norepinephrine transporter inhibition with desipramine
TRANSCRIPT
MOL #93302
1
Norepinephrine transporter inhibition with desipramine exacerbates L-DOPA-induced dyskinesia: role for synaptic dopamine regulation in denervated nigrostriatal terminals Tanya Chotibut, Victoria Fields, Michael F. Salvatore Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
2
Running title: Norepinephrine transporter and L-DOPA dyskinesia Corresponding Author: Michael F. Salvatore, Ph.D. Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130 Email: [email protected] Document statistics: number of text pages: 31 figures: 8 references: 81 words in Abstract: 245 Introduction: 801 Discussion: 1688 Abbreviations used: AIMS: abnormal involuntary movements DA: dopamine DAT: dopamine transporter DMI: desipramine ERK: extracellular signal-regulated protein kinase L-DOPA: L-dihydroxyphenylalanine LID: L-DOPA-induced dyskinesia NE: norepinephrine NET: norepinephrine transporter PD: Parkinson’s disease
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
3
Abstract
Pharmacological dopamine (DA) replacement with Levodopa (L-DOPA) is the gold standard
treatment for Parkinson’s disease (PD). However, long term L-DOPA treatment is complicated
by eventual debilitating abnormal involuntary movements termed L-DOPA induced dyskinesia
(LID), a clinically significant obstacle for the majority of patients who rely on L-DOPA to alleviate
PD-related motor symptoms. The manifestation of LID may in part be driven by excessive
extracellular DA derived from L-DOPA, but potential involvement of DA reuptake in LID severity
or expression is unknown. We recently reported that in 6-OHDA-lesioned striatum,
norepinephrine transporter (NET) expression increases and may play a significant role in DA
transport. Furthermore, L-DOPA preferentially inhibits DA uptake in lesioned striatum.
Therefore we hypothesized that desipramine (DMI), a NET antagonist, could affect the severity
of LID in an established LID model. While DMI alone elicited no dyskinetic effects in lesioned
rats, DMI + L-DOPA treated rats gradually expressed more severe dyskinesia compared to L-
DOPA alone over time. At the conclusion of the study, we observed reduced NET expression
and norepinephrine-mediated inhibition of DA uptake in the DMI + L-DOPA group compared to
L-DOPA alone group in lesioned striatum. LID severity positively correlated with striatal ERK
phosphorylation among the three treatment groups, with increased ppERK1/2 in DMI + L-DOPA
group compared to the L-DOPA- and DMI-alone groups. Taken together, these results indicate
that the combination of chronic L-DOPA and NET-mediated DA reuptake in lesioned
nigrostriatal terminals may have a role in LID severity in experimental Parkinsonism.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
4
INTRODUCTION Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder
and its incidence is likely to only increase with the impending growth of the aging population
(Siderowf and Stern, 2003). PD is primarily characterized by the loss of dopamine (DA)
neurons in the substantia nigra pars compacta. Therapeutically, the primary loss in nigral
dopamine necessitates its replacement through the exogenous administration of L-DOPA, a
dopamine precursor or with DA agonists (Hornykiewicz and Kish, 1987; Steiger and Quinn,
1995). L-DOPA in particular, has remained the drug of choice for treating PD for nearly half a
century (Calne and Sandler, 1970). Despite the ability of L-DOPA to significantly improve motor
function, it is not without considerable side effects that severely limit its use long-term. L-DOPA-
induced dyskinesia (LID) is a debilitating movement disorder brought on by chronic L-DOPA
use. Approximately 90% of patients within the first 10 years of treatment develop LID (Ahlskog
& Meunter 2001; Marsden, 1994; Mones et al., 1971; Olanow and Koller, 1998). Not only is the
onset of dyskinesia a significant setback for the patient, its presence is often permanent,
occurring with every subsequent exposure to L-DOPA, which limits the clinical efficacy of what
has long been considered our gold standard in PD treatment.
Evidence suggests chronic L-DOPA leads to major adaptive molecular changes
occurring within the basal ganglia that may underlie LID pathophysiology (for review see Cenci
and Konradi, 2010). Loss of nigrostriatal dopaminergic neurons not only impairs pre-synaptic
control of DA regulation, but also leads to large variances in extracellular levels of DA that
parallel L-DOPA dosing regimens (Cenci and Lundblad, 2006). It is these supra-physiological
fluctuations in extracellular DA content that are thought to underlie the induction of L-DOPA
induced dyskinesia (Chase, 1998). LID has been associated with plastic changes in post-
synaptic neuronal targets within the striatum, including abnormal trafficking of the DA D1
receptor (Berthet et al., 2009; Guigoni et al., 2007). This has also been shown clinically as well:
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
5
positron emission tomography (PET) imaging studies reveal an association between peak-dose
dyskinesia with abnormally high levels of synaptic DA in the caudate-putamen of L-DOPA
induced dyskinetic patients (de la Fuente-Fernandez et al., 2004; Pavese et al., 2006). In line
with these studies, pre-clinical data show that dyskinetic rats exhibit higher levels of extracellular
DA after L-DOPA administration than those seen in non-dyskinetic animals (Meissner et al.,
2006; Lindgren et al., 2010). These data raise the possibility that not only does striatal
extracellular DA play a pivotal role in the onset of dyskinesia, but could be the triggering
element of the post-synaptic alterations identified in both clinical and pre-clinical models.
However, despite considerable efforts directed at delineating the role L-DOPA plays in altering
dopaminergic striatal signaling, the exact mechanism of how this occurs remains conflicting and
inconclusive (Cenci & Konradi 2010).
In PD, the loss of the dopamine transporter (DAT) likely impairs its ability to maintain DA
bioavailability, yet most studies indicate motor symptoms are seen only when ~70% of striatal
DAT is lost (Bernheimer et al., 1973; Bezard et al., 2001), indicating an alternate mechanism
through which DA may still be effective to produce normal locomotion. Indeed, many studies
have concluded that serotonergic terminals may transport L-DOPA or DA, and subsequently
release DA in order to maintain DA signaling, albeit in a dysregulated fashion (Arai et al., 1995;
Miller and Abercrombie et al., 1999; Tanaka et al., 1999; Kannari et al. 2001, Carta et al., 2007).
However, it is a comparatively neglected observation that in sparsely dopaminergic innervated
regions, such as the frontal cortex, the norepinephrine transporter (NET) can also transport DA
(Moron et al., 2002), and NE uptake inhibitors which can result in increased extracellular DA
levels within the prefrontal cortex (Carboni et al., 1990; Di Chiara et al., 1992; Tanda et al.,
1997; Gresch et al., 1995; Masana et al., 2012; Yamamoto and Novotney, 1998; Wayment et
al., 2001). In line with these observations, we have recently reported that DA transport still
occurs in the 6-OHDA-lesioned striatum, despite major loss of DAT, and that a potential role for
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
6
NET in DA uptake is evident (Chotibut et al., 2012). In these experiments, L-DOPA
preferentially inhibits DA reuptake when striatal DAT loss exceeds the level necessary for PD
symptom appearance, and preferentially inhibits norepinephrine over DA uptake in striatal
tissue. Therefore, based upon these observations, chronic L-DOPA could exacerbate the L-
DOPA induced-dysregulation of synaptic DA by its influence on a NET-dominated regulation of
DA uptake that evolves with the gradual loss of DA terminals.
In an effort to further explore this possibility, we examined how the blockade of NET in a
chronic desipramine (DMI) paradigm would affect dyskinesia expression, NET expression, DA
uptake, and an established post-translational event in striatum associated with dyskinesia,
extracellular signal-regulated protein kinase phosphorylation (ppERK), in an established 6-
OHDA LID rodent model.
MATERIALS and METHODS:
Animals
Male Sprague Dawley rats purchased from Charles-River were used in all experiments.
A total of 21 test subjects were used at the start of the study, were 4-8 months of age, and were
housed 1 per cage and under controlled lighting conditions (12:12 light:dark cycle) with food and
water available ad libitum. All animals were used in compliance with federal and the institutional
Animal Care and Use Committee guidelines at LSU Health Sciences Center-Shreveport. All
behavioral testing was performed between 10:00 and 16:00 h. Behavioral data obtained from
test subjects that did not have >70% TH loss, as discovered after completion of LID
assessments and tissue analysis, was excluded from the results. We note that protein recovery
was limited in some rats due to the fact we analyzed DA uptake, TH, NET, tERK, and ppERK all
from the same striatal tissues and as such, the treatment group numbers are not of equal size.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
7
In all experiments, rats were rendered briefly unconscious with isoflourane and
immediately decapitated on day 40 of the experiment (1 day post last behavioral test and
treatment (L-DOPA + DMI, L-DOPA or vehicle) for dissection of striatal tissues.
