The Role of Synaptic Inhibition in the Primary Motor Cortex of an ALS Mouse Model

by

Kara Marie Place

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Cell & Systems Biology University of Toronto

© Copyright by Kara Marie Place 2018

ii

The Role of Synaptic Inhibition in the Primary Motor Cortex of an

ALS Mouse Model

Kara Marie Place

Master of Science

Department of Cell & Systems Biology University of Toronto

2018

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease that involves progressive muscle

weakness and atrophy resulting from the degeneration of motor neurons in the cortex and spinal

cord. Cortical hyperexcitability is a hallmark feature of ALS, and recent studies report reductions

in synaptic inhibition in the primary motor cortex (M1) presymptomatically. Rescuing

interneuron-mediated inhibition is a promising strategy for reducing hyperexcitability and

slowing the onset of ALS. This study aimed to determine whether chemogenetic or

pharmacological tools could decrease pyramidal neuron hyperexcitability in Layer 5 of the M1

(L5-M1) in the SOD1G93A ALS mouse model. The principle novel findings of my work are: (i)

increasing GABAergic interneuron-mediated inhibition onto L5-M1 pyramidal neurons rescues

hyperexcitability of M1 pyramidal neurons and (ii) riluzole reduces hyperexcitability and affects

intrinsic membrane properties of M1 pyramidal neurons.

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Acknowledgments

First and foremost, I would like to thank my supervisor Melanie Woodin, who not only provided

me the incredible opportunity to work as a master’s student in her lab, but also allowed me to

work alongside her as an undergraduate student through her collaboration with the Gairdner

Foundation. She provided me invaluable support, insight, mentorship and guidance through the

year and I am extremely grateful for that. I am also grateful for the input and suggestions given

to me by my supervisory committee members Dr. John Peever and Dr. Junchul Kim.

I would like to acknowledge Dr. Sahara Khademullah who supervised and mentored not only

myself, but many others, with a great deal of patience, enthusiasm and with the desire to help

each one of us learn. The development of my project was through her work as a PhD student and

the technical skills I gained are through her mentorship.

I would also like to thank Zahra, Danielle, Melissa, Xinyi, and Jessica for all of their feedback

and advice on my project. With their support and guidance, I was able to learn laboratory skills

as well as develop my critical thinking skills. Thanks for always putting up with my crazy

thoughts and tangents!!! I am also thankful for the experimental assistance from Faiza,

Chinmaya and Simon. It is thanks to everyone in the Woodin Lab that I was able to grow as a

scientist.

My experience at the Ramsay Wright Laboratories would not have been possible without the

administrative assistance from Ian, Genna, Baljit, Sue, Peggy and Janet. Moreover, my

endeavors with my numerous mice would not have been as successful without the hard work and

dedication from Christine, Kelly, Kelsie, Suzi, Akbar, Stephanie and Raymond.

Outside of the lab, I am thankful for all the support from my friends, but mostly the unending

love and encouragement from my Mom, Dad and the occasional pep talk from my brother in the

form of, “You got this”. J

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Table of Contents

Acknowledgments ......................................................................................................................... iii

Table of Contents ............................................................................................................................ iv

List of Figures ................................................................................................................................. vi

List of Abbreviations ................................................................................................................... viii

Chapter 1 Introduction ..................................................................................................................... 1

1.1 Amyotrophic Lateral Sclerosis ............................................................................................ 1

1.1.1 General Introduction ................................................................................................ 1

1.1.2 Overview of clinical ALS ........................................................................................ 1

1.1.3 Genetic basis of ALS- Human mutations and mouse models ................................. 2

1.2 Synaptic Transmission: Inhibition and Excitation ............................................................... 5

1.2.1 Glutamatergic Excitotoxicity ................................................................................... 6

1.3 Riluzole ................................................................................................................................ 7

1.4 The Motor Cortex (M1) ....................................................................................................... 8

1.4.1 Inhibitory neurons in M1 ......................................................................................... 8

1.5 Cortical hyperexcitability in ALS ...................................................................................... 13

1.5.1 Disruption of inhibitory circuits in ALS ................................................................ 13

1.6 Chemogenetics ................................................................................................................... 14

1.7 AAV Technology .............................................................................................................. 15

1.8 Aim 1- Rationale, Objective, and Hypothesis ................................................................... 16

1.9 Aim 2- Rationale, Objective, and Hypothesis ................................................................... 18

Chapter 2 Materials and Methods .................................................................................................. 20

Materials and Methods .............................................................................................................. 202

2.1 Materials ............................................................................................................................ 20

2.1.1 Animals .................................................................................................................. 20

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2.1.2 Drugs ..................................................................................................................... 20

2.2 Methods ............................................................................................................................. 21

2.2.1 M1 Slice Preparation ............................................................................................. 21

2.2.2 Electrophysiology .................................................................................................. 21

2.2.3 Surgical Procedures ............................................................................................... 22

2.2.4 Statistics ................................................................................................................. 22

Chapter 3 Results ........................................................................................................................... 23

Results ....................................................................................................................................... 233

3.1 Aim 1: To determine if increasing inhibition from GABAergic forebrain interneurons reduces pyramidal neuron hyperexcitability in the primary motor cortex of a symptomatic ALS mouse ................................................................................................... 23

3.1.1 hDlx-Gq-hM3D Reduces Cortical Hyperexcitability ............................................ 24

3.2 Aim 2: To determine the electrophysiological effects of bath applying riluzole onto neurons in the M1 .............................................................................................................. 35

3.2.1 Action potential frequency is altered in presymptomatic and symptomatic mice ....................................................................................................................... 35

3.2.2 Riluzole modifies intrinsic membrane properties in SOD1 and control mice ....... 41

Chapter 4 Discussion ..................................................................................................................... 50

Discussion ................................................................................................................................. 504

4.1 Summary and interpretation of results ............................................................................... 50

4.1.1 Aim 1: hDlx-Gq-hM3D reduces cortical hyperexcitability ................................... 50

4.1.2 When should this treatment be given to patients? ................................................. 53

4.1.3 Aim 2: Riluzole significantly reduces action potential firing and modifies intrinsic membrane properties in L5-M1 pyramidal neurons ................................ 54

4.1.4 How much riluzole actually crosses the BBB? ...................................................... 56

4.2 Future directions ................................................................................................................ 57

References ..................................................................................................................................... 59

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List of Figures

Chapter 1 Figures

Figure 1.1 Interneuron mediated inhibition of pyramidal neurons in L5 of the M1.

Figure 1.2 PV+ Interneuron development in the neonatal cortex.

Figure 1.3 Electrophysiological recordings show a reduced strength of synaptic transmission in the SOD1G93A pre-symptomatic mouse.

Figure 1.4 Activation of PV+ interneurons in L5 of M1 can reduce cortical hyperexcitability, delay the onset of neurodegeneration, and improve motor performance in presymptomatic SOD1G93A mice.

Chapter 3 Figures and Table

Figure 3.1.1 Excitability of L5-M1 pyramidal neurons at P90-95.

Figure 3.1.2 Representative traces showing activation of hDlx-Gq-hM3D in interneurons reduces pyramidal action potential firing in WT mice.

Figure 3.1.3 Activation of hDlx-Gq-hM3D in interneurons reduces pyramidal action potential firing in WT mice.

Figure 3.1.4 Activation of hDlx-Gq-hM3D does not alter intrinsic properties of L5-M1 pyramidal neurons in WT mice.

Figure 3.1.5 Representative traces showing activation of hDlx-Gq-hM3D in interneurons reduces action potential firing of pyramidal neurons in SOD1 mice.

Figure 3.1.6 Activation of hDlx-Gq-hM3D in interneurons reduces action potential firing of pyramidal neurons in SOD1 mice.

Figure 3.1.7 Activation of hDlx-Gq-hM3D does not alter intrinsic properties of L5-M1 pyramidal neurons in SOD1 mice.

Figure 3.1.8 Activation of GABAergic interneurons in L5 of M1 can restore pyramidal neurons excitability in symptomatic SOD1-hDlx-Gq-hM3D mouse.

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Figure 3.2.1 Pyramidal neurons are hyperexcitable at the presymptomatic and symptomatic stage.

Figure 3.2.2 Representative traces showing riluzole significantly reduces the frequency of action potential firing in presymptomatic SOD1 and control mice.

Figure 3.2.3 Riluzole significantly reduces the frequency of action potential firing in presymptomatic SOD1 and control mice.

Figure 3.2.4 Representative traces showing riluzole significantly reduces the frequency of action potential firing in symptomatic SOD1 and control mice

Figure 3.2.5 Riluzole significantly reduces the frequency of action potential firing in symptomatic SOD1 and control mice.

Figure 3.2.6 Membrane properties are not significantly different between WT and SOD1 presymptomatic mice under aCSF conditions.

Figure 3.2.7 Membrane properties are not significantly different between WT and SOD1 symptomatic mice under aCSF conditions.

Figure 3.2.8 Riluzole significantly alters AP threshold and AP maximum in presymptomatic (P60-65) control mice.

Figure 3.2.9 Resting membrane potential and AP threshold are significantly altered after riluzole application in symptomatic (P90-95) control mice.

Figure 3.2.10 Input resistance significantly decreased after riluzole application in presymptomatic (P60-65) SOD1 mice.

Figure 3.2.11 AP threshold and input resistance are significantly increased after riluzole application in symptomatic (P90-95) SOD1 mice.

Table 1 Electrophysiological properties of pyramidal neurons from presymptomatic and symptomatic SOD1 and control mice exposed to aCSF and riluzole.

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List of Abbreviations

5HT3A Serotoninergic receptor

aCSF Artificial cerebrospinal fluid

AD Alzheimer’s disease

ALS Amyotrophic lateral sclerosis

AMPA α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid

AP Action potential

BAC Bacterial artificial chromosome

BBB Blood brain barrier

C9ORF72 Chromosome 9 open reading frame 72

Ca2+ Calcium

CGE Caudal ganglionic eminence

Cl- Chloride

CNO Clozapine-N-oxide

CNS Central nervous system

CR Calretenin

CSN Corticospinal tract neurons

Dlx5/6 Distalless homeobox 5 and 6

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DREADDs Designer receptors exclusively activated by designer drugs

EPSP Excitatory postsynaptic potential

fALS Familial amyotrophic lateral sclerosis

FDA Food and Drug Administration

FTD Frontotemporal dementia

FUS Fused in Sarcoma

GABA γ-aminobutyric acid

GE Ganglionic eminence

H-MRS Proton-magnetic resonance spectroscopy

HM Hypoglossal motoneurons

iPSC Induced pluripotent stem cell

K+ Potassium

KA Kainate acid

L5 Layer 5

LGE Lateral ganglionic eminence

LMN Lower motor neuron

M1 Primary motor cortex

MGE Medial ganglionic eminence

Na+ Sodium

x

NMDA N-methyl-D-aspartic acid

NPY Neuropeptide Y

PD Parkinson’s disease

PMC Premotor cortex

PV Parvalbumin

rAAV Recombinant adeno-associated virus

RMP Resting membrane potential

sALS Sporadic amyotrophic lateral sclerosis

SICI Short-interval intracortical inhibition

sIPSC Spontaneous inhibitory postsynaptic potential

SMA Supplementary motor area

SOD1 Superoxide dismutase 1

SOM Somatostatin

TARDBP TAR DNA binding protein

TDP-43 Transactive response DNA-binding protein 43

TMS Transcranial magnetic stimulation

UMN Upper motor neuron

VIP Vasointestinal-peptide

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Chapter 1 Introduction

1.1 Amyotrophic Lateral Sclerosis

1.1.1 General Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by

progressive muscle weakness resulting from degeneration of upper motor neurons (UMNs) in the

cortex, and lower motor neurons (LMNs) in the brain stem and spinal cord (Schütz 2005; Martin

& Chang 2012). The most common symptoms experienced are weakness in the hands and legs,

as well as slurred speech or dysphagia (Traub et al. 2011). ALS (also known as Charcot’s disease

or Lou Gherig’s disease) was originally described by Jean-Martin Charcot in 1869 (Nijssen et al.

2017), and since then two hypotheses have emerged to explain the pathogenic mechanisms

involved in the disease progression: the “dying-forward” and “dying back” hypotheses (Vucic et

al. 2008). The “dying-forward” hypothesis proposes that motor neuron degeneration is a result of

hyperexcitability in cortical cells, which causes a cascade of events that lead to the degeneration

of LMNs in the spinal cord (Saba et al. 2016). In contrast, the “dying-back” hypothesis views

pathological changes occurring in more distal targets such as at the nerve terminal or at

neuromuscular junctions before progressing towards the cell body (Dadon-Nachum et al. 2011).

While it seems that the “dying-back” hypothesis is more popular (Dadon-Nachum et al. 2011;

Eisen & Weber 2001), as I will explain below, the focus of my thesis will examine the “dying-

forward” aspect of ALS specifically within the motor cortex, due to the compelling emerging

evidence that cortical activity is profoundly altered in the presymptomatic phase of the disease.

1.1.2 Overview of clinical ALS

The prevalence of ALS in European populations is 2.6 – 3.0 cases per 100,000, with the risk of

developing the disease peaking between 50-75 years of age, and is higher in men than in women

(van Es et al. 2017); Laferrièrea & Polymenidou 2015). The incidence and prevalence of ALS is

thought to be lower in populations of mixed ancestral origin (van Es et al. 2017). Though this

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prevalence is fairly low, a 2017 meta-analysis determined that there are nearly 450,000 ALS

cases worldwide (Kuźma-Kozakiewicz 2018; Marin et al. 2017). Moreover, ALS is the most

common form of motor neuron disease, and is the third most frequent neurodegenerative disease

following Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Talbot 2014). Patients with

ALS have a mean survival of 3-5 years after the disease onset, with denervation of respiratory

muscles typically being the fatal event (Schütz 2005; Cleveland & Rothstein 2001). Any

voluntary muscle can be affected, while motor neurons that control eye movement and sphincter

control appear resistant and remain unaffected (van Es et al. 2017; Nijssen et al. 2017). In terms

of cognitive and behavioral changes, 5-15% of ALS patients also have frontotemporal dementia

(FTD). ALS and FTD are phenotypic extremes on the ALS-FTD spectrum, as about 50% of

patients with ALS show some degree of cognitive or behavioral changes without meeting the

diagnostic criteria for FTD (van Es et al. 2017; Ravits et al. 2013). Apart from life support

management, riluzole is the only Food and Drug Administration (FDA)-approved treatment that

prolongs mechanical ventilation-free survival of patients for ~2-3 months (Laferrièrea &

Polymenidou 2015; Do-Ha et al. 2017). It is thought to do so by decreasing the concentration of

glutamate at the synaptic cleft, increasing glutamate re-uptake, and blocking its receptors

(detailed in section 1.3) (Kalia et al. 2008). However, riluzole has no effect on muscle strength

and does not improve the clinical state of patients (Kuźma-Kozakiewicz 2018).

1.1.3 Genetic basis of ALS- Human mutations and mouse models

In 5-10% of cases, the disease is familial (fALS) and inherited in a dominant or recessive

fashion, while the other 90-95% of cases have no known genetic linkage and are considered

sporadic (sALS) (Cleveland & Rothstein 2001; Martin & Chang 2012). Though the onset of

fALS cases typically occurs around 10 years earlier than sALS, the majority of cases occur

within the same age group of sALS cases (55-75 years) (Talbot 2014). ALS is more commonly

seen in men than women (affecting 1.2-1.5 men to every woman) and disease onset occurs at a

younger age in men. However, gender has no evident effect on survival (van Es et al. 2017;

McCombe & Henderson 2010). More than 30 genes have been implicated in fALS, with

mutations in TAR DNA binding protein (TARDBP), Chromosome 9 open reading frame 72

(C9ORF72) and Superoxide dismutase 1 (SOD1) being the most common (Do-Ha et al. 2017).

