomotor
activity during a 30-minute open field test (Figure 3-15). When we examined the animals’
locomotor activity over time, we found no significant interaction between treatment and time
(p>0.05, 2-way Independent Groups ANOVA), indicating that while the amount of locomotor
activity was decreased in animals that received imipramine, TAT peptide or D1-D2 interfering
peptide compared with saline, the behavioral patterns over time did not change dramatically
(Figure 3-16).
In the previous study done in our laboratory, direct microinjections of the D1-D2
interfering peptide or the TAT-peptide to the PFC, NAc or hippocampus did not significantly
alter locomotor activity during a 30-minute open field test.3 Animals in the imipramine treatment
group displayed decreased locomotor activity in the open field test, a result consistent with other
studies of imipramine’s sedative effects.3,221
Thus, the difference in our results may be due to
differences in testing methodology between this study and our laboratory’s previous study.3
These include the method of administration (Intranasal administration vs. direct microinjections),
the intranasal doses of the D1-D2 interfering peptide and TAT peptide were much larger than
those administered directly to the PFC, and the repeated use of isoflurane anesthetic in all
groups.
Repeated use of isoflurane anesthetic can result in changes in animals’ behavior, and
studies have demonstrated changes in animals’ performance on learning and memory tasks but
not on locomotor activity tests.222,223
What may be more likely is that repeated exposure to
isoflurane anesthesia is an aversive, stressful experience for the animal that, coupled with
exposure to the FST, could result in changes in the animal similar to those that occurring in
animals exposed to a chronic mild stress (CMS) paradigm.224
Some CMS protocols induce
decreases in spontaneous locomotor activity and exploratory behavior in the open field test.225-227
102
Although this hypothesis may support our observation of decreased activity in the open field test,
not all chronic mid stress paradigms cause a decrease in locomotor activity and this effect has not
be replicated.224,228
Additionally, if combined exposure to anesthetic, intranasal administration
and the FST resulted in a CMS-like response in the animals, we would expect the locomotor
activity of animals to be equivalent in all treatment groups, which was not the case in our
experiment.
Although it is likely that aversive experiences from repeated intranasal administrations
under anesthetic and exposure to the FST cause stress in the experimental animals and
contributes to these results, it may not be the only factor at play. To determine if this is the case,
we could test this hypothesis by repeating the locomotor activity experiment using
experimentally naive animals. If all groups have the same locomotor activity profiles, then it can
be concluded that the stress caused by the previous experiments resulted in the observed decrease
in locomotor activity.
A second possibility is that the D1-D2 interfering peptide has an effect on the function of
dopamine in the striatum and reduces the occurrence of motor movements by inhibiting the
functions of the nigrostriatal dopamine pathway. As discussed above, it is likely that after
intranasal administration and transport to the CNS, the D1-D2 interfering peptide and other
substances are delivered to numerous brain areas in a diffuse manner, thus it is probable that the
D1-D2 peptide was present in some amount in the striatum after intranasal administration.
Furthermore, D1 and D2 receptors are known to complex in a specific subset of medium spiny
neurons in the basal ganglia and striatum.108
It is possible that this subset of neurons, as Perrault
et al.108
suggest, are responsible for mediating some aspects of dopaminergic signaling within the
nigrostriatal pathway. Thus, signaling through the D1-D2 receptor complex may mediate
103
locomotor activity, and disruption of this signaling may reduce the incidence of locomotor
activity, although no studies have been done directly address this hypothesis.
This hypothesis could account for the decreased locomotor activity observed in the D1-
D2 interfering peptide treatment group but it does not address the decrease in locomotor activity
observed in animals treated with the TAT peptide. Secondly, this explanation may be unlikely
because the D1-D2 interfering peptide had a robust antidepressant effect (i.e. increased mobility)
in the FST. If it had caused a reduction in locomotor activity through the nigrostriatal pathway,
then it is unlikely we would have observed an anti-immobility effect in the FST, as the
occurrence of motor movements would have been reduced in a global manner.
Another possible explanation for our results is that they are an artifact of the experimental
design we used. In order to maintain consistency between the studies, we followed the protocol
used in our laboratory’s previous study of the D1-D2 interfering peptide’s antidepressant effect
(see Section 2.7 for detailed description).3 Briefly, animals were administered D1-D2 interfering
peptide, TAT-Peptide, saline or imipramine 24 hours, 19 hours and 1 hour before the 30-minute
locomotor activity test. This may not have been the best approach, as the locomotor activity
changes observed in the treatment groups could have been due to the animal’s exploration of a
novel environment and not due to any real changes in locomotor behavior across the treatment
groups.
