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FUNCTIONAL CONTRIBUTIONS OF L-TYPE CALCIUM CHANNELS TO BASOLATERAL AMYGDALA EXCITABILITY AND PATHOPHYSIOLOGY

Yiming Zhang

B.Sc., Henan Agricultural University, China, 2009 M.Sc., Soochow University, China, 2012

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

The Faculty of Graduate and Postdoctoral Studies

(Neuroscience)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

December 2020

The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:

Functional contributions of L-type calcium channels to basolateral amgydala excitability and pathophysiology

Submitted by Yiming Zhang in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience

Examining Committee:

Terrance P. Snutch, Neuroscience, UBC Supervisor

Brian MacVicar, Neuroscience, UBC Supervisory Committee Member

Anthony Phillips, Psychiatry, UBC University Examiner

Filip Van Petegem, Biochemistry, UBC University Examiner

Additional Supervisory Committee Memmbers:

Shernaz Bamji, Cellular and Physiological Sciences, UBC Supervisory Committee Member

Yu-Tian Wang, Neurology, UBC Supervisory Committee Member

Abstract

Calcium influx via neuronal L-type calcium channels (LTCCs) has been implicated in regulating activity-dependent gene transcription, synaptic plasticity, and synaptogenesis. While gain-of-function mutations in neuronal LTCCs have been linked to neurodevelopmental diseases, including autism spectrum disorders (ASDs), the role of LTCCs in regulating neuronal electrophysiological properties during early development remains unclear. The amygdala complex contributes toward emotional processes such as fear, anxiety and social cognition and studies suggest that increased excitability of basolateral amygdala (BLA) principal neurons underlie certain neuropsychiatric disorders. While LTCCs are expressed throughout the BLA, direct evidence for increased LTCC activity affecting BLA excitability and potentially contributing to disease pathophysiology is lacking. In Chapter Ⅰ of my study I investigated the contributions of LTCCs to the excitability and synaptic activity of BLA principal neurons at early developmental stages (postnatal day 7 (P7) and P21). By directly applying LTCC agonist (S)-Bay K8644 (BayK) onto brain slices, I found that BLA principal neurons displayed distinct alterations between P7 and P21 in intrinsic excitability properties, including firing frequency response, spike- frequency adaptation and altered spontaneous neurotransmission. These results suggested the possibility that the functional increase of LTCC activity at different stages of neurodevelopment may lead to alterations to BLA neuronal network activity. To investigate the effects of increased LTCC activity as it might relate to the underlying mechanism of developmental disorders such as ASD, in Chapter Ⅱ I examined the effects of increased LTCC function in early development on long-lasting neuronal excitability, synaptic plasticity and behavioral phenotypes. Bilateral injection of BayK into the BLA at different early stages (P7 or P14) followed by recovery and testing at P28 showed enhanced BLA neuronal excitability, long-term potentiation, as well as altered social behaviors, anxiety and repetitive behaviors. Whereas P28 animals that received BayK injection at P21 did not iii display any differences compared to DMSO control. These results provide evidence for the contributions of LTCCs at different stages of neurodevelopment, as well as their role in inducing long-lasting alterations in neuronal networks and behavioral phenotypes. They also provide new insights into LTCC dysfunction as it is potentially related to amygdala- related neurological disorders.

Lay Summary

L-type calcium channels (LTCCs) are a group of proteins that selectively mediate calcium influx across the cellular membrane. Calcium ions then regulate multiple cellular functions. LTCCs are abundant in the basolateral amygdala (BLA), a brain region involved in social engagement. Morphological and functional changes of the BLA contribute to the impairment of social interaction abilities associated with autism. To date, the potential contributions of LTCCs in BLA neuronal function and networks in physiological or pathological conditions are unclear. In my study using rats, I first found that the functional increase in LTCC activity differentially modified the electrical properties of BLA neurons at different ages of early development. In addition, by inducing LTCC enhancement at early ages after birth I found resulting long-lasting changes in BLA neuronal functions and in animal behavioral traits. The results provide a new approach towards understanding the mechanisms of LTCC dysfunction in neurodevelopmental disorders.

Preface

The dissertation is an original interllectual product of the author, Yiming Zhang. All of the work presented was conducted in the Michael Smith Laboratories and the Centre for Brain Health at the University of British Columbia, Point Grey campus.

A version of Chapter 1 has been published: Zhang Y, Garcia E, Sack A-S & Snutch TP (2020). L-type calcium channel contributions to intrinsic excitability and synaptic activity during basolateral amygdala postnatal development. Journal of Neurophysiology 123, 1216–1235.

Table of Contents

Abstract ...... iii Lay Summary ...... v Preface ...... vi Table of Contents ...... vii List of Tables ...... x List of Figures ...... xi List of Abbreviations ...... xiv Acknowledgements ...... xvi 1 Introduction ...... 1 1.1 Voltage-gated calcium channels ...... 1 1.1.1 VGCC structure and functional domains ...... 1 1.1.2 Alternative splicing in VGCCs ...... 6 1.1.3 VGCC Distribution and functional relevance of VGCC ...... 10 1.1.3.1 Cav1 family ...... 10 1.1.3.1 Cav2 family ...... 10 1.1.3.3 Cav3 family ...... 14 1.2 Neuronal L-type calcium channels ...... 20 1.2.1 Pharmacological ligands of LTCCs ...... 20 1.2.2 Biological functions of neuronal LTCCs ...... 23 1.2.2 Distinct contributions of Cav1.2 and Cav1.3 to neuronal function ...... 25 1.2.4 LTCC neuronal channelopathies ...... 27 1.3 Cav1 channels and Autism Spectrum Disorders ...... 35 1.3.1 Genetic factors related to ASD ...... 36 1.3.2 Animal Models of ASD ...... 39 1.4 Brain regions associated with ASD; involvement of the basolateral amygdala 47 1.5 Thesis Objectives ...... 53 2 CHAPTERⅠ: Contribution of L-type calcium channels to firing activity in the rat basolateral amygdala at different stages of postnatal development ...... 55 vii

2.1 Intruduction ...... 55 2.2 Methods ...... 58 2.3 Results ...... 63 2.3.1 Enhancement of L-type channels differentially regulates intrinsic membrane properties of immature and juvenile BLA neurons...... 63 2.3.2 Increase of L-type calcium channel activity reduces spike frequency adaptation of P7 BLA neurons...... 75 2.3.3 L-type calcium channels mediate a plateau potential in P7 BLA neurons...... 79 2.3.4 Increased L-type calcium channel activity enhances initial bursting and rebound firing in P21 BLA neurons ...... 82 2.3.5 Spontaneous firing activities were modified by (S)-Bay K8644 ...... 92 2.3.6 Increased L-type calcium channel activity modifies the functional properties of quantal post-synaptic currents...... 94 2.3.7 L-type calcium channel activity does not affect evoked GABAergic or glutamatergic responses in BLA principal neurons ...... 100 2.4 Discussion ...... 106 3 CHAPTER Ⅱ : A pharmacological rat model of autism spectrum disorder: Behavioral and neuronal alterations caused by neonatal exposure of the basolateral amygdala to an L-type calcium channel agonist ...... 114 3.1 Introduction ...... 114 3.2 Methods ...... 119 3.3 Results ...... 126 3.3.1 Bilateral injection of BayK at early post-natal ages produced long-lasting alterations in BLA that manifest in juvenile behavioral patterns...... 126 3.3.2 BayK differentially affects long-term potentiation in BLA neurons at different postnatal stages...... 132 3.3.3 Bilateral injection of BayK at P14 modified passive electrophysiological properties of BLA neurons recorded at P28 ...... 134 3.3.3 Neuronal excitability increased in animals that received BayK injection at P7 ...... 137 3.3.4 BayK-associated induction of LTP in the LA to BLA synapse is enhanced but not determined by prior behavioural experience...... 140 3.3.5 BayK injection does not produce long-term change in CREB and BDNF expression...... 142 3.4 Discussion ...... 145

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4 Conclusion ...... 152 4.1 Summary and discussion of research findings ...... 152 4.2 Research significance and potential limitations ...... 157 4.3 Future directions ...... 161 Bibliography: ...... 164

List of Tables

Table 1.1 Classifications of calcium channel α1 subunits ...... 7 Table 1.2 Voltage-gated calcium channel genes implicated in autism spectrum disorders and related pathologies ...... 46 Table 2.1 Passive membrane properties of BLA neurons ...... 69 Table 3.1 Parameters in the exploratory social behavior test ...... 128 Table 3.2 Parameters of the passive properties of BLA neurons ...... 135

List of Figures

Figure 1.1 Multimeric assembly of voltage-gated calcium channels and functional domains of the α1 pore-forming subunit ...... 16 Figure 1.2 Comparison of kinetics and voltage range of activation between high- voltage and low-voltage activated calcium channels ...... 18 Figure 1.3 Gain of function mutations in Cav1.2 and Cav1.3 channels associated with neuropsychiatric disorders ...... 34 Figure 1.4 Brain areas associated with ASD ...... 51 Figure 1.5 Organization of the amygdala functional nuclei and local networks...... 52 Figure 2.1 Post-hoc visualization of patch-clamped BLA principal neuron from rat brain coronal slices using biocytin labeling ...... 66 Figure 2.2 BLA principal neurons exhibit distinct electrophysiological characteristics at different developmental stages ...... 67 Figure 2.3 BayK increases the excitability of P7 BLA principal neurons with no significant effect on P21 neurons ...... 71 Figure 2.4 DMSO exposure had no effect on the electrophysiological phenotype of BLA neurons ...... 72 Figure 2.5 mRNA expression of LTCCs in BLA during early postnatal period of development ...... 74 Figure 2.6 Increased LTCC activity reduces spike frequency adaptation in P7 neurons ...... 78 Figure 2.7 LTCC mediated calcium influx induces a depolarizing plateau potential in P7 neurons ...... 80 Figure 2.8 Increased LTCC activity enhances the initial burst in P21 BLA neurons 85 Figure 2.9 Increased LTCC activity enhances rebound firing in P21 neurons ...... 87 Figure 2.10 Modulation of rebound responses by BayK involves the

xi hyperpolarization-activated cation conductance Ih – analysis based on the magnitude of membrane hyperpolarization ...... 89 Figure 2.11 Modulation of rebound responses by BayK involves the hyperpolarization-activated cation conductance Ih – time dependent effect of membrane hyperpolarization ...... 90 Figure 2.12 (S)-Bay K8644 increases spontaneous activity evoked by sustained depolarization of BLA neurons ...... 92 Figure 2.13 Increased LTCC activity modulates miniature post-synaptic inhibitory activity in P7 BLA neurons ...... 96 Figure 2.14 Spontaneous release at GABAergic and glutamatergic synapses are differentially regulated by BayK in P21 BLA neurons ...... 98 Figure 2.15 BayK does not affect evoked post-synaptic responses in BLA neurons 102 Figure 2.16 Paired-pulse ratio of synaptic responses are not affect by the application of (S)-Bay K8644 at both P7 and P21 ...... 104 Figure 3.1 Time course of the neurodevelopmental critical period in the rat BLA during the first four postnatal weeks ...... 117 Figure 3.2 Timeline diagram of the experimental procedures performed on experimental groups of rats at different ages ...... 118 Figure 3.3 BayK injection at P14 selectively produced long-term social affiliation impairment at P28...... 127 Figure 3.4 Exploratory social behaviors in an open field test were not affected by BayK treatment ...... 130 Figure 3.5 Bilateral injection of BayK at P7 induced increased anxiety and enhanced repetitive behavior at an older age ...... 131 Figure 3.6 Bilateral BayK injection at P7 and P14 induce LTP in BLA neurons at P28 ...... 133 Figure 3.7 Bilateral injection of BayK at P14 produced long-lasting alterations in the

xii

I-V relationship and resting membrane potential recorded at P28 ...... 135 Figure 3.8 BayK injection at P7 produced a long-lasting increase in the firing response of P28 BLA neurons as indicated by the f-I relation ...... 138 Figure 3.9 Changes in synaptic strength induced after BayK treatment without preceding behavioral tests ...... 141 Figure 3.10 BayK injection did not induce long-term alterations in the mRNA expression of LTCCs, CREB and BDNF...... 143

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List of Abbreviations

ACSF Artificial cerebral spinal fluid AHP Afterhyperpolarization AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ASD Autism spectrum disorder BLA Basolateral amygdala Ca2+ Calcium CeL Central lateral amygdala CeM Central medial amygdala CNS Central nervous system CNV Copy number variation DHP Dihydropyridine DSM-5 The Diagnostic and Statistical Manual of Mental Disorders EPSC Excitatory post-synaptic current FHM Familial hemiplegic migraine GABA Gamma-Aminobutyric acid HVA High voltage activated IPSC Inhibitory post-synaptic current LTCC L-type calcium channel LTD Long-term depression LTP Long-term potentiation LVA Low voltage activated NMDA N-Methyl-D-aspartic acid OFC Orbitofrontal cortex PFC Prefrontal cortex

xiv

PPA Propionic acid RT-qPCR Reverse transcription quantitative polymerase chain reaction PTX Picrotoxin TS Timothy syndrome TTX Tetrodotoxin VGCC Voltage-gated calcium channel VPA Valproic acid

Acknowledgements

I would like to thank my supervisor, Dr. Terry Snutch, for his patient guidance and mentorship. He opens the door for my research career, and his passion for research has been truly inspiring. Thank you for providing me with opportunities to attend and present my work in scitific meetings and international conventions, which have greatly benefitted my growth as a scientist.

I would like to thank Dr. Esperanza Garcia for training me in the field of electrophysiology and neuroscience, and also inspiring me in enjoying both science and real life. Many thanks to Lucy Yang for preforming numerous animal surgeries to support my study. Many thanks to Karen Jones and Sophie Sack for their help in the experiments. And thanks to all of the past and present members of the Snutch lab, our scientific collaborations and discussions were excellent and essential for my study.

I would also like to thank my supervisory committee: Dr. Brian MacVicar, Dr. Yu-Tian Wang, and Dr. Shernaz Bamji, for sharing their expertise and providing invaluable feedback for my project.

My PhD training and project were financially supported by Canadian Institutes of Health Research grant.

Lastly, a special thanks to my family, especially my wife Kelly (Xi) Chen, for your uncoditioanl support and encouragement at every stage of the process. Dedication

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Dedication

To my beautiful wife Xi Chen

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1 Introduction

1.1 Voltage-gated calcium channels Voltage-gated calcium channels (VGCCs) are expressed on the membrane of many different cell types, mediating Ca2+ influx in response to action potentials and subthreshold depolarization. Ca2+ entering cells through VGCCs both affects electrical properties and serves as an important second messenger for excitation-contraction coupling in cardiac, smooth and skeletal muscle cells (Reuter, 1979; Catterall, 1991; Tanabe et al., 1993; Bers, 2002), and inducing secretion of hormone in endocrine cells (Yang & Berggren, 2006). In neurons, Ca2+ influx via VGCCs regulates enzyme activities, gene expression and synaptic transmission (Dunlap et al., 1995; Catterall & Few, 2008; Tyson & Snutch, 2013).

1.1.1 VGCC structure and functional domains High voltage-activated (HVA) calcium channels are formed as a complex of several different subunits: α1 (~200 kDa), α2δ (~170 kDa), β (~55 kDa) and γ (~33 kDa) in a

1:1:1:1 stoichiometric ratio (Figure 1.1a). The α1 subunit forms the calcium ion- conducting pore while the associated subunits have different functions including modulation of channel gating and trafficking to the plasma membrane. The VGCC α1 subunits are organized in 4 homologous domains (I-IV), each containing a motif of 6 transmembrane helices (S1-S6) arranged in tandem (Figure 1.1c) (Dolphin, 2006; Tombola et al., 2006; Tyson & Snutch, 2013). Interdomain intracellular loops as well as the carboxyl terminus of various sizes bridge the four repeats and serve as interaction sites for auxiliary subunits and regulatory molecules that modulate channel activity and connect Cav channels to other macromolecular complexes and cellular signaling pathways (Kobayashi et al., 2007). 1

Despite having distinct ion selectivity properties, mammalian VGCCs and voltage gated sodium channels (Nav) exhibit a high structural homology, and share common architectures including voltage-sensing and the pore modules (Figure 1.1b) with members of the bacterial sodium channel family, such as NavAb from Arcobacter butzleri (Payandeh et al., 2011) and NaChBac from Bacillus halodurans (Ren et al., 2001). As such, studies to date on the bacterial channels and the mammalian Navs have provided important insights into the structural basis for Cav channel functions.

The calcium channel selective filter has four acidic side chains surrounding the narrowest part of the ion conduction pathway, and this unique structure forms a high field-strength anionic coordination site. It confers Ca2+ selectivity through partial dehydration via direct interaction with the glutamate side chain, which contains a cluster of four glutamate residues (EEEE locus) (Sather & McCleskey, 2003). Since NavAb and NaChBac channels are selective to Na+ but the amino acid sequence of the pore is more similar to VGCCs and they are sensitive to Ca2+ channel blockers, site-directed mutations were made in the bacterial channels to introduce new negatively charged residues on the extracellular side of the high field-strength site in order to understand the permeation of calcium ions and examine which residues in the P-loop confer Ca2+ selectivity. Substitution of Asp residues located one and four positions on the extracellular side of the high field-strength site plus substitution of Asp for the key Glu at the high field strength site in the selectivity filter confer high calcium selectivity on NaChBac and NavAb, with

PCa:PNa ∼400, which is similar to mammalian calcium channels (Yue et al., 2002; Tang et al., 2014). Structural and functional analyses revealed that the key substitution of Asp for Ser in the position just on the extracellular side of the Glu at the high field-strength site (Glu177 in NavAb) forms a second calcium-binding site in the pore. Remarkably, this single substitution is sufficient to increase calcium selectivity ∼1000-fold from PCa:PNa

∼0.03 to PCa:PNa ∼30 (Tang et al., 2014). One subfamily of VGCC, the T-type calcium channels differ subtly from other VGCCs in selectivity and permeability, with cloning showing that two of the four selectivity filter positions in T-type channels are occupied by aspartate residues rather than glutamate residues. The theoretical structural descriptions only provided a simple link between the channel structure and the high Ca2+ permeability over other ions, and nowadays, more and more studies employing advanced computational model approaches have been conducted to provide a better understanding of the underlying mechanisms of the Ca channel selectivity.