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1.1.3.1 Mutations in TDP-43

Known mutations in patients have been identified in proteins involved in RNA binding and

modulation, such as the 43-kDa transactive response DNA-binding protein (TDP-43), the protein

product of TARDBP (Traub et al. 2011). Normally, TDP-43 localizes to the nucleus where it

plays a role in transcription. Misfolded TDP-43 forms aggregates in the cytosol, which leads to

transcription deficits and toxic features such as the generation of oxidative species (van Es et al.

2017). Abnormal TDP-43 aggregation is a common pathological feature in nearly all patients

with ALS, excluding patients that carry mutations in SOD1 or fused in sarcoma (FUS) (van Es et

al. 2017). Clinical studies have also identified cortical hyperexcitability in patients, and that

glutamate alterations both functionally and structurally are present in sporadic and familial ALS

cases (Fogarty et al. 2016). Mutations in the gene that encodes for TDP-43 have been identified

as causing ALS- frontotemporal dementia (ALS-FTD), and most hereditary forms of ALS-FTD

are characterized by cytoplasmic aggregation of TDP-43 (White et al. 2018).

In a TDP-43Q311K mouse model, increased spontaneous excitatory synaptic inputs were detected

alongside an increase in dendritic spine density in motor cortex layer 5 (L5) pyramidal neurons

presymtomatically. These changes in both dendritic spines and electrophysiological recordings

are consistent with clinical cases. Additionally, these researchers also detected similar TDP-43+

protein inclusions, a pathological feature seen in a large proportion of sALS patients (Fogarty et

al. 2016). Another study using the same mouse model detected an ~25% reduction in

parvalbumin (PV+)-expressing inhibitory interneurons in the frontal cortex, as well executive

dysfunction and memory impairment. Though this model exhibits cortical hyperexcitability and

FTD-related cognitive deficits, it does not display significant motor impairment (White et al.

2018).

1.1.3.2 Mutations in C9ORF72

In humans, though still poorly characterized, repeat expansions of GGGGCC intronic

hexanucleotide in the C9ORF72 gene accounts for 33-40% of fALS and up to 7% of sALS cases

(Ravits et al. 2013). In healthy subjects, this repeat expansion is shorter than 25 units while in

ALS patients it can expand between 800-4400 units. Patients with large expansions in C9ORF72

have the most complex pathology of all ALS cases because they not only have the presence of

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TDP-43 protein inclusions but also an accumulation of RNA foci in both neurons and glial cells

(Ravits et al. 2013; Laferrièrea & Polymenidou 2015). Moreover, there was a recent

identification of this GGGGCC repeat expansion in a collection of families with combined ALS-

FTD phenotype and TDP-43 based pathology (DeJesus-Hernandez et al. 2011). After this

identification, it was soon discovered this repeat expansion in C9ORF72 is the most common

cause of fALS and sporadic TDP-43-positive ALS-FTD to date (Todd & Petrucelli 2016).

Since the identification of repeat expansions of C9ORF72 in ALS is more recent, the generation

of animal models is ongoing with models still being created to understand the molecular events

that lead to ALS/FTD. One bacterial artificial chromosome (BAC) transgenic mouse model that

contains a full length C9ORF72 gene with a flanking sequence that drives the expression of

sense and antisense transcripts, shows decreased survival as well as neuropathological and

phenotypic markers of C9ORF72 ALS/FTD. Specifically, degeneration of L5 pyramidal neurons

in the motor cortex was detected (Liu et al. 2016). However, a different BAC transgenic mouse

was able to display behavioral deficits and mild neurodegeneration, but had no TDP-43

inclusions (Batra & Lee 2017). Evidence for neuronal hyperexcitability has been observed in

symptomatic ALS patients with mutated C9ORF72 (Schanz et al. 2016), and though this has yet

to be reported in a C9ORF72 mouse model, it has been demonstrated in patient induced

pluripotent stem cells (iPSC)-derived neurons (Selvaraj et al. 2017).

1.1.3.3 Mutations in SOD1

For quite some time, the SOD1 gene was the only gene known to be associated with ALS

patients (van Es et al. 2017). Mutations in the gene encoding the SOD1 enzyme responsible for

the detoxification of free radicals, account for around 20% of the fALS and 1-2% of sALS cases

(Kuo 2003; Traub et al. 2011; Ravits et al. 2013). The role of the ubiquitously expressed SOD1

enzyme is to convert the free radical superoxide into water and hydrogen peroxide, which helps

protect cells from reactive oxygen species toxicity (Cleveland & Rothstein 2001; Bunton-

Stasyshyn et al. 2014). Under normal conditions, the SOD1 stable homodimer can be found in

the cytoplasm or mitochondrial intermembrane space. When this gene carries an ALS-causing

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mutation, SOD1 misfolds and forms ubiquitinated protein inclusions (Laferrièrea &

Polymenidou 2015).

My proposed research will focus on the SOD1G93A ALS mouse model, because it was one of the

first models created that recapitulated the disease phenotype consistently and reliably, and since

it’s establishment in 1994, has been used extensively in research and thus a great deal of our

current understanding of the disease mechanisms involved in ALS is based on this model (Van

Damme et al. 2017). Moreover, this mouse model exhibits increases in cortical excitability that

depend on the disease stage similar to that seen in ALS patients (Kim et al. 2017). This is in

contrast to C9ORF72 mouse models, where cortical hyperexcitability has not yet been reported

(Selvaraj et al. 2017) and TDP-43 mouse models where cortical hyperexcitability is reported, yet

no significant motor deficits develop (White et al. 2018). In the SOD1 G93A mouse, imbalances in

excitation and inhibition are observed long before (2-3 months) significant motor neuron death is

detected, and clinical symptoms appear. Specifically, cortical hyperexcitability was detected

neonatally in hypoglossal motoneurons and superior colliculus interneurons (van Zundert et al.

2008). Early measurement of SOD1 activity in ALS patient blood points to the loss of enzymatic

activity being central to the disease progression. However, the transgenic mouse expressing the

fALS mutant, SOD1G93A, develops progressive motor neuron disease despite having elevated

SOD1 activity which results in an enhanced free radical-generating capacity (Martin & Chang

2012). Moreover, three sets of mice expressing different SOD1 mutations have the disease with

either elevated or unchanged SOD1 activity. This suggests that SOD1 mutants have acquired

toxic properties regardless of the amount of SOD1 activity each retains, most likely mediated by

misfolded SOD1 protein aggregates (Cleveland & Rothstein 2001; Taylor et al. 2016; Traub et

al. 2011; Martin & Chang 2012).

1.2 Synaptic Transmission: Inhibition and Excitation

In healthy brains, the balance between excitation and inhibition is critical for numerous functions

such as sleep, cognitive processes, and motor control (Cline 2001). Rapid signal transmission

between neurons in the mammalian central nervous system (CNS) occurs through two main

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modes of neurotransmission: excitation and inhibition. Excitatory synaptic transmission is

largely mediated by glutamate, while γ-aminobutyric acid (GABA) and glycine are principal

neurotransmitters at inhibitory synapses (Xiang et al. 2002; Choi 1988). Glutamate is an

excitatory neurotransmitter in the CNS that acts on G protein-coupled (metabotropic) and ligand-

gated (ionotropic) receptors found on both the presynaptic and postsynaptic terminals at synapses

in the brain and spinal cord (Choi 1988). There are three ionotropic receptors classified

according to their affinity to the exogenous agonists α-amino-3-hydroxy-5-methyl-4-

isoxazoleproprionic acid (AMPA), N-methyl-D-aspartic acid (NMDA) and kainate acid (KA)

receptors. NMDA receptors, as well as AMPA receptors (that lack a GluR2 subunit) are

permeable to calcium (Ca2+) and sodium (Na+), while KA receptors are not, and also potassium

(K+) in the case of NMDA receptors (Young et al. 2007). GABA acts on chloride (Cl-)

permeable ionotropic GABAA receptors and metabotropic GABAB receptors, which are located

both pre-synaptically and post-synaptically (Allain et al. 2011). Despite that GABAergic

interneurons represent a minority (~20%) of neurons in the neocortex when compared to

glutamatergic neurons (~80%), they are key regulators of neuronal excitability and are involved

in the generation of temporal synchronous and oscillatory behavior within networks of pyramidal

neurons (Di Cristo et al. 2011). Given the important role of GABAergic interneurons in the

function of neuronal circuits, alterations in cortical GABAergic interneuron function leads to an

imbalance between excitation and inhibition. A consequence of this imbalance can be excessive

synaptic glutamatergic transmission, which leads to the over activation of glutamate receptors,

and can result in degeneration (Corona et al. 2007).

1.2.1 Glutamatergic Excitotoxicity

In healthy conditions, appropriate glutamate release is required for the brain to function

normally, and is involved in learning and memory formation (Wang et al. 2004). Excitotoxicity

is a phenomenon that is caused by a massive release of glutamate. Overstimulation of glutamate

receptors, such as NMDA, AMPA and KA receptors can overexcite neurons and trigger a

cascade of events that result in cell death (Kalia et al. 2008). Neuronal death by excitotoxicity

has been implicated in numerous neurological diseases such as Alzheimer’s disease,

Huntington’s disease and ALS (Pose-Utrilla et al. 2017). Several recent studies have

demonstrated profound alterations in glutamatergic neurotransmission within the ALS motor

cortex, which will be the area of focus throughout my thesis (Corona et al. 2007). Thus,

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disrupting the mechanisms that lead to excitotoxicity may protect the brain and prevent pro-death

cascades from being triggered. Impeding excitotoxic effects has been an appealing therapeutic

target for ALS, and is thought to be one of the mechanisms behind the only approved treatment,

riluzole, for ALS patients worldwide.

1.3 Riluzole

Riluzole, originally developed in the 1950s as an anticonvulsant and muscle relaxant, has been

the only FDA approved treatment given to ALS patients worldwide (Foerster et al. 2013). It has

been demonstrated to delay the onset of respiratory complications modestly and increase the

median survival of patients for 2-3 months (Lu et al. 2016). Recently, a retrospective analysis

revealed that riluzole prolongs the last clinical stage of ALS in patients that take a dose of

100mg/day (Fang et al. 2018). It is thought that riluzole protects motor neurons against death

induced by glutamate excitotoxicity by disrupting glutamate neurotransmission and decreasing

the concentration of glutamate. Specifically, it is reported to decrease glutamate release from

nerve terminals by stabilizing the inactivated form of voltage-gated Na+ channels, as well as

having an effect on the NMDA receptor-glutamate system by acting as a noncompetitive

antagonist at NMDA and AMPA receptors (Kalia et al. 2008; Gurney et al. 1985; Vucic, Lin, et

al. 2013). Additional studies have reported that riluzole decreases repetitive firing of action

potentials in response to sustained current injection in cultured rat cerebellar granule cells and

spinal motor neurons, in rat striatal and cortical neurons, and cultured mouse cortical and spinal

neurons (Belluzzi & Urbani 2000; Bellingham 2011). In rat striatal and cortical neurons, and

mouse spinal neurons, the effects of riluzole on current-induced firing were not linked to changes

in resting membrane potential or input resistance. However, in some instances action potential

amplitude, duration, and the threshold for action potential initiation were increased (Bellingham

2011). Though there have been several steps to understanding the mechanisms behind the

neuroprotective role and electrophysiological effects of riluzole in certain models of ALS, what

has yet to be characterized is the effect riluzole has specifically on pyramidal neurons located

within L5 of the primary motor cortex in the SOD1G93A ALS mouse.

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1.4 The Motor Cortex (M1)

The motor cortex, important for the initiation and execution of voluntary movements, is divided

into three regions: the supplementary motor area (SMA), premotor cortex (PMC), and primary

motor cortex (M1) (Park et al. 2015; Ueta et al. 2014). M1 plays a role in both internally

generated and sensory driven behaviours, and is innervated by axons projecting from the

thalamus, as well as from the frontal and parietal cortex. These cortical and thalamic inputs

interdigitate and overlap to provide excitation across the layers of M1 (Hooks et al. 2013).

M1 provides a major source of descending motor output for voluntary movement, with some of

these commands originating from corticospinal tract neurons (CSNs) in L5 with axons that

terminate in the ventral horn of the spinal cord where they monosynaptically connect with

motoneurons (Rathelot & Strick 2009). Because of this direct connection, CSNs (also called Betz

cells) (Rivara et al. 2003), are believed to play a special role in the generation and control of

skilled movements (Rathelot & Strick 2009). Corticospinal pyramidal neurons directly target

spinal motor neurons in humans, though in mice are found to do so more indirectly through

polysynaptic contacts (Rathelot & Strick 2009; D’Acunzo et al. 2014). Though pyramidal cells

constitute roughly 75-80% of the total population of neurons in all cortical areas, M1 is also

made up of inhibitory interneuron populations (DeFelipe & Fariñas 1992).

1.4.1 Inhibitory neurons in M1

Inhibitory neurons are also known as interneurons (or local circuit neurons) because in the

cortex, their axons make only short-range and local connections (Shipp 2007). Inhibitory circuits

are mediated by cortical interneurons that can be divided into around 20 different subtypes based

on characteristics such as morphology, molecular markers, connectivity, as well as

electrophysiological properties (Tanaka et al. 2011; Kelsom & Lu 2013a; Nakazawa et al. 2012).

PV+ and somatostatin-expressing (SOM+) GABAergic neurons make up the largest and second

largest subclass of L5 interneurons, representing 40-50% and 20-30% of the GABAergic cortical

interneuron population respectively (Kelsom & Lu 2013). Basket cells and chandelier cells are

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the two types of interneurons that make up the PV+ interneuron group. Most PV+ cells in L5 of

the M1 are basket cells whose axons target the soma and proximal dendrite of target pyramidal

cells and provide powerful inhibition. Fast-spiking basket neurons are thought to play a large

role in the regulation of excitatory and inhibitory inputs within the cerebral cortex (Kelsom & Lu

2013; Moore et al. 2018; Zecevic et al. 2011). In general this interneuron subtype gives rise to

oscillatory activity in the gamma-frequency range (between 30-80 Hz) (Marín 2012). PV+

interneurons are the main target of projections from the thalamus in the cortex, while also

forming nets of synaptic contacts with adjacent pyramidal neurons. Additionally, PV+ basket

cells are interconnected with each other through chemical and electric synapses, creating a nest

of synchronous interneurons (Di Cristo et al. 2011). In contrast to PV+ interneurons, SOM+ cells

primarily target the distal dendrites of excitatory pyramidal cells, have a low spike threshold, and

are mostly Martinotti cells (Figure 1.1). SOM+ interneurons tend to oscillate within the theta

range (3-9Hz) (Di Cristo et al. 2011). PV+ interneurons mediate a major component of inhibition

within L5 of M1 and are more numerous than SOM+ cells (Naka & Adesnik 2016). Moreover,

paired recordings suggest that the synaptic conductance of PV+ to pyramidal cells in L5 is many

fold larger than that of the synaptic conductance of SOM+ to pyramidal cells (Tanaka et al. 2011;

Naka & Adesnik 2016). Based on the dominant role of PV+ interneurons in inhibiting L5

pyramidal neurons, my project will focus on regulating inhibition from this cell type.

The remaining 30% of neocortical interneurons are a more heterogeneous group that share the

expression of 5HT3A serotoninergic receptors. These include vasointestinal-peptide positive

(VIP+), calretenin-positive (CR+) and neuropeptide-y expressing (NPY) interneurons. VIP+

interneurons are found mostly in superficial cortical layers, targeting pyramidal neuron dendrites

as well as preferentially targeting other interneurons (Marín 2012; Clark et al. 2017). Calretinin

is largely expressed in double bouquet and bipolar cells located in layer I, and are classified as

“regular spiking cells” (Benes & Berretta 2001). NPY-expressing neurons are widely distributed

in the cortical layers, but are found more frequently within layers II-III and in layer VI.

Moreover, these neurons are heterogeneous as they exhibit either fast-spiking or accelerating

firing patterns (Karagiannis et al. 2009).