6-OHDA Lesions
Rats were anesthetized with 40 mg/kg Nembutal intraperitoneal (i.p.) (pentobarbital
Lundbeck Inc, Deerfield, IL) with supplement of 9.0, 0.6, and 0.3 mg/kg ketamine, xylazine, and
acepromazine, respectively. Each animal then underwent survival surgery to deliver the
neurotoxin 6-OHDA unilaterally to the medial forebrain bundle while immobilized in a stereotaxic
frame (coordinates ML +1.5, AP -3.8, DV -8.0 relative to Bregma). All stereotaxic coordinates
are cited according to the stereotaxic atlas of Paxinos and Watson rat brain atlas, 4th ed.
(Academic Press, 1998). A total of 16 µg of 6-OHDA in a total of 4 µl in 0.02% ascorbic acid
(concentration of 4 mg/ml) was infused unilaterally at a rate of 1 µl/minute. The contralateral
medial forebrain bundle was infused with vehicle (.02% ascorbic acid) and infused at the same
rate and coordinates. The syringe was left in place for 10 min before removal to allow for
maximal diffusion of drug and to avoid further mechanical damage to the tissue. Body
temperature was maintained at 37º during surgery using a temperature monitor with probe and
heating pad (FHC, Bowdoingham, ME). Animals were kept warm after surgery and monitored
closely after anesthesia. In our hands (Chotibut et al., 2012, Fig 7)), we have previously shown
that after 6-OHDA infusion, there was no significant difference in lesioned striatal tissue NE
content compared to contralateral striatum and as such, have based these experiments from
these observations. As such, we did not pre-treat these rats with DMI.
Amphetamine Testing
The extent of the lesion was evaluated 7 days post-surgery based on the net ipsilateral
rotations measured over a 60 minute period following an injection of 2.5 mg/kg D-amphetamine
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
8
i.p (in 0.9% saline) (Ungerstedt and Arbuthnott, 1970). 100 net full turns on the ipsilateral side
to the lesion was necessary to be included in the study. In our hands, we have established this
time frame as enough to consistently catch >60% of TH loss (Chotibut et al., 2012; 2014;
Salvatore, et al., 2014). Ultimately however, post-mortem verification of the lesion severity was
determined by assessment of tyrosine hydroxylase protein as previously reported (Chotibut et
al., 2012; 2014). And thus, any further exclusions were due to rats that did not also have >70%
TH loss after western blot analysis at the end of the study. The behavioral AIMS data reflects
test subjects with >70% TH protein loss.
Desipramine and L-DOPA Administration
9 days post lesion, rats were randomly divided into 3 groups. 2 groups received a
treatment of DMI (Tocris, cat #3067) (12mg/kg) and 1 group received Vehicle (.9% saline
12mg/kg) i.p for 30 consecutive days. Then, at 19 days post lesion, an additional treatment of
either L-DOPA (12mg/kg) and Benserazide-hydrochloride (15mg/kg) or Vehicle (.9% saline) was
given once daily for 20 consecutive days (Fig 1). In summary, 3 treatment groups were created:
(1) DMI Pretreatment + L-DOPA/Benserazide, (2) Vehicle Pretreatment + L-DOPA/Benserazide
and (3) DMI Pretreatment + Vehicle (0.9% NaCl Saline, Hospira, Lake Forest, IL). With 6-
OHDA lesion present in each group, these three groups were used to evaluate the impact of
DMI on LID behavior caused by chronic L-DOPA, with the DMI alone group controlling for
whether NET-blockade alone could produce LID.
Behavioral AIMS ratings
L-DOPA-induced abnormal involuntary movements (AIMs) were then rated at 6 discrete
time points (days 19, 23, 27, 31, 35, 39) during the 20-day administration of L-DOPA starting on
day 19-post lesion until the end of the study. AIMs were assessed by an investigator blind to
treatment. Twenty minutes after L-DOPA administration, rats were placed in separate cages
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
9
and individual dyskinetic behaviors were quantified based on severity and frequency during a 1-
minute observation period performed every 20 minutes over a period of 160 minutes. AIMs
were classified into three subtypes as follows: axial AIMs (dystonic posturing of the upper trunk
towards the side contralateral to the lesion), limb AIMs (hyperkinetic or jerky movements of the
forelimb contralateral to the lesion), and orolingual AIMs (abnormal jaw movements, facial
twitching and tongue protrusion). Each subtype was scored based on severity and frequency
from 0 to 4, with 4 being the most severe and/or occurring continuously during the entire 1-
minute observation period (for review, see Cenci et al., 1998 and Lundblad et al., 2004). Global
AIMs scores were calculated by multiplying the severity score x frequency score for each
observation period and combining the scores on all monitoring periods. Theoretically, the
highest AIMs score achievable in one behavioral test day (with 8 observation periods) would be
384.
Preparation of Synaptosomes
In order to ascertain uptake properties in conjunction with protein and protein
phosphorylation expression levels present in the rats immediately following the last LID
assessment, synaptosomes were prepared according to the protocol previously described
(Salvatore et. al. 2003) with the following modifications: Tissue dissected from dorsal striatum
was homogenized in 5 mL of 0.32 M sucrose solution using a Teflon/glass homogenizing wand
(Glas-Col, Terre Haute, IN) then spun at 1000 x g for 10 minutes in a chilled (4° C) centrifuge.
The resulting pellet was stored as the P1 fraction while the supernatant was spun further at
16,500 x g for 30 minutes at 4° C, yielding the P2 fraction. An aliquot of the P1 fraction was
saved for determination of TH protein, ppERK, and total ERK protein from the 6-OHDA-lesioned
and contralateral (control) striatum against a standard curve of TH protein standard (Salvatore
et al., 2009). We have determined in previous experiments that this fraction is sufficient for
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
10
precise assessment of cytosolic proteins (Chotibut et al., 2012, 2014), reflecting the relative
quantities recovered in fresh frozen preparations (Salvatore et al., 2009). An aliquot of P2
fraction was saved for determination of NET protein from the 6-OHDA lesioned and contralateral
(control) striatum. The supernatant was aspirated and resuspended in 1 mL of Kreb’s buffer
(118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 1.0 mM Na2EDTA, 1.7 mM
CaCl2, 10 mM glucose, 100 μM parglyline, 100 μM ascorbic acid). Protein concentration was
determined using a BCA colormetric assay (Thermo Scientific, Rockford, IL). All tissue was
kept on ice or at 4 °C from the moment of brain excision until the uptake assay took place.
[3H]DA uptake into Synaptosomes
Synaptosomes were distributed in ice-cold test tubes to prepare for DA uptake. The
determination of [3H]DA uptake in the crude synaptosomes from dorsal striatum harvested from
the contralateral and 6-OHDA-infused hemispheres was conducted simultaneously and included
assessments of DA uptake capacity in the presence of unlabeled 1 µM NE and DA. We
previously determined that this concentration of NE or DA had differential impact on DA uptake
(Chotibut et al., 2012) and consistent with previous observations. Each determination was done
in triplicate for each assay condition and uptake was determined comparing the lesioned
striatum with the contralateral control striatum. Non-specific uptake was determined by counts
obtained in synaptosomes incubated with 500 nM DA (all as labeled DA) on ice during the time
period of uptake. Background was determined and subtracted in the same manner as in the DA
uptake studies.
Synaptosomes (30 μg protein per replicate) were added to 4 °C oxygenated Kreb's
buffer and test ligand (if indicated) to reach a total volume of 100 μL. The synaptosomes were
then warmed to 35°C for 5 min, then 100 μL of pre-warmed 1µM 3H-dopamine, prepared from
ViTrax, [7-, 8- 3H-DA], specific activity of 25 Ci/mmol was added to the synaptosome
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
11
preparations (giving a 500 nM final [3H]DA concentration), allowed to incubate for uptake, and
terminated after 120 seconds with an excess volume of ice-cold Kreb’s buffer and re-immersing
the tubes in the ice-bath. The uptake time for DA was chosen to be as close as technically and
practically possible to the approximately 2-minute uptake time of striatal dopamine observed in
vivo as seen by Sabeti et. al. (2002) and where differences in uptake capacity between lesioned
and intact striatum have been previously reported (Chotibut et al., 2012). Synaptosomes were
washed extensively to remove excess labeled-dopamine with equal-osmolarity PBS buffer
through a Brandel M24-TI (Gaithersburg, MD) cell harvester using Brandel GF/C filter paper
pretreated with a 2% polyethylenimine solution to reduce non-specific binding of label. The filter
paper containing the rinsed synaptosomes was transferred into scintillation vials containing 5
mL of biodegradable scintillation cocktail (Research Products International, Mount Prospect, IL)
and counted with a Beckman Coulter LS6500 scintillation counter (Brea, CA).