Initially, it was proposed that the positively charged residues of the S4 transmembrane helixes serve as the gating charges, moving outward across the membrane in response to depolarization and thereby initiating the activation process (Catterall, 1986a, 1986b; Guy & Seetharamulu, 1986). Further studies confirmed that the S1-S4 segments serve as the voltage-sensing domain (VSD) while the S5 and S6 segments serve as the pore-forming module, and eventually led to the now-familiar six-transmembrane-segment structural model for the functional domains of VGCCs (Figure1b and c). This mechanistic model has been refined in recent studies on the Cav1.1 complex from rabbit skeletal muscle, as well as a recombinant human Cav3.1 channel using cryo-electron microscopy (cryo-EM)

(Wu et al., 2015; Zhao et al., 2019b). The high-resolution structure of Cav1.1 indicates a four-fold symmetry disrupted by the distinct extracellular loops and by the slightly different conformations of the S5 and S6 segments among the four repeats. The S6Ⅳ is immediately followed by a cytosolic C-terminal domain. The selective filter is constituted by the side chains of four critical Glu residents and the carbonyl oxygen atoms of the two preceding residues in each repeat. The clockwise arrangement of the four repeats in the extracellular view may be conserved in all eukaryotic Cav and Nav channels (Wu et al., 2015). The cryo-EM of Cav3.1 revealed that certain cytosolic segments observed in

Cav1.1 α1 (Protein Data Bank (PDB) code 5GJV), such as the α1-interacting domain

(AID) helix, the elongated S6Ⅱ, the Ⅲ-Ⅳ linker between S6Ⅲ and the VSDⅣ are absent in Cav3.1, which instead shows a noticable globular carboxy terminal domain (Zhao et al., 2019b). Further, Cav3.1 shows a unique disulfide bond between Cys104 on the S1-S2 linker and Cys889 on extracellular loop Ⅱ that the authors suggest stabilizes the interaction between VSDⅠ relative to the pore domain and may be responsible for the T- type-specific redox modulation of activation and inactivation kinetics. Overall, the structural observations suggest that different conformations of the VGCCs can support the same functional state, indicating that transitions between functional states are more dynamic than previously thought.

Channel functional studies on voltage gating detected the transmembrane charge movement as an outward capacitive gating current, whose magnitude is equivalent to the movement of 2–3 gating charges across the transmembrane electrical field per voltage sensor (Bezanilla, 2000). Upon depolarization, the S4 segment moves outward, exchanging ion pair partners and transporting the arginine (Arg) gating charges through the hydrophobic constriction site (HCS) according to a sliding-helix model (Yarov- Yarovoy et al., 2012). The presence of the large side chain of the Arg gating charges serves to seal the voltage sensor and prevent transmembrane movement of water and ions. Changes in membrane potential drive the S4 segment inward and outward in response to hyperpolarization and depolarization, moving the gating charges through the HCS and across the complete transmembrane electric field (Yarov-Yarovoy et al., 2012). These voltage-driven conformational changes provide electromechanical coupling of depolarization and repolarization to the opening and closing of the pore, respectively. The pore opening takes place at the intracellular ends of the S6 segments, which cross and interact closely to form the closed activation gate (Catterall & Zheng, 2015). The bundle of S6 helices opens in an iris-like motion in response to voltage-dependent conformational changes in the voltage sensor.

VGCC ancillary subunits modify HVA channel surface expression (trafficking) and also impact channel biophysical and second-messenger-dependent modulation properties. The

α2δ subunit is composed of two peptides (α2 and δ) encoded by the same gene, with δ acting as a glycosyl-phosphatidylinositol anchor to the plasma membrane (Davies et al.,

2010). The α2 subunit binds to the δ subunit through disulfide bonds and with predicted structural domains involved in protein-protein interactions, particularly between the extracellular matrix and cell-adhesion proteins (Whittaker & Hynes, 2002).

Bioinformatic-based studies of the α2δ subunit primary structure identified both a structural domain homologous to the cell adhesion von Willebrand factor A (VWA) domain, which is involved in binding to the extracellular matrix proteins, as well as two bacterial chemosensory-like domains (CSD) (Dolphin, 2013). The cryo-EM of the

Cav1.1 complex revealed that the α2δ subunit interacts with the extracellular loops of repeats Ⅰ to Ⅲ through its VWA and CSD domains (Wu et al., 2015). Co-expression of

α2δ subunit increases the surface expression of functional Cav channels and decreases channel turnover resulting in significant alterations in Ca2+ current density and affecting also the inactivation rates (Cantí et al., 2005; Bernstein & Jones, 2007).

The β subunits are cytosolic proteins that bind to a proximal region of the intracellular linker between domains I-II of the HVA α1 subunits. Interaction of a  subunit with the pore-forming  subunit increases trafficking to the plasma membrane, alters kinetic and voltage-dependent properties, and affects channel modulation via several distinct intracellular signaling pathways (Stea et al., 1994; Buraei & Yang, 2010).

The γ1 ancillary subunit was first purified and characterized in skeletal muscle; however, the knowledge of γ subunit interaction domains with the pore-forming α1 subunit is very limited compared to other ancillary subunits, partially due to its preferential expression in

5 striated muscle. Also, γ subunits are associated with different molecular complexes besides VGCCs, playing numerous functions, including modulation of AMPA receptor trafficking (Chen et al., 2007). Studies performed in γ subunit null mice show an increase in current amplitude, a slowing of inactivation, and a positive shift of steady-state inactivation of VGCC in skeletal muscle, indicating the important involvement of γ1 subunit in regulating Cav1.1 functions (Kang et al., 2001).

In general, the electrophysiological and pharmacological functions of HVA VGCCs are determined by a combination of distinct 1 subunit properties and different ancillary subunits. The functional characteristics of VGCCs are also fine-tuned at the mRNA level by alternative splicing of all subunit types to result in a multitude of distinct VGCC conductances.

1.1.2 Alternative splicing in VGCCs The VGCC family contains several subtypes that have been classified according to their voltage-dependence (high voltage-activated channels or HVA, and low voltage-activated channels or LVA), kinetic properties (e.g. L-type calcium channel for its long-lasting current, and T-type channel for its transient current), and their sensitivities toward specific pharmacological agents (Catterall et al., 2005). The properties of calcium channel subtypes are defined according to the α1 subunits (Table 1.1). In total, there are 10 different α1 subunits encoded by 10 genes. HVA channels have a relatively positive membrane potential threshold for opening (approximately -40 mV) and include Cav1.1,

Cav1.2, Cav1.3 Cav1.4, Cav2.1, Cav2.2, and Cav2.3, whereas LVA channels with a low threshold for activation (approximately -60 mV) include Cav3.1, Cav3.2 and Cav3.3 (Figure 1.2) (Cain & Snutch, 2011; Zamponi et al., 2015).

Table 1.1 Classifications of calcium channel α1 subunits

The table shows the correspondence between gene name, channel protein name, and the nomenclature based on the functional characterization of the calcium currents; only some of the main physiological roles are included.

In addition to the 10 different α1 subunit genes in mammals, numerous structural and functional Cav variants for each subtype are known to result from alternative splicing mechanisms. These mRNA splice variation further expands the repertoire of unique Cav channels. Upon estimation, alternative splicing in the human genome results in the production of 4~10 unique mRNA products from each individual genomically encoded loci (Modrek & Lee, 2002). Furthermore, significant changes in Cav splice variant composition occurs between tissues, cell types, developmental time points, and disease states (Gray et al., 2007). These different levels of regulated complexity add further the diversity of functional Cav channels. Sites of alternative splicing in the α1 subunit genes (Cacna1) are typically, although not exclusively, located in regions encoding hyper- variable domains of Cav proteins that presumably can accommodate changes in protein 7 structures. These include the intracellular C-termini and the II–III intracellular linker (Lipscombe et al., 2002). The location of certain alternatively spliced exons is conserved among Cacna1 genes. For example, all but one Cacna1 gene contain an alternatively spliced exon that encodes a peptide in the putative extracellular linker between transmembrane spanning helices S3 and S4 in domain IV of Cav channels. In some genes such as Cacna1a and Cacna1b, a homologous alternatively spliced exon also encodes a short peptide sequence in the S3–S4 linker of domain III (Allen et al., 2010). The composition and/or length of the S3–S4 extracellular linker exons influences voltage- dependent gating of Cav channels, perhaps unsurprisingly given their proximity to the putative S4 voltage-sensors (Lin et al., 1999). Modulation of Cav channel activation induced by these exons can result in only 5~7 mV change in activation, but such changes can significantly impact the total movement of calcium during action potential-like stimuli (Lin et al., 1999). Thus, different Cav splice isoforms might be selected to fine- tune the coupling efficacy between membrane depolarization and calcium entry according to cell needs. On the other hand, while the importance of a given apparent low frequency of occurrence, care must be taken as large changes in abundance between tissues or developmental stages can also occur.

Furthermore, many studies have located the importance of splice variants of VGCC linking aberrant expressions with disease. The tissue-selective expression of the VGCC alternatively spliced exon that contains a genetic mutation associated with a particular disease could determine the severity of the disease. For example, the association of alternative spliced Cav1.2 gene with disease was previously discovered, a pair of developmentally regulated exons 31/32 was found to change in their relative proportion of expression in a rat model of myocardial infarction (Gidhjain et al., 1995). From the studies in Timothy syndrome, point mutations have been found both in the mutually exclusive exon8 and 8a that code for the IS6 region of Cav1.2 channel, with the most

8 lethal symptoms found in the cardiac exon 8a (Splawski et al., 2005). This will be discussed more thoroughly in section 1.2.4.

Alternative splicing events have also been extensively studied in the Cav2 family.

Researchers initially identified multiple versions of the Cav2.1 channel with sequence differences in the intracellular loop between domains Ⅰ and Ⅱ a crucial site for G-protein mediated regulation of channel inactivation, and in the C-terminus, a major interface between Ca influx and downstream response machinery (Mori et al., 1991; Starr et al.,

1991). Since then, many other alternative splicing events in the Cav2.1 channel have been identified in neuronal tissues from mammals, many of which have functionally distinct biophysical and modulatory properties. For example, an insertion (+NP) or exclusion (- NP) of a dipeptide segment occurs within the IVS3-IVS4 extracellular loop was identified in human and rodent brain (Hans et al., 1999; Bourinet et al., 1999; Toru et al., 2000; Soong et al., 2002). Inclusion/exclusion of the dipeptide NP segment determines

Cav2.1 sensitivity to ω-Aga IVA (Bourinet et al., 1999). It is suggested that –NP corresponds to native P-type currents with a high sensitivity to ω-Aga IVA (2 nM), whereas +NP corresponds to Q-type currents with a lower sensitivity to ω-Aga IVA (100 nM).

Thus, alternative splicing provides a level of fine-tuning of channel activity to adapt to specific cellular or tissue conditions or in response to various signals or stimuli. The assortment of alternatively spliced exons is not random, but it is still intriguing as to how the alternative splicing may be regulated in physiological or pathological conditions.

1.1.3 VGCC Distribution and functional relevance of VGCC

1.1.3.1 Cav1 family Although a high level of similarity takes place in protein structure, the distribution and function vary significantly across the 10 VGCC subtypes. The four subtypes in the Cav1 family are known as LTCC which was first isolated from cardiac myocytes and neurons (Nowycky et al., 1985; Nilius, 1986). One feature that distinguishes LTCC from all other VGCCs is their sensitivity to dihydropyridines (DHPs), which are a group of compounds that serve as agonists or antagonists of LTCC. These DHPs and their derivatives are not only used as a tool to isolate LTCC current in vitro, but have also been used clinically to treat hypertension (Mukherjee & Spinale, 1998; Sinnegger-Brauns et al., 2009). The four LTCC subtypes show similar pharmacological properties but differ in distribution and biological properties in mammals. The expression of Cav1.1 is more restricted to the transverse tubule membranes of skeletal muscle cells, where the channels interact directly with ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and active them to initiate contraction (Tanabe et al., 1993). Cav1.4 is mainly expressed in the retina for visual function. It is localized in the synapses of the outer and inner plexiform layer, and on the photoreceptor cell bodies (Morgans, 2001; Regus‐Leidig et al., 2009). The release of glutamate from the ribbon synapse is Ca2+ dependent and Cav1.4 is the predominant source of Ca2+ entry. The other two isoforms Cav1.2 and Cav1.3 are expressed in many organs and cell types, such as neurons, cardiomyocytes, and adrenal chromaffin cells (Olson et al., 2005; Chan et al., 2007; Dragicevic et al., 2014). In the brain, both channels play different roles in maintaining the neuronal function, which will be discussed in subsection 1.2.

1.1.3.1 Cav2 family

The Cav2 family consists of three subtypes: Cav2.1, Cav2.2 and Cav2.3 encoded by CACNA1A, CACNA1B and CACNA1E genes, respectively (Mori et al., 1991; Williams et

10 al., 1992; Dubel et al., 1992). Cav2 channels are primarily expressed in the presynaptic side of neuromuscular junctions and neuronal terminals to control and initiate synaptic transmission. Cav2.1 channels conducting P-type currents were first recorded in Purkinje cells, and are distinguished by high sensitivity to the spider toxin ω-Agatoxin IVA, while Q-type currents were discovered from cerebellar granule cells, and are blocked by ω- Agatoxin IVA with lower affinity (Kd ~2nM for P-type versus 100 nM for Q-type) (Llinás & Yarom, 1981; Mintz et al., 1992; Randall & Tsien, 1995). For the inactivation kinetics, P-type currents show a non-inactivating waveform during prolonged membrane depolarization, whereas Q-type currents show a pronounced inactivation (Bourinet et al.,

1999). The different current properties between these two Cav2.1 channels are likely due to alternative splicing (Bourinet et al., 1999) and/or the co-expression of the associated β- subunit (Richards et al., 2007). In contrast, Cav2.2 channels, also known as N-type calcium channels, are sensitive to ω-Conotoxin GVIA. They are mainly expressed at excitatory synaptic terminals, and are known to regulate the synchronous release of neurotransmitters (Weber et al., 2010). The Cav2.3 channel mediates the so called R-type currents, which are sensitive to the synthetic peptide toxin SNX-482. The role of Cav2.3 in synaptic transmission is different from the role of Cav2.1 and Cav2.2 (Ricoy &

Frerking, 2014). For example, Cav2.3 channels boost the accumulation of presynaptic Ca2+ triggering presynaptic LTP and post-tetanic potentiation in the mossy fiber synapses without affecting the release probability (Dietrich et al., 2003).

2+ In the nervous system, Cav2.1 and Cav2.2 are the predominant pathway of Ca entry for triggering the fast release of classical neurotransmitters like glutamate, acetylcholine and GABA; their functions in regulating synaptic transmissions are extensively controlled by interacting with membrane fusion machinery, including SNARE protein, a complex of syntaxin 1A, SNAP-25, and VAMP/synaptobrevin (Bajjalieh & Scheller, 1995; Südhof,

1995, 2004). Presynaptic Cav2 channels interact directly with the SNARE proteins in the

2+ large intracellular loop connecting domain Ⅱ and Ⅲ, Ca entry through Cav2 channels initiate the fusion of synaptic vesicles membranes with the cell membrane and release the neurotransmitters into the synaptic cleft (Dolphin & Lee, 2020). In the peripheral nerve system, P/Q-type channels have been shown to regulate synaptic transmission and affect the nociceptive response of dorsal horn neurons (Malmberg & Yaksh, 1994; Nebe et al., 1999).

Besides the functional role of interaction between Cav2 channels and SNARE proteins in synaptic neurotransmission, their interaction also serves a regulatory role on Cav2 channel function. Previous studies showed that co-expression of the SNARE protein syntaxin or SNAP-25 in the plasma with Cav2.1 and Cav2.2 channel proteins reduces the level of channel expression and down-regulate channel activity by shifting the voltage dependence of steady-state inactivation during long depolarization prepulse towards more negative membrane potentials, but the formation of a mature SNARE complex containing syntaxin and synaptotagmin at synaptic terminal could reactivated the channels (Bezprozvanny et al., 1995; Wiser et al., 1996; Zhong et al., 1999). Therefore, in the synaptic terminal, this mechanism would ensure that Ca2+ entry through calcium channels occurs primarily near active zone with docked synaptic vesicles and efficiently evokes neurotransmitter release.

In addition to their contribution to the synaptic release, Cav2 channels also play a role in regulating neuronal excitability. Blockade of P/Q-type calcium channels in spontaneously firing Purkinje neurons results in a significant increase in firing rate and bursting

2+ (Womack & Khodakhah, 2002). Ca entry through Cav2.1 and Cav2.2 channels also activates large conductance calcium-activated potassium channels (BK channels) and in turn hyperpolarize membrane potential which in turn supress neuronal firings (Berkefeld et al., 2006; Berkefeld & Fakler, 2008). Cav2.3 channels are expressed throughout the

12 central and peripheral nervous system and they play a role as a source of calcium for calcium-dependent potassium channels, including small-conductance calcium-activated potassium channels (SK channels) (Gutzmann et al., 2019). Therefore, multiple membrane conductances are cooperating together with Cav2 channels in regulating excitability, which is an essential neuronal function to integrate and transmit signals within the neural circuitry, and to help maintaining the homeostasis of the membrane.

The dysfunction of Cav2 channels has been implicated in numerous severe human pathologies. To date, mutations in human gene CACNA1A encoding Cav2.1 have been associated with three autosomal dominantly inherited neurological disorders: episodic ataxia type 2, spinocerebellar ataxia type 6 and familial hemiplegic migraine type 1 (FHM-1) (Ophoff et al., 1996; Zhuchenko et al., 1997; Jen et al., 2004). In the case of

FHM-1, there have been 21 mutations in the Cav2.1 channel gene identified in human patients; these mutations are all substitutions in conserved functional domains. Two FHM-1 knock-in mouse strains (R192Q and S218L) have been widely used to study the consequences of FHM-1 mutations on endogenous Cav2.1 channels (van den Maagdenberg et al., 2004; Vecchia et al., 2015). R192Q mice display mild phenotype of FHM, however, homozygous S218L mice show the most severe clinical features identified in humans, including spontaneous attacks of hemiparesis and/or generalized seizures (van den Maagdenberg et al., 2004; Maagdenberg et al., 2010). Furthermore,

Cav2.1 knockout mice display ataxia and absence seizures and die in early life (Jun et al., 1999).

Recently, a mutation has been found in Cav2.2 gene that cause a unique dominant myoclonus-dystonia-like syndrome with cardiac arrhythmias (Groen et al., 2015). Further analysis on the single-channel level showed that the mutant Cav2.2 channel carried less current compared with wild-type channels, but the activation or inactivation kinetics are

13 not significantly altered. The functional changes could be consistent with a gain-of- function causing the observed hyper-excitability characteristic of this unique myoclonus- dystonia-like syndrome associated with cardiac arrhythmias.