10

Figure 1.1 Interneuron mediated inhibition of pyramidal neurons in L5 of the M1. PV+ Basket cells in L5 synapse onto the soma of pyramidal neurons and chandelier cells onto the proximal dendrites where they provide powerful inhibition. SOM+ Martinotti cells, in contrast, synapse onto the distal dendrites of L5 pyramidal neurons. Figure from (Marin et al. 2012).

11

Neocortical GABAergic interneurons are derived primarily from a structure called the ganglionic

eminence (GE), a transient brain structure present during embryonic development located in the

ventral telecephalon (Kelsom & Lu 2013).In total, there are three GEs: the medial ganglionic

eminence (MGE), caudal ganglionic eminence (CGE) and lateral ganglionic eminence (LGE).

The MGE and CGE are the main courses of cortical GABAergic interneurons in the developing

nervous system. The MGE produces around 70% of neocortical interneurons, and gives rise to

both PV+ fast-spiking interneurons and SOM+ populations (Kepecs & Fishell 2014) (Figure

1.2A). In vitro and in vivo studies demonstrated that around 65% of MGE-derived interneurons

express parvalbumin, while 35% express somatostatin. The CGE gives rise to the other ~30% of

neocortical interneurons, producing a more heterogeneous population of cortical interneurons

that share the expression of 5HT3A serotoninergic receptors (Di Cristo et al. 2011). There are

numerous transcription factors that play a role in regulating the development and specification of

MGE interneuronal populations, including Dlx homeobox genes. The Dlx family of homeobox

genes, comprised of Dlx1, 2, 5 and 6 all influence the specification of MGE-derived cortical

interneurons. However, it has been shown that Dlx5/6 has a preferential expression in PV+

mature interneurons (Kelsom & Lu 2013;Wang et al. 2010) (Figure 1.2B). This finding is of

particular relevance to my proposed research because I plan to use a Dlx5/6 enhancer to target

chemogenetic tools to predominantly PV+ interneurons, in order to regulate their activity.

12

Figure 1.2 PV+ Interneuron development in the neonatal cortex. A) PV+ interneurons in the M1 are derived from the MGE. B) Transcription signals of Dlx5/6 affect the development of PV+ interneurons. Taken from (Kelsom & Lu 2013).

A)

B)

13

1.5 Cortical hyperexcitability in ALS

There have been many mechanisms implicated in the pathogenesis of ALS that underlie the

selective degeneration of both upper and lower motor neurons, including oxidative stress,

improper neurofilament transport, mitochondrial dysfunction and glutamatergic excitotoxicity

(Schütz 2005; Cleveland & Rothstein 2001). As ALS is a multifaceted disease, the pathologies

that cause motor neuron degeneration between patients may differ, but cortical hyperexcitability

is a well-documented pathophysiological feature seen in fALS patient cohorts with SOD1, FUS,

and C9ORF72 mutations (Vucic, Lin, et al. 2013). Additionally, cortical hyperexcitability has

been demonstrated in the SOD1G93A and TDP43 mouse models (Saba et al. 2016; Zhang et al.

2016). Some proposed mechanisms of hyperexcitability involve ionic channel dysfunction in

motor neurons, dysregulation of K+ homeostasis by astrocytes, and the disruption of inhibitory

circuits in the cortex, which is the focus of this thesis (Do-Ha et al. 2017).

1.5.1 Disruption of inhibitory circuits in ALS

Complex brain circuits are comprised of networks of excitatory and inhibitory neurons that

require a fine balance in their transmission crucial for cortical function (Marín 2012). When this

dynamic equilibrium is disrupted aberrant changes can occur and interfere with synaptic

communication, and can contribute to the emergence of neuropsychiatric disorders such as

epilepsy, schizophrenia, and ALS (Schütz 2005; Kelsom & Lu 2013; Nakazawa et al. 2012;

Ramamoorthi & Lin 2011). In healthy individuals, generally a sub-threshold stimulus leads to

the activation of GABAergic interneurons that then reduce subsequent excitability, or short-

interval intracortical inhibition (SICI) as demonstrated through transcranial magnetic stimulation

(TMS) studies. In ALS patients, SICI is reduced or absent, suggesting either the dysfunction or

loss of inhibitory interneurons (Vucic, Ziemann, et al. 2013; Do-Ha et al. 2017; Zanette et al.

2002). Specifically, the reduction of SICI is evident in both the M1 and motor cortex (Menon et

al. 2017). These human studies are supported by animal studies, for example, the reduction of

inhibitory currents was shown to precede defects in motor neurons in presymptomatic SOD1G93A

mice (Geevasinga et al. 2016). Additionally, an increase in hyperexcitability was demonstrated

in cultured spinal motor neurons from the same mouse model (Kuo 2003). One study revealed a

14

substantial loss of CR-expressing interneurons located in the symptomatic SOD1G93A motor

cortex, with a varying vulnerability of NPY-expressing interneurons at a later disease stage

(Clark et al. 2017). More recently, impairments in GABAergic signaling in the TDP-43A315T ALS

mouse model were shown to contribute to cortical hyperexcitability (White et al. 2018). With

respect to human studies, post mortem histological analyses have revealed a reduction in both

cortical pyramidal neurons and PV+ inhibitory interneurons (Do-Ha et al. 2017; Nihei et al.

1993). Moreover, two proton-magnetic resonance spectroscopy (H-MRS) studies in small ALS

patient cohorts revealed a decrease in GABA levels in motor cortex, suggesting reduced

inhibitory transmission (Foerster et al. 2012; Foerster et al. 2013). My proposed research will

examine the disruptions in inhibition involved in ALS. The rationale for studying inhibition in

ALS is primarily based on studies on human ALS patients where decreases in cortical inhibition

are observed. Thus it is clear that inhibition is in disrupted in ALS patients and mouse models,

but despite this understanding the role of this inhibition in contributing to cortical

hyperexcitability has not been fully characterized. In this thesis, I will selectively target

inhibitory interneurons to regulate neural activity using a recently developed genetic technique:

chemogenetics.

1.6 Chemogenetics

Chemogenetics is an approach involving the introduction of proteins that have been engineered

to interact with small drug-like compounds into a region of interest (Dobrzanski & Kossut 2017).

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)-based chemogenetic

tools have been widely used by neuroscientists to identify the circuitry and signals that specify

cortical function in multiple species, as well as gain selective control of neural activity (Roth

2016). Since its discovery in 2007, DREADDs have been successfully employed in basic

research aimed at determining neural circuits that underlie behaviors and more recently, has been

used in research on neurological conditions such as Parkinson’s Disease, epilepsy, and

schizophrenia (Dobrzanski & Kossut 2017). DREADDs are modified human muscarinic

receptors that are activated by the inert and orally bioavailable designer drug Clozapine-N-oxide

(CNO) (Dobrzanski & Kossut 2017; Dimidschstein J et al. 2016). Studies have supported the

15

successful expression of DREADDs in nonhuman primates with no apparent toxicity, and that

CNO-DREADDs can modulate electrophysiology, circuitry and even behavior in nonhuman

primates (Roth 2016). Along with the original invention of DREADDs came the creation of three

Gq-coupled DREADDs: hM1D1, hM3Dq, and hM5Dq, each based on a different human

muscarinic receptor. The hM3Dq DREADD is a commonly used excitatory DREADD for

enhancing neuronal firing and activity when activated by CNO (Roth 2016; Dimidschstein et al.

2016), and will be used in my project as a means of increasing the activity of GABAergic

interneurons. DREADDs are commonly delivered to cell types of interest using viral delivery

methods (Dimidschstein et al. 2016), and in this thesis I will be using a viral vector to drive

DREADD expression in inhibitory interneurons.

1.7 AAV Technology

Inhibitory GABAergic interneurons, though fewer in abundance than glutamatergic projection

neurons, are critical for regulating the balance in cortical circuits (Shipp 2007). While certain cell

types such as principal excitatory cells have been targeted successfully, the ability to target

inhibitory interneurons had previously been limited to transgenic mice (Taniguchi et al. 2011).

Gene delivery using recombinant adeno-assiociated virus (rAAV) technology has shown

increased promise for treating complex diseases, and has generated encouraging results in many

phase I-III clinical trails (Dimidschstein et al. 2016). The recent development of a novel rAAV

for targeting and manipulating interneurons is based on the expression of the distalless

homeobox 5 and 6 (Dlx5/6) genes that are specifically expressed during the development of

forebrain GABAergic interneurons (Wang et al. 2010; Stühmer et al. 2002). This rAAV-Dlx was

modified to gain chemogenetic control of interneurons using the excitatory Gq-DREADD

hM3Dq, and was successfully used to manipulate the activity of interneurons in various

vertebrates including ferrets, gerbils, and zebra finches (Dimidschstein et al. 2016).

16

1.8 Aim 1- Rationale, Objective, and Hypothesis

An electrophysiological characterization of L5-M1 pyramidal cells and interneurons in the

SODG93A mouse was completed by the Woodin lab (S. Khademullah). This characterization in

presymptomatic SOD1 mice, revealed that: (i) PV+ interneurons are hypoactive, (ii) pyramidal

neurons are hyperactive, and (iii) pyramidal neurons have a significant decrease in the frequency

of spontaneous inhibitory postsynaptic currents (sIPSCs) in SOD1G93A mice compared to WT

mice (Figure 1.3 A-C). Additionally, through the use of rAAV technology in SOD1 mice

expressing cre-recombinase in PV+ interneurons, the Woodin lab (S. Khadmullah) targeted the

excitatory DREADD Gq-hM3D to PV+ interneurons in L5-M1. The data from these experiments

indicated that following bath-application of CNO onto M1 slices, excitability of the pyramidal

neurons decreased, and after chronic administration of CNO (via drinking water), there was a

delayed onset of neurodegeneration and improved motor performance (Figure 1.4 A-E). To

translate these findings into a therapeutic treatment, the focus of my thesis is to use a viral

strategy for the delivery of chemogenetics that does not depend on cre-recombinase technology.

17

Figure 1.3 Electrophysiological recordings show a reduced strength of synaptic transmission in the SOD1G93A pre-symptomatic mouse. SOD1 mice have a lower frequency but unaltered amplitude of sIPSCs when compared to wild-type mice. A) Representative traces of sIPSCs in wild-type and SOD1 neurons from L5-M1 of pre-symptomatic (P60-65) adult mice, and B) the frequency (WT, n = 8; SOD1, n= 7) and C) amplitude (WT, 8 cells from 3 animals; SOD1, 7 cells from 5 animals) of events were quantified with a two-tailed t-test in the insets. Taken from (Khademullah C.S. 2018).

Figure 1.4 Activation of PV+ interneurons in L5 of M1 can reduce cortical hyperexcitability, delay the onset of neurodegeneration, and improve motor performance in presymptomatic SOD1G93A mice. A) Representative traces at 280 pA current injection steps in PV-Cre and PV-Cre x SOD1 neurons from L5-M1 of pre-symptomatic (P60-65) adult mice are quantified with the number of action potentials (APs) before (black circles) and after CNO (grey circles) bath application shown. B) PV-Cre (6 cells from 3 animals) and C) PV-Cre x SOD1 (7 cells from 3 animals). (i) PV-Cre x SOD1 pyramidal cells are hyperexcitable when compared to PV-Cre cells and D) this hyperexcitability can be abolished and returned to the PV-Cre baseline (n=6-7 cells from 3 animals). Number of action potentials were quantified with a Bonferroni’s Multiple Comparisons test). Taken from (Khademullah C.S. 2018).

18

Two questions stemming from this are: 1) could the DREADDs be delivered in mice in the

absence of Cre? 2) How does this strategy compare to the current treatment of riluzole in

reducing hyperexcitability? The main objective of my project is to characterize the use of

chemogenetic and pharmacological tools to reduce the cortical hyperexcitability of pyramidal

neurons within L5 of the M1 in the SOD1G93A mouse.

Aim 1: To determine if targeting GABAergic interneurons to express DREADDs using a

novel viral strategy can reduce the hyperexcitability of pyramidal neurons in the M1

Previously, a similar strategy was implemented by the Woodin Lab (S. Khademullah) using PV-

Cre mice, but to translate it to clinical application I adopted a viral strategy for targeting

interneurons derived from the Dlx5/6 lineage. The regulatory element Dlx5/6 contained within

the rAAV-hDlx vector has been shown to specifically target cortical GABAergic interneurons

(Dimidschstein et al. 2016). It has been shown that Dlx5/6 has a preferential expression in PV+

interneurons (Kelsom & Lu 2013), which in L5 of M1 synapse frequently on the soma of

adjacent pyramidal cells and influence their activity by modulating action potential firing (Naka

& Adesnik 2016). I hypothesize that increasing predominantly PV+ interneuron activity by

delivering the excitatory Gq-hM3D DREADD through a viral strategy, thus increasing GABA

release, will lead to a decrease in pyramidal cell firing and hyperexcitability.

1.9 Aim 2- Rationale, Objective, and Hypothesis

Riluzole has been an FDA-approved drug given to ALS patients since 1995, and still remains a

treatment given to patients over a decade later (Bellingham 2011). Though there have been

several steps to understanding the mechanisms behind its neuroprotective role and its effect

electrophysiologically in certain models of ALS, what has yet to be characterized is the effect

riluzole has specifically on pyramidal neurons located within layer 5 of the M1 in the SOD1G93A

ALS mouse.

19

Aim 2: To determine the electrophysiological effect of bath applying riluzole onto neurons

in the M1

Riluzole is a drug that disrupts glutamate neurotransmission, as well as decreases the repetitive

action potential firing rate in various neurons such as rat striatal neurons, mouse and rat cultured

cortical neurons and cultured rat spinal motor neurons (Bellingham 2011). I hypothesize that the

repetitive firing rate of action potentials of pyramidal neurons from L5-M1 will also be

decreased after bath application of riluzole. Moreover, I expect not to see changes in membrane

properties such as resting membrane potential and input resistance, but to see an increase in

action potential amplitude and the threshold for action potential initiation as reported in previous

studies with rodent ALS models (Bellingham 2011).

20

Chapter 2 Materials and Methods

Materials and Methods 2

2.1 Materials

2.1.1 Animals

Adult female and male Wild-Type (WT) and mutant SOD1G93A (Jackson Laboratories; B6SJL-

Tg (SOD1G93A) 1Gur/J) mice were used (90-95 days) to investigate the effects of chemogenetic

increased activation of inhibition during the symptomatic phase. The same mice strains (aged 60-

65 and 90-95 days) were used o investigate the effect of Riluzole during the pre-symptomatic

and symptomatic phase respectively. Mice were housed under a 12-h/12-h light/dark cycle with

ad libitum access to food and water. All experiments were approved by the University of Toronto

Animal Care Committee and conform with the guidelines of the Canadian Council on Animal

Care.

2.1.2 Drugs

Clozapine N-oxide/CNO (Obtained from the NIH as part of the Rapid Access to Investigate

Drug Program funded by the NINDS) was dissolved in a solution of 10% DMSO. To investigate

the effects of increased activation of inhibition, CNO at a concentration of 1 µM was perfused

onto slices of primary motor cortex at a rate of 2mL/min.

Riluzole was dissolved in 10% DMSO to yield a stock solution of 200mM. For

electrophysiological recordings, 100 µM of Riluzole in aCSF was perfused onto slices at a rate of

2mL/min. Riluzole was purchased from HelloBio (Catalog number: HB6093).