Calculating DA uptake
To determine the quantity of DA uptake, the percent of [3H]DA recovered in the
synaptosomes against the total amount of [3H]DA added during the uptake experiment was first
determined. The total pmole of recovered [3H]DA was then determined based upon the percent
[3H]DA recovery in the synaptosomes after subtracting the non-specific binding value, and the
result was normalized to synaptosome protein and expressed as pmole DA per mg protein per
minute.
Analyses of proteins and ERK phosphorylation
Synaptosome fraction (P1) and the processed preparatory sample (P2) were sonicated
in a 1% sodium dodecyl sulfate solution (pH ~8) using a Branson Sonifier 150 (Danbury, CT).
Protein concentration was determined using the bicinchoninic acid colormetric assay.
Following gel electrophoresis, proteins were transferred for 500 volt hours in a
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
12
Tris/glycine/methanol buffer onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules,
CA).
The nitrocellulose membrane was stained with Ponceau S to further normalize staining
in each sample lane. These lanes were scanned and quantified by Image J to normalize protein
in each sample. This relative total level then served as an additional normalizing value to
determine the quantity of each protein assayed (Salvatore et al., 2012). To continue
processing, the membranes were blocked in PVP buffer (1% polyvinylpyrrolidone and 0.05%
Tween 20) for a minimum of two hours to reduce nonspecific antibody binding. The membrane
was soaked in primary antibody for 1-3 hours. Specific primary antibodies were as follows: NET
(Alpha Diagonistics, cat NET11-A) and TH (Millipore, cat #AB152), D1 receptor (Santa Cruz, cat
#14001), ERK1/2 (Millipore cat #442704), ppERK1/2 (Sigma, cat #M8159). Protein loads for
linear detection were 50ug total protein for NET, 10ug for TH and total ERK1/2, and 40ug for D1
and ppERK1/2. After primary treatment, blots were exposed to secondary antibody (swine anti-
rabbit IgG for TH and NET) signal enhancement, followed by 1 hour incubation with [125I] protein
A (PerkinElmer, Waltham, MA).
Statistical Analysis
Data were analyzed using SPSS (Chicago, IL, USA) with correction for multiple
comparisons and p-values < 0.05 considered significant. A two-way ANOVA (time x treatment)
was used to analyze AIM scores over time within each session. Total AIM scores (axial + limb +
oral (ALO)) summed across the 160-minute sessions were analyzed by two-way ANOVA. One-
way ANOVA and t-paired student tests were used in instances with more than 3 or more groups
or 2 groups, respectively. One-way ANOVA analyses was followed by Post-Hoc Tukey multi-
comparison test.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
13
Surgeries were performed in triplicates with the assumption that any irregularities in
efficacy of the 6-OHDA lesion may occur in all animals lesioned with the same 6-OHDA on that
day. Specifically, surgeries were performed in multiples of three each day to accommodate for
the three treatments (L-DOPA, L-DOPA + DMI, DMI). Thus, any variances that may be
encountered on each day that may affect lesion severity is assumed to occur to each rat on that
given date of surgery. Additionally, the data was also analyzed using a paired t test when
comparing contralateral and lesioned striatum or one way ANOVA between the 3 treatment
groups.
Due to no dyskinesia elicited in DMI treatment group alone, area under the curve
dyskinesia values (Fig 3A, B) plots only included DMI + L-DOPA and L-DOPA treatment groups.
As such, a paired t-test (Fig 3A) was performed to examine for differences in dependent
measures between these two treatments as well as regression analyses to determine if LID
differed over the course of L-DOPA treatment (Fig 3B).
RESULTS:
LID severity increases with desipramine (DMI) pretreatment + L-DOPA compared to L-
DOPA alone
L-DOPA-induced abnormal involuntary movements (AIMs) were rated at 6 discrete time
points (days 19, 23, 27, 31, 35, 39 post 6-OHDA lesion, or days 1, 5, 9, 13, 17, 21 of daily L-
DOPA) during the 20-day administration of L-DOPA starting on day 1 (19 days post 6-OHDA
lesion). As L-DOPA treatment duration increased, the norepinephrine transporter inhibitor,
desipramine (DMI), exacerbated dyskinesia expression in 6-OHDA lesioned male Sprague
Dawley rats compared to L-DOPA alone beginning on day 27 and through day 39 post-6-OHDA
lesion induction (Fig 2A-F). However, on day 19, when the first dose of L-DOPA was
administered, DMI significantly attenuated LID severity (Fig 2A). However, this attenuation of
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
14
LID was not evident after day 5 of L-DOPA (or 23 days post-lesion) (Fig. 2B). Thereafter, DMI
exacerbated LID severity (Fig. 2C-F). By the end of the study on day 39, DMI pre-treatment with
L-DOPA significantly exacerbated LID at all time periods of observation compared to L-DOPA
treatment alone.
Desipramine alone (no chronic L-DOPA, 6-OHDA lesion only) did not elicit dyskinesia,
supporting previous findings that DA replacement (with its precursor, L-DOPA) is a necessary
component of L-DOPA induced dyskinesia, despite lesion severity.
The area under the curve (AUC) reflects the accumulative LID score attained between
the two groups. L-DOPA + DMI elicited greater dyskinesia than L-DOPA alone over the course
of the 20 day treatment regimen (Fig 3A). Because dyskinesia was not observed with DMI
alone (Fig 2A-F, average abnormal involuntary movement score = 0), a comparison of L-DOPA
+ DMI versus L-DOPA alone was made.
Additionally, LID severity did not change over time in the L-DOPA alone group.
However, in the L-DOPA+DMI group there was a significant increase in dyskinesia over the 20-
day course of treatment with L-DOPA + DMI, with the severity increasing as the study
progressed (Fig. 3B).
Tyrosine Hydroxylase loss in treatment groups
There were no significant differences in tyrosine hydroxylase (TH) loss among the three
treatment groups between lesioned and contralateral control striatum (Fig. 4A, B). Therefore,
the exacerbation of LID severity in DMI + L-DOPA treated rats most likely was not due to
increased dopaminergic terminal destruction or lesion severity (L-DOPA: Average TH loss
80.2% + 4.9; DMI: 79% + 5.3; L-DOPA + DMI: 77% + 4.1).
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
15
Relative abundance of NET: lesioned vs. contralateral striatum
Either DMI alone or DMI + L-DOPA treatment significantly decreased NET abundance in
the lesioned striatum compared to contralateral control striatum in the respective groups (Fig
5A, B, D). No difference in NET expression between control and lesioned striatum was
observed in the L-DOPA group (Fig 5C, D).
ERK phosphorylation in treatment groups
Levels of phosphorylated and total ERK1/2 were assessed in tissue fractions prepared
from the 6-OHDA lesioned striatum from rats from each treatment group. Both antibodies
revealed two bands with the expected molecular weights of ERK1 (44kDa) and ERK2 (42kDa).
Accounting for ppERK ½ in lesioned striatum, when compared to DMI alone, L-DOPA and DMI
+ L-DOPA treatment groups showed increased ppERK/ERK1 and ppERK/ERK2 (Fig 6A, B
respectively; representative western blot Fig 6E). Interestingly, these changes were blunted in
the control striatum (Fig 6C, D). ppERK/ERK1 increased compared to DMI alone in both L-
DOPA and L-DOPA + DMI treatments but there were no differences between L-DOPA and L-
DOPA + DMI groups (Fig 6C). Conversely, ppERK/ERK2 was increased only in L-DOPA
treated rats compared to DMI (Fig 6D). No differences were observed in total ERK between
treatments (data not shown).
ERK phosphorylation and dyskinesia severity
The levels of ppERK1 are increased in L-DOPA preclinical studies (Santini et al., 2007;
Westin et al., 2007). We also observed a correlation between the severity of dyskinesia (as
seen in the L-DOPA + DMI treatment group) and the ppERK1 signal, as measured in the
lesioned striatum in each treatment group (Fig 7), indicating that NET blockade in combination
with L-DOPA may increase DA signaling associated with LID manifestation as observed at the
end of the study (day 21 of L-DOPA). Specifically, simple regression of phospho-ERK1 levels
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
16
on the cumulative axial, limb and orolingual global AIM scores recorded from chronically L-
DOPA, DMI + L-DOPA and DMI treated rats had a positive correlation with dyskinesia severity
(Fig 7).
DA uptake and NE inhibition
DA uptake was determined in synaptosomes from lesioned versus control striatal tissue
in the presence of a concentration of DA or NE (1 µM) previously shown to differentially inhibit
uptake of labeled [3H]-DA (Chotibut et al., 2012). In the L-DOPA group, we did not observe a
significant difference in inhibition [3H]-DA uptake by unlabelled DA between the lesioned and
control striatum (% inhibition in control ~70%) (Fig. 8A). However, NE inhibited DA uptake in
lesioned striatum to a greater extent compared to that in control striatum (% inhibition in control
~50%) (Fig. 8B). In the DMI + L-DOPA group, unlabelled DA did not differentially inhibit [3H]-DA
uptake between lesioned and control striatum (% inhibition in control ~70%) (Fig. 8C).