De Novo pathogenic variants in the gene encoding the Cav2.3 channel have been found recently; the variants cluster within the cytoplasmic ends of all four S6 segments that form the presumed Cav2.3 channel activation gate, which leads to facilitated voltage- dependent activation and slow inactivation. These molecular alterations cause developmental and epileptic encephalopathies with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death (Helbig et al., 2018).

1.1.3.3 Cav3 family

Cav3 family is also known as LVA or T-type channels, which includes three different subtypes: Cav3.1, Cav3.2, and Cav3.3 (Perez-Reyes et al., 1998; Cribbs et al., 1998; Lee et al., 1999). Cav3 currents are activated at more negative membrane potentials compared to other VGCCs, and their voltage-dependent inactivation is faster (Nowycky et al.,

1985). Unlike the high voltage-activated calcium channels, functional expression of Cav3 channels in heterologous systems does not require assembly of the ancillary subunits, but these subunits still can provide regulatory functions. Co-expression of β1b and α2δ3 subunits enhanced the membrane expression level of Cav3 channel in various expression systems (Dubel et al., 2004) with a two-fold increase in T-type current with no change in the electrophysiological properties.

Cav3 channels are thought to regulate neuronal excitability due to their distinct voltage dependence for activation and inactivation. A current window is formed because of the overlap of activation and inactivation curve, thus at resting membrane potential, a fraction of channels have the probability to open and contribute to the regulation of neuronal excitability (Chevalier et al., 2006; Dreyfus et al., 2010). In addition, as a result 14 of their hyperpolarized range of activation and inactivation, the majority of Cav3 channels are basically inactivated at neuronal resting membrane potential. When an inhibitory input produces a brief membrane hyperpolarization, it is sufficient to recover these calcium channels from inactivation, thus increasing the fraction of channel that is available for opening, and in turn, cause membrane depolarization that triggers rebound bursting firing (Coulter et al., 1989; Huguenard & Prince, 1992; McCormick &

Huguenard, 1992). Cav3 channels are crucial for thalamic delta rhythms and they determine the generation of the rhythmic bursts of action potentials that drive sleep spindles and control sleep (Lee et al., 2004).

Mutations in the Cav3.2 channel have been identified in patients with absence epilepsy and also other types of idiopathic generalized epilepsies (Chen et al., 2003b; Heron et al., 2007). Studies on transgenic mice containing these mutations revealed a gain-of-function in Cav3.2 channel, and an increased level of channel protein expression at the cell surface

(Khosravani et al., 2004, 2005; Vitko et al., 2005, 2007). Mutations in human Cav3.1 and Cav3.3 channel genes have also been associated with the pathophysiology of neuropsychiatric diseases: a mutational analysis of Cav3.1 gene from patients with idiopathic generalized epilepsies revealed 13 variants, and five of them involved amino acid substitutions (Singh et al., 2007). More recently, two de novo missense variations, T797M and R1346H, of Cav3.3 gene were identified in individuals with schizophrenia (Andrade et al., 2016). In order to further investigate the pathophysiological function of

Cav3 channels, gene knockout and knock-in models have been established in mice.

Cav3.1 null mice display increased visceral pain sensation due to alterations in thalamic neuron firing (Kim et al., 2003), whereas mice overexpressing Cav3.1 show absence epilepsy (Ernst et al., 2009). Cav3.2 knockout mice display abnormal developmental of the trachea and reduced relaxation of vascular tissue in response to acetylcholine, and also show lower peripheral pain sensitivity (Chen et al., 2003a; Choi et al., 2007). Cav3.3

15 knockout mice show increased susceptibility to drug-induced spike and wave discharges (Lee, 2014).

Figure 1.1 Multimeric assembly of voltage-gated calcium channels and functional domains of the α1 pore-forming subunit

Panel A shows a schematic representation of the subunit composition of high voltage- gated calcium channels. The white tetramer represents the α1 subunit, the purple peptides linked by a cysteine bound represent the α2δ subunit and the intracellular  subunit is shown in red, while the blue transmembrane peptide represents the γ subunit. Panel B 16 shows a ribbon diagram of the 3D structural model of a rabbit Cav1.1 calcium channel α1 subunit from the top view (Protein Data Bank, code: 6BYO; Matinez Ortiz, 2018). The four wings are the voltage sensor domains and the pore is located in the central region lined by the S5-S6 segments. In panel C, a diagram of the transmembrane topology of the α1 subunit shows the four repeated homologous domains (I-IV) and the structural determinants of ion permeation and voltage dependence. The S5-S6 linker (P-loop) from all four domains form the calcium permeating domain (depicted in green); the S4 segments (yellow) contain positively charged amino acids, interspersed every three residues, that form part of the voltage sensor.

Figure 1.2 Comparison of kinetics and voltage range of activation between high- voltage and low-voltage activated calcium channels

Macroscopic calcium currents recorded from HEK293 cells transfected with recombinant

Cav1.2 α1, β4 and α2δ-1 subunits (upper left panel) or Cav3.1 α1 subunit (upper right panel) display distinct inactivation kinetics. Whole-cell voltage clamp recordings were obtained using extracellular [Ca]=2mM. Representative traces were obtained by applying depolarizing pulses of 180 ms duration and 5 mV increment from -60 to +30 mV for the

HVA Cav1.2 channel and from -90 to +10 mV for the LVA Cav3.1 channel. Lower panel shows the normalized voltage-current relationship plots showing the different range of voltage activation. The continuous lines correspond to the fitting curve with a Boltzmann function: ICa= [Imax (V-Vrev)]/[1+exp((V1/2-V)/k)], where V=membrane potential,

Vrev=reversal potential, V1/2=membrane potential at half maximal current, k=Boltzmann factor. (Unpublished data by Y. Zhang & E. Garcia)

1.2 Neuronal L-type calcium channels

As mentioned above, the unique pharmacological sensitivity of Cav1 channels to LTCC blockers has been used for their identification in physiological assays and also for their biochemical isolation (Kanngiesser et al., 1988). Cav1.1 channel was the first VGCC cloned as an LTCC blocker receptor from skeletal muscle; sequence information form this landmark study was then used to screen for, and clone the cDNAs of Cav1.2 and

Cav1.3, the main L-type calcium channels in the nervous system (Tanabe et al., 1987; Mikami et al., 1989; Biel et al., 1990; Koch et al., 1990; Perez-Reyes et al., 1990; Hui et al., 1991; Williams et al., 1992).

The Cav1.2 and Cav1.3 isoforms display similar expression patterns in many tissues, including neurons and other electrically excitable tissues, such as heart and smooth muscle (Catterall et al., 2005); however, a differential profile during development and subcellular localization of each subtype has been reported. Cav1.2 and Cav1.3 channels share significant sequence homology and similar structure; the main differences in the amino acid sequence occur in the linker between domains II and III, and the C-terminus (Zuccotti et al., 2011). The structural and sequence variations between these two channels likely determine the different voltage range of activation and drug sensitivity.

1.2.1 Pharmacological ligands of LTCCs There are three main classes of selective LTCC ligands for clinical use, which are the phenylalkylamines (PAA), the benzothiazepine derivatives (BTZ), and the dihydropyridines (DHP). These drugs bind to different sites on the channel structure that are allosterically linked (Tang et al., 2019). Verapamil is the only drug in the PPA class in clinical use for the treatment of hypertension. It binds to Cav1 channels in a use- dependent manner: the affinity depends on the voltage-dependent conformational changes in the functional state of the channel protein (Lee & Tsien, 1983; Sanguinetti M C & Kass R S, 1984; Uehara & Hume, 1985). Biochemical and electrophysiological studies 20 implicated the transmembrane segment IVS6 as a critical component of the PAA receptor site (Lipkind & Fozzard, 2003). In the BTZ class, Diltiazem is used clinically for the treatment of muscle hypertension and arrhythmias (Triggle, 1991). The BTZ-binding site on Cav1 channels is allosterically linked to the binding sites for PAA and DHP: interestingly, BTZ binding inhibits PAA binding but stimulates DHP binding (DePover et al., 1982; Ferry & Glossmann, 1982; Boles et al., 1984). These three drug classes have different mechanisms of action; for example, upon a train of depolarization, Diltiazem displays more frequency-dependent blockade than the DHPs, but less than methoxyverapamil. Biochemical studies have indicated that the possible binding sites of

BTZ on Cav1 channels are located around or adjacent to the transmembrane segments IIIS6 and IVS6 (Tang et al., 2016).

The DHPs can act on Cav1 channels as either agonist (eg. (S)-Bay K8644 or BayK for short, and PN 202 791) or antagonist (eg. Nifedipine and Nitrendipine) (Bechem & Schramm, 1987; McDonald et al., 1994). Their specific ability depends mainly on drug structure, but it is also affected by membrane potential and the channel conformational state; thus the modulation of Cav1 channel function by DHPs is more complex and depends on the specific interaction of each individual drug. Electrophysiological experiments revealed that the DHP binding site is close to the extracellular surface of the cell membrane (Strübing et al., 1993). Multiple experimental approaches have been used to localize the DHP binding site, such as photoreactive DHPs, chimeric Cav1 channels, or site-directed mutagenesis. Results from all approaches indicate that the core of the receptor binding site for DHP drugs is shared between transmembrane segments IIIS6 and IVS6; in addition, IIIS5 and some adjacent amino acid residues have also been implied in DHP binding (Cataldi & Bruno, 2012).

Radioligand binding studies have identified binding sites for DHP antagonists in the brain

21 and in cardiac and smooth muscle membranes with similar binding properties and similar comparative binding pharmacology (Ehlert et al., 1982; Huber et al., 2000; Takahashi et al., 2008). However, the DHP antagonists are weakly active or inactive in many neuronal preparations (Miller & Freedman, 1984). For example, acetylcholine release from the electrically stimulated guinea pig ileal myenteric plexus preparation was unaffected by verapamil, diltiazem or nicardipine at concentrations much higher than their potent relaxant effects on depolarized intestinal smooth muscle (Rosenberger et al., 1979). On the other side, DHP activators show potent effects on neuronal LTCC current, as well as multiple functional alterations in neurons. BayK, a close structural analog of nifedipine, is one of the frequently used agonists for LTCCs. Its pharmacologic effects are competitively antagonized by nifedipine, consistent with agonist and antagonist acting at the same site (Zhao et al., 2019a). BayK was proved to increase calcium influx by prolonging channel opening and burst activity during membrane depolarizations (Kass,

1987a). Mechanistically, single-channel recordings showed that BayK promotes Cav1 channels switching from gating mode 1, characterized by low open probability and short open time, to gating mode 2, with higher open probability and longer open times; while In whole-cell patch clamp configuration, BayK strongly increased the peak current of LTCC in both guinea pig and frog ventricular cells (Hess et al., 1984). Therefore, BayK is also widely used as a tool to investigate the involvement of LTCCs in physiological processes (Dolphin, 2006; Espinosa-Parrilla et al., 2015).

Currently, drugs from DHP class are traditionally used for the treatment of hypertension, and there is a growing interest in their anti-inflammatory activity as well as metabolic effects (Rubio-Guerra et al., 2009). However, despite the fact that LTCCs are identified as a high risk factor in neuropsychiatric disorders, which will be discussed in section 1.2.4 and 1.3, DHPs have not yet been proven to be clinically relevant for the treatment of neuropsychiatric diseases. Thus, it is still necessary to improve our understanding of

DHPs mechanisms of action on neuronal LTCCs under pathological conditions, which will shed light on the future drug development research for the therapy of neuropsychiatric diseases (Godfraind, 2017).

1.2.2 Biological functions of neuronal LTCCs Based on the application of LTCC binding ligands combined with multidisciplinary experimental approaches, researchers have revealed the cellular distribution and function of neuronal Cav1 channels. In the nervous system, LTCC Cav1.2 and Cav1.3 are mainly present on the nerve cell soma, dendrites and postsynaptic membranes (Tippens et al.,

2008; Leitch et al., 2009). In contrast to Cav2 channels, which are expressed in the presynaptic side to control neurotransmitter release, Cav1.2 and Cav1.3 are involved in processing the input synaptic signals on the postsynaptic terminals. Calcium influx into the postsynaptic cell membrane via these channels has been implicated in regulating neuronal firing, activity-dependent gene transcription, synaptic plasticity, synaptogenesis and dendritic growth (Perrier et al., 2002a; Moosmang et al., 2005a; Yang et al., 2018; Kamijo et al., 2018a; Wild et al., 2019).

The role of LTCCs in the induction of synaptic plasticity has been suggested previously in many brain nucleus, such as in rat hippocampus (Norris et al., 1998). The application of DHP antagonist nifedipine showed that short-term plasticity of GABA release is controlled by LTCCs at presynaptic firing rates in the gamma-frequency (40Hz) range in the cerebral cortex (Jensen & Mody, 2001a). In addition, the activation of LTCCs by BayK, combined with modest postsynaptic depolarization and synaptic activation, is sufficient to induce robust long-term depression (LTD) in rat striatal medium spiny neurons (Adermark & Lovinger, 2007). By using DHP ligands, researchers have also found a significant contribution of Cav1 channels in neuronal excitability. Nimodipine was found to decrease the amplitude of afterhyperpolarization (AHP), with a subsequent increase in excitability in rabbit CA1 neurons (Moyer et al., 1992). Furthermore, BayK 23 significantly increased the slow AHP amplitude and caused a reduction in γ–frequency oscillation in mice hippocampal neurons (Driver et al., 2007). In rat substantia nigra GABAergic neurons, the plateau potentials, a long-lasting depolarization potential following a train of action potentials evoked by depolarizing current, were found to be mediated by a calcium-activated nonselective cation conductance that is activated by calcium entry predominantly through L-type calcium channels (Lee & Tepper, 2007a).

Activity-dependent gene expression and synaptic plasticity of neurons allow the brain to respond to changes in its environment; during brain development, sensory-driven neural activity is essential for the processes involved in synapse development (Chaudhury et al., 2016). Various studies have proved that calcium entry through LTCCs preferentially drives and regulate gene transcription via calcium-signaling pathways (Wheeler et al., 2008). Most commonly, calcium bound to calmodulin or other calcium sensors activates any of a number of calcium-regulated intracellular signaling cascades, which include kinases of the calcium-calmodulin kinase (CaMK) and Ras/mitogen-activated protein kinase (MAPK) signaling pathways as well as the calcium-regulated phosphatase calcineurin (West & Greenberg, 2011). The calcium-signaling pathways mediating activity-dependent transcription and their relationship to the mechanisms that regulate synapse development will be discussed further in chapter two.

Since LTCCs mediate critical processes in the brain, interest has arisen about their participation in regulating the cellular basis of behavior under normal and pathological conditions. Clinical and in vivo animal model studies conducted to investigate the contribution of Cav1 channels in behavior have shown that both activation and inhibition of LTCCs cause changes in behavioral phenotypes. Intracerebroventrical (icv) administration of BayK is known to induce convulsions and seizures in mice, which were potently blocked by DHP antagonist (Shelton et al., 1987; Palmer et al., 1993). BayK

24 dose-dependently enhanced somatic signs and hyperalgesia response in nicotine- withdrawn mice, while the blocker nimodipine attenuated these signs, suggesting an important role of LTCCs in nicotine withdrawal (Jackson, 2008). For DHP antagonists (eg. Nimodipine, isradipine, or diltiazem), clinical reviews show that they do not affect brain function in human patients during chronic treatment of hypertension. Nevertheless, one experimental clinical study claimed that DHPs might induce some subtle changes in corticospinal metaplasticity (Wankerl et al., 2010).

1.2.2 Distinct contributions of Cav1.2 and Cav1.3 to neuronal function Interestingly, while Cav1.2 and Cav1.3 are normally co-expressed in many of the same cells, their overall contributions to Ca2+ influx are not equal with approximately 90% of the LTCC currents in the brain being from Cav1.2 and the remaining 10% Cav1.3 (Hell et al., 1993a; Olson et al., 2005; Chan et al., 2007; Sinnegger-Brauns et al., 2009; Dragicevic et al., 2014). This raises the question as to whether Cav1.2 and Cav1.3 serve different cellular functions. Previous studies showed that they do not have a significant difference in the sensitivity to DHP blockers, and all amino acid residues known to participate in the formation of the DHP-binding site are conserved between these two α1 subunits (Striessnig et al., 1998). However, DHPs block LTCCs in a voltage-dependent manner, and this voltage-dependence differs for Cav1.2 and Cav1.3; therefore Cav1.2 channels are blocked with approximate 10-fold lower IC50 values at negative resting membrane potential (Xu & Lipscombe, 2001a). Subsequently, it was shown that DHPs block both channels more slowly and with higher IC50 values upon stimulation by high frequency action potential-like waveforms, which are supposed to mimic neuronal firing (Helton et al., 2005). These findings imply that the relative contribution of Cav1.2 and Cav1.3 to the physiological properties of a neuron could not be dissected by pharmacological tools; thus genetic manipulations such as Cav1.2- or Cav1.3-null mice

25 are used for this purpose.

Studies on conditional Cav1.2 knockout mice show that Cav1.2 is required for hippocampal spatial memory formation involving protein synthesis-dependent, NMDA receptor-independent, late-phase long-term potentiation (L-LTP) in CA3-CA1 synapses, and for activation of the microtubule-associate protein kinase/cAMP/calcium cascade (Moosmang et al., 2005b). Whereas Cav1.3 does not show contribution to the CA3-CA1 hippocampal LTP, and the Cav1.3-null mice display normal behaviors in spatial memory encoding in the Morris water maze (McKinney & Murphy, 2006). A study of fear memory also revealed the different roles of the two Cav1 channels; Cav1.2, but not Cav1.3 is involved in the process of acquisition and extinction of conditioned contextual fear memory, while Cav1.3 is required for fear consolidation (McKinney & Murphy, 2006;

Busquet et al., 2008). Cav1.3 null mice displayed impaired consolidation and reduced LTP in the basolateral amygdala (BLA) synapse receiving input from the entorhinal cortex, as well as an increase in BLA neuronal excitability (McKinney et al., 2009a).

Other study using Cav1.2-deficient mice showed that Cav1.2 accounts for most of the LTCC currents in the amygdala neurons, and the DHP antagonist isradipine administration decreased thalamus-amygdala LTP and auditory cued fear memory acquisition (Langwieser et al., 2010). In addition, these two Cav1 channels differentially affect anxiety- and depression-like behaviors. For instance, the conditional forebrain

Cav1.2 elimination mice have enhanced anxiety-like behavior, whereas the Cav1.3 null mice show an antidepressant-like behavior (Dao et al., 2010; Lee et al., 2012).