21

2.2 Methods

2.2.1 M1 Slice Preparation

Brains were extracted rapidly after decapitation and placed into ice-cold oxygenated (95% O2

and 5% CO2) cutting solution containing the following (in mM): 205 sucrose, 2.5 KCL, 1.25

NaH2PO4, 25 NaHCO3, 25 glucose, 0.4 ascorbic acid, 1 CaCl2, 2 MgCl2 and 3 sodium pyruvate

in double-distilled water (ddH2O). The solution was brought to a pH of 7.4 with NaOH and an

osmolarity of 300-305 mOsm. Slices cut in transversal plane (300µm) were prepared from 9 or

12-week old SOD1G93A and wild-type littermates using a Vibratome 1000 plus. Slices were

incubated at 37° C in a 50:50 mixture of cutting saline and artificial cerebrospinal fluid (aCSF)

containing the following (in mM): 123 NaCl, 2.5 KCl, 1. 25 NaH2PO4, 25 NaHCO3, 25 glucose,

2 CaCl2, and 1 MgCl2 in ddH2O and saturated with 95% O2/5% CO2, pH 7.4 with NaOH,

osmolarity 300-305 mOsm, for 30 min and then placed in oxygenated aCSF alone for 30 min and

maintained at room temperature.

2.2.2 Electrophysiology

All recordings were obtained from primary motor cortex L5 pyramidal neurons. Recordings

pipettes were pulled from thin-walled boroscillate (TW-150 F, World Precision Industries;

Sarasota, Florida) to resistances of 4-6 MΩ with Sutter Intruments P-87 (Novato, CA, USA).

Micropipettes were backfilled with an intracellular solution (ICS) containing the following (in

mM): 130 potassium gluconate, 10 KCL, 10 HEPES, 0.2 EGTA, 4 ATP, 0.3 GTP and 10

Phosphocreatine, pH 7.4 and osmolarity of 300 mOsm kg-1. Recordings were initiated ~5-10 min

after membrane penetration and signals were amplified using an Axon Instruments Multiclamp

700B and digitized using an Axon Intruments Digidata 1322a (Molecular Devices; Sunnyvale,

CA, USA). For all current-induced action potential analysis in pyramidal neurons in the presence

of aCSF, CNO, and riluzole, recordings were performed on minimum of two animals per group.

The resting membrane potential was recorded (in current-clamp mode) in normal aCSF and a

series of 500ms currents at increasing steps (-100- 300pA) were injected to neurons before and

after the addition of 1uM CNO or 100uM riluzole for 15 minutes at a perfusion rate of 2mL/min.

22

Action potentials and membrane properties were analyzed using Clampfit 10.7 and verified

manually for precision.

2.2.3 Surgical Procedures

AAV2/5-hDLx-hM3D(Gq)-tdTomato (pAAV-hDlx-GqDREADD-dTomato-Fishell-4 – Addgene

Plasmid #83897) was packaged by the Molecular Tool Platform at the CERVO Brain Research

Center at Laval University. Stereotaxic surgery was conducted on mice maintained on

isofluorane anesthesia. For Gq-hM3D experiments, viral vectors were bilaterally infused into the

M1 (AP: + 2.22, ML: ± 1.95, DV: -1.7) at a volume of 0.2-0.3µl. All infusions were made at a

rate of 0.1µl min-1 by pressure ejection by means of a cannula connected by Tygon tubing to a

10µl Hamilton syringe mounted on an infusion pump. Surgery was performed at p45 and

electrophysiological recordings were taken at P60-65 or P90-95.

2.2.4 Statistics

All data are presented as a mean ± SEM, and each datum point represents an individual

experiment. Tests of statistical difference were performed with GraphPad Prism 6 software.

Action potential frequency recordings from WT and SOD1 mice (in aCSF, CNO or riluzole

conditions) were analyzed using a two-way ANOVA with Bonferroni post-hoc test (repeated

measure, when comparing responses before and after washing on CNO or riluzole, and a non-

repeated measure when comparing WT to SOD1 in aCSF, CNO or riluzole). For all intrinstic

membrane properties analyzed under CNO conditions, a Students paired t-test was used as all

data passed the D’Agostino-Pearson and Shapiro-Wilk normality tests. For all intrinsic

membrane properties analyzed under riluzole conditions, either an unpaired or paired t-test was

used for data that passed the D’Agostino-Pearson and Shapiro-Wilk normality tests. A Mann-

Whitney u-test was used for all unpaired data sets that did not pass either the D’Agostino-

Pearson or Shapiro-Wilk normality tests, and a Wilcoxon signed rank-test was used for all paired

data sets that did not pass either normality test. For all experiments, the criterion α level was set

at 0.05.

23

Chapter 3 Results

Results 3

3.1 Aim 1: To determine if increasing inhibition from GABAergic forebrain interneurons reduces pyramidal neuron hyperexcitability in the primary motor cortex of a symptomatic ALS mouse

ALS is a multifaceted neurodegenerative disease, with cortical hyperexcitability being a

hallmark feature (Schütz 2005; Do-Ha et al. 2017). Human studies have revealed that this

hyperexcitability is accompanied by decreases in cortical inhibition, which has been supported

by studies involving various ALS mouse models such as the wobbler, TDP-43A315T and

SOD1G93A mice (Kuo 2003; Geevasinga et al. 2016; Clark et al. 2017). It was previously

demonstrated that increasing PV+ interneuron activity using the Gq-hM3D DREADD reduces

pyramidal neuron hyperexcitability in a presymptomatic ALS mouse model (Khademullah

2018). However, this study was performed using the cre-lox system to target the DREADD to

PV+ interneurons and thus has limited potential for clinical translation. An important preclinical

experiment is to determine a method for delivering DREADDs to PV+ interneurons using non-

cre technology. In this current set of experiments I tested whether delivery of Gq-hM3D using a

novel virus that specifically targets GABAergic forebrain interneurons would be sufficient to

reduce pyramidal neuron excitability in the L5-M1 of a symptomatic SOD1G93A ALS mouse

(hereafter referred to as SOD1 or SOD1 mouse).

The Dlx5/6 genes are expressed during embryonic development by all GABAergic forebrain

interneurons (Dimidschstein J et al. 2016). Recently, the rAAV-hDlx viral vector containing an

hDlx enhancer has been shown to specifically target cortical GABAergic interneurons, and gain

chemogenetic control of interneurons (Dimidschstein J et al. 2016). PV+ interneurons represent

40-50% of interneurons present in L5-M1 compared to SOM+ interneurons, which make up 20-

24

30%, and thus make up a larger proportion of interneurons targeted by the hDlx enhancer

(Kelsom & Lu 2013). In L5-M1, corticospinal pyramidal neurons whose projections directly

target spinal motor neurons in humans (Rathelot & Strick 2009), and indirectly in mice, are

powerfully inhibited at the soma and proximal dendrites by PV+ inhibitory interneurons. In a

recent study conducted in the TDP-43 ALS mouse model, both cortical hyperexcitability and

PV+ interneuron hypoactivity was reported (Zhang et al. 2016). Thus, I hypothesized that

activation of the excitatory DREADD Gq-hM3D in GABAergic interneurons (predominantly

PV+) would reduce pyramidal neuron hyperexcitability in L5-M1.

3.1.1 hDlx-Gq-hM3D Reduces Cortical Hyperexcitability

Neuronal excitability refers to the tendency of a neuron to generate an action potential in

response a certain stimulus or given input signal (such as an excitatory post-synaptic potential

(EPSP)) (Daoudal 2003). In contrast, neuronal hyperexcitability has been defined as an increased

or exaggerated response to a stimulus. Pyramidal neuron hyperexcitability in the motor cortex

has been well characterized in the SOD1G93A ALS mouse model (Kim et al. 2017). In order to

see if I could reduce pyramidal neuron hyperexcitability using the hDlx-Gq-hM3D virus, I first

wanted to confirm the neuronal hyperexcitability of L5-M1 pyramidal neurons that has been

previously reported in both SOD1 mice and their WT littermates. To measure the frequency of

action potential firing using patch-clamp electrophysiology, I applied a current injection protocol

that delivered 500 ms of current at increasing steps from -100 to 300pA. Electrophysiological

recordings from the motor cortex of symptomatic SOD1 mice (P90-95, n=9 from 2 mice)

revealed that L5-M1 pyramidal neurons were hyperexcitable compared to their WT littermates at

300pA of current injected (p< 0.05, n= 9 from 3 mice, Fig. 3.1.1).

After determining that pyramidal neurons in the SOD1 mouse are hyperexcitable during the

symptomatic stage of the disease, I wanted to test the effect of increasing GABAergic

interneuron activity. Based on the fact that PV+ interneurons provide powerful inhibition at the

soma and proximal dendrites of L5-M1 pyramidal neurons, I hypothesized that increasing their

activity should decrease pyramidal neuron activity and restore their firing pattern to that of their

WT littermates. To test this, a virus containing the chemogenetic activator Gq-hM3D and an

hDlx enhancer was bilaterally injected into L5-M1 of SOD1 and WT mice by Afif Aqrabawi

from the Junchul Kim Lab (Department of Psychology, University of Toronto).

25

To see if this viral strategy could be effective to reduce the hyperexcitability in SOD1 mice, I

first wanted to see if activation of hM3D in interneurons is strong enough to elicit a change in

WT neurons. The rationale being that if there is no significant change in neuronal excitability in

WT mice, then the effect in SOD1 mice would also be insignificant. To test if pyramidal neuron

activity could be altered by the increased inhibition I again recorded the excitability on L5-M1

pyramidal neurons in WT mice by injecting various steps of current. To activate Gq-hM3D and

increase the activity of GABAergic interneurons, I bath applied 1µM of CNO (a synthetic ligand

for Gq-hM3D) onto my slices and measured their response. Pyramidal neuron activity in WT

mice upon exposure to CNO decreased, with an overall ~21% reduction in the number of action

potentials fired (***p < 0.001, n=9 from 3 mice, Fig 3.1.2, 3.1.3).

To establish if this decrease in activity was a result of increased inhibition, or due to alterations

in the intrinsic properties of the pyramidal neuron themselves, I measured the effect of CNO

application on the following properties: input resistance, Action potential (AP) threshold, AP

maximum, and resting membrane potential (RMP). Current injected at -100, -80, -60, -40 and -

20pA and the corresponding voltage changes were used to calculate input resistance following

Ohm’s Law (V=IR). In the WT mice, L5-M1 pyramidal neurons had no significance alteration in

AP threshold, AP maximum or RMP, while input resistance was decreased significantly after

CNO was added to the slices (*P=0.0392, n=9 from 3 mice, Fig 3.1.4).

26

Figure 3.1.1 Excitability of L5-M1 pyramidal neurons at P90-95. Quantification of action potential recordings in WT neurons (n=9 from 3 mice) and SOD1 neurons (n=9 from 4 mice) from L5-M1 from symptomatic mice aged P90-95. Two-way ANOVA with a Bonferroni’s Multiple Comparison post-hoc test (*p <0.05). Bars represent mean ± SEM.

0

5

10

15

20 WT aCSF

SOD1 aCSF

40 100 180 240 300

*

Current Injected (pA)

# of

APs

27

Figure 3.1.2 Representative traces showing activation of hDlx-Gq-hM3D in interneurons reduces pyramidal action potential firing in WT mice. Representative traces of the action potential firing rate of L5-M1 pyramidal neurons (n=9 from 3 mice) at 300 pA of current injected in WT adult mice after activation of Gq-hM3D with CNO. Top) Represents before, and Bottom) represents after CNO (orange line) is bath applied.

300pA

300pA

1µM CNO

Before CNO

20mV 200ms

After CNO

-70 mV

-72 mV

28

Figure 3.1.3 Activation of hDlx-Gq-hM3D in interneurons reduces pyramidal neuron excitability in WT mice. Quantification of all action potential recordings (n= 9 from 3 mice) in WT neurons from L5-M1 of adult mice (P90-95) expressing Gq-hM3D are shown before (black circles) and after CNO (grey circles) bath application. Two-way repeated measures ANOVA with a Bonferroni’s Multiple Comparison post-hoc test (*** p < 0.001). Bars represent mean ± SEM.

0

5

10

15

20WT aCSFWT CNO

40 100 180 240 300

***

*********

Current Injected (pA)

# of

APs

29

Figure 3.1.4 Activation of hDlx-Gq-hM3D in interneurons significantly decreases input resistance of L5-M1 pyramidal neurons in WT mice. A) RMP (p=0.4295), B) AP maximum (p=0.0949) and C) AP Threshold (p=0.2499) were not significantly altered, while D) input resistance (*p=0.0392) increased after bath application of CNO in WT mice (n=9, from 3 mice) using a Student’s paired t-test. Circles indicate values from single samples and bars represent the mean ± SEM.

WT (aC

SF)

WT (CNO)

-80

-70

-60

-50

-40

RM

P (

mV

)

WT (aC

SF)

WT (CNO)

-50

-45

-40

-35

-30

-25

-20

AP

Thr

esho

ld (

mV

)

WT (aC

SF)

WT (CNO)

100

110

120

130

140

150

AP

Max

imum

(m

V)

WT (aC

SF)

WT (CNO)

0

50

100

150

200

250 *In

put

Res

ista

nce

(MΩ

)*

A)

C) D)

B)

30

Having established that the activation of Gq-hM3D in interneurons can lead to a reduction in

action potential firing of pyramidal neurons in WT mice, I wanted to then test the effect of

applying CNO in SOD1 symptomatic mice. Specifically, I wanted to determine if the increase in

inhibition was enough to reduce the excitability of the SOD1 pyramidal neurons back to the level

that was detected in WT mice. First, I recorded the activity of L5-M1 pyramidal neurons under

the current injection protocol previously described before and after application of CNO.

Compared to WT mice, I was able to detect a greater reduction in the firing frequency whereby

there was an ~27% decrease in firing in the SOD1 mice (p<0.001, n=9 from 2 mice, Fig 3.1.5,

3.1.6). I then examined the changes in the intrinsic properties mentioned previously, and in

contrast to WT mice whereby input resistance decreased, there was no significant alteration in

any of the four properties (n= 9 from 2 mice, Fig 3.1.7). Finally, to see if this reduction in firing

frequency after CNO application was enough to reduce the excitability of pyramidal neurons in

SOD1 mice to levels comparable to their WT littermates, I compared the firing frequency of

SOD1 (with CNO) and WT (with aCSF) L5-M1 pyramidal neurons under various current

injections and found their to be no significant difference between the two groups at any current

step (n=9 from 2 mice, Fig 3.1.8).

31

Figure 3.1.5 Representative traces showing activation of hDlx-Gq-hM3D in interneurons reduces action potential firing of pyramidal neurons in SOD1 mice. Representative traces of the average action potential firing rate of L5-M1 pyramidal neurons (n=9 from 2 mice) at 300 pA of current injected in symptomatic (P90-95) SOD1 mice after activation of Gq-hM3D with CNO. Top) Represents before, and Bottom) represents after CNO (orange line) is bath applied.

300pA

300pA

1µM CNO 20mV

200ms

Before CNO

After CNO

-71 mV

-70 mV

32

Figure 3.1.6 Activation of hDlx-Gq-hM3D in interneurons reduces pyramidal neuron excitability in SOD1 mice. Quantification of all action potential recordings (n= 9 from 2 mice) in SOD1neurons from L5-M1 of symptomatic adult mice (P90-95) expressing Gq-hM3D are shown before (black circles) and after CNO (grey circles) bath application. Two-way repeated measures ANOVA with a Bonferroni’s Multiple Comparison post-hoc test (*p <0.05, *** p < 0.001). Bars represent mean ± SEM

0

5

10

15

20SOD1 aCSFSOD1 CNO

40 100 180 240 300

*

******

***

Current Injected (pA)

# of

APs

33

Figure 3.1.7 Activation of hDlx-Gq-hM3D in interneurons does not alter intrinsic properties of L5-M1 pyramidal neurons in SOD1 mice. A) RMP (p=0.4980), B) AP maximum (p=0.999) C) AP Threshold (p=0.0506) D) input resistance (p=0.3929) were similar under aCSF conditions and after bath application of CNO in SOD1 mice (n=9 from 2 mice) using a Student’s paired t-test. Circles indicate values from single samples and bars represent the mean ± SEM.