However, NE was significantly less effective to inhibit DA uptake in lesioned compared to
control striatum (% inhibition in control ~70%) (Fig. 8D). NE also inhibited DA uptake to a
significantly greater extent in lesioned striatum in the DMI alone group (data not shown).
DISCUSSION:
Dysregulation of extracellular (DA) in the DA-denervated Parkinsonian striatum is
associated with LID (Carta et al., 2006; 2007; Cenci and Lundblad, 2006). However, the relative
contribution of DA uptake in LID onset or severity has not been established. The 6-OHDA
lesion may increase striatal NET expression and NET may affect DA uptake therein (Chotibut et
al., 2012). In intact CNS tissue, such as prefrontal cortex, there is evidence for NET-mediated
DA reuptake (Carboni et al., 1990; Tanda et al., 1997; Moron et al., 2002). Accordingly, in
lesioned striatum, the tissue content level of NE is comparable to that of DA (Chotibut et al.,
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
17
2012), suggesting that remaining DAT and inherent NET are potentially in comparable
abundance and NET may therefore assume a greater role in DA uptake. Our results are limited
to striatal analysis because of the relative abundance of DA neuropil (although lesioned) over
other DA regions like substantia nigra. DA uptake in the substantia nigra could also affect LID
severity (Navailles et al., 2014), given significant noradrenergic innervation in this region.
However, there is comparatively less DAT protein and DA uptake in the substantia nigra, which
precluded us from examining this possibility.
L-DOPA may inhibit DA uptake in lesioned striatum (Chotibut et al., 2012; Hashimoto et
al., 2005), which could lead to accumulation of extracellular DA, and therefore affect LID onset
or severity. Increased extracellular DA in DA-denervated striatum is observed following DMI
(Arai et al., 2008). This finding is congruent with our observations that reuptake of DA, derived
from L-DOPA, may be modulated by NET. Thus, reducing NET function, either by L-DOPA-
blockade or reduced NET expression, could reduce DA clearance and exacerbate LID severity.
Accordingly, we found that NET inhibition (with DMI) and L-DOPA, gradually exacerbated LID
severity over the 20-day course of L-DOPA administration, compared to L-DOPA alone (Fig. 3).
These differences in LID severity were unrelated to differences in lesion severity (Fig. 4). Our
previous finding that striatal NET expression increases at a comparatively earlier time point in
lesion progression (Chotibut et al., 2012), coupled with the present findings, give credence to
involvement of NET in DA uptake and regulating LID expression or severity.
Our results also indicate that extracellular signal-regulated kinases 1 and 2 (ERK)
phosphorylation was increased in the DMI + L-DOPA group compared to L-DOPA alone group
(Fig. 6). Increased signaling through D1 receptors is implicated in the molecular and synaptic
responses in striatal neurons associated with LID onset (Aubert et al. 2005; Konradi et al. 2004;
Picconi et al. 2003; Westin et al., 2007) and enhanced ERK1/2 phosphorylation in DA
denervated striatum occurs with selective agonists for D1 or D2 receptors (Cai et al. 2000;
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
18
Pavon et al., 2006; Zhen et al. 2002). Thus, increased ERK1/2 activity in striatal neurons is at
the very least a biochemical marker of L-DOPA–induced dyskinesia through D1 or D2 over-
activation. As such, phosphorylation of ERK1/2 may provide a molecular counterpart for
increased D1 activity and be involved in LID induction. Reduced ERK1/2 phosphorylation dose-
dependently decreases LID and other molecular correlates causally linked to LID development
(Santini et al., 2007). Thus, the observation that DMI pretreatment with L-DOPA both
exacerbates LID and increases ERK1/2 phosphorylation over that of L-DOPA alone supports a
dopaminergic mechanism in LID severity wherein NET-mediated DA uptake and L-DOPA
together modulate LID expression. Given that chronic DMI also reduced NET expression in
lesioned striatum (Fig. 5), this observation suggests that increasing NET function or expression
could reduce LID severity.
Compensatory changes in DA regulation do occur during the loss of DA-regulating
proteins in 6-OHDA rodent models (Snyder et al., 1990; Sarre et al., 2004; Perez et al., 2008),
but the potential involvement of NET function is a relatively novel concept. We have reported
increased NET expression in lesioned striatum (Chotibut et al., 2012). Increased locomotion is
observed in monkeys with DAT inhibitors with high NET, but low serotonin transporter, affinity in
cases of severe DAT loss (80%) compared to those with moderate DAT loss (46%) (Madras et
al., 2006). LID severity could also be diminished by other interactions with noradrenergic inputs
to the basal ganglia (Lundblad et al., 2002; Dekundy et al., 2007; Gomez-Mancilla and Bedard
1993). Furthermore, rats with combined noradrenergic and dopaminergic lesions have greater
LID severity, compared to dopaminergic lesions alone (Fulceri et al., 2007; Shin et al., 2014).
Indeed, noradrenergic lesions can produce dyskinesias through DA-mediated locomotor
impairment (Donaldson et al. 1976, Rommelfanger et al. 2007). As such, NET blockade via
DMI may not be the only factor in worsening LID behavior. For example, in LID pathology,
norepinephrine has the ability to act as a D1 dopaminergic agonist (Kubrusly et al., 2007).
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
19
Thus, it may be possible that agents which decrease NET expression could lead to increased
extracellular norepinephrine, thereby increasing D1 activation and worsening LID.
Reduced NET expression in the lesioned striatum therefore appears to play one of two
critical components of LID and its severity. The other component of LID relates to the impact of
chronic L-DOPA. DMI alone did not produce LID, but the combination of DMI and L-DOPA
gradually worsened LID compared to L-DOPA alone. The neurobiological background of
nigrostriatal lesion is also an important component. Chronic DMI reduced NET expression in
lesioned striatum (Fig. 5), arguably offsetting any lesion-induced increase (Chotibut et al.,
2012). Chronic DMI can reduce NET expression in other CNS regions such as amygdala,
striatum (Jeannotte et al., 2009) and hippocampus (Kitayama et al., 2006). We also point out
that the serotonin terminals or transporter (SERT) can affect LID severity in preclinical models
(Carta et al., 2007; Eskow et al., 2009; Bishop et al., 2012). However, chronic DMI is not
reported to alter SERT expression or function (Hyttel,1994; Mantovani et al., 2009), thus making
it unlikely that changes in SERT expression or SERT-mediated uptake are associated with our
behavioral observations. From a clinical perspective, this leads to questions as to whether an
antidepressant with NET-affinity could produce, hasten the onset of, or worsen the severity of
LID, given the prevalence of PD-related depression and that depression commonly precedes
motor manifestations (Burn 2002; Aarsland et al., 2012; Brichta et al., 2013). DMI or other
tricyclics are often prescribed for this patient population. In small-scale clinical trials (17 test
subjects completing each study), methylphenidate (which also affects NET function (Pan et al.,
1994)), tended to increase LID (Devos et al., 2007; Espay et al., 2011). Exacerbation of
dyskinesia in a clinical setting may go unnoticed given the absence of baseline readings before
DMI administration. Therefore, further study could answer to the possibility that NET-blocking
drugs could exacerbate LID severity or its frequency.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
20
Desipramine may also interfere with alternate receptors such as muscarinic, adrenergic
and histamine. There is a functional interaction between the cholinergic and dopaminergic
systems (Quik and Wonnacott 2011), and DA denervation can increase acetylcholine levels in
striatum. Given that acetylcholine mediates its effects via muscarinic and nicotonic receptors
and that DMI may affect the sensitivity of post and/or pre-synaptic muscarinic receptors
(Murugaiah and Ukponmwan, 2003), it is possible DMI may be mediating its effects through
muscarinic receptors, which may contribute to development of motor signs.
Behaviorally, we observe an initial delay in LID in the L-DOPA + DMI treatment group
compared to L-DOPA, which may be related to increased NET expression with 6-OHDA lesion
(Chotibut et al., 2012). Therefore, this initial increase in NET in the lesioned compared to the
contralateral unlesioned striatum may explain why dyskinetic behavior was not worsened on the
first day of L-DOPA (day 19 post lesion) and that subsequent L-DOPA, in conjunction with DMI
treatments, offset this increase in NET expression, revealing increased LID severity. Our DA
uptake experiments (at the end of the LID assessment) also indicated that DA uptake is more
inhibited by NE in the L-DOPA alone group, but less inhibited by NE in the DMI + L-DOPA
group (Fig. 8). We speculate that these differences may be related to NET expression.