Furthermore, researchers have developed another mouse model to dissect the physiological role of Cav1.2 and Cav1.3 channels: it was achieved by mutating Thr1066 to tyrosine in the DHP binding site of Cav1.2 α1 subunit (Cav1.2DHP-/-) to reduce the sensitivity of DHP blockers, allowing to examine directly the mechanisms mediated by

Cav1.2 channel (Sinnegger-Brauns et al., 2004). In a study using the forced swim test, where animal immobility indicates a level of depression, the absence of DHP-binding site decreased immobility, suggesting that an antidepressant-like effect could be achieved by blocking Cav1.2 channels (Sinnegger-Brauns et al., 2004). In addition, Cav1.3-selective channel activation by BayK induces depression-like effect, suggesting that Cav1.2 and Cav1.3 channels control different mood behaviors in mice. Also, in Cav1.2DHP-/- mouse, the DHP agonist-induced dystonic neurobehavioral syndrome is completely absent, suggesting that this syndrome is mediated by activation of Cav1.2 channels (Sinnegger- Brauns et al., 2004).

At the present time, these genetic mouse models suitable to dissect Cav1.2 and Cav1.3 function in the brain and other tissues are revealing distinct, non-overlapping, functional contributions of these two channels: these studies may provide a rational basis for the developmental of novel LTCC modulators, and provide insight into the underlying mechanisms of LTCC-related channelopathies.

1.2.4 LTCC neuronal channelopathies Early studies showing elevated calcium levels in platelets or lymphoblasts of patients with bipolar affective disorders hypothesized that dysregulation of intracellular calcium homeostasis could explain these psychiatric illnesses (Dubovsky et al., 1994; Emamghoreishi et al., 1997). Since LTCC family is one of the main sources of intracellular calcium level, thus, unsurprisingly, LTCC dysfunction can result from structural aberrations within their pore-forming α1 subunits, like in retinal Cav1.4 found in patients with incomplete congenital stationary night blindness (CSNB2), or in skeletal muscle Cav1.1 found in patients with hypokalemic periodic paralysis or malignant hyperthermia susceptibility. To date, however, human diseases resulting from mutations in the two neuronal LTCCs genes (CACNA1C encoding Cav1.2, and CACNA1D encoding Cav1.3) are very rare compared to those associated to other VGCCs. This could 27 be due to the fact that loss-of-function or gain-of-function mutations cause no phenotype in the heterozygous state, but are lethal in the homozygous state. For instance, it is known that heterozygous Cav1.2 or Cav1.3 knock-out mice were not distinguishable from wild type, suggesting that heterozygous loss-of-function mutations could also be clinically silent in humans; homozygous Cav1.2 knock-out mice die during embryonic development, before day 14.5 post-coitum, which may be due to their prominent role in the cardiovascular system (Seisenberger et al., 2000). However, a few spontaneous gain- of-function mutations may cause a clinical syndrome compatible with life. In addition, emerging evidence from genome-wide association studies (GWAS) support the association of polymorphisms in the Cav1 gene with psychological disorders (Lotan et al., 2014). These studies extend the understanding of the LTCC dysfunction in the pathophysiology of neuropsychiatric disorders.

Naturally occurring mutations of human Cav1.2 channel gene were identified only recently from patients with a rare developmental disorder, Timothy Syndrome (TS) (Splawski et al., 2004a), which affects many parts of the body including the heart, digits (fingers and toes), and the nervous system. It is first characterized by a heart condition with long QT syndrome, which causes the cardiac muscle to take longer than usual to recharge between beats. This abnormality in the heart can cause irregular heartbeats (arrhythmia), which can lead to sudden death. As a result of these serious heart problems, patients with TS live only into childhood. So far, fewer than 20 people with this disorder have been reported worldwide. In addition to the heart long QT symptom, central nervous system development is delayed in TS patients and lead to autism spectrum disorder (ASD), a condition in which the patients’ social, communicative, behavior functions are affected (Splawski et al., 2004a, 2005). The detailed mechanisms are discussed below.

TS is caused by missense point mutations in mutually exclusive exons of the CACNA1C gene and causes single amino acid substitutions in the Cav1.2 protein. The location of the mutations is illustrated in Figure 1.3 (upper panel). Initially, two different point mutations were identified in exon 8 of CACNA1C: G406R, a Gly-to-Arg mutation, and G402S, a Gly-to-Ser mutation. The exon 8, which undergoes alternative splicing into exon 8 or 8a, encodes the peptide sequence located in the S6 segment of the first homologous repeat and forms part of the activation gate (Splawski et al., 2004a). The so-called typical or TS type 1 (TS1) mutation is the G406R in exon 8a (coding VNDAV, low (~20%) expression in brain and heart) causes a mild phenotype. However, if this Gly-to-Arg mutation occurs in the highly (~80%) expressed alternative exon 8 (coding MQDAM, 5’ of exon 8a), it causes a more severe variant TS type 2 (TS2). These TS mutations generally cause a gain-of-function in Cav1.2 channels, and a subsequent delay in cardiomyocytes repolarization, with increased risk of severe ventricular arrhythmias. However, unlike the heart, where the majority of calcium influx is through Cav1.2 channels, the nervous system is more complex and other VGCC channels also contribution to Ca2+ entry into the neurons. Therefore, animal models containing the TS Cav1.2 mutant gene selectively targeted to the brain would be a promising tool to study the functional consequences of the mutation in neuronal circuits and the pathogenesis of ASD.

An initial attempt to generate a TS2-like mice model produced non-viable heterozygous and homozygous animals (Bader et al., 2011). Nevertheless, by inserting a reversed neomycin cassette aligned with the mutational exon (TS2-neo) the heterozygous mice survived through adulthood, probability due to the reduced expression level of the G406R Cav1.2 protein via transcriptional interference. The TS2-neo mice showed normal locomotor activity and anxiety level, but displayed evident restricted, repetitive, and perseverative behavior, altered social behavior, impaired ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning (Bader et al.,

2011). On a macroscopic level, the brains of TS2-neo mice were found relatively normal, the dimensions of the cerebral cortex and the cerebellum showed no systematic differences in shape or size (Bett et al., 2012).

Heterologous systems have been used to characterize the electrophysiological properties of the recombinant TS-Cav1.2 channels. After identifying the TS mutation from human patients, Splawski and his colleagues first expressed wild-type (wt) and G406R forms of Cav1.2 channel in Chinese hamster ovary (CHO) cells and Xenopus oocytes (Splawski et al., 2004a). Compared to wt channel, TS mutation produces sustained inward Ca2+ currents by causing nearly complete loss of voltage-dependent channel inactivation without significantly shifting the normalized I/V relationship. By combining different proportions of recombinant channels it was found that even a small fraction of TS-Cav1.2

2+ channel mixed in a wt Cav1.2 channel background will result in a sustained Ca current that prolonged the action potential duration by 17%; this is consistent with the role of Cav1.2 channels mediating the plateau phase of the cardiac action potential. Furthermore, TS-Cav1.2 (G406R) expressed in HEK-293 cells also showed a marked slowdown of the Ca2+ current inactivation kinetics at all the depolarizing pulses tested (Bidaud & Lory, 2011). The I-V relationship showed a negative shift in the activation threshold for TS- Cav1.2 channels but no change in the peak current membrane potential as well as in the reversal potential values. On the other hand, recovery from inactivation, an important mechanism that controls cellular excitability, was largely facilitated in TS-Cav1.2 channel.

To date, TS mutations are the only evidence for a direct link between the CACNA1C gene and ASD. However, by using the genome-wide association approach, there is increasing information on the association between CACNA1C SNPs and psychiatric disorders including bipolar disorders, schizophrenia, and major depression (Ferreira et

30 al., 2008). One SNP (rs1006737) located in the third intron of the Cav1.2 gene was found to be strongly linked to bipolar disorder, a mental disorder characterized by episodes of mania hypomania and depression (Sklar et al., 2008). Human brain imaging and behavioral studies have demonstrated morphological and functional alterations in individuals carrying the CACNA1C risk allele (Yoshimizu et al., 2015). However, the cellular impact of this SNP on channel function in human neurons is still waiting to be explored. By utilizing the technology of rapid neuronal programming that converts fibroblasts into functional induced neurons, Yoshimizu’s team derived human neuron-like cells from a relatively large library of fibroblasts obtained from individuals with and without the risk SNP rs1006737, to examine its functional consequences on a cellular level. Their results show that the CANCNA1C mRNA expression level is higher, and the LTCC current density is larger in the cells from individuals carrying the SNP compared to the control group. In the meantime, another team reported that the same SNP rs1006737 also has a significant association with schizophrenia (Nyegaard et al., 2010). The association of CACNA1C with bipolar disorders and schizophrenia, therefore, suggests that the two disorders are, at least in part, ion-channelopathies. Additionally, a study conducted in a cohort of schizophrenia patients and healthy control Chinese subjects shows that, besides re1006737, significant differences in the allele frequencies were found for another SNP rs4765905 marker in CACNA1C gene between the patients and controls (Guan et al., 2014). Further analysis of the haplotype rs1006737-rs4765905- rs882194 in CACNA1C showed significant association with schizophrenia, and two haplotypes (ACC and ACT) in the block were significantly increased in the patient tested.

Similar to Cav1.2, mutations in the human Cav1.3 gene are rare; loss-of-function Cav1.3 mutations or gene silencing in knockout mice model does not result in overt neurological manifestations. Nevertheless, the specific acute activation of Cav1.3 channels cause depression-like behaviors and lead to activation of brain regions involved in anxiety and

31 fear (Sinnegger-Brauns et al., 2004; Hetzenauer et al., 2006). These studies were performed in Cav1.2DHP-/- mice, in which the application of BayK selectively increased the function of Cav1.3 in the brain. The results from this mice model suggest that Cav1.3 gain-of-function mutation could potentially increase the risk of CNS disease. Recently, two gain-of-function mutations in the Cav1.3 gene (A749G and G406R) have been identified in human patients affected by neuropsychiatric disease including ASD and epilepsy (Pinggera et al., 2015a) (Fig 1.3 lower panel). The G406R point mutation is identical to the TS mutation in Cav1.2. Model analysis of the two mutations in Cav1.3 α1 subunit predicted pronounced changes of the interaction of the affected distal S6 helices with adjacent S6 helices of the activation gate (G407R, A749G) and of the S4-S5 linker with the voltage sensor (G407R) (Pinggera et al., 2015a). By expressing the two mutant channel genes into tsA-201 cells, it was found that both mutations strongly affect channel gating. Mutation A749G largely increased peak current density, and shifted steady-state activation and inactivation voltage dependence to more negative potentials. In contrast, mutation G407R reduced maximal current amplitude and had no effect in activation voltage dependence. However, similarly to the effect of G406R mutation on Cav1.2 channel, mutation on glycine 407 significantly slowed down the inactivation time course, resulting in a larger Ca2+ influx during prolonged depolarization compared to wt channels. Another study using exome sequencing in autistic patients has confirmed the de novo A749G and G407R mutations, and also identified an additional de novo mutation V594I and three other risk variants (De Rubeis et al., 2014). None of the newly identified mutations has been functionally characterized so far.

Furthermore, two other mutations in the CACNA1D gene were found as germline de novo mutations in two patients with a severe congenital syndrome presenting not only with primary aldosteronism but also with neurodevelopmental deficits and seizures at an early age (Scholl et al., 2013). One patient carrying the G403D mutation developed sinus

32 bradycardia, prolonged QT-interval, primary aldosteronism, hypokalaemia, failure to thrive and a global developmental delay. This patient also displayed epilepsy and was diagnosed with cortical blindness, spasticity and cerebral palsy. EEG recordings showed recurrent spikes emanating predominantly from the right temporoparietal occipital region, and to a lesser extent, from the left temporal region without a clinical correlate. The second patient, carrying an I750M mutation, was diagnosed with cerebral palsy, spastic quadriplegia and mold athetosis, severe generalized intellectual disability, complex partial seizures and generalized seizures. A common finding is the presence of seizures and generalized intellectual disability and spasticity, suggesting the association of Cav1.3 gain-of-function with these risks.

In comparison, these point mutations found in Cav1.2 and Cav1.3 share some similarities in terms of the location and functional consequences, but also possess differences in the disease symptoms, which probably due to the differences in tissue expression pattern, as well as the different functional roles of these two LTCCs.

Figure 1.3 Gain of function mutations in Cav1.2 and Cav1.3 channels associated with neuropsychiatric disorders

Schematic shows the simplified topological structure of Cav1.2 (upper panel) and Cav1.3 channels (lower panel) and the location of some point mutations. Among them, four point mutations have identified at the intracellular end of segment 6 of domain I, and two other mutations were located at the transmembrane region of segment 6 of domain II. They presumably affect the activation gate and/or voltage sensor of the channels. Red star: G402S and G406R in Cav1.2 channel. Red triangle: G403D, G407R, A749G, and I750M in Cav1.3 channel. 34

1.3 Cav1 channels and Autism Spectrum Disorders Autism Spectrum Disorders (ASD) are a complex condition characterized and diagnosed on the basis of a triad of behavioral traits: impaired social interaction, impaired social communication and repetitive and restricted activities/interests (DSM-5) (American Psychiatric Association, 2013). ASD are further associated with lifetime consequences related to the quality of life and social integration. Furthermore, ASD are one of the most serious neurodevelopmental disorders, with significant caregiver, family and social financial burdens.

The current prevalence of ASD reported in a recent survey is about ~2 % with a male predominance (Kim et al., 2011). Siblings born in families with ASD have a much greater risk of ASD, with a recurrence rate of 5~8 % (Szatmari et al., 1998). The prevalence has been increasing in the last decades, partially due to the changes in DSM-5 criteria, but an increase in risk factors cannot be ruled out (Mattila et al., 2011; Kim et al., 2011; Elsabbagh et al., 2012; Fisch, 2012). Commonly referred as a single disorder, ASD is rather a complex and heterogeneous group of disorders, arisen as a consequence of environmental and/or genetic factors. Nowadays, there is growing attention to environmental risk factors contributing to the etiology of ASD, due to a high incidence in association with in-utero valproic acid exposure or congenital rubella (Chess et al., 1978; Rodier et al., 1997). Additional evidence for the role of environmental factors in the rising numbers has emerged, including an incomplete monozygotic concordance, differences related to geography, occupation and time of birth, environmental toxins, and also unanticipated evidence of plasticity and improvement in response to environmental modulation (Herbert, 2010). Some other factors have been also considered as the cause, such as the use of assisted reproductive technologies, and tocolytic drugs such as terbutaline (Connors et al., 2005; Knoester et al., 2007).

1.3.1 Genetic factors related to ASD The high heritability of ASD indicates a strong contribution of genetic factors. Genetic alterations that have been associated to ASD mainly include three aspects: chromosomal abnormalities, copy number variants (CNV), and single-gene mutations/SNPs (Herman et al., 2007; Miles, 2011). In this section I discuss these three genetic mechanisms, with emphasis on single-gene mutations relevant to the calcium channel family. Although genetic associations could be identified in 20~25 % of ASD patients using standard genetic evaluation techniques, the etiology for the remaining 75~80 % of children remains unknown. However, the analysis of these genetic factors, especially the study of single-gene mutations, has revealed a common theme since many of these genetic alterations can potentially affect the development of neural networks and/or synaptic functions (Gilman et al., 2011), processes also related to LTCCs.

Using fluorescence in situ hybridization techniques, it has been shown that roughly 3~5 % of children with ASD carry chromosomal abnormalities. The maternally derived 15q11-q13 interstitial duplication is the most commonly observed cause of ASD and the phenotype correlates with the number of 15q; trisomy causes subtle effects on the physical phenotype, whereas children with four copies of 15q including those with a supernumerary isodicentric 15 are typically more impaired and might exhibit hypotonia, seizures, microcephaly, and severe developmental delay (Borgatti et al., 2001; Dykens et al., 2004; Wang et al., 2008; Hogart et al., 2010).

The most common autism-related CNVs are the 15q11.2-11.3 duplications, and the reciprocal 16p11.2 micro-deletions and duplications (Dennis et al., 2006; Conant et al., 2014). The 16p11.2 micro-deletion and micro-duplications of approximately 555 kb are located at a hot spot of genomic instability caused by duplicated blocks of DNA, which lead to unequal crossing over during meiosis. There are still limitations to our

36 understanding of CNVs as causes of autism; for example, unaffected parents and family members may carry the same CNV as the ASD proband. Also, patients with different neurocognitive disorders such as mental retardation, seizures, schizophrenia, and bipolar disorder have been reported to carry the same CNVs (Sebat et al., 2009; de Kovel et al., 2010; Kirov, 2010).

In addition, a number of single-gene mutations have been identified in patients with ASD in the past two decades. As described in the previous section, Timothy Syndrome is one of the rare mutations that cause ASD, yet other VGCCs have been considered as ASD candidate genes (Strom et al., 2010; Daghsni et al., 2018) (see Table 1.2). Besides the point mutations found in LTCC genes linked to ASD, which was discussed in section 1.2, genetic abnormalities have been identified in other subfamilies of VGCC genes. Two high-risk SNPs are found in CACNA1A encoding Cav2.1 channel linked to ASD from the Autism Genetic Resource Exchange (AGRE) studies (Skafidas et al., 2014). A recent study in Chinese Han population reported the association of two markers (rs7249246 and rs12609735) in CACNA1A gene with patients with ASD (Li et al., 2015). Furthermore, variations in the CACNA1G gene (rs12603122, rs757415, rs12603112, and rs198547) have also been associated with patients with ASD (Lu et al., 2012). However, unlike the mutations in Cav1.2 and Cav1.3 genes, the functional consequences of these variants on channel properties are still unclear, waiting to be explored.

As described in sections 1.1 and 1.2, calcium entry through VGCCs activates multiple signaling pathways that regulate important neuronal functions such as synaptogenesis and neuronal differentiation. Dysfunction in these pathways is known to be responsible for abnormalities observed in patients with ASD, which include dendritic and axonal branching alterations, as well as disrupted neuronal connectivity (Krey & Dolmetsch, 2007). While some susceptibility genes identified in monogenic ASD-like diseases

37 encode for synapse-related proteins, additional proteins that regulate chromatin remodeling and gene transcription have also been reported (De Rubeis et al., 2014; Rylaarsdam & Guemez-Gamboa, 2019). Approximately 1~3 % of children diagnosed with ASD can be shown to have Fragile X syndrome, which occurs as a result of an expansion of a CGG trinucleotide repeat in the FMR1 gene to 200 or more repeats (Reddy, 2005). Many children with fragile X syndrome exhibit autistic behaviors, including avoidance of eye contact, language delays, repetitive behaviors, sleep disturbances, tantrums, and can be self-injurious. Molecular studies have suggested that the FMR1 gene may cause ASD by inducing RNA toxicity and gene silencing, which potentially affects neuronal connectivity (Schenck et al., 2003; Handa et al., 2005; Hagerman et al., 2008).