SOD1 (aC

SF)

SOD1 (CNO)

-90

-80

-70

-60

-50

RM

P (m

V)

SOD1 (aC

SF)

SOD1 (CNO)

-60

-40

-20

0

AP T

hres

hold

(mV)

SOD1 (aC

SF)

SOD1 (CNO)

100

120

140

160

AP M

axim

um (m

V)

SOD1 (aC

SF)

SOD1 (CNO)

0

200

400

600In

put R

esis

tanc

e (MΩ

)

A) B)

C) D)

34

Figure 3.1.8 Activation of GABAergic interneurons in L5 of M1 can restore PN excitability in the symptomatic SOD1-hDlx-Gq-hM3D mouse. Quantification of action potential recordings (n= 9 from 4 mice) in WT neurons under aCSF conditions (black circles) and in SOD1 neurons (n=9 from 2 mice) from L5-M1 of symptomatic (P90-95) adult mice (grey circles) after expression of Gq-hM3D. Hyperexcitability is abolished with CNO application. Two-way ANOVA with a Bonferroni’s Multiple Comparison post-hoc test. Bars represent mean ± SEM.

WT (aCSF) vs SOD1 (CNO)

0

5

10

15WT aCSF

SOD1 CNO

40 100 180 240 300Current Injected (pA)

# of

APs

35

3.2 Aim 2: To determine the electrophysiological effects of bath applying riluzole onto neurons in the M1

Riluzole, an FDA approved treatment for ALS, disrupts glutamate neurotransmission and

decreases the repetitive action potential firing rate in various neurons such as rat striatal neurons,

mouse and rat cultured cortical neurons and cultured rat spinal motor neurons (Bellingham

2011). While its effects on electrophysiological properties of cortical neurons in rats and cultured

mouse neurons have been reported, its effect on properties in cortical neurons in the SOD1G93A

ALS mouse model has yet to be characterized. In this set of experiments, I aimed to determine

the effect riluzole has in altering action potential frequency and intrinsic membrane properties in

pyramidal neurons from L5 of the primary motor cortex.

3.2.1 Action potential frequency is altered in presymptomatic and

symptomatic mice

Cortical hyperexcitability has been reported in the hSOD1G93A mouse model at two disease time

points, presymptomatically (P60-65) and symptomatically (P90-95) (Kim et al. 2017;

Khademullah 2018). To measure the frequency of action potential firing using patch-clamp

electrophysiology, I applied a current injection protocol that delivered 500 ms of current at

increasing steps from -100 to 300pA. Electrophysiological recordings from the primary motor

cortex of presymptomatic and symptomatic SOD1 mice (P60-65 and P90-95) revealed that L5-

M1 pyramidal neurons were hyperexcitable at both the presymptomatic and symptomatic stage

compared to their WT littermates at 300pA of current (**p<0.01, Fig 3.2.1). Next, I tested the

effect of bath applying riluzole onto my slices at a concentration of 100µM for a period 10

minutes. Comparison of current-induced action potential firing before and after bath application

of riluzole revealed a significant reduction in response to injected current in both WT and SOD1

mice at the presymptomatic (*p<0.05, **p<0.01, ***p<0.001, Fig 3.2.2, Fig 3.2.3) and

symptomatic phase (*p<0.05, **p<0.01, ***p<0.001, Fig 3.2.4, Fig 3.2.5).

36

Figure 3.2.1 Pyramidal neurons are hyperexcitable at the presymptomatic and symptomatic stage. A) Quantification of action potential recordings (n= 7 from 4 mice) in WT neurons and in SOD1 neurons (n=10 from 6 mice) from L5-M1 of presymptomatic (P60-65) adult mice in aCSF. B) Quantification of action potential recordings (n= 9 from 4 mice) in WT neurons and in SOD1 neurons (n=9 from 4 mice) from L5-M1 of symptomatic (P90-95) adult mice in aCSF. Two-way ANOVA with a Bonferroni’s Multiple Comparison post-hoc test. Bars represent mean ± SEM. ** p<0.01

0

5

10

15WT aCSFSOD1 aCSF

40 100 180 240 300

**

Current Injected (pA)

# o

f A

Ps

0

5

10

15 WT aCSF

SOD1 aCSF

40 100 180 240 300

**

Current Injected (pA)#

of

AP

s

A) B)

37

Figure 3.2.2 Representative traces showing riluzole significantly reduces the frequency of action potential firing in presymptomatic SOD1 and control mice. Representative traces of the action potential firing rate of L5-M1 pyramidal neurons in WT (n=7 from 4 mice) and in SOD1 (n=10 from 7 mice) at 300 pA of current injected. Top) Represents before and Bottom) after the addition of riluzole

Before riluzole

After riluzole

WT SOD1

20mV

200ms

300pA

300pA 300pA

300pA

100µMriluzole 100µMriluzole

-65mV

-66mV -74mV

-82mV

38

Figure 3.2.3 Riluzole significantly reduces the frequency of action potential firing in presymptomatic SOD1 and control mice. Quantification of action potential recordings in aCSF (black and green circles) and in riluzole (blue circles) in WT neurons (n= 7 from 4 mice) and in SOD1 neurons (n=10 from 6 mice) from L5-M1 of presymptomatic (P60-65) adult mice. Two-way repeated measures ANOVA with a Bonferroni’s Multiple Comparison post-hoc test. Bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001

0

2

4

6

8

10SOD1 aCSFSOD1 riluzole

**

***

40 100 180 240 300

***

Current Injected (pA)# o

f A

Ps

A) B)

0

2

4

6

8

10WT aCSFWT riluzole

40 100 180 240 300

* ***

Current Injected (pA)

# o

f A

Ps

39

Figure 3.2.4 Representative traces showing riluzole significantly reduces the frequency of action potential firing in symptomatic SOD1 and control mice. Representative traces of the action potential firing rate of L5-M1 pyramidal neurons in WT (n=9 from 4 mice) and in SOD1 (n=9 from 4 mice) at 300 pA of current injected. Top) Represents before and Bottom) after the addition of riluzole.

300pA

300pA 100µMriluzole

20mV

200ms

-55mV

-55mV 300pA

300pA 100µMriluzole

-59mV

-61mV

Before riluzole

After riluzole

WT SOD1

40

Figure 3.2.5 Riluzole significantly reduces the frequency of action potential firing in symptomatic SOD1 and control mice. Quantification of action potential recordings in aCSF (black and green circles) and in riluzole (blue circles) in WT neurons (n= 9 from 4 mice) and in SOD1 neurons (n=9 from 4 mice) from L5-M1 of symptomatic (P90-95) adult mice. Two-way repeated measures ANOVA with a Bonferroni’s Multiple Comparison post-hoc test. Bars represent mean ± SEM.*p<0.05, **p<0.01, ***p<0.001

0

5

10

15WT aCSFWT riluzole

40 100 180 240 300

*** *** ****

Current Injected (pA)

# o

f A

Ps

0

5

10

15SOD1 aCSFSOD1 riluzole

40 100 180 240 300

*****

***

Current Injected (pA)# o

f A

Ps

A) B)

41

3.2.2 Riluzole modifies intrinsic membrane properties in SOD1 and control mice

The effects of riluzole on neuronal firing and membrane properties have been reported in various

mouse and rat models (Bellingham 2011). For instance, in rat striatal and cortical neurons

riluzole significantly decreased repetitive firing of action potentials (Belluzzi & Urbani 2000;

Pieri et al. 2009). In rat striatal neurons, changes in current-induced firing were not linked to

changes in resting membrane potential or input resistance (Centonze et al. 1998). However, in

some instances action potential amplitude, duration, and the threshold for action potential

initiation were increased (Bellingham 2011).

Since the effect of riluzole on intrinsic membrane properties has yet to be characterized in the

primary motor cortex of the SOD1G93A mouse model, I sought to evaluate the effect riluzole has

on the following membrane properties: RMP, AP threshold, AP Max and input resistance. Under

aCSF conditions, there were no significant differences in intrinsic membrane properties between

WT and SOD1 pyramidal neurons during the presymptomatic (Fig 3.2.6) or the symptomatic

phase (Fig 3.2.7).

Next, I evaluated the effect of bath applying riluzole onto slices of L5-M1 in SOD1

presymptomatic (P60-65) and symptomatic (P90-95) mice along with their aged-matched WT

controls. Interestingly, riluzole differentially altered membrane properties between the control

mice and the SOD1 mice at the two stages. In the presymptomatic control mice the RMP was

unaffected, the AP threshold (***p=0.001) increased and AP max (*p=0.0365) decreased

significantly, while input resistance (p=0.1704) was unaltered after riluzole was added (Fig

3.2.8, n=7 from 4 mice). In the symptomatic control mice, the RMP (***p=0.0009) became more

hyperpolarized and AP threshold increased (***p=0.0001) after adding riluzole, while AP

maximum (p=0.0742) and input resistance (p=0.6713) remained unchanged (Fig 3.2.9, n=9 from

4 mice). In the SOD1 presymptomatic mice cohort input resistance significantly decreased after

the addition of riluzole (*p=0.0116), while the RMP (p=0.1674), AP maximum (p=0.9219), and

AP threshold (p=0.0827) remained similar before and after riluzole was washed on (Fig 3.2.10, n=10 in 6 mice). In the symptomatic SOD1 mice both AP threshold (***p<0.0001) and input

resistance (**p=0.0044) significantly increased upon exposure to riluzole, while RMP

(p=0.3428) and AP maximum (p=0.8203) were unchanged (Fig 3.2.11, n=9 from 4 mice). All

42

intrinsic membrane values in WT and SOD1 presymptomatic and symptomatic mice under aCSF

and riluzole conditions are summarized below (Table 1).

43

Figure 3.2.6 Membrane properties are not significantly different between WT and SOD1 presymptomatic mice under aCSF conditions. A) RMP (P= 0.8030, Mann-Whitney test), B) AP Threshold (P=0.827, Mann-Whitney test), C) AP Maximum (P=0.335, unpaired t-test), and D) Input Resistance (P=0.988, unpaired t-test) were similar under aCSF conditions between WT mice (n=7 from 4 mice) and SOD1 mice (n=10 from 6 mice). Circles indicate values from single samples and bars represent mean ± SEM.

WT

SOD1

-90

-80

-70

-60

-50

-40

RM

P (

mV

)

WT

SOD1

-50

-40

-30

-20

-10

0

AP

Th

resh

old

(m

V)

WT

SOD1

0

50

100

150

AP

Max

imu

m (

mV

)

WT

SOD1

0

100

200

300

400

500In

pu

t R

esis

tan

ce (

MΩ

)

A)

C) D)

B)

44

Figure 3.2.7 Membrane properties are not significantly different between WT and SOD1 symptomatic mice under aCSF conditions. A) RMP (P= 0.2888, Mann-Whitney test), B) AP Threshold (P=0.835, unpaired t-test), C) AP Maximum (P=0.6270, Mann-Whitney test), and D) Input Resistance (P=0.851, unpaired t-test) were similar under aCSF conditions between WT mice (n=9 from 4 mice) and SOD1 mice (n=9 from 5 mice). Circles indicate values from single samples and bars represent mean ± SEM.

WT

SOD1 -80

-60

-40

-20

0R

MP

(m

V)

WT

SOD1-50

-40

-30

-20

-10

0

AP

Th

resh

old

(m

V)

WT

SOD10

50

100

150

AP

Max

imu

m (

mV

)

WT

SOD10

100

200

300In

pu

t R

esis

tan

ce (

MΩ

)

A)

C) D)

B)

45

Figure 3.2.8 Riluzole significantly alters AP threshold and AP maximum in presymptomatic (P60-65) control mice. A) RMP (p= 0.5314) was not significantly different while B) AP Threshold (***p=0.0001) increased, C) AP Maximum (*p=0.0365) decreased significantly and D) Input Resistance (p=0.1704) was not altered after application of 100µM riluzole in WT mice (n=10from 4 mice) using a Student’s paired t-test. Circles indicate values from single samples and bars represent mean ± SEM.

WT (aC

SF)

WT (rilu

zole)

-85

-80

-75

-70

-65

-60

RM

P (m

V)

WT (aC

SF)

WT (rilu

zole)

-50

-40

-30

-20

-10

0***

AP T

hres

hold

(mV)

WT (aC

SF)

WT (rilu

zole)

60

80

100

120

140

*

AP M

axim

um (m

V)

WT (aC

SF)

WT (rilu

zole)

0

100

200

300

400

500*

Inpu

t Res

ista

nce

(MΩ

)

***

*

A)

C) D)

B)

46

Figure 3.2.9 Resting membrane potential and AP threshold are significantly altered after riluzole application in symptomatic (P90-95) control mice. A) RMP (***P= 0.0009, paired t-test) decreased, and B) AP Threshold (***P=0.001, paired t-test) increased significantly after application of 100µM riluzole, while, C) AP Maximum (P=0.0742, Wilcoxon test) and D) Input Resistance (P=0.6713, paired t-test) were unchanged in WT mice (n=9 from 4 mice). Circles indicate values from single samples and bars represent mean ± SEM.

WT (aC

SF)

WT (rilu

zole)

-80

-60

-40

-20

0

***

RM

P (m

V)

WT (aC

SF)

WT (rilu

zole)

0

50

100

150

AP

Max

imum

(mV

)

WT (aC

SF)

WT (rilu

zole)

0

100

200

300In

put R

esis

tanc

e (MΩ

)

A)

C) D) WT (

aCSF)

WT (rilu

zole)

-50

-40

-30

-20

-10

0***

AP

Thr

esho

ld (m

V)

*** B) ***

47

Figure 3.2.10 Input resistance significantly decreased after riluzole application in presymptomatic (P60-65) SOD1 mice. A) RMP (p=0.1674, Wilcoxon test), B) AP Threshold (p=0.0827, Wilcoxon test), and C) AP Maximum (p=0.9219, Wilcoxon test) remained unchanged while D) Input Resistance (p=0.0185, paired t-test) was decreased significantly after application of 100µM riluzole in SOD1 mice (n=10 in 6 mice). Circles indicate values from single samples and bars represent mean ± SEM

SOD1 (aC

SF)

SOD1 (rilu

zole)

-90

-80

-70

-60

-50

-40

RM

P (m

V)

SOD1 (aC

SF)

SOD1 (rilu

zole)

-50

-40

-30

-20

-10

0

AP T

hres

hold

(mV)

SOD1 (aC

SF)

SOD1 (rilu

zole)

0

50

100

150

AP M

axim

um (m

V)

SOD1 (aC

SF)

SOD1 (rilu

zole)

0

100

200

300

400*

Inpu

t Res

ista

nce

(MΩ

)*

A)

C) D)

B)

48

Figure 3.2.11 AP threshold and input resistance are significantly increased after riluzole application in symptomatic (P90-95) SOD1 mice. A) RMP (p=0.3428, Wilcoxon test) remained unchanged, B) AP Threshold (***p<0.001, paired t-test) significantly increased while, C) AP Maximum (p=0.8203, Wilcoxon test) was unaffected and D) Input Resistance (**p=0.0044, paired t-test) significantly increased after application of 100µM riluzole in SOD1 mice (N=4, n=9). Circles indicate values from single samples and bars represent mean ± SEM

SOD1 (aC

SF)

SOD1 (rilu

zole)

-80

-70

-60

-50

-40

-30

RM

P (m

V)

SOD1 (aC

SF)

SOD1 (rilu

zole)

0

50

100

150

AP

Max

imum

(mV

)

SOD1 (aC

SF)

SOD1 (rilu

zole)

-40

-30

-20

-10

0***

AP

Thr

esho

ld (m

V)

SOD1 (aC

SF)

SOD1 (rilu

zole)

0

100

200

300

400**

Inpu

t Res

ista

nce

(MΩ

)

***

**

A)

C) D)

B)

49

Table 1. Electrophysiological properties of pyramidal neurons from presymptomatic and symptomatic SOD1 and control mice exposed to aCSF and riluzole

WT (7 cells, 4 mice) SOD1G931A (10 cells, 6 mice)

L5-Pyramidal Neurons

(P60-65)

L5-Pyramidal Neurons

(p60-65)

aCSF riluzole p-value aCSF riluzole p-value

RMP (mV) -71.32 ±

1.628

-70.54 ±

2.155

0.514 -68.36 ±

2.575

-70.43 ±

3.079

0.1674

AP threshold (mV) -34.62 ±

2.122

-25.90 ±

2.271

0.0001 -33.39 ±

2.089

-26.67 ±

2.018

0.0827

AP maximum (mV) 101.3 ±

4.616

93.14 ±

5.340

0.0365 99.0 ±

4.325

93.93 ±

7.729

0.9219

Input resistance (MΩ) 221.1 ±

41.04

194.8 ±

29.71

0.1704 201.9 ±

19.85

160.0 ±

19.26

0.0185

L5-Pyramidal Neurons

(P90-95)

RMP (mV) -51.61 ±

3.312

-56.90 ±

3.422

0.0009 -56.02 ±

2.575

-57.50 ±

1.973

0.3428

AP threshold (mV) -27.26 ±

2.401

-14.24 ±

2.418

0.0001 -27.19 ±

2.295

-13.43 ±

2.144

<0.0001

AP maximum (mV) 112.7 ±

7.748

109.1 ±

7.361

0.0742 112.9 ±

9.096

109.9 ±

6.265

0.8203

Input resistance (MΩ) 179.7 ±

18.75

174.2 ±

14.82

0.6713 180.2 ±

14.38

216.2 ±

20.97

0.0044

50

Chapter 4 Discussion

Discussion 4

4.1 Summary and interpretation of results

ALS is a devastating multifaceted neurodegenerative disease with cortical hyperexcitability

being a well-documented and hallmark feature in patients (Vucic, Ziemann, et al. 2013; Menon

et al. 2017; Schanz et al. 2016; Zanette et al. 2002) and in mouse models of the disease (Zhang et

al. 2016; Khademullah 2018; van Zundert et al. 2008). Alongside this, alterations in cortical

inhibition recorded using TMS and H-MRS on ALS patients, revealed decreases in SICI

(suggestive of the degeneration or loss of function of inhibitory interneurons) and reduced levels

of GABA in the motor cortex (Do-Ha et al. 2017; Vucic, Ziemann, et al. 2013; Zanette et al.