Whereas, NET expression is not changed relative to control striatum in the L-DOPA group, it is
decreased in the groups with DMI. Thus, this decrease in NE-sensitivity in DMI groups could be
due to decreased NET availability in lesioned striatum. We also speculate that the lack of
difference in NET expression in rats treated with L-DOPA alone may be due to increased NET
with 6-OHDA lesion that is offset by possibility that L-DOPA could reduce NET expression over
time. As such, this is an important unresolved question moving forward.
The cellular sources of NET are an important point of consideration. Noradrenergic
innervation of dorsal striatum is relatively sparse, although NE levels are comparable with
remaining DA in lesioned striatum (Chotibut et al., 2012). In prefrontal cortex, NET transports
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
21
DA and conversion of DA to NE may be slower than DA uptake via NET. As such, DA could be
co-released along with NE (Ahn and Klinman 1989). Therefore, pharmacologically-induced
alteration of the noradrenergic system, such by as chronic DMI, could also influence
extracellular DA levels (Devoto and Flore, 2006). Thus our results may also be related
compensatory alterations in DA release. Another possible source of NET-mediated DA
transport may be glia, as glial express NET (Takeda et. al., 2002). Astrocyte and microglia cell
numbers may increase with DA neuron loss (Teismann and Schulz, 2004), possibly increasing
NET protein abundance and DA uptake. Future studies examining alterations in DA uptake in
glial preparations from dyskinetic rats may further delineate the cellular basis for NET-mediated
DA uptake in LID pathophysiology
CONCLUSION:
In summary, our results indicate the possibility that increasing NET expression within the
striatum may be a novel therapeutic avenue to attenuate LID severity. Our previous work has
shown an upregulation of NET expression in lesioned striatum compared to contralateral
striatum after DA denervation in the absence of L-DOPA treatment, but further work could
establish whether this increase in NET is transient or alters with L-DOPA administration. Our
results also indicate that L-DOPA must be present for any development of LID following 6-
OHDA lesion. Our results add to a growing body of literature that NET regulates dopamine
uptake dynamics in the CNS, including pathophysiological mechanisms of L-DOPA induced
dyskinesia.
Authorship Contributions:
Participated in research design: Chotibut, Salvatore Conducted experiments: Chotibut, Fields Performed data analysis: Chotibut, Salvatore Wrote or contributed to writing of the manuscript: Chotibut, Salvatore
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
22
References:
Aarsland D, Pahhagen S, Ballard CG, Ehrt U, Svenningsson P (2012) Depression in Parkinson disease – epidemiology, mechanisms and management. Nat Rev Neurol 8:35–47
Ahlskog JE, Muenter MD (2001) Frequency of levodopa-related dyskinesias and motor fluctuations as estimated from the cumulative literature. Mov Disord 16: 448–458.
Ahn NG, Klinman JP (1989) Nature of rate-limiting steps in a compartmentalized enzyme system. Quantitation of dopamine transport and hydroxylation rates in resealed chromaffin granule ghosts. J Biol Chem, 264: 12259-12265.
Arai R, Karasawa N, Geffard M, Nagatsu I (1995) L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study. Neurosci Lett 195: 195–198.
Arai A, Tomiyama M, Kannari K, Kimura T, Suzuki C, Watanabe M, Kawarabayashi T, Shen H, Shoji M (2008) Reuptake of L-DOPA-derived extracellular DA in the striatum of a rodent model of Parkinson’s disease via norepinephrine transporter. Synapse 62: 632–635. Aubert I, Guigoni C, Hakansson K, Li Q, Dovero S, Barthe N, Bioulac BH, Gross CE, Fisone G, Bloch B, Bezard E (2005) Increased D1 dopamine receptor signaling in levodopa-induced dyskinesia. Ann Neurol 57:17–26. Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F (1973) Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurological Sci 20; 415–455. Berthet A, Porras G, Doudnikoff E, Stark H, Cador M, Bezard E, Bloch B (2009) Pharmacological analysis demonstrates dramatic altera- tion of D1 dopamine receptor neuronal distribution in the rat analog of L-DOPA-induced dyskinesia. J Neurosci 15: 4829–4835.
Bezard E, Dovero S, Prunier C, Ravenscroft P, Chalon S, Guilloteau D, Crossman AR, Bioulac B, Brotchie JM, Gross CE (2001) Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- lesioned Macaque model of Parkinson’s disease. J Neurosci 21: 6853–6861. Bishop C, George JA, Buchta W, Goldenberg AA, Mohamed M, Dickinson SO, Eissa S, Eskow Jaunarajs KL (2012) Serotonin transporter inhibition attenuates L-DOPA induced dyskeinsia without compromising L-DOPA efficacy in hemi-parkinsonian rats. Eur J Neurosci 36: 2839-2848. Brichta L, Greengard P, Flajolet M (2013) Advances in the pharmacological treatment of Parkinson’s disease: targeting neurotransmitter systems. Trends Neurosci: 36: 543-554. Burn, DJ. (2002) Beyond the iron mask: towards better recognition and treatment of depression associated with Parkinson’s disease. Mov Disord 17: 445–454.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
23
Cai G, Zhen X, Uryu K, Friedman E. (2000) Activation of extracellular signal- regulated protein kinases is associated with a sensitized locomotor re- sponse to D(2) dopamine receptor stimulation in unilateral 6-hydroxy- dopamine-lesioned rats. J Neurosci 20:1849 –1857.
Calne DB, Sandler M. L-DOPA and Parkinsonism. (1970) Nature 226: 21-24.
Carboni E, Tanda GL, Frau R, Di Chiara G (1990) Blockade of the noradrenaline carrier increases extracellular dopamine concentrations in the prefrontal cortex: evidence that dopamine is taken up in vivo by noradrenergic terminals. J Neurochem 55: 1067-1070.
Carta M, Lindgren HS, Lundblad M, Stancampiano R, Fadda F, Cenci MA (2006) Role of striatal L-DOPA in the production of dyskinesia in 6-hydroxydopamine lesioned rats. J. Neurochem 96: 1718–1727.
Carta M, Carlsson T, Kirik D and Bjorklund A (2007) Dopamine released from 5-HT terminals is the cause of L-DOPA-induced dyskinesia in parkinsonian rats. Brain 130: 1819–1833.
Cenci MA, Lee CS, Bjorklund A (1998) L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA. Eur J Neurosci 10: 2694–2706
Cenci MA, Konradi C (2010) Maladaptive striatal plasticity in L-DOPA-induced dyskinesia. Prog Brain Res 183: 209–233.
Cenci MA and Lundblad M (2006) Post- versus presynaptic plasticity in L-DOPA-induced dyskinesia. J Neurochem 99: 381–392.
Chase TN (1998) Levodopa therapy: Consequences of the nonphysiologic replacement of dopamine. Neurology 50(5 Suppl. 5): S17–S25.
Chotibut T, Apple DM, Jefferis R, Salvatore MF. (2012) Dopamine transport loss in 6-OHDA Parkinson’s model is unmet by parallel reduction in dopamine uptake. PLoS ONE 7: e52322.
Chotibut T, Davis RW, Arnold JC, Frenchek Z, Gurwara S, Bondada V, Geddes JW, Salvatore MF (2014) Ceftriaxone increases glutamate uptake and reduces striatal tyrosine hydroxylase loss in 6-OHDA Parkinson’s model. Mol Neurobiol 49: 1282-1292.
Dekundy A, Lundblad M, Danysz W, Cenci MA (2007) Modulation of L- DOPA-induced abnormal involuntary movements by clinically tested com- pounds: further validation of the rat dyskinesia model. Behav Brain Res 179: 76–89.
Devos D, Krystkowiak P, Clement F, Dujardin K, Cottencin O, Waucquier N, Ajebbar K, Thielmans B, Kroumova M, Duhamel A, Destee A, Bordet R, Defebvre L (2007) Improvement of gait by chronic high dosese of methylphenidate in patients with advanced Parkinson’s disease. J Neural Neurosurg Psychiatry 78: 470-475.
Devoto P, Flore G. (2006) On the origin of cortical dopamine: is it a co-transmitter in noradrenergic neurons? Curr Neuropharmacol, 2: 115-25.
de la Fuente-Fernandez R, Sossi V, Huang Z, Furtado S, Lu JQ, Calne DB, Ruth TJ, Stoessl AJ (2004) Levodopa-induced changes in synaptic dopamine levels increase with progression of Parkinson’s disease: Implications for dyskinesias. Brain 127: 2747–2754.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
24
Di Chiara G, Tanda GL, Frau R, Carboni E (1992)Heterologous mono- amine reuptake: lack of transmitter specificity of neuron-specific carriers. Neurochem Int 20:231S–235S.
Donaldson I, Dophin A, Jenner P, Marsden CD, Pycock C (1976) The roles of noradrenaline and dopamine in contraversive circling behavior seen after unilateral electrolytic lesions of the locus coeruleus. Eur J Pharmacol 39:179-91.