While the phosphatase and tensin homolog (PTEN) gene was initially described as a tumor suppressor gene, heterozygous mutations in PTEN have been identified in a subset of patients with autism and macrocephaly (Butler et al., 2005; Buxbaum et al., 2007; Varga et al., 2009). PTEN haploinsufficiency has been reported to play a role in brain development, including neuronal survival and synaptic plasticity. Further, researchers have shown that PTEN haploinsufficiency and the serotonin transporter gene SLC6A4 act synergistically to increase brain size and decrease sociability in a mouse model (Page et al., 2009). A recent study used multiple model systems, including yeast, fly, HEK293, to assess the impact of PTEN variants on molecular function, neuronal morphogenesis and behavior, and have revealed protein instability which results in loss-of-function (Post et al., 2020).

Rett syndrome (RS), another single-gene disorder related to ASD, has been linked to mutations in the X-linked MECP2 (methyl CpG binding protein 2) gene (Amir et al., 1999). RS patients often have a period of normal development followed by a loss of

38 language and with certain stereotypic behaviors, progressive gait disturbance, and hand wringing. Further intensive genetic screening studies have identified risk genes such as NLGN3X/4X encoding the postsynaptic cell-adhesion molecules neuroligin 3/4 essential for synapse formation and function, SHANK3 encoding a synaptic scaffolding protein that binds neuroligins and is involved in the organization of the postsynaptic density, and the SLC9A9 gene encoding the Na+/H+ exchanger isoform 9 (Miles, 2011). However, more models and clinical studies are needed to explore the underlying mechanism of the pathology of these mutations.

1.3.2 Animal Models of ASD In order to investigate the neural mechanisms underlying the impairments observed in ASD a number of animal models have been established. At this point in time however, the validation of animal models for their direct relationship to most human neuropsychiatric diseases cannot be based exclusively on the presence of the same diagnostic markers to the human situation, as compared to animal models of diabetes that show metabolic dysfunctions (Belzung & Lemoine, 2011). A difficulty in modeling ASD, a group of complex disorders whose diagnosis is largely based on behavioral phenotypes, is that the behavior of rodents and other common model animals do not replicate the spectrum of behavioral features of an autistic child. Nevertheless, the behavioral pattern of each category in the animal model could potentially mimic the pattern observed in children with ASD, such as the engagement of social interaction or repetitive behaviors. In these models, neurobehavioral tests can normally identify specific autistic-like behaviors as well as morphological and/or functional changes in the brain, offering a suitable system to examine fundamental mechanisms of disease. Generally, there are two types of ASD-like animal models: pharmacological models, mainly produced by exposure of pregnant animals to a chemical agent, and genetic models, generated by introducing the corresponding human mutant gene into animal embryos. The most

39 common models of ASD have been developed using mice and rats, due to the availability of standardized behavioral tests and the possibility to apply genetic modification approaches.

To date, there exists a limited number of pharmacological models established for ASD with the only one exhaustively examined the VPA-induced rat model. In humans, VPA is a commonly used antiepileptic drug for the treatment of a series of different neurological diseases including bipolar disorder, migraine, headaches and neuropathic pain (Lammer et al., 1987). It is known to inhibit histone deacetylases, modulate epigenetics and increase oxidative stress (Phiel et al., 2001). Clinical studies have revealed that exposure to VPA in pregnancy, especially during the first trimester, is associated with a higher risk for ASD among offspring (Williams et al., 2001; Rasalam et al., 2005; Christensen et al., 2013; Evatt & Mahlon DeLong, 2019). In the rat, the neuronal tube closes on day 11 and within the 12th day of gestation the production of motor nuclei of trigeminal, abducens and hypoglossal nerves are completed (Altman & Bayer, 1980). Offspring of female rats injected with VPA on the 12.5th day of gestation show brain abnormalities similar to those observed at autopsy and in brain-imaging studies in autistic human patients; the number of motor neurons is decreased in the oculomotor, trigeminal, abducens and hypoglossus nuclei of cranial nerves, and the overall size of cerebellum is reduced with a decrease in Purkinje cell number compared to control animal (Rodier et al., 1996, 1997; Ingram et al., 2000). Behaviorally in this model, offspring display lower sensitivity to pain but higher sensitivity to non-painful stimuli, diminished acoustic prepulse inhibition, locomotor and repetitive hyperactivity along with lower exploratory activity, and co- occurring with increased latency and lower frequency of social behaviors (Schneider & Przewłocki, 2005). In addition, offspring exhibit delayed maturation, lower body weight, delayed nest-seeking response mediated by the olfactory system, and normal negative geotaxis. Other studies performed with the VPA-exposed model have found structural and

40 behavioral changes similar to those reported in human ASD patients (Ingram et al., 2000; Wagner et al., 2006; Ornoy, 2009; Bambini-Junior et al., 2011).

Based upon the prenatal VPA exposure model, some researchers have subsequently employed a postnatal model which exposes mice or rats to VPA on postnatal day 14 (P14), thought to be a sensitive period wherein cerebellar organization and neuronal proliferation are essentially complete, but neuronal differentiation, myelination, synaptogenesis and glia genesis are emerging or continuing in the cerebellum, striatum and hippocampus (Wagner et al., 2006; Yochum et al., 2008). Results from the postnatal VPA models show that the postnatal administration causes a delay of maturation, regression of acquired skills, decreased anogenital sniffing, crawl-under/over behavior and allogrooming. Thus, a single postnatal exposure to VPA has long-lasting effects mimicking ASD. In most of these studies evidence of gender-difference has also been demonstrated. While in some studies there seemed to be no gender difference in ASD- like behavioral phenotype, other researchers report a larger decrease in social interaction in male VPA animals compared to females (Stanton et al., 2007; Tsujino et al., 2007; Markram et al., 2008; Dufour-Rainfray et al., 2010; Roullet et al., 2010; Gandal et al., 2010; Mehta et al., 2011; Kataoka et al., 2013; de Theije et al., 2014; Olde Loohuis et al., 2015). In the regard, many investigators use only males to eliminate the possible sex- dependent difference (Schneider & Przewłocki, 2005; Narita et al., 2010; Bringas et al., 2013; Lucchina & Depino, 2014; Cheaha et al., 2015; Hara et al., 2016; Gao et al., 2016).

There are a few other pharmacological animal models that undergo investigation for ASD but most have not been thoroughly characterized as yet. In rats, exposure to thalidomide on embryonic days E2, E4, E7, E9 and E11 increased serotonin levels in the hippocampus and dopamine in the frontal cortex and induced impaired achievement of learning, but no other autistic behavioral effects were observed (Narita et al., 2010). A couple of have

41 studies demonstrated that propionic acid (PPA), a short-chain fatty acid, can induce autistic-like behaviors in rats, including impaired social and play behaviors, restricted behavioral interest to a specific object, and impaired reversal in a T-maze task (Shultz et al., 2008; MacFabe et al., 2011). The results from the PPA animal model are revealing although the relevance to the human condition remains to be demonstrated.

A number of genetic models representing known human diseases have been established and characterized for the study of ASD. Most rodent genetic models have been generated in mice due to the well-developed embryo microinjection techniques compared to the more difficult situation in rats, even though rat behavioural studies are far more advanced compared to mice. For Fragile X syndrome, a frequent inherited cause of ASD, knockout mice models that mimic the abnormal expansion resulting in loss-of-function of FMR1 exhibit certain autistic traits (Spencer et al., 2005; Restivo et al., 2005; Mines et al., 2010). Some studies also demonstrate structural and physiological changes including abnormal dendritic spine morphology and exaggerated protein synthesis in the hippocampus (Bhattacharya et al., 2012; Oddi et al., 2015). The validation of the PTEN mutant knock-in or PTEN null mice as ASD models has been unsuccessful since the animals die during embryogenesis, although one study successfully employed a Cre- driven neuron-specific PTEN knock-in mice model finding that mutant mice display multiple autistic traits (Kwon et al., 2006). Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in the TSC gene and induces intellectual disability, seizures and an increased incidence of ASD (Crino et al., 2006). Most mouse strains have no cortical tubers thus they do not suffer from seizures but still display autistic traits (Tsai et al., 2012; Reith et al., 2013). While the study of ASD in animals has been intensely studied in genetic mice models, there is a growing interest and effort in developing and evaluating rat models of ASD (Waltereit et al., 2011; Kisko et al., 2018) as compared to mice, rats exhibit a rich and complex pattern of behavioral expression

42 making them more promising towards modeling neurobehavioral diseases associated to social impairment.

While the gene products associated with genetic ASD appear to serve distinct neuronal functions, the genetic rodent models all display at least some traits linked to the human phenotypes. Interestingly, abnormalities in synapse morphology and/or function are described in many of the animal models suggesting a convergence in the underlying pathophysiology of ASD.

The development and characterization of pharmacological and genetic animal models have contributed our understanding of cellular mechanisms of ASD, and ideally will be useful to evaluate diagnostic methods and potential treatments that are effective in humans. However to date, the detailed electrophysiological, synaptic and circuit mechanisms underlying the impairments observed in ASD remain largely unknown. A now widely accepted hypothesis for ASD was initially proposed by Rubenstein and Merzenich in 2003; they hypothesized that ASD and related disorders might be caused by the alteration in the ratio between excitation and inhibition (E/I balance) leading to hyperexcitability in brain networks (Rubenstein & Merzenich, 2003). The theory has been supported by clinical and animal model studies in ASD and both excitation and inhibition have been investigated accordingly. However, conflicting results have raised the notion that the E/I imbalance cannot be unambiguously evaluated and/or interpreted without a clear notion of how such balance is established and regulated. Studies on some animal models of ASD reveal a shift in the E/I balance away from excitation. For instance, an increase of inhibition has been suggested to cause learning deficits in Down syndrome, a disorder sharing many symptoms with ASD (Belichenko et al., 2009). Both enhanced inhibition and reduced excitation have been observed in a mouse model of Rett syndrome, and the reduced excitation has been replicated in different experimental

43 contexts including the in vitro synaptic connection in neocortical pyramidal neurons, and hippocampal neurons from knockout mice, and induced pluripotent stem cells from human Rett patients (Dani et al., 2005; Nelson et al., 2006; Dani & Nelson, 2009; Marchetto et al., 2010).

The contributions of excitatory and inhibitory inputs to maintaining E/I balance has been studied by examining electrophysiological properties, including synaptic plasticity, in an effort to understand how functional networks are affected in different brain circuits when balance is disturbed. Alterations of excitatory synaptic function were found in Angelman syndrome, including changes in LTP and LTD threshold, decreased mEPSC frequency and reduced AMPA/NMDA ratio (Mabb et al., 2011). Interestingly, studies in a TS model initially found a reduction in LTP, but later studies revealed a profound loss of protein synthesis-dependent, mGluR-mediated LTD, which causes enhanced excitatory synaptic transmission (Bateup et al., 2011; Auerbach et al., 2011). In part, the cell-type-specific knockout data raise the notion that the changes in excitation may be secondary to reduced inhibition (Nelson & Valakh, 2015).

More and more research suggests that instead of enhanced excitation, neurodevelopmental disorders such as ASD are directly associated with alterations of GABAergic inhibitory signaling causing E/I imbalance in selective neuronal circuits (Cellot & Cherubini, 2014; Nelson & Valakh, 2015). Consistent with this neurodevelopmental hypothesis of the etiology of ASD, early-onset seizures is one of the most frequently observed complications in ASD syndromes, including TSC, Fragile X, Angelman and Rett syndromes. Considering that GABA-mediated neurotransmission is known to play a critical role in synaptic tuning and neuronal wiring in pre- and postnatal periods (Ben-Ari et al., 2012), some authors have hypothesized that early-onset seizures reflect a primary deficit in inhibition because of the lack of an asymptomatic period

44 during which homeostatic compensation could develop. The involvement of GABAA receptors in ASD was suggested by genetic studies that have identified CNV in chromosomal loci 15q11-q13, which contains a number of genes encoding for GABAA receptor subunits (Coghlan et al., 2012). Systematic changes in GABAA receptor subunit expression have been found in the superior frontal cortex, parietal cortex and cerebellum of ASD patients. In addition, many autism-related genes have been found to affect or be expressed predominantly in GABAergic interneurons (Xu et al., 2014). This is consistent with animal model studies demonstrating that interneuron-specific knock-out of even broadly expressed syndromic autism genes, such as the Mecp2 gene responsible for Rett syndrome, can result in autism-like phenotypes, and complete knock-out of other ASD candidate genes can result in a specific loss of interneurons (Chao et al., 2010; Saunders et al., 2013; Sgadò et al., 2013). The VPA animal model showed an altered E/I balance due to decreased GABAergic signaling, which affects both pre- and post-synaptic sites (Banerjee et al., 2013). In addition, this model exhibits an asymmetric reduction of parvalbumin-positive cells and a loss of oxytocin-mediated GABAA-mediated inhibition during the transition from fetal to postnatal life (Gogolla et al., 2009; Tyzio et al., 2014).

To date, many pieces of evidence concerning ASD point to the E/I imbalance mechanism, potentially paving the way towards the development of new drugs targeting excitatory and/or inhibitory signaling pathways. Since ASD has been suggested to be an early neurodevelopmental disorder, animal models allowing to test early pharmacological interventions are essential and will aid in the design of therapeutical tools while minimizes human risks.

Table 1.2 Voltage-gated calcium channel genes implicated in autism spectrum disorders and related pathologies

Modified from (F. Breitenkamp et al., 2015)

1.4 Brain regions associated with ASD; involvement of the basolateral amygdala Numerous neuroanatomical studies have been performed in ASD patients and animal models to investigate the potential relationship between specific autistic symptoms and neurodevelopmental differences associated with brain anatomy, functioning and connectivity. Various neuroimaging techniques used to study the brain in vivo, such as magnetoencephalography, positron emission tomography (PET), and magnetic resonance imaging (MRI), have largely expanded our understanding of brain circuits involved in ASD. The brain areas shown most likely underlie ASD are well established, including the frontotemporal and frontoparietal regions, hippocampal complex, cerebellum, basal ganglia, and anterior and posterior cingulate regions (Amaral et al., 2008) (Fig 1.4). Within these, the amygdala complex has been considered as a key core area related to ASD pathophysiology (Baron-Cohen et al., 2000a; Amaral et al., 2003).

The amygdala is an almond-shaped region located deep in the medial temporal lobe across vertebrates. The amygdala complex encompasses several heterogeneous nuclei with distinct network connectivity and functional characteristics in humans and other animals (Bzdok et al., 2013); these include lateral, basolateral, cortical, medial, central and intercalated cell clusters. The intrinsic connectivity in the amygdala complex is unidirectional and topographically organized (Fig 1.5). The lateral (LA) and basolateral amygdala nucleus (BLA) are the primary targets of cortical and subcortical input, where multiple axons form excitatory synapses with principal neurons and interneurons within the two nuclei, allowing for the integration of sensory information. The BLA principal neurons send glutamatergic projections to the central lateral amygdala (CeL) and intercalated cells (IC), both of which provide inhibitory input to the central medial amygdala (CeM), which in turn provides the main output of the amygdala to the hypothalamus, brainstem and other brain regions (Figure 1.4) (Aggleton et al., 1980; Fudge & Tucker, 2009).

The amygdala has classically been linked to emotional processing, particularly fear and anxiety (Adamec & Morgan, 1994). Partial kindling of the amygdala or ventral hippocampus permanently increases fearful response in cats and rats, and the kindling of the amygdala also decreases the threshold to elicit feline defensive responses. Moreover, kindling of the CeM of either the left or right hemispheres in Wistar rats increases restraint stress-induced stomach ulceration, and also increases anxiety as measured in the elevated plus maze 1 week after the completion of kindling (Henke & Sullivan, 1985; Nieminen et al., 1992). Humans also exhibit a freezing response to a potentially threatening situation, probably reflecting activation of the CeM (Roelofs et al., 2010) and functional neuroimaging has shown activation of the entire human amygdala during fear conditioning paradigms (Petrovic et al., 2008). Increasing evidence has further extended our understanding of the amygdala roles and its links it to a host of processes, including classical conditioning, emotion regulation, reward processing and social cognition (Baxter & Murray, 2002; Adolphs, 2008, 2010; Müller et al., 2011).

Early in the 1930s, classic studies described that a large bitemporal lesion in monkeys caused a propensity to rapidly shift in exploring different objects and profound changes of emotional and social behaviors (Klüver & Bucy, 1937, 1939). Although the lesions included the entire temporal lobe without targeting specific nuclei, the work inspired current studies using more selective pharmacological lesions of the amygdala (Machado & Bachevalier, 2006; Mason et al., 2006; Machado et al., 2009). A commonly used method is to induce a reversible lesion by injecting drugs such as muscimol, a GABAA receptor agonist that temporarily silences the electrical activity of amygdala neurons through inhibition. Monkeys with amygdala lesion show less caution in approaching potential danger to which they normally have a fear response (Muller et al., 1997). In addition, amygdala-silenced monkey exhibited a complex impairment in social behaviours: they showed increased prosocial cues and fewer avoidance behaviors toward

48 other healthy monkeys, and further showed increased approach behavior towards unfamiliar humans. Further complexities were shown to arise when lesions were generated neonatally in monkeys and included exaggerated social fear monkeys (Bauman et al., 2004). A study in rats also reported that the amygdala with ibotenic acid lesion in early life (postnatal day 7) resulted in a variety of behavioral disturbances later in life, such as decreased juvenile play, diminished the frequency of social exploration and approaching/following behavior, increased ambulation in the small open field, and decreased investigatory activities in the large open field (Wolterink et al., 2001).

The temporal lobes are markedly affected areas in patients with ASD. Thus, the role of the amygdala in ASD has been suggested and proved in many neuropathological studies. The findings in individuals with amygdala lesions are similar to the phenomena in ASD. Small neuronal size and increased cell density in several nuclei of the amygdala were also detected in ASD patients (Bauman & Kemper, 2005). The individuals with temporal lobe tumors involving the amygdala have experienced autistic symptoms, which provide another evidence of the correlation between the amygdala and ASD (Hoon & Reiss, 1992; Taylor et al., 1999). In addition, individuals with tuberous sclerosis experienced autistic symptoms including facial expression due to a temporal lobe hamartoma (Bolton & Griffiths, 1997). Although many previous researches using MRI studies failed to find structural abnormalities in the temporal lobe of autistic subjects, recent development in neuroimaging techniques help researchers to identify an increase in bilateral amygdala volume and reduction in hippocampal volume in ASD individuals (Howard et al., 2000).