2002; Foerster et al. 2013; Foerster et al. 2012). Cortical interneurons play a critical role in

maintaining a balance between excitation and inhibition, yet their role in the pathogenesis of

ALS remains to be fully explored. The aim of this project was to examine both cortical

hyperexcitability and disrupted inhibition in an ALS mouse model, and compare the efficacy of a

novel viral strategy in reducing cortical hyperexcitability to the current FDA approved drug,

riluzole, that is given to patients. In brief, the principle findings of my work are: (i) increasing

forebrain GABAergic interneuron activity using hDlx-Gq-hM3D can reduce L5-M1 pyramidal

neuron excitability to a level comparable to healthy controls, (ii) riluzole significantly reduces

the frequency of action potential firing in presymptomatic and symptomatic mice, and (iii)

riluzole differently alters intrinsic membrane properties in both WT and SOD1 mice and depends

on disease stage (presymptomatic vs symptomatic).

4.1.1 Aim 1: hDlx-Gq-hM3D reduces cortical hyperexcitability

For this part of the project, L5-M1 GABAergic interneurons were targeted with an rAAV

containing an hDlx5/6 enhancer in addition to the chemogenetic activator hM3D. I showed that

activation of the hM3D DREADD, by bath application of CNO onto brain slices containing M1,

51

reduces cortical hyperexcitability of L5 pyramidal neurons to a level comparable to that detected

in age-matched healthy control mice (Fig 3.1.8).

I hypothesized that expression of this viral vector would be predominately in PV+ interneurons

based on the fact that PV+ interneurons make up the largest group of interneurons (~40-50%) in

L5-M1, and that Dlx5/6 is preferentially expressed in PV+ mature interneurons (Kelsom & Lu

2013; Wang et al. 2010). This has important implications based on the fact that PV+

interneurons, such as basket and chandelier cells, have axons that target the soma and proximal

dendrites of L5 pyramidal neurons providing more powerful inhibition than SOM+ interneurons

that target the distal dendrites (Tanaka et al. 2011; Naka & Adesnik 2016). Moreover, it is

important because PV+ interneurons have been reported to be hypoactive in both SOD1 and

TDP-43 mice, and in SOD1 patients (Nihei et al. 1993; Khademullah 2018; Zhang et al. 2016).

Though PV+ interneurons represent the largest class of interneurons in L5-M1, SOM+

interneurons make up roughly 30-40% with Dlx5/6 also playing a role in their development

(Kelsom & Lu 2013). Moreover, CR+ and NPY-expressing interneurons are also found within

the motor cortex, and are vulnerable to degeneration during the symptomatic phase of the

SOD1G93A mouse (Clark et al. 2017). In contrast to PV+ interneurons that are hypoactive, SOM+

interneurons are hyperactive in the TDP-43 ALS mouse model (Zhang et al. 2016). Since the

hDlx-Gq-hM3D virus targets all forebrain GABAergic interneurons (Dimidschstein et al. 2016),

it is possible that other interneuron subtypes could express hM3D as well. What this effectively

means is that this strategy could enhance the activity of SOM+ interneurons, already described as

hyperactive, and lead to a further increase in pyramidal cell activity. However, it is quite possible

that the activity of these hyperactive SOM+ interneurons cannot be increased further (due to a

ceiling effect of activity levels). Moreover, since PV+ interneurons provide powerful inhibition to

pyramidal neurons at the proximal dendrites, I predict that the inhibition provided by PV+ upon

exposure with CNO would outweigh the inhibition by SOM+ interneurons. If this prediction were

proved wrong however, it would be important to test the following: is there an improvement in

the survivability or a delay in the onset of motor deficits of mice transduced with this virus? If

yes, then perhaps increasing inhibition in multiple interneuron subtypes still yields a feasible

therapeutic strategy. These questions were addressed in experiments conducted by the Woodin

lab (S. Khademullah) whereby SOD1G93A mice injected with hDlx-Gq-hM3D virus showed a

delay in motor deficits, preserved neuronal health and an extended survival compared to

52

untreated SOD1G93A mice. Moreover, it was also shown by the Woodin lab that this viral strategy

has a selective preference for targeting PV+ interneurons, whereby there was an ~80%

transduction of PV+ interneurons, compared to an ~20% transduction of SOM+ interneurons

(Khademullah 2018).

Another important consideration of this study is the use of CNO as a ligand for the chemogenetic

activator hM3D. It was recently reported that CNO rapidly metabolizes to clozapine in vivo, and

that clozapine has a high affinity for hM3D in the brain (Manvich et al. 2018; MacLaren et al.

2016). Clozapine, used clinically as an antipsychotic, has the ability to cross the blood brain

barrier (BBB) and is an FDA approved drug (Gomez et al. 2017). While this drug could provide

another means for controlling neuronal activity in humans, a concern with this conversion in vivo

is that if clozapine levels are high enough, then clozapine could act on both DREADDs and

additional off-target binding sites. Known side effects of clozapine include sedation, weight gain,

drooling and hypotension (De Fazio et al. 2015). In terms of using CNO or clozapine as a means

of targeting neuronal activity through DREADDs in patients, it would be important to keep

clozapine doses at lower levels. However, it should be noted that clozapine is able to activate

DREADDs at a dosage 10-fold less than is required for antipsychotic effects (Gomez et al.

2017). Moreover, these recent findings stress the need for a CNO-only control group that lacks

DREADDs when designing and conducting DREADD-based research (MacLaren et al. 2016).

For my particular set of experiments, this rapid conversion is not as high a concern due to the

main mechanism behind this metabolic conversion involving cytochrome P450 enzymes

primarily found in the liver (Mahler & Aston-Jones 2018). Low levels of cytochrome P450 are

present in the brain, and there is speculation that CNO may be metabolized into clozapine

directly in the brain (bypassing the liver) (Gomez et al. 2017). Nonetheless, CNO has been

reported to be effective in both mammalian neuronal and non-neuronal cell cultures (Mahler &

Aston-Jones 2018; Dimidschstein J et al. 2016).

Since the discovery of the first adeno-associated virus in the 1960s, AAV technology has been

extensively researched, and the potential to use recombinant AAV vectors for gene therapy was

explored over the next three decades (Asokan et al. 2012). To date, there are several completed

or in progress phase I clinical trials for gene therapy of acquired or inherited diseases, including

the use of recombinant AAV vectors in the treatment of cystic fibrosis, Parkinson’s disease, and

hemophilia B (Asokan et al. 2012; Mingozzi & High 2011). Concerns about using AAV-

53

mediated gene therapy strategies in clinical settings include gene silencing, immunotoxicity, and

random insertion in the host DNA (Mingozzi & High 2011). A recent publication identified

acute toxicity of AAV9 used to treat spinal muscular atrophy in piglets and non-human primates.

Hinderer et al. concluded that the systemic and neuron toxicity could be results from the

intravenous delivery of AAV at large doses, and is regardless of the capsid serotype or transgene

(Hinderer et al. 2018). With that in mind, in order to develop AAV-mediated therapies to treat

human disease, investigators should be careful to follow the rules imparted by the FDA and

European Medicines Agency (EMA), which oversee human clinical research conducted which

follow guidance from the International Committee on Harmonization (ICH) for Good Clinical

Practice (GCP) (Byrne 2018). Moreover, it is imperative that researchers conduct informative

basic and clinical investigation with the goal of bringing innovative medical therapies to patients

who are highly burdened by disease.

4.1.2 When should this treatment be given to patients?

Changes in hyperexcitability as the disease progresses have been reported both in animal and

human models, which has implications for when viral treatment should be given to patients. In

the hSOD1G93A mouse changes in both inhibitory and excitatory cortical neuron excitability were

detected a few days after birth, normalized in juvenile mice, and became hyperexcitable again at

the end stage of the disease. Both corticospinal and corticocortical neurons in symptomatic mice

(P90-129) are hyperexcitable. Moreover, despite an overall loss in PV+ cells in ALS, PV+

interneuron activity was increased in this mouse model at age P90-101 (Kim et al. 2017).

Cortical hyperexcitability is a common phenotype seen in both familial and sporadic variants of

SOD1 and C9ORF72 cases (Kim et al. 2017). This has important implications with respect to the

timing of a therapeutic intervention in ALS. However, most patients aren’t formally diagnosed

with ALS until they start showing signs or symptoms. By the time a diagnosis is made, ~50% of

motor neurons are already lost, indicating the necessity of early intervention (Laferrièrea &

Polymenidou 2015).

54

4.1.3 Aim 2: Riluzole significantly reduces action potential firing and modifies intrinsic membrane properties in L5-M1 pyramidal neurons

In this part of my study, I sought to investigate the effect of bath applying riluzole to brain slices

containing M1 in WT and SOD1 mice at two disease stages: presymptomatic and symptomatic. I

showed that there was a large reduction in action potential frequency in both groups regardless of

the disease stage (Figure 3.2.2, 3.2.3). This is in line with previous reports that riluzole

decreased the repetitive firing of action potentials in rat cortical and striatal neurons, as well as in

cultured mouse cortical neurons (Bellingham 2011; Pieri et al. 2009). In contrast to what I

reported, in one study riluzole application at concentrations between 2-10µM in cultured mouse

spinal cord completely abolished repetitive firing (Kuo et al. 2006).

I show that both presymtomatically and symptomatically, there are no significant differences in

intrinsic properties between WT and SOD1 pyramidal neurons in aCSF (Figure 3.2.4, 3.2.5).

These results are in line with electrophysiological data collected from cultured SOD1G93A

cortical neurons where RMP and input resistance were comparable between control and SOD1

cortical neurons (Pieri et al. 2009). However, a further finding in the same study was that the AP

threshold in SOD1 neurons was significantly reduced compared to control neurons, which was

not detected in my data. This group concludes that these cultured neurons demonstrate intrinsic

hyperexcitability, displaying a higher firing frequency and reduced threshold potential, due to a

higher persistent Na+ current density (Pieri et al. 2009). The authors also attribute the lack of a

difference in RMP values between control and SOD1 cortical neurons to a potential

compensatory mechanism that increases Na+/K+ pump activity, which could counter the Na+

influx. Another group reported that hyperexcitability in cultured SOD1 iPSC-derived motor

neurons taken from ALS patients is caused by reductions in voltage-gated K+ channels (Wainger

et al. 2014). While it could be possible that the cells I recorded from also had alterations to

persistent Na+ current density or voltage-gated K+ channels (albeit, small enough changes that

intrinsic membrane properties were not significantly impacted), it was not specifically

investigated in my set of experiments.

Of important consideration is the difference between sample preparations, specifically those that

arise between acute slices relative to in vitro cell cultures. While cell cultures provide a useful

tool for gaining insight on cellular process in an isolated system, the pattern of synaptic

connections is altered at the time of harvest (Humpel 2015; Lein et al. 2011). It is possible that

55

differences arise between intrinsic properties reported in SOD1G93A cortical neurons because the

cytoarchitecture and synaptic circuits are largely preserved in my experiments (Lein et al. 2011).

This could highlight the fact that perhaps SOD1 L5 pyramidal neurons of the M1 are

hyperexcitable, not due to alterations in intrinsic membrane properties, but rather because of

alterations in synaptic transmission or inputs onto them (ie, decreased inhibition).

It was previously reported that the effect of riluzole on action potential firing was not associated

with changes in subthreshold membrane properties such as RMP or input resistance in rat striatal

neurons or cultured rat spinal motoneurons (Bellingham 2011). However, in some cases riluzole

decreased AP amplitude and duration, as well as AP threshold (Del Negro et al. 2002). What I

saw in this set of experiments was alterations in intrinsic membrane properties that varied in the

WT and SOD1 mice at two different time points. In WT mice aged P60-65 (controls for the

presymptomatic SOD1 mice), AP threshold increased and AP maximum decreased after the

addition of riluzole whereas in WT aged P90-95 (controls for the symptomatic control mice) AP

threshold again increased but RMP became significantly more hyperpolarized (Figure 3.2.6,

3.2.7). In presymptomatic SOD1 mice, only a change in input resistance was noted, whereby it

decreased after the addition of riluzole (Figure 3.2.8). In contrast, the input resistance in

symptomatic SOD1 mice increased along with the AP threshold (Figure 3.2.9).

When high concentrations of riluzole (>50µM) are applied to cortical neurons, fast transient Na+

currents and K+ currents are inhibited (Pieri et al. 2009). Voltage-dependent sodium channels

play a key role in the generation of action potentials and the fast inactivating Na+ current and

persistent Na+ current have functional significance in the generation of an AP, as well as setting

the membrane potential in a range below threshold (Kiss 2008). The fact that riluzole acts to

inhibit fast transient Na+ currents could explain increases in AP threshold detected in WT mice

(aged P60-65 and P90-95) and SOD1 at the symptomatic stage, making it harder for those

neurons to elicit an AP. Though AP threshold was not significantly increased in the

presymptomatic SOD1, there is a general trend showing an increase after the addition of riluzole.

Thus, increasing the sample size of those recordings could eventually yield similar conclusions.

Riluzole addition influenced RMP only in the WT pyramidal neurons of mice aged P90-95

leading to a more hyperpolarized RMP. In support of this, 20µM of riluzole in hypoglossal

motoneurons (HMs) in rat brain stem slices led to a hyperpolarized RMP caused by inhibited

56

persistent Na+ current and Ca2+ currents. Additionally, the repetitive firing of these HMs was

also strongly inhibited, which increase the action potential threshold voltage (Bellingham 2014).