Espay AJ, Dwivedi AK, Payne M, Gaines L, Vaughan JE, Maddux BN, Slevin JT, Gartner M, Sahay A, Revilla FJ, Duker AP, Shukla R (2011) Methylphenidate for gait impairment in Parkinson disease. A randomized clinical trial. Neurology 76: 1256-1262.
Eskow K, Gupta V, Alam S, Park J, Bishop C (2007) The partial 5-HT1A agonist buspirone reduces the expression and development of l-DOPA-induced dyskinesia in rats and improves l-DOPA efficacy (2007) Pharmacol Biochem Behav 87: 306–314.
Fulceri F, Biagioni F, Ferrucci M, Lazzeri G, Bartalucci A, Galli V, Ruggieri S, Paparelli A, Fornai F (2007) Abnormal involuntary movements (AIMs) following pulsatile dopaminergic stimulation: severe deterioration and morphological correlates following the loss of locus coeruleus neurons. Brain Res 1135: 219–229.
Gomez-Mancilla B, Bedard PJ (1993) Effect of nondopaminergic drugs on L-dopa- induced dyskinesias in MPTP-treated monkeys. Clin Neuropharmacol 16: 418-27.
Gresch PJ, Sved AF, Zigmond MJ, Finlay JM (1995) Local influence of endogenous norepinephrine on extracellular dopamine in rat medial prefrontal cortex. J Neurochem 65: 111–116.
Guigoni C, Doudnikoff E, Li Q, Bloch B, Bezard E (2007) Altered D(1) dopamine receptor trafficking in parkinsonian and dyskinetic non-human primates. Neurobiol Dis 26: 452–463.
Hashimoto W, Kitayama S, Kumagai K, Morioka N, Morita K, Dohi T (2005) Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH. Life Sciences 76:1603-1612.
Hornykiewicz O, Kish SJ. Biochemical pathophysiology of Parkinson’s disease. In: Yahr, M., Bergmann, K. Eds. Advances in Neurology, Parkinson’s disease, vol. 45. Raven Press, New York, 1987; p 19–34.
Hyttel J. (1994) Pharmacological characterization of selective serotonin reuptake inhibitors (SSRIs). Int Clin Psychopharmacol 9 suppl 1: 19-26.
Jeannotte AM, McCarthy JG, Redei EE, Sidhu A (2009) Desipramine modulation of alpha-, gamma-synuclein, and the norepinephrine transporter in an animal model of depression, Neuropsychopharmacology 34: 987–998.
Kannari K, Yamato H, Shen H, Tomiyama M, Suda T, Matsunaga M (2001) Activation of 5-HT1A but not 5-HT1B receptors attenuates an increase in extracellular dopamine derived from exogenously administered l-DOPA in the striatum with nigrostriatal denervation. J Neurochem 76:1346-1353.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
25
Kitayama T, Song L, Morita K, Morioka N, Dohi T (2006) Down- regulation of norepinephrine transporter function induced by chronic administration of desipramine linking to the alteration of sensitivity of local-anesthetics-induced convulsions and the counteraction by co-administration with local anesthetics. Brain Res 1096: 97–103.
Konradi C, Westin JE, Carta M, Eaton ME, Kuter K, Dekundy A, Lundblad M, Cenci MA (2004) Transcriptome analysis in a rat model of L-DOPA-induced dyskinesia. Neurobiol Dis 17: 219 –236.
Kubrusly RC, Ventura AL, de Melo Reis RA, Serra GC, Yamasaki EN, Gardino PF, de Mello MC, de Mello FG (2007) Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue. Neurochem Int 50: 211–218.
Lindgren HS, Andersson DR, Lagerkvist S, Nissbrandt H, Cenci MA (2010) L-DOPA-induced dopamine efflux in the striatum and the substantia nigra in a rat model of Parkinson’s disease: temporal and quantitative relationship to the expression of dyskinesia. J Neurochem 112:1456–1476.
Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N, Cenci MA. (2002) Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson’s disease. Eur J Neurosci 15:120–132
Lundblad M, Picconi B, Lindgren H, Cenci MA (2004) A model of L-DOPA-induced dyskinesia in 6-hydroxydopamine lesioned mice. Re- lation to motor and cellular parameters of nigrostriatal function. Neurobiol. Dis 16: 110 –123
Madras BK, Fahey MA, Goulet M, Lin Z, Bendor J, Goodrich C, Meltzer PC, Elmaleh DR, Livni E, Bonad AA, Fischman AJ (2006) Dopamine transporter (DAT) inhibitors alleviate specific parkinsonian deficits in monkeys: association with DAT occupancy in vivo. J Pharmacol Exp Ther 319: 570–585.
Mantovani M, Dooley DJ, Weyerbrock A, Jackisch R, Feuerstein TJ (2009) Differential inhibitory effects of drugs acting at the noradrenaline and 5-hydroxytryptamine transporters in rat and human neocortical synaptosomes. Br J Pharmacol 158: 1848-1856.
Marsden CD (1994) Problems with long-term levodopa therapy for Parkinson’s disease. Clin Neuropharmacol 17(suppl 2): S32– 44.
Masana M, Santana N, Artigas F, Bortolozzi A (2012) Dopamine Neurotransmission and Atypical Antipsychotics in Prefrontal Cortex: A Critical Review. Curr Top Med Chem 21: 2357-2374.
Meissner W, Ravenscroft P, Reese R, Harnack D, Morgenstern R, Kupsch A, Klitgaard H, Bioulac B, Gross CE, Bezard E, Boraud T (2006) Increased slow oscillatory activity in substantia nigra pars reticulata triggers abnormal involuntary movements in the 6-OHDA-lesioned rat in the presence of excessive extracellular striatal dopamine. Neurobiol Dis 22: 586–598.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
26
Miller GW, Abercrombie ED (1999) Role of high-affinity dopamine uptake and impulse activity in the appearance of extracellular dopamine in striatum after administration of exogenous L-DOPA: studies in intact and 6-hydroxydopamine-treated rats. J Neurochem 72: 1516–1522.
Mones RJ, Elizan TS, Siegel GJ (1971) Analysis of L-dopa induced dyskinesias in 51 patients with Parkinsonism. J. Neurol. Neurosurg. Psychiatry 1971; 34: 668–673
Moron JA, Brockington A, Wise RA, Rocha BA, Hope BT (2002) Dopamine uptake through the Norepinephrine Transporter in Brain Regions with Low Levels of Dopamine Transporter: Evidence from Knock-Out Mouse Lines. J Neurosci 22: 389-395.
Murugaiah KD, Ukponmwan OE (2003) Functional reactivity of central cholinergic systems following desipramine treatments and sleep deprivation. Archives of Pharmacology 368: 294-300.
Navailles S, Milan L, Khalki H, Di Giovanni G, Lagiere M, De Deurwaedere P (2014) Noradrenergic terminals regulate L-DOPA-derived dopamine extracellular levels in a region-dependent manner in Parkinsonian rats. CNS Neurosci Ther 20:671-8.
Olanow CW, Koller WC (1998) An algorithm (decision tree) for the management of Parkinson's disease: treatment guidelines. American Academy of Neurology. Neurology 50: S1–S57.
Pan D, Gatley SJ, Dewey SL, Chen R, Alexoff DA, Ding Y-S, Fowler JS (1994) Binding of bromine-substituted analogs of methylphenidate to monoamine transporters. Eur J Pharmacol 264: 177-182.
Pavese, N., Evans, A. H., Tai, Y. F., Hotton, G., Brooks, D. J., Lees, A. J.,(2006) Clinical correlates of levodopa induced dopamine release in Parkinson disease: A PET study. Neurology, 2006; 67: 1612–1617.
Pavon, N., Martin, A. B., Mendialdua, A., and Moratalla, R. (2006). ERK phosphorylation and FosB expression are asso- ciated with L-DOPA-induced dyskinesia in hemiparkinso- nian mice. Biological Psychiatry, 59(1), 64–74.
Paxinos G, Watson C. Rat Brain in Stereotaxic Coordinates, 1998. 4th edition. Waltham, MA: Academic Press.
Perez XA, Parameswaran N, Huang L, O’Leary KT, Quik M. Presynaptic dopaminergic compensation after moderate nigrostriatal damage in non-human primates. J Neurochem 2008; 105: 1861–1872.
Picconi B, Centonze D, Håkansson K, Bernardi G, Greengard P, Fisone G, Cenci MA, Calabresi P. Loss of bidirectional striatal synaptic plasticity in L-DOPA-induced dyskinesia. Nat Neurosci 2003; 6: 501–506.
Quik, M. and Wonnacott, S. a6b2* and a4b2* nicotinic acetylcholine receptors as drug targets for Parkinson’s disease. Pharmacol. Rev. 2011; 63: 938–966.