.The BLA nuclei in the amygdala complex were suggested to have a solid association with ASD. The BLA confers the amygdala a role as a node connecting sensory stimuli to higher social cognition levels. As briefly mentioned above, it links the CeM and superficial groups, and it has reciprocal connections with the medial prefrontal cortex

(mPFC), orbitofrontal cortex (OFC), anterior cingulate cortex (ACC) (Adolphs, 2010). The BLA area was suggested to have responses to faces and actions of others, which is not found in other subnuclei of the amygdala (Anon, 1992). Under resting conditions the BLA has a basal level of tonic inhibition by the local GABA networks, and disruption of this inhibitory tone in the BLA could result in abnormal response to a social stimulus, whereas poor coordination of this amygdala inhibitory tone with PFC/OFC circuits could result in faulty emotional assessment and poor ability to modulate ongoing responses appropriately (Rainnie et al., 1991a, 1991b; Bachevalier & Málková, 2006). Therefore, one can hypothesize that an enhanced responsiveness of the BLA could result in assigning an exaggerated salience to a novel social stimulus, which causes social anxiety- like conditions (Amaral, 2002; Shekhar et al., 2005). Furthermore, one clinical study showed that activation of BLA by bilateral deep brain stimulation (DSB) resulted in substantial improvement of both self-injurious behavior and autism-related symptoms, including deficits in social contacts, affect-modulation and speech, fear and anxiety as well as sleep disorder (Sturm et al., 2013). The same DSB in the paralaminar, central amygdala, or in the supra-amygdaloid projection system respectively could not produce any of these behavioral changes. In an animal study, the pharmacologically increased BLA network excitability due to targeted damage of specific GABAergic cells, induced long-term reductions in social interaction, such as a significant reduction in the amount of SI time with novel partners, and an increase in anxiety-like behavior as measured by the social interaction test (Truitt et al., 2007). Another study has investigated the area of BLA in the VPA rat model of ASD, and a significant reduction of size was found in BLA at P21 and P35 without change at P70 (Sosa-Díaz et al., 2014). Taking all reviewed hypotheses and findings together, an appropriate animal model of ASD targeting BLA and a thorough investigation on the molecular, cellular and behavioral levels is required in order to examine the functional contribution of BLA to ASD.

Figure 1.4 Brain areas associated with ASD

The diagram shows a sagittal section of the rat brain, illustrating the relative localization of ASD-relevant brain regions and their interconnections, represented in one single plane. The red arrows indicate excitatory synaptic inputs, and blue arrows indicate inhibitory synaptic inputs. The schematic shows the main direct and indirect neural pathways between the amygdala (green circle) and other brain regions. PFC: prefrontal cortex. NAc: nucleus accumbens. VTA: ventral tegmental area.

Figure 1.5 Organization of the amygdala functional nuclei and local networks

Schematic of a coronal view of the rodent amygdala showing the main structures of the amygdala subregions: lateral amygdala (LA), basolateral amygdala (BLA), central amygdala (CeA), lateral paracapsule intercalated cells (LPICs). Arrows (or flathead arrows) indicate major excitatory (or inhibitory) synaptic connections in the intra- amygdalar circuit.

1.5 Thesis Objectives

Objective 1 – to examine the functional roles of LTCC towards the electrophysiological properties of BLA neurons Increased excitability of BLA principal neurons underlies certain neuropsychiatric disorders. Gain-of-function mutations in LTCCs are linked to neurodevelopmental diseases including ASDs. While LTCCs are expressed throughout the BLA, direct evidence for increased LTCC activity affecting BLA excitability and potentially contributing to disease pathophysiology is lacking. In this part of the study, we hypothesize that functional increased LTCC activity might differentially affect the electrophysiological properties of BLA principal neurons at different developmental stages.

Objective 2 – to evaluate whether an increase in LTCC activity during the perinatal period of neurodevelopment has long-lasting effects on rat behavior phenotypes and BLA neuron electrophysiological properties The gain-of-function TS mutation in the CACNA1C gene, as well as the newly found point mutations in the CACNA1D gene, have provided a promising association between neuronal LTCCs and core symptoms of ASD. Pathological alterations during critical periods of neurodevelopment can lead to excitation: inhibition imbalances that predispose individuals to neurological disorders such as ASD. As such, we hypothesized that alterations in LTCC activity during a critical period might impact the balance in neural circuitries. In order to examine the potential role of LTCCs in the pathology of ASD, I evaluated whether a local increase of L-type calcium channel activity in ASD-related circuits translated into ASD-like behavioral traits. I further examined the neurodevelopmental impact of increased LTCC activity in neuronal and synaptic functions, which could provide support for the use this strategy as an ASD animal

53 model. I tested whether applying LTCC agonist into the BLA of rat pups at different developmental stages resulted in pathological alterations in the amygdala electrophysiological functions and social behavior phenotypes.

2 CHAPTERⅠ: Contribution of L-type calcium channels to firing

activity in the rat basolateral amygdala at different stages of

postnatal development

2.1 Intruduction

The high voltage-gated L-type calcium channels (LTCCs) Cav1.2 and Cav1.3 are widely expressed on nerve cell soma, dendrites and postsynaptic membranes in the mammalian central nervous system (Hell et al., 1993b; Tippens et al., 2008; Leitch et al., 2009). Calcium influx via LTCCs has been implicated in regulating activity-dependent gene transcription, synaptic plasticity, synaptogenesis and dendritic growth (Perrier et al., 2002a; Moosmang et al., 2005a; Yang et al., 2018; Kamijo et al., 2018a; Wild et al., 2019). While some reports have suggested that LTTCs make minimal direct contributions towards neuronal excitability (Diaz & Dickenson, 1997), there is emerging evidence that for a subset of neurons LTCCs in fact play key roles in modulating excitability. For example, LTCCs regulate firing patterns in both subthalamic neurons (Beurrier et al., 1999) and cultured hippocampal pyramidal cells (Lacinová et al., 2008; Geier et al., 2011). Further, distinct LTCC subtypes differentially contribute to burst-firing and single- firing modes in dopaminergic neurons from the ventral tegmental area (Liu et al., 2014). Overall however, the evidence for direct LTCC effects on neuronal excitability is limited to a few specific cell types wherein their functional effects seem to be context-dependent.

Morphological and functional alterations of the neuronal circuitry of the amygdala have been correlated with the impairment of social interaction abilities associated with autism spectrum disorders (ASDs), a range of neurodevelopmental disorders characterized by challenges with social skills, repetitive behaviors and restricted interests (Baron-Cohen et al., 2000b; Ha et al., 2015). Gain of function mutations in LTCCs have been shown to underlie ASD (Splawski et al., 2004b; Pinggera et al., 2015b), providing a potential

55 association among LTCCs, the amygdala and ASDs (Kass, 1987b). It has been suggested that disruption of activity-dependent regulation of synaptic development contributes to the pathogenesis of ASDs (Ebert & Greenberg, 2013; Doll & Broadie, 2014). LTCCs are expressed in rat basolateral amygdala (BLA) principal neurons, which are pyramidal glutamatergic projection neurons. They are the main neuronal type in the BLA, and are thought to be necessary for higher order behaviors related to fear extinction, emotion processing and social communication (Davis & Bauer, 2012a). The functional enhancement of LTCCs under physiologically relevant conditions has previously been reported in neurons and neurosecretory cells; glucocorticoids, which mediate the effect of stress on the functional role of the amygdala in emotion processing and fear behaviors (Roozendaal et al., 2009), are known to increase the magnitude of L-type currents (Karst et al., 2002a) in BLA principal neurons. Also, upregulation of Cav1.2 channel expression has been reported in the hippocampus and the central and basolateral nucleus of the amygdala in alcohol-dependent rats (Uhrig et al., 2017); this study also showed a correlation between an increment in L-type Ca2+ currents in hippocampal CA1 neurons and the display of alcohol seeking behavior. Furthermore, a recent study has provided strong evidence for the direct link between a rise in Ca2+ influx through LTCCs, increased intrinsic excitability, and secretory dysfunction in chromaffin cells in a mouse model of Timothy syndrome (TS), a multi-systemic disorder resulting from a missense mutation in the gene encoding Cav1.2 channel (Calorio et al., 2019). In rodent models, an increased excitability of BLA principal neurons has been suggested to contribute to the anxiogenic phenotype (Rau et al., 2015). Contrastingly, decreased excitability alleviates anxiety measured in humans and rodents (McEwen & Olié, 2005). While evidence suggests that the excitability and synaptic strength of BLA neurons may contribute to social perception and emotional engagement (Baron-Cohen et al., 2000b), the functional implications of LTCCs in modulating BLA activity and synaptic homeostasis in the context of a neurodevelopmental model has yet to be explored.

In both humans and animal models there exist critical periods of vulnerability during neurodevelopment (Rice D & Barone S, 2000; Hensch & Bilimoria, 2012; Arain et al., 2013). During postnatal development in rat amygdala the overall electrophysiological properties of BLA principal neurons change dramatically between postnatal day 7 (P7) and P21 (Ehrlich et al., 2012), implying a critical period of amygdala maturation. In order to help define the contributions of LTCC dysfunction towards ASDs, it is important to examine the functional roles of LTCC at specific stages of BLA development.

The dihydropyridine (DHP) LTCC agonist BayK interferes with the LTCC inactivation gating mechanism, increasing calcium influx during membrane depolarization (Kass, 1987b), resembling the effect of the TS mutation on the biophysical properties of LTCC channels (Barrett & Tsien, 2008). I hypothesized that BayK could serve as a candidate tool to mimic the functional increase of LTCC activity caused by gain-of-function mutations. As such, BayK was employed to acutely manipulate LTCC activity in BLA principal neurons in brain slices as an initial strategy to study the direct, short-term functional consequences of L-type activation on neuronal membrane excitability. Electrophysiological analyses revealed that BayK causes a significant increase in excitability in immature P7 BLA neurons, while at P21 BayK promotes bursting activity with no changes in the frequency response to somatic stimulation. Interestingly, at both P7 and P21 increased LTCC activity results in differential effects on synaptic properties through enhancing the frequency of miniature inhibitory synaptic events with no significant change in evoked postsynaptic currents. The susceptibility of BLA neurons to enhanced LTCC activity at the P7 immature period may have a significant impact on the functional maturation of BLA local circuitry, possibly shedding light onto the underlying mechanisms of certain neurodevelopmental disorders.

2.2 Methods

Animals All experimental procedures were approved by the University of British Columbia Animal Care Committee (UBC ACC Protocol A16-0127) and were in accordance with the regulations and guidelines of the Animal Care and Use Program, according to the standards of the Canadian Council on Animal Care. Male Sprague Dawley rats were purchased from Charles River Laboratories (Cat: Crl:CD(SD) IGS; Strain Code #001;

Stock #400) and housed with the dam in a 12:12-h day-night cycle at the Animal Resource Unit of the University of British Columbia. Rat pups were weaned at P21. Rat pups of P7/P8 and P21–P23 were used in this study.

Acute Brain Slice Preparation Animals were anesthetized with isoflurane [5% in oxygen (vol/vol)] using a VetEquip inhalation system and decapitated by a rodent guillotine. The brain was quickly removed and transferred to ice-cold sucrose cutting solution (in mM): 214 sucrose, 26 NaHCO3,

1.25 NaH2PO4, 11 glucose, 2.5 KCl, 0.5 CaCl2, 6 MgCl2, bubbled constantly with a 95%

O2:5% CO2 gas mix. Trimmed brain tissue was glued to a cutting chamber of a vibratome (VT 1200, Leica). Coronal brain slices (300 µm thick) containing the amygdala complex (from Bregma -2.0 to -3.0) were incubated at 32 oC in artificial cerebral spinal fluid

(ACSF) (in mM): 130 NaCl, 30 NaHCO3, 3.5 KCl, 1.1 KH2PO4, 1.3 MgCl2, 2.5 CaCl2, 10 glucose, 0.4 Sodium Ascorbate, 0.8 Thiourea, 2 Sodium Pyruvate, saturated with 95%

O2:5% CO2.

Electrophysiological Recordings After 1 hour incubation in ACSF at 32 oC, individual slices were transferred into a recording chamber (Warner Instruments) mounted in the fixed stage of a microscope

(Zeiss Axioscop 2 FSPlus) and the slices were maintained at 32 oC (Temperature controller Warner TC-344B), and perfused with carbogen saturated ACSF at a flow rate of 2 ml/min. The recording chamber was grounded with an Ag/AgCl pellet. The BLA pyramidal neurons were visually identified using infrared differential interference contrast (IR-DIC) in combination with a 40X water immersion objective. In some experiments, neurons were labeled with biocytin upon completion of current clamp recordings in order to confirm their localization within the BLA. Recorded neurons were filled with biocytin (0.05% in the internal whole-cell recording solution) by applying hyperpolarizing current pulses for 20min; the electrode was then withdrawn and the slice was fixed in 4% paraformaldehyde at 4 oC. Biocytin tracer was visualized using the avidin-biotinylated-HRP (ABC) reaction (Nambu & Llinás, 1997). Patch-clamp recordings were performed using a Multiclamp 700B amplifier and an Axon DigiData 1550B acquisition System with pClamp software version 11 (Molecular Devices). Current-clamp recordings were obtained and low-pass filtered at 10 kHz and digitized at 50 kHz, while voltage-clamp recordings were obtained and low-pass filtered at 2 kHz and digitized at 50 kHz.

All electrophysiological recordings were performed in normal ACSF solution. Patch pipettes were pulled from borosilicate capillary glass with a P-1000 micropipette puller (Sutter Instrument). For action potential recordings in current clamp, 3-5 MΩ resistance electrodes were filled with internal solution (in mM): 130 K gluconate, 2 KCl, 10

HEPES, 3 MgCl2, 2 K-ATP, 0.2 Na-GTP, and 5 Tris-Phosphocreatine; the pH was adjusted to 7.3 with KOH, and osmolality was adjusted to 290 mOsm/kg with D- Mannitol. Cells were discarded if they had a resting membrane potential less than -55 mV, or access resistance higher than 30 MΩ, or action potentials with no overshoot. Access resistance and bridge balance values were checked throughout the recording, and cells with over 15% changes were also excluded. The liquid junction potential was

59 calculated as 11.4 mV, but data shown were not corrected. To obtain current-voltage (I-V) relationships, a 1 s long square pulse current was injected from -100 pA to +100 pA with an increment of 10 pA for P7 animals, and from -200 pA to +200 pA with an increment of 20 pA for P21 animals.

Spontaneous synaptic activity was recorded in voltage clamp mode in the presence of 1 µM tetrodotoxin (TTX) in the bath and using the following internal solution (in mM): 134.63 Cs-Methanesulfonate, 5 CsCl, 5 TEA-Cl, 0.4 EGTA, 10 HEPES, 2.5 Mg-ATP, 2.5 Na-GTP, 5 Phosphocreatine Tris, the pH was adjusted to 7.3 with CsOH, and osmolality was adjusted to 290 mOsm/kg with D-Mannitol. For miniature inhibitory post-synaptic (mIPSC) recordings, slices were incubated in the presence of AMPA receptor antagonist

CNQX (10 µM), NMDA receptor antagonist D-AP5 (50 µM), and GABAB receptor antagonist CGP52432 (1 µM) at least 20 minutes prior to recording. Cells were held at 0 mV during a 2 min gap-free recoding. For miniature excitatory post-synaptic (mEPSC) recordings, slices were incubated in the presence of CGP52432 (1 µM), and GABAA receptor blocker picrotoxin (PTX, 100 µM) at least 20 minutes, and cells were held at -70 mV during a 1 min gap-free recording. Slices were maintained in the corresponding blockers throughout the mIPSC/mEPSC recordings.

Afferent electric stimulation to examine evoked synaptic responses was performed with a concentric bipolar stimulating electrode (CBAPC100 from FHC Inc.). Pulses were generated with an S48 Stimulator via a Stimulus Isolation Unit SIU5 (Grass Instruments). Evoked inhibitory post-synaptic currents (eIPSCs) were elicited in BLA pyramidal neurons when the stimulating electrode was placed on the lateral cluster of paracapsular intercalated cells (lPICs) (see schematic in Fig. 2.16A). eIPSCs were recorded in ACSF containing CNQX, D-AP5, and CGP52432. Excitatory post-synaptic currents (eEPSCs) were evoked by placing the stimulating electrode in the LA (see schematic in Fig. 2.16D).

60 eEPSCs were recorded in ACSF containing PTX and CGP52432. A pair of stimuli was delivered at increasing time intervals to measure short-term plasticity by analyzing the paired-pulse ratio.

Quantitative real-time PCR Rat brain samples were collected by the Palkovits microdissection technique using Micropunches from Leica Biosystem (0.51 mm for P7 and 0.96 mm for P21) in the area of BLA, and stored in TRIzolTM Reagent (Thermal Fisher cat#: 15596026). The real time qPCR reactions were set up in triplicates using KAPA RPOBE FAST qPCR Master Mix (2x) (KAPA Biosystems; KM4702) in hard-shell 384-well PCR plates (Bio-Rad Laboratories; HSP3805). Probes were purchased from Invitrogen Co. (CACNA1C: Rn00709287_m1; CACNA1D: Rn00692157_m1; CACNA1S: Rn01490941_m1; CACNA1F: Rn00671367_g1). The machine used for detection was the C1000 Touch Thermal cycler CFX384 TouchTM Real-Time (PCR Detection) System (Bio-Rad). The following protocol was used for qPCR: 95oC for 3 minutes, (95oC for 15 seconds, 60oC for 45 seconds) x 40.

Quantification cycle (Cq) values were obtained using the function of regression in the bio-Rad CFX Manager version 3.1 (Bio-Rad Laboratories) and were normalized to GAPDH. Values of each sample were averaged between the triplicates, and the results were then averaged between the different animals to generate Figure 2.6.

Data Analysis Electrophysiological data analysis was performed using Clampfit version 11 (Molecular Devices). Statistical analyses and data plotting were performed with Origin version 8.6 (OriginLab). For current-voltage (I-V) relationship analyses, steady-state voltage changes were measured and averaged in a 400 ms period at the end of the 1 s current square pulse,

61 and the input resistance (Rin) calculated from the linear part of the I-V curve around the resting membrane potential. The steady-state frequency of action potentials was obtained from the last 400 ms period of the depolarizing pulses, and plotted as the function of normalized current injection for f-I relationship. Gain was measured as the slope of the initial linear part of f-I curve. Quantitative evaluation of dynamic changes of action potentials within the initial burst were obtained by the phase plot analysis; briefly, changes in the membrane potential with time (dVm/dt measured as mV/ms) were plotted as a function of instantaneous membrane potential. Spike frequency adaptation level was calculated as the fractional increase in the inter-spike interval (ISI) during a train of action potentials evoked by twice threshold depolarizing current injection, relative to the third ISI. To examine the post-spike afterhyperpolarization (AHP) following a train of action potentials a 20-pulse stimulus was applied at 20 Hz, and the AHP amplitude was measured as the maximal negative membrane potential following the last action potential. The paired-pulse ratio (PPR) was calculated from the amplitude of the synaptic response to the second pulse divided by the first and plotted as a function of the inter-pulse interval (IPI). Data are reported as mean + SEM. Statistical comparison was performed with paired Student’s t-test, or non-parametric Kolmogorov-Smirnov test (K-S test), as appropriate. Differences between control and treatment conditions were considered statistically significant at P<0.05. The n value represents for number of cells tested.