Of particular interest were the changes in input resistance that occurred in the SOD1 recordings

after the addition of riluzole. At the presymptomatic phase, input resistance significantly

decreased after adding riluzole, while at the symptomatic phase there was a significant increase

in input resistance. Riluzole significantly reduced AP firing in presymptomatic mice, and since

AP threshold was not greatly increased, the decrease in input resistance could account in part for

this decrease in firing. A lower input resistance implies that the current required to drive intrinsic

firing must be larger than required to drive the firing of high input resistance neurons (Russo et

al. 2007). However, when looking at the changes in the symptomatic SOD1 mice input resistance

significantly increased. Reasons for this increase in input resistance could be due to loss or

regression of dendrites in aged neurons, which is what was seen in layer 2/3 pyramidal neurons

in aged monkeys (Chang et al. 2005). Not only are symptomatic SOD1 mice older than

presymptomatic mice, they also have more neurodegeneration. In support of the studies

conducted on monkey pyramidal neurons, profound apical dendrite degeneration has been

demonstrated in Betz cells of M1 in patients with sALS, fALS and FTD-ALS (Genç et al. 2017).

Additionally, in the study examining the effects of riluzole in HMs (mentioned above), there was

a small but significant increase in input resistance. Bellingham attributes this increase in input

resistance to the membrane hyperpolarization caused by the inhibited Na+ current. He argues that

the inhibition of an active inward current by riluzole will produce hyperpolarization, as well as

an increase in steady-state input resistance (Bellingham 2014). In contrast, Siniscalichi et al.

observed no significant effect on input resistance after applying 3-100µM of riluzole onto rat

cortical neurons (Siniscalchi et al. 1997).

4.1.4 How much riluzole actually crosses the BBB?

Riluzole given to patients is typically done so at a dosage of 100mg/day and at clinically

effective concentrations, human plasma concentrations of riluzole range between 0.5-2 µM.

However, animal pharmacokinetic studies indicate that the levels of riluzole in the brain can be

up to four-fold higher than plasma levels (Bellingham 2011). So how much riluzole actually

enters the brains of patients? The BBB provides a major obstacle for penetrating the CNS and

thus makes it harder for drugs to gain access to the brain in therapeutic concentrations

57

(Mohamed et al. 2017). In my experimental design, I exposed L5 pyramidal neurons in the M1 to

a concentration of 100 µM. This concentration is likely much higher than what would be found

in patient brain, however, multiple animal studies using riluzole concentrations ranging from 0.5-

20 µM also see similar prominent decreases in AP firing (Schuster et al. 2012; van Drongelen et

al. 2006; Bellingham 2014; Kuo et al. 2006; Kuo et al. 2005). My data showed that once riluzole

was applied, pyramidal neurons largely decreased their firing frequency to the point where most

neurons under the current-injection protocol only fired one AP. If there were a way to localize

the effects of riluzole, versus having a systemic effect, behavioral studies on mice would need to

be conducted to assess the motor function, as decreasing the frequency of firing in M1 pyramidal

neurons to that degree may lead to impairments in motor function.

4.2 Future directions

This thesis demonstrates that using chemogenetic tools as a means of regulating GABAergic

interneuron activity is an effective strategy for reducing pyramidal neuron hyperexcitability in

L5 of the M1. Additionally, application of the pharmacological drug riluzole onto these

hyperexcitable neurons also significantly reduces their activity, to an even higher degree than the

chemogenetic strategy. Despite the large reduction in hyperexcitability seen in L5 of M1 in

SOD1G93A mouse brain slices, riluzole given to patients doesn’t lead to pronounced effects and

improvements in clinical symptoms such as deficits in motor function (Lacomblez et al. 1996;

Bellingham 2011). Since riluzole is given orally to patients, and thus acts systemically on

multiple cells types, the effect seen in my experiments in the M1 may be minimal compared to

other effects riluzole is exerting throughout the body. Though riluzole aims to minimize

degeneration caused by excess glutamate through various mechanisms, evidence shows that

though low concentrations of riluzole could enhance GABAA currents, higher concentrations will

inhibit them (Bellingham 2011). Moreover, a study in rat hypoglossal motoneurons detected an

83% reduction in the amplitude of glycinergic IPSCs (Umemiya & Berger 1995). A potential

future direction of this project is to assess the motor degeneration and survivability of mice given

both the viral treatment and regular doses of riluzole. If increasing inhibition through

chemogenetic manipulations outweighs the negative effects of riluzole on GABA and glycine-

related currents, then perhaps the beneficial mechanisms of riluzole in reducing glutamatergic

58

excitotoxic effects could be more prominent. The main goal from these proposed experiments

would be to assess if there is a synergistic effect of the two strategies combined. The reason this

is critical to assess in mouse models is because if the viral delivery of the chemogenetic activator

hM3D is translated into a clinical setting, it is likely patients will have already been taking

riluzole, and could continue to do so.

59

References

Allain, A.E. et al., 2011. Maturation of the GABAergic transmission in normal and pathologic

motoneurons. Neural Plasticity, 2011.

Asokan, A., Schaffer, D. V. & Samulski, R.J., 2012. The AAV vector toolkit: Poised at the clinical

crossroads. Molecular Therapy, 20(4), pp.699–708. Available at:

http://dx.doi.org/10.1038/mt.2011.287.

Batra, R. & Lee, C.W., 2017. Mouse Models of C9orf72 Hexanucleotide Repeat Expansion in

Amyotrophic Lateral Sclerosis/ Frontotemporal Dementia. Frontiers in Cellular Neuroscience,

11(July). Available at: http://journal.frontiersin.org/article/10.3389/fncel.2017.00196/full.

Bellingham, M.C., 2011. A Review of the Neural Mechanisms of Action and Clinical Efficiency of

Riluzole in Treating Amyotrophic Lateral Sclerosis: What have we Learned in the Last Decade?

CNS Neuroscience and Therapeutics, 17(1), pp.4–31.

Bellingham, M.C., 2014. Pre- and postsynaptic mechanisms underlying inhibition of hypoglossal motor

neuron excitability by riluzole Pre- and postsynaptic mechanisms underlying inhibition of

hypoglossal motor neuron excitability by riluzole. Journal of neurophysiology, 110(June 2013),

pp.1047–1061.

Belluzzi, O. & Urbani, A., 2000. Riluzole inhibits the persistent sodium current in rat cortical neurones.

Pflügers Archiv-European Journal of Physiology, 440(5), p.16-.

Benes, F.M. & Berretta, S., 2001. GABAergic interneurons: Implications for understanding schizophrenia

and bipolar disorder. Neuropsychopharmacology, 25(1), pp.1–27.

Bunton-Stasyshyn, R.K.A. et al., 2014. SOD1 Function and Its Implications for Amyotrophic Lateral

Sclerosis Pathology: New and Renascent Themes. The Neuroscientist : a review journal bringing

neurobiology, neurology and psychiatry, 21(5), pp.1–11. Available at:

http://nro.sagepub.com/cgi/doi/10.1177/1073858414561795\nhttp://nro.sagepub.com/cgi/doi/10.117

7/1073858414561795\nhttp://www.ncbi.nlm.nih.gov/pubmed/25492944.

Byrne, B.J., 2018. Safety First: Perspective on Patient-Centered Development of AAV Gene Therapy

Products. Molecular Therapy, 26(3), pp.669–671. Available at:

60

https://doi.org/10.1016/j.ymthe.2018.02.009.

Centonze, D. et al., 1998. Electrophysiology of the neuroprotective agent riluzole on striatal spiny

neurons. Neuropharmacology, 37(8), pp.1063–1070.

Chang, Y.M. et al., 2005. Increased action potential firing rates of layer 2/3 pyramidal cells in the

prefrontal cortex are significantly related to cognitive performance in aged monkeys. Cerebral

Cortex, 15(4), pp.409–418.

Choi, D.W., 1988. Glutamate neurotoxicity and diseases of the nervous system. Neuron, 1(8), pp.623–

634.

Clark, R.M. et al., 2017. Calretinin and Neuropeptide y interneurons are differentially altered in the motor

cortex of the SOD1 G93A mouse model of ALS. Scientific Reports, 7(November 2016), pp.1–13.

Cleveland, D.W. & Rothstein, J.D., 2001. From Charcot to Lou Gehrig: deciphering selective motor

neuron death in ALS. Nature reviews. Neuroscience, 2(11), pp.806–819.

Cline, H., 2001. Synaptogenesis: A balancing act. Nature Reviews Neuroscience, 2(10), p.680.

Corona, J.C., Tovar-y-Romo, L.B. & Tapia, R., 2007. Glutamate excitotoxicity and therapeutic targets for

amyotrophic lateral sclerosis. Expert Opinion on Therapeutic Targets, 11(11), pp.1415–1428.

Available at: http://www.tandfonline.com/doi/full/10.1517/14728222.11.11.1415.

Di Cristo, G. et al., 2011. GABAergic circuit development and its implication for CNS disorders. Neural

Plasticity, 2011.

D’Acunzo, P. et al., 2014. A conditional transgenic reporter of presynaptic terminals reveals novel

features of the mouse corticospinal tract. Frontiers in Neuroanatomy, 7(January), pp.1–12.

Available at: http://journal.frontiersin.org/article/10.3389/fnana.2013.00050/abstract.

Dadon-Nachum, M., Melamed, E. & Offen, D., 2011. The “dying-back” phenomenon of motor neurons in

ALS. Journal of Molecular Neuroscience, 43(3), pp.470–477.

Van Damme, P., Robberecht, W. & Van Den Bosch, L., 2017. Modelling amyotrophic lateral sclerosis:

progress and possibilities. Disease Models & Mechanisms, 10(5), pp.537–549. Available at:

http://dmm.biologists.org/lookup/doi/10.1242/dmm.029058.

Daoudal, G., 2003. Long-Term Plasticity of Intrinsic Excitability: Learning Rules and Mechanisms.

61

Learning & Memory, 10(6), pp.456–465. Available at:

http://www.learnmem.org/cgi/doi/10.1101/lm.64103.

DeFelipe, J. & Fariñas, I., 1992. The pyramidal neuron of the cerebral cortex: morphological and

chemical characteristics of the synaptic inputs. Prog Neurobiol, 39(6), pp.563–607. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/1410442.

DeJesus-Hernandez, M. et al., 2011. Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region

of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS. Neuron, 72(2), pp.245–256. Available

at: http://dx.doi.org/10.1016/j.neuron.2011.09.011.

Dimidschstein J, Chen Q, Tremblay R, Rogers SL, Saldi GA, Guo L, Xu C, Liu R, Lu C, Chu J, Avery

MC, Rashid SM, Baek M, Jacob AL, Smith GB, Wilson DE, Kosche G, Kruglikov I, Rusielewicz T,

Kotak VC, Mowery TM, Anderson SA, Callaway EM, Dasen JS, Fitzpatri, F.G., 2016. A viral

strategy for targeting and manipulating interneurons across vertebrate species. Nature Neuroscience,

19(12), p.accepted. Available at: http://www.nature.com/neuro/journal/v19/n12/pdf/nn.4430.pdf.

Do-Ha, D., Buskila, Y. & Ooi, L., 2017. Impairments in Motor Neurons, Interneurons and Astrocytes

Contribute to Hyperexcitability in ALS: Underlying Mechanisms and Paths to Therapy. Molecular

Neurobiology, pp.1–9.

Dobrzanski, G. & Kossut, M., 2017. Application of the DREADD technique in biomedical brain research.

Pharmacological Reports, 69(2), pp.213–221.

van Drongelen, W. et al., 2006. Role of Persistent Sodium Current in Bursting Activity of Mouse

Neocortical Networks In Vitro. Journal of Neurophysiology, 96(5), pp.2564–2577. Available at:

http://jn.physiology.org/cgi/doi/10.1152/jn.00446.2006.

Eisen, a & Weber, M., 2001. The motor cortex and amyotrophic lateral sclerosis. Muscle and Nerve,

24(4), pp.564–573. Available at:

/home/michael/Dokumente/Exp.Ortho/Literatur\nAwiszus/pdf/0994.pdf.

van Es, M.A. et al., 2017. Amyotrophic lateral sclerosis. The Lancet, 390(10107), pp.2084–2098.

Available at: http://dx.doi.org/10.1016/S0140-6736(17)31287-4.

Fang, T. et al., 2018. Stage at which riluzole treatment prolongs survival in patients with amyotrophic

lateral sclerosis: a retrospective analysis of data from a dose-ranging study. The Lancet Neurology,

4422(18), pp.1–7. Available at: http://linkinghub.elsevier.com/retrieve/pii/S1474442218300541.

62

De Fazio, P. et al., 2015. Rare and very rare adverse effects of clozapine. Neuropsychiatric Disease and

Treatment, 11, pp.1995–2003.

Foerster, B.R. et al., 2013. An imbalance between excitatory and inhibitory neurotransmitters in

amyotrophic lateral sclerosis revealed by use of 3-t proton magnetic resonance spectroscopy. JAMA

Neurology, 70(8), pp.1009–1016.

Foerster, B.R. et al., 2012. Decreased motor cortex gamma-aminobutyric acid in amyotrophic lateral

sclerosis. Neurology, 78(20), pp.1596–1600.

Fogarty, M.J. et al., 2016. Cortical synaptic and dendritic spine abnormalities in a presymptomatic TDP-

43 model of amyotrophic lateral sclerosis. Scientific Reports, 6(October), pp.1–13. Available at:

http://dx.doi.org/10.1038/srep37968.

Geevasinga, N. et al., 2016. Pathophysiological and diagnostic implications of cortical dysfunction in

ALS. Nature reviews. Neurology, 12(11), pp.651–661. Available at:

http://www.nature.com/doifinder/10.1038/nrneurol.2016.140\nhttp://www.ncbi.nlm.nih.gov/pubmed

/27658852.

Genç, B. et al., 2017. Apical dendrite degeneration, a novel cellular pathology for Betz cells in ALS.

Scientific Reports, 7, pp.1–10. Available at: http://dx.doi.org/10.1038/srep41765.

Gomez, J.L. et al., 2017. Chemogenetics revealed: DREADD occupancy and activation via converted

clozapine. Science, 357(6350), pp.503–507.

Gurney, M.E. et al., 1985. Benefit of Vitamin E, Riluzole, and Gabapentin in a Transgenic Model of

Familial Amyotrophic Lateral Sclerosis. Annals of Neurology, 51, pp.546–551.

Hinderer, C. et al., 2018. Severe toxicity in nonhuman primates and piglets following high-dose

intravenous administration of an AAV vector expressing human SMN. Human Gene Therapy, 29(3),

p.hum.2018.015. Available at: http://online.liebertpub.com/doi/10.1089/hum.2018.015.

Hooks, B.M. et al., 2013. Organization of Cortical and Thalamic Input to Pyramidal Neurons in Mouse

Motor Cortex. Journal of Neuroscience, 33(2), pp.748–760. Available at:

http://www.jneurosci.org/cgi/doi/10.1523/JNEUROSCI.4338-12.2013.

Humpel, C., 2015. Neuroscience forefront review organotypic brain slice cultures: A review.

Neuroscience, 305, pp.86–98. Available at: http://dx.doi.org/10.1016/j.neuroscience.2015.07.086.

63

Kalia, L. V., Kalia, S.K. & Salter, M.W., 2008. NMDA receptors in clinical neurology: excitatory times

ahead. The Lancet Neurology, 7(8), pp.742–755.

Karagiannis, A. et al., 2009. Classification of NPY-Expressing Neocortical Interneurons. Journal of

Neuroscience, 29(11), pp.3642–3659. Available at:

http://www.jneurosci.org/cgi/doi/10.1523/JNEUROSCI.0058-09.2009.

Kelsom, C. & Lu, W., 2013a. Development and specification of GABAergic cortical interneurons. Cell &

Bioscience, 3(1), p.19. Available at:

http://cellandbioscience.biomedcentral.com/articles/10.1186/2045-3701-3-19.