Rommelfanger KS, Edwards GL, Freeman KG, Liles LC, Miller GW, Weinshenker D. , Norepinephrine loss produces more profound motor deficits than MPTP treatment in mice. Proc Natl Acad Sci U S A 2007; 104:13804-9, 2007
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
27
Sabeti J, Gerhardt GA, Zahniser NR. Acute cocaine differentially alters accumbens and striatal dopamine clearance in low and high cocaine locomotor responders: Behavioral and electrochemical recordings in freely moving rats. J Pharmacol Exper Ther 2002; 302: 1201–1211. Salvatore MF. Ser31 tyrosine hydroxylase phosphorylation parallels differences in dopamine recovery in nigrostriatal pathway following 6-OHDA lesion. J Neurochem 2014; 129: 548-558.
Salvatore MF, Apparsundaram S, Gerhardt GA. Decreased plasma membrane expression of striatal dopamine transporter in aging. Neurobiol Aging 2003; 24: 1147-1154.
Salvatore MF, Pruett BS, Spann LS, Dempsey C. Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation. PLoS ONE 2009; 4: e8466.
Salvatore MF, Pruett BS, Dempsey C, Fields V. Comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area. J Vis Exp 2012; doi: 10.3791/4171.
Santini E, Valjent E, Usiello A, Carta M, Borgkvist A, Girault JA, Hervé D, Greengard P, Fisone G. Critical involvement of cAMP/ DARPP-32 and extracellular signal-regulated protein kinase signaling in L-DOPA-induced dyskinesia. J Neurosci 2007; 27: 6995–7005.
Sarre S, Yuan H, Joners N, Van Hemelrijck, Ebinger G, Michotte Y. In vivo characterization of somatodendritic dopamine release in the substantia nigra of 6-hydroxydopamine-lesioned rats. J Neurochem 2004; 90: 29-39.
Shin E, Rogers JR, Devoto P, Bjorklund A, et al. Noradrenaline neuron degeneration contributes to motor impairments and development of L-DOPA-induced dyskinesia in a rat model of Parkinsons disease. Exp Neurol 2014; 257: 25-38.
Siderowf A, Stern M. Updated on Parkinson’s disease. Ann Intern Med 2003; 138: 651-8.
Snyder GL, Keller RW, Zigmond MJ. Dopamine efflux from striatal slices after intracerebral 6-hydroxydopamine: evidence for compensatory hyperactivity of residual terminals. J Pharmacol Exp Ther 1990; 253: 867–876.
Steiger, M.J., Quinn, N.P. Levodopa-based therapy. In: Koller, W.C., Paulson, G. (Eds.), Therapy of Parkinson’s Disease, 2nd ed. Marcel-Dekker, New York, 1995: 105–143.
Takeda H, Inazu M, Matsymiya T. Astroglial dopamine transport is mediated by norepineprine transporter. Arch Pharmacol 2002; 366: 620-623.
Tanaka H, Kannari K, Maeda T, Tomiyama M, Suda T, Matsunaga M. Role of serotonergic neurons in L-DOPA-derived extracel- lular dopamine in the striatum of 6-OHDA-lesioned rats. Neuroreport 1999; 10: 631–634. Tanda G, Pontierri FE, Frau R, Di Chiara G. Contribution of blockade of the noradrenaline carrier to the increase of extracellular dopamine in the rat prefrontal cortex by amphetamine and cocaine. Eur J Neurosci 1997; 9: 2077-2085.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
28
Teismann P, Schulz, JB. Cellular pathology of Parkinson’s disease: astrocytes, microglia and inflammation. Cell Tissue Res 2004; 318: 149-161. Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res 1970; 24: 485–493.
Wayment H, Schenk JO, Sorg BA. Characterization of extracellular dopamine clearance in the medial prefrontal cortex: role of monoamine uptake and monamine oxidase inhibition. J Neurosci 2001; 21: 35-44.
Westin, JE., Vercammen, L., Strome, EM., Konradi, C., Cenci, MA. Spatiotem- poral pattern of striatal ERK1/2 phosphorylation in a rat model of L-DOPA- induced dyskinesia and the role of dopamine D1 receptors. Biol. Psychiatry 2007; 62, 800–810.
Yamamoto BK, Novotney S. Regulation of extracellular dopamine by the norepinephrine transporter. J Neurochem 1998; 71:274–280. Zhen X, Torres C, Cai G, Friedman E. Inhibition of protein tyrosine/ mitogen-activated protein kinase phosphatase activity is associated with D2 dopamine receptor supersensitivity in a rat model of Parkinson’s disease. Mol Pharmacol 2002; 62: 1356 –1363.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
29
Footnotes:
This work was funded in part by the Ike Muslow Predoctoral Fellowship Award to TC and an award to MFS through the Edward P. Stiles Trust Fund-Louisiana State University Health Sciences Center-Shreveport and The Biomedical Research Foundation of Northwest Louisiana.
Reprint requests:
Michael F. Salvatore, Ph.D. Department of Pharmacology, Toxicology, & Neuroscience Louisiana State University Health Sciences Center 1501 Kings Highway Shreveport, Louisiana 71130 Email: [email protected]
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
30
Legends for Figures
Figure 1. Timeline of treatment paradigm. Male Sprague Dawley rats were lesioned unilaterally with 6-
OHDA and randomly assigned a treatment group. 7 days post lesion, rats were tested with
amphetamine (2.5 mg/kg i.p) and number of turns ipsilateral to lesion turns were assessed to confirm
for a successful lesion. Treatment began on day 9 with 3 treatments: (1) Desipramine + L-DOPA, (2)
Desipramine + Vehicle, (3) Vehicle + L-DOPA. Beginning on day 19, behavioral AIMs were assessed at 6
discrete time points for the remainder of the study (days 19, 23, 27, 31, 35, 39). On Day 39, rats were
sacrificed and dopamine uptake and biochemical markers were assessed.
Figure 2. Abnormal movements induced by the chronic treatment with L-DOPA in animals lesioned
with 6-OHDA. The time course of changes in dyskinesia evaluated from the product of the frequency
and amplitude behavior (orolingual, axial, forelimb) induced by a 20-day treatment with L-DOPA (6
mg/kg plus benserazide 12 mg/kg, i.p). (A) Day 19 (first day of L-DOPA) and (B) Day 23 showed minimal
difference at timepoints in dyskinesia severity. (C) Day 27, (D) Day 31 and (E) Day 35, approximately 1
week after treatment initiation, shows DMI + L-DOPA treatment is worsening dyskinesia severity at
latter timepoints in the observation period. (F) By the end of the study on Day 39, DMI + L-DOPA
treatment worsens dyskinesia severity at all timepoints compared to L-DOPA alone. In all observation
periods, DMI alone did not elicit any dyskinesia. Data analyzed by repeated ANOVA followed by
Bonferroni post hoc test (time course) Significant difference from L-DOPA only treatment: * p<.05,
**p<.01, ***p<.001.
Figure 3. Dyskinesia over time with L-DOPA versus L-DOPA + DMI treatment. (A) Area Under the Curve
(AUC) of dyskinesia elicited between day 4 until end of 20 day treatment regimen between L-DOPA and
L-DOPA + DMI (Paired Student t-Test n = 5 * p<.05) (B) Linear regression of dyskinesia and time with L-
DOPA and L-DOPA + DMI treatment (L-DOPA r2
= .30, p = .24; L-DOPA +DMI r2 = .82, p = .0028).
Percentages reflect the difference in the AUC score between the two treatment groups as a function of
L-DOPA administrations.
Figure 4. (A) Tyrosine Hydroxylase (TH) loss with treatment between control and lesioned striatum.
There was no significant differences between TH loss among treatments (one-way ANOVA, p =.87, n = 3-
5 (DMI n = 3, DMI + L-DOPA = 4, L-DOPA = 5). DMI + L-DOPA vs L-DOPA F(2,9) = .74, DMI + L-DOPA vs DMI
F(2,9) = .42; L-DOPA vs DMI F(2,9) = .24 (B) Tyrosine Hydroxylase loss representative western blot
(Standards of TH shown in ng (0.5, 2, 3, 4); UL = unlesioned, L = lesioned).
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
31
Figure 5. NET Expression after treatment between lesioned and intact striatum. (A) Lesioned striatum
showed less NET expression compared to intact contralateral striatum with chronic DMI + L-DOPA
treatment. (B) DMI treatment also decreased NET expression in lesioned striatum compared to control.