The above is supported by our experimental data, as after 15 minutes in the novel
locomotor activity box, there was no difference in locomotor activity between any of the
treatments groups (Figure 3-16). To test this theory, we could repeat the locomotor activity
experiment and include a sensitization period where the animal is exposed to the locomotor
activity testing apparatus, before the three treatment administrations and subsequent locomotor
activity test. This method may also be a better control for activity in the FST paradigm because
104
animals are replaced in the same plexiglass cylinder on Day 2 of the test as on Day 1, and thus
not exposed to a novel environment during the 5-minute test for antidepressant efficacy.
4.8 Future Directions
The results of this project raise a number of interesting questions for future investigation.
Although this project demonstrated that the intranasal pathway can be used to deliver peptide
therapies to the central nervous system, we did not directly investigate the mechanisms through
which these peptides gain access to the CNS after intranasal delivery. A radioactively-labeled
peptide with the same sequence as the D1-D2 interfering peptide could be used to address this
question, as its pathway to the central nervous system and its presence in various brain areas
could be easily traced and quantified by measuring the resulting levels of radioactivity in these
tissues. This strategy has been successfully used to trace the intranasal transport route of insulin
to the CNS and identify the areas with the most accumulation within the CNS.190,192
A second area warranting further investigation is strategies for improving the efficiency
of D1-D2 interfering peptide transport to the central nervous system after intranasal delivery.
This would allow us to reduce the minimum effective dose of the D1-D2 interfering peptide,
thereby decreasing the potential toxicity of the peptide and accumulation in non-CNS tissues.
One strategy to improve the efficiency of delivery to the CNS is using mucoadhesive solutions to
increase the residence time of the D1-D2 interfering peptide at the olfactory epithelium.229
Mucoadhesive solutions of this type have been shown to increase the proportion of the total dose
of proteins or peptides delivered to the CNS after intranasal administration.229-231
Finally, this project and previous studies done in our laboratory have demonstrated that
the D1-D2 interfering peptide selectively uncouples the D1-D2 receptor heterodimer and has an
antidepressant effect in the FST and other models of depression.3 Other laboratories have
105
investigated the downstream effects of the D1-D2 receptor heterodimerization, and shown that it
causes an increase in cytoplasmic calcium levels via a Gq-dependent mechanism.1,2
Beyond
these observations, there is currently little known about the pathological role of the D1-D2
interaction in MDD, or the exact mechanisms behind the antidepressant effect of the D1-D2
interfering peptide. More studies both in preclinical models and in vitro at the cellular level will
be necessary to fully understand why the D1-D2 interfering peptide has an antidepressant effect.
Although intranasal delivery of the D1-D2 interfering peptide has an antidepressant effect in the
FST, it is unclear whether it will be effective in other preclinical models of MDD when delivered
intranasally. The antidepressant effect of the D1-D2 interfering peptide after intranasal
administration should be tested in other animal models of MDD, such as the LH task and social
aversion paradigms.
Overall, this project has raised a number of interesting questions about the antidepressant
mechanism of the D1-D2 interfering peptide and the intranasal pathway used to deliver these
peptides to the central nervous system. At the same time, the results presented here demonstrate
that the D1-D2 interfering peptide can have an antidepressant effect after intranasal delivery, in a
pharmacologically valid preclinical model. Furthermore, these results have provided important,
clinically relevant results vital to the development of the D1-D2 interfering peptide as a novel
therapeutic for MDD, warranting further preclinical studies should be conducted to conclusively
determine whether the D1-D2 interfering peptide can be tested as a novel antidepressant in the
clinical setting.
106
107
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Appendix 1: A Sufficient intranasal D1-D2 interfering peptide
dose to produce antidepressant effect in the Forced Swimming
Test (Calculation)
Efficiency of Intranasal Delivery to CNS: 1-5%15
of total intranasal dose
Previous D1-D2 Interfering peptide administered directly to the PFC: 5 nmol3
Estimate of Sufficient Intranasal dose: 100 × 5nmol = 500 nmol
Estimated Weight of Rats at time of behavioral testing : 300 – 325 g
Dose as nmol/g body weight: 500 nmol ÷ 300 g = 1.67 nmol/g
Dose as mg/kg body weight : 5.75 mg / kg