Kelsom, C. & Lu, W., 2013b. Development and specification of GABAergic cortical interneurons. Cell &

Bioscience, 3(1), p.19. Available at:

http://cellandbioscience.biomedcentral.com/articles/10.1186/2045-3701-3-19.

Kepecs, A. & Fishell, G., 2014. Interneuron cell types are fit to function. Nature, 505(7483), pp.318–26.

Available at: http://www.nature.com/nature/journal/v505/n7483/full/nature12983.html#abstract.

Khademullah, C.S., 2018. Inhibitory Dysfunction in the SOD1-ALS Motor Cortex.

Kim, J. et al., 2017. Changes in the Excitability of Neocortical Neurons in a Mouse Model of

Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by

Advancing Disease. The Journal of Neuroscience, 37(37), pp.9037–9053. Available at:

http://www.jneurosci.org/lookup/doi/10.1523/JNEUROSCI.0811-17.2017.

Kiss, T., 2008. Persistent Na-channels: Origin and function. Acta Biologica Hungarica, 59(Supplement

2), pp.1–12. Available at: http://www.akademiai.com/doi/abs/10.1556/ABiol.59.2008.Suppl.1.

Kuo, J.J. et al., 2006. Essential role of the persistent sodium current in spike initiation during slowly

rising inputs in mouse spinal neurones. Journal of Physiology, 574(3), pp.819–834.

Kuo, J.J., 2003. Hyperexcitability of Cultured Spinal Motoneurons From Presymptomatic ALS Mice.

Journal of Neurophysiology, 91(1), pp.571–575. Available at:

http://jn.physiology.org/cgi/doi/10.1152/jn.00665.2003.

Kuo, J.J. et al., 2005. Increased persistent Na+ current and its effect on excitability in motoneurones

cultured from mutant SOD1 mice. Journal of Physiology, 563(3), pp.843–854.

Kuźma-Kozakiewicz, M., 2018. Edaravone in the treatment of amyotrophic lateral sclerosis. Neurologia i

64

Neurochirurgia Polska, 52(2), pp.124–128. Available at:

http://linkinghub.elsevier.com/retrieve/pii/S002838431830080X.

Lacomblez, L. et al., 1996. Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Amyotrophic

Lateral Sclerosis/Riluzole Study Group II. Lancet, 347(9013), pp.1425–31. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/8676624.

Laferrièrea, F. & PolymenidouPhD, M., 2015. Advances and challenges in understanding the multifaceted

pathogenesis of amyotrophic lateral sclerosis. Swiss Medical Weekly, 145(January), pp.1–13.

Lein, P.J., Barnhart, C.D. & Pessah, I.N., 2011. Acute Hippocampal Slice Preparation and Hippocampal

Slice Cultures. In Vitro Neurotoxicology: Methods and Protocols, Methods in Molecular Biology,

758(8), pp.115–134. Available at: http://www.springerlink.com/index/10.1007/978-1-61779-170-3.

Liu, Y. et al., 2016. C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of

ALS/FTD. Neuron, 90(3), pp.521–534. Available at:

http://dx.doi.org/10.1016/j.neuron.2016.04.005.

Lu, H. et al., 2016. Current therapy of drugs in amyotrophic lateral sclerosis. Curr Neuropharmacol, 14,

pp.314–321. Available at: http://dx.doi.org/.

MacLaren, D.A.A. et al., 2016. Clozapine N-Oxide Administration Produces Behavioral Effects in Long-

Evans Rats: Implications for Designing DREADD Experiments. eNeuro, 3(5). Available at:

http://eneuro.sfn.org/cgi/doi/10.1523/ENEURO.0219-16.2016.

Mahler, S. V. & Aston-Jones, G., 2018. CNO Evil? Considerations for the Use of DREADDs in

Behavioral Neuroscience. Neuropsychopharmacology, 43(5), pp.934–936. Available at:

http://dx.doi.org/10.1038/npp.2017.299.

Manvich, D.F. et al., 2018. The DREADD agonist clozapine N-oxide (CNO) is reverse-metabolized to

clozapine and produces clozapine-like interoceptive stimulus effects in rats and mice. Scientific

Reports, 8(1), pp.1–10. Available at: http://dx.doi.org/10.1038/s41598-018-22116-z.

Marin, B. et al., 2017. Variation in world wide incidence of amyotrophic lateral sclerosis: A meta-

analysis. International Journal of Epidemiology, 46(1), pp.57–74.

Marín, O., 2012. Interneuron dysfunction in psychiatric disorders. Nature reviews. Neuroscience, 13(2),

pp.107–20. Available at: http://dx.doi.org/10.1038/nrn3155.

65

Martin, L.J. & Chang, Q., 2012. Inhibitory synaptic regulation of motoneurons: A new target of disease

mechanisms in amyotrophic lateral sclerosis. Molecular Neurobiology, 45(1), pp.30–42.

McCombe, P.A. & Henderson, R.D., 2010. Effects of gender in amyotrophic lateral sclerosis. Gender

Medicine, 7(6), pp.557–570. Available at: http://dx.doi.org/10.1016/j.genm.2010.11.010.

Menon, P. et al., 2017. Cortical hyperexcitability and disease spread in amyotrophic lateral sclerosis.

European Journal of Neurology, 24(6), pp.816–824.

Mingozzi, F. & High, K.A., 2011. Therapeutic in vivo gene transfer for genetic disease using AAV:

Progress and challenges. Nature Reviews Genetics, 12(5), pp.341–355.

Mohamed, L.A. et al., 2017. Blood–Brain Barrier Driven Pharmacoresistance in Amyotrophic Lateral

Sclerosis and Challenges for Effective Drug Therapies. The AAPS Journal, 19(6), pp.1600–1614.

Available at: http://link.springer.com/10.1208/s12248-017-0120-6.

Moore, A.K. et al., 2018. Rapid Rebalancing of Excitation and Inhibition by Cortical Circuitry. Neuron,

pp.1–15. Available at: http://linkinghub.elsevier.com/retrieve/pii/S0896627318300709.

Naka, A. & Adesnik, H., 2016. Inhibitory Circuits in Cortical Layer 5. Frontiers in neural circuits,

10(May), p.35. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/27199675%5Cnhttp://www.pubmedcentral.nih.gov/articleren

der.fcgi?artid=PMC4859073.

Nakazawa, K. et al., 2012. GABAergic interneuron origin of schizophrenia pathophysiology.

Neuropharmacology, 62(3), pp.1574–1583. Available at:

http://dx.doi.org/10.1016/j.neuropharm.2011.01.022.

Del Negro, C.A., Morgado-Valle, C. & Feldman, J.L., 2002. Respiratory rhythm: An emergent network

property? Neuron, 34(5), pp.821–830.

Nihei, K., McKee, A.C. & Kowall, N.W., 1993. Patterns of neuronal degeneration in the motor cortex of

amyotrophic lateral sclerosis patients. Acta Neuropathologica, 86(1), pp.55–64.

Nijssen, J., Comley, L.H. & Hedlund, E., 2017. Motor neuron vulnerability and resistance in amyotrophic

lateral sclerosis. Acta Neuropathologica, 133(6), pp.1–23. Available at:

"http://dx.doi.org/10.1007/s00401-017-1708-8.

Park, C. hyun et al., 2015. Which motor cortical region best predicts imagined movement? NeuroImage,

66

113, pp.101–110. Available at: http://dx.doi.org/10.1016/j.neuroimage.2015.03.033.

Pieri, M. et al., 2009. Increased persistent sodium current determines cortical hyperexcitability in a

genetic model of amyotrophic lateral sclerosis. Experimental Neurology, 215(2), pp.368–379.

Available at: http://dx.doi.org/10.1016/j.expneurol.2008.11.002.

Pose-Utrilla, J. et al., 2017. Excitotoxic inactivation of constitutive oxidative stress detoxification

pathway in neurons can be rescued by PKD1. Nature Communications, 8(1), pp.1–18. Available at:

http://dx.doi.org/10.1038/s41467-017-02322-5.

Ramamoorthi, K. & Lin, Y., 2011. The contribution of GABAergic dysfunction to neurodevelopmental

disorders. Trends in Molecular Medicine, 17(8), pp.452–462. Available at:

http://dx.doi.org/10.1016/j.molmed.2011.03.003.

Rathelot, J.-A. & Strick, P.L., 2009. Subdivisions of primary motor cortex based on cortico-motoneuronal

cells. Proceedings of the National Academy of Sciences, 106(3), pp.918–923. Available at:

http://www.pnas.org/cgi/doi/10.1073/pnas.0808362106.

Ravits, J. et al., 2013. Deciphering amyotrophic lateral sclerosis: what phenotype, neuropathology and

genetics are telling us about pathogenesis. Amyotrophic Lateral Sclerosis and Frontotemporal

Degeneration, 14 Suppl 1(S1), pp.5–18. Available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3779649&tool=pmcentrez&rendertype=

abstract\nhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3779649%7B&%7Dtool=pmc

entrez%7B&%7Drendertype=abstract.

Rivara, C.-B. et al., 2003. Stereologic characterization and spatial distribution patterns of Betz cells in the

human primary motor cortex. The Anatomical Record, 270A(2), pp.137–151. Available at:

http://doi.wiley.com/10.1002/ar.a.10015.

Roth, B.L., 2016. DREADDs for Neuroscientists. Neuron, 89(4), pp.683–694.

Russo, M.J., Mugnaini, E. & Martina, M., 2007. Intrinsic properties and mechanisms of spontaneous

firing in mouse cerebellar unipolar brush cells. Journal of Physiology, 581(2), pp.709–724.

Saba, L. et al., 2016. Altered Functionality, Morphology, and Vesicular Glutamate Transporter

Expression of Cortical Motor Neurons from a Presymptomatic Mouse Model of Amyotrophic

Lateral Sclerosis. Cerebral Cortex, 26(4), pp.1512–1528.

67

Schanz, O. et al., 2016. Cortical hyperexcitability in patients with C9ORF72 mutations: Relationship to

phenotype. Muscle and Nerve, 54(2), pp.264–269.

Schuster, J.E. et al., 2012. Effect of prolonged riluzole exposure on cultured motoneurons in a mouse

model of ALS. Journal of Neurophysiology, 107(1), pp.484–492. Available at:

http://jn.physiology.org/cgi/doi/10.1152/jn.00714.2011.

Schütz, B., 2005. Imbalanced excitatory to inhibitory synaptic input precedes motor neuron degeneration

in an animal model of amyotrophic lateral sclerosis. Neurobiology of Disease, 20(1), pp.131–140.

Selvaraj, B.T., Livesey, M.R. & Chandran, S., 2017. Modeling the C9ORF72 repeat expansion mutation

using human induced pluripotent stem cells. Brain Pathology, 27(4), pp.518–524.

Shipp, S., 2007. Structure and function of the cerebral cortex. , 17(22), pp.443–449.

Siniscalchi, A. et al., 1997. Effects of riluzole on rat cortical neurones: an in vitro electrophysiological

study. Br J Pharmacol, 120(2), pp.225–230. Available at:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_ui

ds=9117114.

Stühmer, T. et al., 2002. Expression from a Dlx Gene Enhancer Marks Adult Mouse Cortical GABAergic

Neurons. Cerebral Cortex, 12, pp.75–85.

Talbot, K., 2014. Amyotrophic lateral sclerosis: Cell vulnerability or system vulnerability? Journal of

Anatomy, 224(1), pp.45–51.

Tanaka, Y.H. et al., 2011. Local connections of layer 5 GABAergic interneurons to corticospinal neurons.

Frontiers in neural circuits, 5(September), p.12. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/21994491\nhttp://www.pubmedcentral.nih.gov/articlerender.f

cgi?artid=PMC3182329.

Taniguchi, H. et al., 2011. A Resource of Cre Driver Lines for Genetic Targeting of GABAergic Neurons

in Cerebral Cortex. Neuron, 71(6), pp.995–1013.

Taylor, J.P., Brown, R.H. & Cleveland, D.W., 2016. Decoding ALS: from genes to mechanism. Nature,

539(7628), pp.197–206. Available at: http://www.nature.com/doifinder/10.1038/nature20413.

Todd, T.W. & Petrucelli, L., 2016. Insights into the pathogenic mechanisms of Chromosome 9 open

reading frame 72 (C9orf72) repeat expansions. Journal of Neurochemistry, 138, pp.145–162.

68

Traub, R., Mitsumoto, H. & Rowland, L.P., 2011. Research advances in amyotrophic lateral sclerosis,

2009 to 2010. Current Neurology and Neuroscience Reports, 11(1), pp.67–77.

Ueta, Y. et al., 2014. Multiple layer 5 pyramidal cell subtypes relay cortical feedback from secondary to

primary motor areas in rats. Cerebral Cortex, 24(9), pp.2362–2376.

Umemiya, M. & Berger, A.J., 1995. Inhibition by riluzole of glycinergic postsynaptic currents in rat

hypoglossal motoneurones. British Journal of Pharmacology, 116(8), pp.3227–3230.

Vucic, S., Lin, C.S.Y., et al., 2013. Riluzole exerts central and peripheral modulating effects in

amyotrophic lateral sclerosis. Brain, 136(5), pp.1361–1370.

Vucic, S., Ziemann, U., et al., 2013. Transcranial magnetic stimulation and amyotrophic lateral sclerosis:

pathophysiological insights. Journal of neurology, neurosurgery, and psychiatry, 84(10), pp.1161–

70. Available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3786661&tool=pmcentrez&rendertype=

abstract.

Vucic, S., Nicholson, G.A. & Kiernan, M.C., 2008. Cortical hyperexcitability may precede the onset of

familial amyotrophic lateral sclerosis. Brain, 131(6), pp.1540–1550.

Wainger, B.J. et al., 2014. Intrinsic membrane hyperexcitability of ALS patient-derived motor neurons.

Cell Reports, 7(1), pp.1–11.

Wang, S.J., Wang, K.Y. & Wang, W.C., 2004. Mechanisms underlying the riluzole inhibition of

glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Neuroscience, 125(1),

pp.191–201.

Wang, Y. et al., 2010. Dlx5 and Dlx6 regulate the development of parvalbumin-expressing cortical

interneurons. The Journal of neuroscience : the official journal of the Society for Neuroscience,

30(15), pp.5334–45. Available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2919857&tool=pmcentrez&rendertype=

abstract.

White, M.A. et al., 2018. TDP-43 gains function due to perturbed autoregulation in a Tardbp knock-in

mouse model of ALS-FTD. Nature Neuroscience, 21(4), pp.552–563. Available at:

http://dx.doi.org/10.1038/s41593-018-0113-5.

69

Xiang, Z., Huguenard, J.R. & Prince, D. a, 2002. Synaptic inhibition of pyramidal cells evoked by

different interneuronal subtypes in layer v of rat visual cortex. Journal of neurophysiology, 88(2),

pp.740–750.

Young, K.C., McGehee, D.S. & Brorson, J.R., 2007. Glutamate receptor expression and chronic

glutamate toxicity in rat motor cortex. Neurobiology of Disease, 26(1), pp.78–85.

Zanette, G. et al., 2002. Changes in motor cortex inhibition over time in patients with amyotrophic lateral

sclerosis. Journal of Neurology, 249(12), pp.1723–1728.

Zecevic, N., Hu, F. & Jakovcevski, I., 2011. Interneurons in the developing human neocortex.

Developmental Neurobiology, 71(1), pp.18–33.

Zhang, W. et al., 2016. Hyperactive somatostatin interneurons contribute to excitotoxicity in

neurodegenerative disorders. Nature neuroscience, 19(4), pp.2–6. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/26900927.

van Zundert, B. et al., 2008. Neonatal Neuronal Circuitry Shows Hyperexcitable Disturbance in a Mouse

Model of the Adult-Onset Neurodegenerative Disease Amyotrophic Lateral Sclerosis. Journal of

Neuroscience, 28(43), pp.10864–10874. Available at:

http://www.jneurosci.org/cgi/doi/10.1523/JNEUROSCI.1340-08.2008.