Drugs (S)-Bay K8644, nifedipine, D-AP5, CNQX, CGP52432, apamin, biocytin, ZD7288, and flufenamic acid were purchased from Tocris Bioscience. Picrotoxin (PTX) and Tetrodotoxin (TTX) were purchased from Sigma Aldrich. Z944 was synthesized by the lab as per Tringham et al (2012) (Tringham et al., 2012). (S)-Bay K8644, nifedipine, CNQX were dissolved in DMSO and stored at -20 oC before use. Other drugs were dissolved in nanopure H2O. All drugs were applied by perfusion in the bath solution.

2.3 Results

2.3.1 Enhancement of L-type channels differentially regulates intrinsic membrane properties of immature and juvenile BLA neurons. First, I utilized biocytin labelling (0.05%) technique to verify the location and typical morphology of BLA principal neurons examined upon patch-clamp recordings (Figure 2.1). Rat BLA principal neurons exhibit two main firing patterns in response to a square pulse current stimulation: regular tonic firing (Fig 2.2A, left) and tonic firing with an initial burst (Fig 2.2A, middle). Regular tonic firing is predominant in most P7 neurons (85%), whereas at P21 most principal neurons display tonic firing accompanied with an initial burst (84%) (Fig 2.2A, right). A fraction of neurons displayed spike frequency adaptation (SFA), which is defined as an increasing spike interval during a long sustained square pulse. The SFA is more commonly seen in P7 neurons (70%) compared to P21 neurons (28%) (Fig 2.2B). Additional distinct electrophysiological features were also observed between the two developmental stages including a plateau potential represented as a prolonged depolarization at the end of a train of spikes and only seen in P7 neurons (Fig 2.2C left); and both an initial burst of action potentials (Fig 2.2A, and C right, star) and rebound firing mainly observed in P21 neurons (28%) (Fig 2.2C right, arrow). These distinct properties are consistent with previous studies in BLA principal neurons (Ehrlich et al., 2012; Ehrlich & Rainnie, 2015).

To evaluate whether increased LTCC activity affects passive properties of BLA principal neurons, the LTCC agonist BayK (2 µM) was applied to slices and a 1 s square pulse stimulation protocol used to hyperpolarize and depolarize neurons. As showed in table 2.1, P7 neurons showed no significant change in input resistance (Control: 336.8±17.2 MΩ; BayK: 315.2±9.4 MΩ, n=13, p=0.23), and no change in the resting membrane potential in response to BayK application (Control: -61.1±1.6 mV; BayK: -60.4±1 mV, n=13, p=0.71). Further, the threshold for action potential initiation, measured as the 63 maximal value of the 2nd derivative, was more hyperpolarized in the presence of BayK (Control: -36.1±1.5 mV; BayK: -38.4±1.43 mV, n=13, p=0.035). In P21 neurons there were no significant changes in any of the measured passive properties including input resistance (Control 101.6±11.2 MΩ, BayK 106.8±12.9 MΩ, p=0.19, n=18), action potential threshold (Control -41.2±0.8 mV, BayK -42.4±0.8 mV, p=0.16, n=18), and resting membrane potential (Control -57.8±1.6 mV, BayK -56.5±0.9 mV, p=0.15, n=18).

To examine any effects of increased LTCC activity on BLA firing ability I tested for the effects of BayK on the f-I relation (firing frequency versus current injection). In P7 neurons BayK (2 µM) increased the average frequency of evoked spikes around the threshold with no change in the maximal frequency (Fig 2.3Aa). BayK also significantly reduced the gain (slope) from 0.26±0.12 Hz/pA to 0.12±0.12 Hz/pA (p=0.004, n= 12) as well as the mean value of current threshold (rheobase) to evoke action potential discharge (Control: 31.7±3.7 pA; BayK: 25±3.0 pA, n=12, p=0.05; Fig 2.3Ab). Contrastingly for P21 neurons, in the presence of BayK the maximal firing frequency was only slightly reduced with no significant change in the gain (Fig 2.3Ba; Control: 0.084±0.17 Hz/pA; BayK 0.07±0.17 Hz/pA, p=0.12, n=18). The mean value of rheobase of P21 neurons was also not significantly affected by BayK (Control: 85±11.6 pA; BayK: 68±9.0 pA, p=0.11, n=19; Fig 2.3Bb).

Overall neuronal excitability phenotypes are the combined outcome of intrinsic excitability, which mainly depend on membrane conductances and synaptic inputs which are determined by the neural network circuit. In order to examine the influence of synaptic activity on the BayK-mediated changes in BLA excitability, I incubated brain slices in a cocktail of synaptic receptor antagonists (50 µM D-AP5 for NMDA receptors,

10 µM CNQX for AMPA receptors and 100 µM picrotoxin for GABAA receptors) prior to recording, and maintained in the cocktail during the recordings. Interestingly, in the

64 presence of synaptic blockers BayK produced similar effects on f-I relationships for P7 and P21 neurons compared to that for their respective control ACSF conditions (Fig 2.3C for P7, n=15, and Fig 2.3D for P21, n=13). This suggests that the changes in BLA firing properties induced by enhancing LTCCs via BayK largely rely on intrinsic excitability properties. The application of synaptic blockers reduced current injection threshold and maximal firing frequency in BLA neurons (Fig 2.3), consistent with previous reports showing that synaptic transmission contributes to the regulation of excitability (Semyanov et al., 2003; Rosenkranz & Johnston, 2006).

To examine for possible non-specific effects of vehicle on BLA principal neuron firing properties, I applied the maximal concentration of the vehicle DMSO (0.15%) and did not detect observable changes for either P7 (Fig 2.4B, n=5) or P21 neurons (Fig 2.4D, n=7). The effects on firing frequency persisted after BayK was washed out with ACSF for up to 10 minutes. To test for the selectivity of BayK on LTCCs, the LTCC antagonist nifedipine (20 µM) was examined. Application of nifedipine alone had no significant effect on firing properties both at P7 (Fig 2.5A, B) and P21 (Fig 2.5C, D), but completely reversed the effects of BayK when applied subsequently (see Figs 2.8B and 2.10F).

Furthermore, in order to investigate the expression profile of LTCCs in BLA at the two developmental stage, and to rule out the probability of LTCC agonist not having remarkable impact on P21 cell excitability due to lack of LTCC expression, quantitative PCR was performed on P7 and P21 BLA samples. Figure 2.5 shows the expression profile of mRNA level of the four LTCC channels: mean value of relative expression level of Cav1.2 is 0.0022±0.0004 at P7 to 0.0027±0.0005 at P21, and the mean value for Cav1.3 channel is 0.00192±0.001 at P7 and 0.00195±0.0004 at P21. No Cav1.1 or Cav1.4 mRNA was detected in BLA.

Together, my findings indicate that BLA neurons at an immature stage (P7) are strongly susceptible to changes in excitability mediated via LTCC activity, an effect that could potentially shift signal output patterns of BLA projection neurons to alter local synaptic circuits. Conversely, in more mature P21 BLA neurons the sensitivity to alterations in frequency response via LTCC enhancement appears to have been lost and/or is attenuated.

Figure 2.1 Post-hoc visualization of patch-clamped BLA principal neuron from rat brain coronal slices using biocytin labeling

Photo in the left panel shows a 300 μm thick rat brain coronal slice under a 10x microscope. Yellow square indicates the amygdala region. Scale bar=1 cm. The middle panel shows the amygdala region at 40x magnification. Yellow square indicate the location of biocytin labeled neuron. Scale bar=0.5 cm. The right panel shows an enlarged photo of biocytin labeled BLA principal neuron at 400x magnification. Scale bar=25 μm.

Figure 2.2 BLA principal neurons exhibit distinct electrophysiological characteristics at different developmental stages

Traces show representative examples of evoked action potential discharge patterns from BLA neurons. A high percentage of immature P7 BLA neurons respond with a continuous train of action potentials of regular frequency in response to supra-threshold current 67 injection (A, Left; Bar graph). At this stage, spike-frequency adaptation, defined as the reduction of the instantaneous firing rate over time during a constant amplitude depolarizing pulse, was also commonly observed (B, left). A subpopulation of P7 neurons displayed a distinctive depolarizing plateau potential (double arrows on Panel C, left) that outlasts the current stimulus. In contrast, BLA neurons from P21 juvenile rats often displayed an initial brief burst of action potentials (indicated by a star on Panels A and C, right) followed by regular firing throughout the duration of the depolarizing step. Further, rebound firing (single arrow on Panel C, right) was typically evoked in P21 neurons at the offset of hyperpolarizing pulses. n=20 from P7, n= 21 from P21.

Table 2.1 Passive membrane properties of BLA neurons

Bath application of (S)-Bay K8644 has no significant effects on passive properties of P7 or P21 BLA principal neurons (see text for details)

Figure 2.3 BayK increases the excitability of P7 BLA principal neurons with no significant effect on P21 neurons

The effect of L-type channel activation on intrinsic firing properties was examined on the relationship between firing frequency and the magnitude of current injection (f-I) before (black symbols) and after acute superfusion of slices with 2 µM BayK (red symbols). The frequency of action potentials evoked by depolarizing somatic current injections of increasing amplitude was plotted as a function of stimulus intensity, and the current threshold (rheobase) values are shown as box plots (the box represents the interquartile range from 25% to 75%, horizontal line across the box represents the mean value and whiskers indicate the minimum and maximum values of the data set). The firing response to BayK was analyzed in acute brain slices from P7 (A, n=12; and C, n=15) and P21 (B, n=19; D, n=13) rats. Brain slices were bathed with artificial cerebrospinal fluid (ACSF) (Panel A and B Control) or in ACSF plus glutamatergic and GABAergic antagonists CNQX (10 µM), D-AP5 (50 µM), and Picrotoxin (100 µM) (C and D, Control). BayK reduced the steepness (gain) of the f-I relationship obtained from P7 neurons and decreased neither the rheobase (Box Plots) (Ab, Cb) with no significant change in repetitive firing nor rheobase from P21 neurons (B, D). The Maximal frequency at saturating amplitudes was not affected by the pharmacological increase of L-type channels. The minimum current required to produce repetitive firing was lower when the synaptic drive is pharmacologically reduced (C and D). Similar changes in the f-I curve were obtained when BayK was applied in the presence of synaptic blockers (C and D). Data are presented as mean ± SEM. * P<0.05, paired t-test.

Figure 2.4 DMSO exposure had no effect on the electrophysiological phenotype of BLA neurons

Representative traces of a train of action potentials evoked in P7 neurons (Panel A) or P21 neurons (Panel C) recorded in ACSF (control traces in black) and after bath application of 0.15% DMSO (traces in grey) showing that the highest concentration of

72 vehicle utilized in electrophysiological recordings had no effect on the excitability of BLA neurons. The relationship between the frequency of repetitive firing and the stimulus intensity (B, D) was not altered by DMSO. Data are presented as mean ± SEM.

Figure 2.5 mRNA expression of LTCCs in BLA during early postnatal period of development

Tissue samples were collected from postnatal day 7 (P7; n = 3) and P21 (n = 3) animals. Samples from each animal consisted of 10 micropunches obtained from 5 serial 300-μm fresh brain slices. Top: black arrows indicate the location of micropunches. Bottom: bar graphs represent means ± SD of samples from all animals for the same age (P7, black; P21, gray). Data are LTCC mRNA expression mean values relative to control GAPDH.

2.3.2 Increase of L-type calcium channel activity reduces spike frequency adaptation of P7 BLA neurons. Spike frequency adaptation (SFA) refers to an increase of the inter-spike interval (ISI) during repetitive firing in response to a sustained stimulus and confers neurons with the ability of high-pass filtering which separates fast input signals from slow ones (Benda et al., 2005; Peron & Gabbiani, 2009). In BLA principal neuron recordings, this feature was predominantly observed in P7 cells (Fig 2.2B), suggesting that SFA is more important for immature neurons to filter out slower stimuli during early neurodevelopment. As such, the effect of BayK and underlying mechanisms were further mainly examined on P7 neurons. A small conductance calcium-activated potassium channel (SK channel) expressed in amygdala is thought to be one of the main conductances regulating SFA (Faber & Sah, 2002). To examine this, I first tested the SK channel agonist NS309 (1 µM) and antagonist apamin (100 nM) on P7 neurons. As shown in figure 8A, adaptation was nearly completely attenuated by apamin (green trace), while NS309 significantly prolonged the spike intervals to the extent that cells essentially stop firing (blue trace). Application of BayK resulted in a significant reduction in the adaptation ratio during repetitive firing compared to control albeit not to the same extent as apamin (Fig 2.6B).

Next, I asked if the changes in SFA induced by SK channel modulators and BayK were associated with the magnitude of afterhyperpolarization (AHP). I measured AHP amplitudes at the end of a train of spikes induced by 20 repetitive pulses at 20 Hz (5 ms duration for each pulse) (Fig 2.6C). The SK agonist NS309 significantly increased the AHP by 45.7% (mean values from 2.1±0.3 to 3.1±0.3 mV, p=0.046, n=5; Fig 2.6D, blue trace), while the antagonist apamin significantly reduced the AHP to 62.7% (mean values from 2.8±0.6 to 1.7±0.3 mV, p=0.043, n=6; Fig 2.6D, green trace). Application of BayK resulted in a trend towards reducing the AHP amplitude (reduced to 75.8%, mean values from -3.3±1.1 to -2.5±0.8 mV, n=7; Fig 2.6D, red) but the effect was not statistically significant (p=0.093), hence the robust reduction in SFA produced by BayK was not

75 strictly correlated with the magnitude of change in the post-train AHP. A possible explanation is that the LTCCs, restricted to the somatodendritic compartment, might not be tightly coupled to the channels responsible for the generation of AHP, which are mainly located in the dendritic arbors of BLA neurons (Power et al., 2011). Even though I observed a modest effect of BayK applied in the presence of the SK ligands, the changes evoked in the amplitude of AHP did not reach statistical significance (Apamin: 1.75±0.34 mV; BayK+Apamin: 1.3±0.45 mV; n=6, p=0.09; NS309: 3.11±0.3 mV; BayK+NS309: 3.93±0.58 mV; n=5, p=0.074). Taken together, my results suggest that while SK channels contribute to the BayK-induced SFA reduction, the involvement of other conductances, such as those operating in other nuclei of the amygdala (Faber & Sah, 2005) cannot be discarded thus the underlying mechanism requires further study.

Figure 2.6 Increased LTCC activity reduces spike frequency adaptation in P7 neurons

Under control conditions, repetitive firing evoked by depolarizing pulses of 1 exhibits spike frequency adaptation in a subpopulation of P7 neurons (Panel A, black trace). The spike frequency adaptation was evaluated in P7 neurons using the adaptation ratio (A and B), calculated as the fractional increase in the inter-spike interval during a train of action potentials evoked by a depolarizing current, relative to the third interval. The ratio was plotted as a function of the consecutive intervals during the train. The adaptation level was decreased by SK channel antagonist Apamin (100 nM; Panel A, green trace; n=5), whereas SK-agonist NS309 provoked most cells to stop firing after one or two action potentials (1 µM; Panel A, blue trace; n=5). The application of BayK alone reduced the adaptation ratio compared to the control condition (Panel B, red trace, n=10).

A 20-pulse stimulus at 20 Hz was used to examine the relation between SK-mediated frequency adaptation and afterhyperpolarization (AHP) (C). The inset shows the last two action potentials from a train on expanded time and voltage scales (action potentials truncated for clarity), obtained before (black) or after the application of Apamin (green) or NS309 (blue); the AHP amplitude was measured as the maximal negative potential (star) following the last action potential, horizontal dotted line indicates the baseline. Panel D shows the summary of the effect of SK-channel modulator NS309 (n=5), Apamin (n=6) and BayK (n=7) calculated as the percentage of AHP amplitude relative to the mean value obtained in control condition. Adaptation ratio is presented as mean ± SEM, * P<0.05, paired t-test.

2.3.3 L-type calcium channels mediate a plateau potential in P7 BLA neurons. Another feature of BLA neurons is a prominent plateau potential, a long-lasting depolarizing potential following a train of action potentials evoked by depolarizing current pulses. Plateau potentials were only observed in P7 neurons (Fig2. 2C, left). Bath application of BayK was found to either increase plateau potential decay time (from 201.6±46.0 ms to 501.9±117.1 ms, p=0.0068, n=11; Fig 2.7A), or to elicit a plateau potential in neurons that did not exhibit one under control conditions (Fig 2.4B upper and middle traces). Notably, prolonged plateau potentials induced by BayK were abolished by nifedipine (Control: 180.0±60.1 ms, BayK: 389.1±94.4 ms, 5 µM nifedipine: 161.4±37.0 ms; Control vs BayK p=0.012, BayK vs nifedipine p=0.008; n=4). A similar plateau potential has been reported in rat nigral GABAergic neurons and deep dorsal horn neurons of the spinal cord, and in both cell types the ionic mechanisms generating the plateau potential involve the influx of Ca2+ through LTCCs and subsequent activation of a calcium-activated nonselective cation conductance (ICAN) (Morisset & Nagy, 1999; Lee & Tepper, 2007b; Zhou & Lee, 2011). I also tested if BayK enhanced plateau potential is mediated by TRP channel. However, the application of TRP channel blocker flufenamic acid (FFA, 100 µM) had no effect on either the decay time of plateau potential in P7 BLA neurons (Control: 464±54.6 ms, FFA: 488.1±67.7 ms, p=0.45) or on BayK-induced plateau potentials (BayK 1030.5±120.3 ms, FFA 1054.2±145.2, ms, p=0.33; Fig 2.7C).