(C) Treatment with chronic L-DOPA however, did not show changes in striatal NET expression. Paired
Student t-test (A) t = 2.692, p = .03, (B) t = 12.92,p = .006 (C) t = 2.077, p = .06 (D) Representative
western blot (L = Lesion, UL = Unlesion)
Figure 6. ppERK1/2 in lesioned striatum after treatment. (A) ppERK/ERK1 was significantly increased in
both DMI + L-DOPA and L-DOPA compared to DMI. DMI + L-DOPA treatment also increased ppERK/ERK1
compared to L-DOPA alone in lesioned striatum. One-Way ANOVA n = 3, p = .0001, f = 58.33. (DMI + L-
DOPA vs DMI: F(2, 6) = 15, DMI + L-DOPA vs L-DOPA F(2, 6) = 10, L-DOPA vs DMI F(2, 6) = 5) (# denotes
significance compared to DMI, * compared to L-DOPA). (B) ppERK/ERK2 was significantly increased in
both DMI + L-DOPA and L-DOPA compared to DMI. DMI + L-DOPA treatment also increased ppERK/ERK2
compared to L-DOPA alone in lesioned striatum. One-Way ANOVA n = 3, p = .0007, f = 30.33 (DMI + L-
DOPA vs DMI: F(2, 6) = 5, DMI + L-DOPA vs L-DOPA F(2, 6) = 11, L-DOPA vs DMI F(2, 6) = 6 (# denotes
significance compared to DMI, * compared to L-DOPA). (C) ppERK/ERK1 was significantly increased in
both L-DOPA and L-DOPA + DMI groups compared to DMI alone in unlesioned striatum n = 3, p =.02, f =
9.0 F(2, 6) = 5.20, DMI + L-DOPA vs DMI F(2, 6) = 5.19 (* denotes significance compared to DMI alone). (D)
ppERK/ERK2 was significantly increased in L-DOPA + DMI treated rats compared to DMI alone in the
unlesioned striatum n = 3, p = .02, f = 7.8, F(2, 6) = 5.42, L-DOPA vs DMI F(2, 6) = 3.87, DMI + L-DOPA vs DMI
F(2, 6) = 1.55 (* denotes significance to DMI alone) (E) ppERK 1/2 representative western blot (L = Lesion,
UL = Unlesion)
Figure 7. Correlation analysis of the severity of dyskinesia and the ppERK/ERK1 in all treated animals.
ppERK/ERK1 values with their corresponding Global AIMs over all treatments showing a significant
correlation between ppERK1 and dyskinesia severity. Correlation analysis, n = 7, R = .86, p = .003.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
MOL #93302
32
Figure 8. Uptake of DA in presence of unlabelled DA or NE. DA uptake was measured in preparations
of crude striatal synaptomes from the contralateral control and lesioned (>70% TH loss) striatum.
Results are presented as the percent inhibition of tritiated DA uptake by 1 µM DA or NE. Note, the
assessments of inhibition by unlabelled monoamine were conducted simultaneously in lesioned and
control synaptosome preparations, one test subject per group at a given time. (A) DA inhibition of DA
uptake in L-DOPA group. DA uptake (2 min) was determined by the parameters described in methods
(ns, Student’s two-tailed t-test, t=2.36; df=4). (B) NE inhibition of DA uptake in L-DOPA group. DA
uptake (2 min) was determined by the parameters described in methods. There was a significant
increase (37%) in inhibition of DA uptake in the lesioned striatum by NE (*p<0.05, Student’s two-tailed
t-test, t=3.38; df=4). (C) DA inhibition of DA uptake in DMI + L-DOPA group. DA uptake (2 min) was
determined by the parameters described in methods (ns, Student’s two-tailed t-test, t=3.03; df=3). (D)
NE inhibition of DA uptake in DMI + L-DOPA group. DA uptake (2 min) was determined by the
parameters described in methods. There was a significant decrease (45%) in inhibition of DA uptake in
the lesioned striatum by NE (**p<0.01, Student’s two-tailed t-test, t=8.33; df=3).
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 1
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
0 20 40 60 80 100 120 140 1600
10
20
30
40
50
Desipramine + Vehicle
Vehicle + L-DOPA
Desipramine + L-DOPA
*
D19:Time (min) post-L-DOPA injection
Glo
bal A
IMS
0 20 40 60 80 100 120 140 1600
10
20
30
40
50
Vehicle + L-DOPADesipramine + Vehicle
Desipramine + L-DOPA
D23:Time (min) post-L-DOPA injection
Glo
bal A
IMS
0 20 40 60 80 100 120 140 1600
10
20
30
40
50 Desipramine + VehicleVehicle + L-DOPADesipramine + L-DOPA
*
**
**
***
D27: Time (min) post-L-DOPA injection
Glo
bal A
IMS
0 20 40 60 80 100 120 140 1600
10
20
30
40
50Vehicle + L-DOPA
Desipramine + Vehicle
Desipramine + L-DOPA
*
D31:Time post-L-DOPA injection
Glo
bal A
IMS
0 20 40 60 80 100 120 140 1600
10
20
30
40
50 Vehicle + L-DOPADesipramine + Vehicle
Desipramine + L-DOPA
*
**
******
****
D35: TIme (min) post L-DOPA injection
Glo
bal A
IMS
(Mea
sure
of D
yski
nesi
a)
0 20 40 60 80 100 120 140 1600
10
20
30
40
50Vehicle + L-DOPADesipramine + Vehicle
Desipramine + L-DOPA
********* ***
***
***
**
*
D39:TIme (min) post L-DOPA injection
Glo
bal A
IMS
(Mea
sure
of D
yski
nesi
a)
Figure 2
A B
C D
E F
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 3
A
B
-1 -1
0
1000
2000
3000
4000
5000Ar
ea U
nder
the
Cur
ve
L-DOPA L-DOPA + DMI
*
0 3 6 9 12 15 18 210
1000
2000
3000
4000
5000
6000
L-DOPAL-DOPA + DMI
29%
18%
52% 45%68% 115%
days of L-DOPA
AU
C L
ID s
core
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 4
A
B
TH Loss in Lesioned Striatum Compared to Unlesioned Striatum
-1 -1 -1
0
20
40
60
80
100TH
% L
oss
DMI + L-DOPA L-DOPA DMI
.5 ng 2ng 3ng 4ng UL / L UL / L
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 5
A
B
C
D
NET expression in chronically treated DMI and L-DOPA: Lesioned vs Control Striatum
DMI w/DOPA Control DMI w/L-DOPA Lesion0
10
20
30
40
50
*
NE
T E
xpre
ssio
n (u
g)
NET expression in chronically treated DMI alone:Lesioned vs Control Striatum
Contralaterol Control DMI Alone Lesion0
10
20
30
40
*
NET
Exp
ress
ion
(ug)
NET Expression in chronically treated L-DOPA: Lesioned vs Control Striatum
L-DOPA Contralateral Control L-DOPA Lesion0
10
20
30
40
50
NET
Exp
ress
ion
(ug)
DMI L-‐DOPA L-‐DOPA + DMI UL / L UL / L UL / L
80 kDA
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 6
A B
C D
E
ppERK/ERK1 in Lesioned Striatum
DMI + L-DOPA L-DOPA DMI 0.00
0.02
0.04
0.06
0.08
0.10
#
#
*
pp
ER
K / E
RK
1
ppERK/ERK2 in Lesioned Striatum
DMI + L-DOPA L-DOPA DMI 0.00
0.02
0.04
0.06 #
#
*
pp
ER
K/E
RK
2
ppERK/ERK1 in Control Striatum
-1 -1 -1
0.00
0.02
0.04
0.06* *
pp
ER
K /
ER
K1
DMI + L-DOPA L-DOPA DMI
L / UL L / UL L / UL
DMI L-DOPA + DMI L-DOPA
44 kDA ERK1
42 kDA ERK2
ppERK/ERK2 in Control Striatum
-1 -1 -1
0.00
0.01
0.02
0.03
0.04
0.05
*
pp
ER
K / E
RK
2
ppERK/ERK1 in Control Striatum
-1 -1 -1
0.00
0.02
0.04
0.06* *
pp
ER
K /
ER
K1
DMI + L-DOPA L-DOPA DMI
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
Figure 7
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from
L-DOPA + Vehicle with DA
Con
trol
Lesion
ed
0
10
20
30
40
50
60
70
80
90
% inhib
itio
n o
f [3
H]-
DA
upta
ke b
y D
A
L-DOPA + Vehicle with NE
Con
trol
Lesion
ed
0
10
20
30
40
50
60
70
80
90*
% inhib
itio
n o
f [3
H]-
DA
upta
ke b
y N
E
Desip + L-DOPA with DA
Con
trol +
DA
Lesion
ed +
DA
0
10
20
30
40
50
60
70
80
90
% inhib
itio
n o
f [3
H]-
DA
upta
ke b
y D
A Desip + L-DOPA with NE
Con
trol +
NE
Lesion
ed +
NE
0
10
20
30
40
50
60
70
80
**
% inhib
itio
n o
f [3
H]-
DA
upta
ke b
y N
E
Figure 8
A. B.
C. D.
This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2014 as DOI: 10.1124/mol.114.093302
at ASPE
T Journals on D
ecember 1, 2021
molpharm
.aspetjournals.orgD
ownloaded from