Figure 2.7 LTCC mediated calcium influx induces a depolarizing plateau potential in P7 neurons

Bay K enhanced (A) or induced (B) a plateau potential following a suprathreshold 80 depolarizing pulse. In P7 neurons exhibiting a plateau potential under control conditions (action potential traces in black), the application of BayK (red traces) prolonged significantly the duration of the depolarization (Bar graph Panel A; n=11). The effect of BayK on the plateau potential was reversed by the L-type antagonist nifedipine (20 µM; panel B; n=5). Note that the recording solution containing BayK was replaced by nifedipine. C. Application of the TRP channel blocker FFA (violet trace) did not reverse the effect of BayK (n=5). Data are presented as mean ± SEM. * P<0.05, paired t-test.

2.3.4 Increased L-type calcium channel activity enhances initial bursting and rebound firing in P21 BLA neurons Enhanced burst firing may strongly influence the input signal transmission through BLA neurons and affect local networks (Brumberg et al., 2000; Anstrom & Woodward, 2005). Initial burst firing evoked at the beginning of a depolarizing pulse was largely observed in P21 neurons (Fig 2.2A and C). BayK was found to enhance burst firing as measured by an increased number of action potentials within the initial burst (Fig 2.8A, B, and C, Control 2.4±0.2, BayK 4.8±0.8, p=0.004, n=20). The interval between the initial burst and subsequent repetitive action potentials was also found to be significantly prolonged by BayK (Fig 2.8F, Control 126.1±17.2 ms, BayK 221.8±38.1 ms, p=0.003, n=20). Phase plot analysis was performed on the initial burst to investigate the potential involvement of other membrane conductances that shape action potentials. Application of BayK did not show a significant effect on the rate of membrane potential change of either the depolarization or repolarization phases, or on the consecutive action potential threshold (Fig 2.8D, E, G, H), suggesting that voltage-gated sodium and potassium channels are not involved in BLA cell excitability changes induced by the LTCC agonist.

A large proportion of P21 BLA neurons display a moderate sag depolarization in response to hyperpolarizing current injections, and a subsequent rebound depolarization that might lead to a single spike or a burst of action potentials (Fig 2.2C, single arrow). When rebound neurons receive an inhibitory input (or intrinsic hyperpolarization), they respond with a burst of spikes that acts as a signal for rhythmic network activity (Bevan et al., 2000; Alviña et al., 2008). I found these characteristic rebound responses in P21 BLA pyramidal neurons to be enhanced by application of BayK (Fig 2.9). In a subgroup of P21 neurons displaying rebound burst firing, BayK significantly increased the number of action potentials within the rebound burst (Fig 2.9A and C, Control: 2.0±0.2 to 3.2±0.5 BayK: p=0.043, n=6). Further, there was a small but non-significant reduction in the latency of the first rebound action potential (Fig 2.9D right, Control: 60.4±7.2 ms; BayK:

51.5±5.3 ms, p=0.32, n=6). Finally, in another subgroup of P21 neurons, rebound firing was not present under control conditions but could be elicited by BayK (Fig 2.9B, n=7) and was accompanied by a non-significant increase in the Sag amplitude (Fig 2.9E, Control 4.2±0.7 mV; BayK; 6.3±0.6 mV, p=0.11, n=7). The enhancement of rebound burst firing induced by BayK was abolished by nifedipine (Fig 9F; number of action potentials in the rebound firing: Control: 2.2±0.3, BayK: 3.39±0.4, nifedipine: 2.05±0.7; Control vs BayK, P=0.041; BayK vs nifedipine, P=0.044; n=4).

The amplitude of the rebound depolarization in P21 neurons was positively correlated to the magnitude of membrane hyperpolarization (Fig 2.9). Further increase of the hyperpolarizing pulse duration induced a gradual increase in the rebound depolarization and reduction of latency to first spike discharge. With the pulse duration increased from 20 ms to 400 ms, rebound depolarization amplitude increased from 1.69±0.23 mV to 3.76±0.92 mV (n=7), while the latency to first spike decreased from 119.2±23.1 ms to 59.3±13.2 ms (n=6). To attempt to understand the underlying mechanisms of BayK effects, I examined the voltage-dependence of rebound depolarization and the relationship between the sag amplitude and the latency of the first rebound action potential. However, BayK did not significantly affect these rebound parameters (data not shown).

The non-selective cation conductance Ih and low threshold activated calcium currents IT have been identified as ionic mechanisms responsible for hyperpolarizing-evoked rebound responses in CNS neurons (Engbers et al., 2011; Wang et al., 2016; Shah, 2018).

The hyperpolarization-activated channel HCN underlying Ih and voltage-gated T-type channels have been reported to be expressed in the BLA (Karst et al., 2002a; Park et al., 2011). As such, I examined the BayK-sensitive components of the rebound responses in the presence of the selective antagonists ZD7288 and Z944, which potently block Ih and

IT currents, respectively. Application of ZD7288 (10 µM) significantly hyperpolarized the resting membrane potential (Control: -57.6±6.5 mV; ZD7288: -68.4±5.1mV, p=0.0002, n=8), consistent with previous reports that HCN channels contribute to the regulation of resting membrane potential (Park et al., 2011; Shah, 2018). ZD7288 also completely attenuated the ability of P21 neurons to exhibit rebound bursting and further that BayK was unable to induce rebound firing in the presence of ZD7288 (Fig. 2.10A, n=5). The IT inhibitor Z944 (10 µM) significantly but partially decreased the ability of P21 neurons to rebound burst fire, an effect that was reversed by application of BayK (Fig. 2.10C, n=5). In a fraction of neurons, the increase of hyperpolarization intensity (Fig 2.10) and/or longer polarization duration (Fig 2.11) can only induce rebound depolarization. In these cells, ZD7288 completely abolished the rebound depolarization and occluded the effect of BayK (Fig 2.10B, Fig 2.12A and B, n=10), whereas Z944 slightly reduced the rebound depolarization induced by hyperpolarizing step pulses but did not prevent the effect of BayK (Fig 2.10D, Fig 2.12 C and D, n=6).

Overall, Ih channel inhibition altered the effect of BayK as both rebound depolarization and spike discharges were completely abolished by ZD7288 application, which also occluded the activity of BayK. Z944 abolished the rebound spikes in a subset of neurons, indicating T-type currents might play a role in regulating rebound firing, which has been reported in other cell types; however, the BayK effect was insensitive to blockade of low threshold calcium currents. Collectively, the results suggest that the voltage sag and the rebound depolarization in P21 BLA neurons are primarily due to the activation of Ih.

Figure 2.8 Increased LTCC activity enhances the initial burst in P21 BLA neurons

P21 BLA principal neurons commonly displayed a short burst, consisting of 2 - 3 action potentials at the beginning of the evoked train of spikes; expanded scale traces of initial bursts (dotted rectangles) are shown as inserts in A and B. Bath application of BayK increased the number of action potentials in the initial burst (C), and prolonged the interval between the initial burst and the subsequent repetitive action potentials (F). Phase plot was performed on the initial burst to evaluate the changing rate of membrane potential (D, control; E, BayK). Each loop represents one action potential. The maximal 85 dVm/dt for the depolarization phase (left Y-axis in G), minimal dVm/dt for repolarization phase (right Y-axis in G), and voltage threshold of the first two action potentials (H) exhibit no changes in the presence of BayK.

Figure 2.9 Increased LTCC activity enhances rebound firing in P21 neurons

In a subpopulation of P21 neurons, voltage responses to hyperpolarizing current pulses were followed by a rebound depolarization (asterisks, Panel B), which might lead to action potential firing at the offset of the current injection (arrow, panel A). Bath application of BayK increased the number of action potentials in the rebound firing (Panel C); insert traces shown on an expanded scale on the left of panel A correspond to control (black) and BayK (red) rebound bursts. BayK also produced a small but non- statistically significant decrease on the firing latency from the end of the hyperpolarization to the peak of the first action potential (D, p =0.16; n=7). On a subset of P21 neurons displaying only a rebound depolarization under control condition (B, black trace), BayK application elicited rebound firing (arrowhead, B, red, n=7). In the second group of neurons the hyperpolarizing-induced Sag showed an increase in amplitude in the presence of BayK (E, n=6). When the LTCC blocker nifedipine (20 µM) was applied in the presence of BayK (F) the effect on rebound firing was reduced (n=4). Note that nifedipine was applied in the absence of BayK. Data are presented as mean ± SEM. * P<0.05, paired t-test.

Figure 2.10 Modulation of rebound responses by BayK involves the hyperpolarization-activated cation conductance Ih – analysis based on the magnitude of membrane hyperpolarization

To examine membrane conductances contributing to the rebound response, the amplitude of rebound depolarization as a function of the magnitude of hyperpolarizing pulses was measured in the presence of selective blockers. Generation of rebound action potentials was abolished in the presence of the Ih blocker ZD7288 (10 uM; n=5) (Panel A). Application of ZD7288 (10 uM; n=10) also abolished rebound depolarization and occluded the effect of BayK (B). Blockade of T-type calcium channels with the selective blocker Z944 (10 uM; n=6) reduced the rebound depolarization induced by

89 hyperpolarizing step pulses (D) but did not prevent the effect of BayK in facilitating rebound firing (C, n=5). Data are presented as mean ± SEM.

Figure 2.11 Modulation of rebound responses by BayK involves the hyperpolarization-activated cation conductance Ih – time dependent effect of membrane hyperpolarization

To further examine membrane conductances contributing to the rebound response, the amplitude of rebound depolarization as a function of the duration of hyperpolarizing pulses was measured in the presence of selective blockers. The rebound depolarization 90 was completely abolished in the presence of the Ih blocker ZD7288 (10 uM; n=5) which also occluded the effect of BayK (Panel A and B). Blockade of T-type calcium channels with the selective blocker Z944 (10 uM; n=6) did not modify the amplitude of the rebound depolarization induced by hyperpolarizing step pulses (Panel A and B). Data are presented as mean ± SEM.

2.3.5 Spontaneous firing activities were modified by (S)-Bay K8644 At resting membrane potential BLA neurons are quiescent. For P7 neurons, when a tonic current was applied to hold the membrane potential at the action potential firing threshold cells discharge burst of action potentials. The subsequent application of BayK decreased burst duration while increasing the inter-burst spike frequency (Fig 2.12C, control: 0.59±0.06 Hz; BayK: 1.36±0.17 Hz; n=12, p=0.002), in 12 out of 14 (85.7%) and with no change in spike frequency during bursts (Fig 2.12 D, control: 14.9±1.2 Hz; BayK: 16.6±1.3 Hz; n=12, p=0.45). In contrast, for the majority of the P21 neurons (17 out of 19; 89.5%) sustained depolarizing current injection evoked spontaneous single spikes accompanied with a significantly increased frequency in the presence of BayK (Fig 2.12 G, control: 2.04±0.2 Hz; BayK: 3.59±0.5 Hz; n=17, p=0.0027).

Figure 2.12 (S)-Bay K8644 increases spontaneous activity evoked by sustained depolarization of BLA neurons

In the absence of exogenous stimulation, pyramidal BLA neurons are silent. Tonic depolarization of immature (P7) BLA neurons at firing threshold (A, control) results in membrane oscillations underlying bursts of action potentials. Superfusion with 2 μM (S)- Bay K8644 increased bursting activity consisting of more frequent short duration bursts 92

(B, C) without modifying the discharge frequency within each burst (D). Sustained current injection in P21 neurons under control conditions give rise to spontaneous regular firing of single action potentials (E, F). The frequency is significantly increased by application of the LTCC agonist (G). *p<0.05. Data are presented as mean ± SEM.

2.3.6 Increased L-type calcium channel activity modifies the functional properties of quantal post-synaptic currents. LTCCs are highly expressed at post-synaptic terminals and regulate both long-term potentiation of thalamic inputs into the amygdala (Weisskopf et al., 1999a) and synaptic immediate early gene expression (Murphy et al., 1991). As such, I was interested in whether enhanced LTCC activity impacted inhibitory and excitatory synaptic inputs in the BLA.

In order to investigate the impact of LTCC activity on GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs), slices were incubated in TTX (1 µM), the

GABAB receptor blocker CGP52432 (1 µM) and the glutamate receptor blockers D-AP5 (50 µM) and CNQX (10 µM). The results from distribution plots, cumulative probability and mean value analyses showed that bath application of BayK increased mIPSC frequency both in P7 (Fig 2.13A and C; mean value, Control: 1.6±0.1 Hz; BayK: 2.4±0.1 Hz; p=0.0008; K-S test value: 0.12; n=9) and P21 neurons (Fig 2.14A and C; mean value, Control: 2.0±0.2 Hz; BayK: 4.5±0.2 Hz; p=0.0006; K-S test value: 0.39; n=6). Additionally, in P7 neurons, smaller quantal events were found to be more predominant around the mean value in the total population of the BayK treated group compared to the control population (Fig 2.13A and B), which is also reflected in the cumulative probability plot (K-S test value: 0.18). However, the mean value of amplitude was not affected by BayK (Control: 29.6±1.2 pA; BayK: 26.3±0.69 pA; p=0.58; n=9). No change was found in the amplitude population for P21 neurons (mean value, Control: 30.8±1.2 pA; BayK: 28.2±0.48 pA; p=0.78; K-S test value: 0.07; n=6). Together with the increase in frequency, the results suggest a presynaptic enhancement of inhibitory neurotransmission induced by BayK. No significant changes were observed in the mIPSC rise time and decay time constants at either P7 or P21 (P7, mean value of rise time: Control: 12.7±0.2 ms; BayK: 12.6±0.1 ms, p=0.61, n=9; decay tau: Control: 37.1±1.1 ms; BayK: 38.3±0.8 ms, p=0.88, n=9; P21, mean value of rise time: Control: 13.8±0.3 ms;

BayK: 12.6±0.1 ms, p=0.42, n=6; decay tau: Control: 32.6±1.7 ms; BayK: 33.6±0.9 ms; p=0.72, n=6). The mIPSC decay kinetics were found to be different between P7 (slow) and P21 (fast), consistent with a previous report (Ehrlich et al., 2013). I next examined excitatory post-synaptic currents (mEPSCs) in BLA neurons. Recordings were performed in the presence of TTX (1 µM), CGP52432 (1 µM), and the GABAA receptor blocker picrotoxin (100 µM). Data showed that BayK increased the amplitude of the mEPSCs only in P21 neurons (Fig 2.14D and E; mean value in Control: 18.2±0.2 pA; BayK: 22.6±0.3 pA, p=0.033; K-S test value: 0.33; n=6) without any significant change in frequency (Fig 2.14F; mean value in Control: 3.1±0.1 Hz; BayK: 3.5±0.4 Hz, p=0.16; K- S test value: 0.07, n=6), indicating postsynaptic enhancement of excitatory neurotransmission selectively in more mature neurons. No effect of BayK on mEPSCs was found for P7 neurons (Fig 2.13D, E, and F; amplitude mean value, Control: 20.2±1.3 pA; BayK: 19.5±1.3 pA; p=0.7; K-S test value: 0.1; n=5. Frequency mean value, Control: 0.97±0.1 Hz; BayK: 1.0±0.2 Hz; p=0.82; K-S test value: 0.06; n=5).

Figure 2.13 Increased LTCC activity modulates miniature post-synaptic inhibitory activity in P7 BLA neurons

Action potential-independent miniature post-synaptic inhibitory (mIPSCs) and excitatory 96 currents (mEPSCs) were examined for P7 neurons in the presence of TTX (1 µM ). Representative traces are shown in panel A and D. For mIPSCs cells were held at a holding potential of 0 mV, whereas for mEPSCs holding was at -70 mV. In addition, mIPSCs were recorded in the presence of CNQX (10 µM) and D-AP5 (50 µM). mEPSC histograms show the distribution of amplitudes (B and E) and frequencies (C and F) of inhibitory (B and C) or excitatory (E and F) miniature currents. Cumulative probability plots (insets) comparing miniature events obtained in ACSF (control, black) and in the presence of BayK (red) are compared with the Kolmogorov-Smirnov test. Mean values of postsynaptic spontaneous activity are shown in bar graphs (insets). The plots show that the application of BayK increased the frequency of IPSC transmission (Panel A, B and C, n=9), particularly the number of small events (Panel B). In contrast, no significant change was observed in miniature EPSCs (Panel D, E and F, n=5). Calibration bar for mIPSCs: vertical 40 pA, horizontal 0.5 s. Calibration bar for mEPSCs: vertical 40 pA, horizontal 0.2 s. Data are presented as mean ± SEM.

Figure 2.14 Spontaneous release at GABAergic and glutamatergic synapses are differentially regulated by BayK in P21 BLA neurons

Action potential-independent miniature post-synaptic inhibitory (mIPSCs) and excitatory 98 currents (mEPSCs) were examined for P21 neurons in the presence of TTX (1 µM ). Representative traces are shown in panels A and D. For mIPSCs cells were held at a holding potential of 0 mV, whereas for mEPSCs holding was at -70 mV. Bath application of BayK increased the frequency of IPSC transmission (Panel A, B and C, n=6) without shifting the distribution of current amplitude (B). Conversely, the amplitude of miniature EPSCs was increased by BayK (Panel D and E, n=6) whereas no change was observed in the mEPSC frequency (Panel F). Calibration bar for mIPSCs: vertical 40 pA, horizontal 0.5 s. Calibration bar for mEPSCs: vertical 40 pA, horizontal 0.2 s. Data are presented as mean ± SEM.

2.3.7 L-type calcium channel activity does not affect evoked GABAergic or glutamatergic responses in BLA principal neurons Calcium entry through LTCCs is generally thought to play a minor role in triggering neurotransmitter release in central synapses under basal conditions (Meir et al., 1999). However, activation of LTCCs is necessary for the induction of long-term potentiation (LTP) in the lateral amygdala (Fourcaudot et al., 2009), contribute to short term plasticity in the dentate gyrus (Jensen & Mody, 2001b), and exert a frequency-dependent differential regulation of spontaneous and evoked GABAergic transmission in the medial preoptic nucleus (Malinina et al., 2010). Hence, I examined whether increased LTCC activity affects synaptic strength and/or paired-pulse facilitation in BLA principal neurons.

BLA principal neurons receive inhibitory inputs from various sources including local interneurons, prefrontal cortex, thalamus, lateral amygdala and the lateral cluster of paracapsular intercalated (lPICs) (Marowsky et al., 2005). Here, I examined direct inhibitory inputs from lPICs (Fig 2.15A). Slices were incubated with D-AP5 (50 µM), CNQX (10 µM) and CGP53432 (1 µM). Notably, a single pulse evoked IPSC in the presence of BayK failed to impact amplitude in either P7 or P21 BLA neurons (Fig 2.15B). The mean value of IPSC amplitude showed no significant change in the presence of BayK in P7 neurons (Control: 165.1±31.4 pA; BayK: 192.4±33 pA; p=0.15, n=9; Fig 2.15C) or in P21 neurons (Control: 420.4±96.2 pA; BayK: 413.3±88.2 pA, p=0.86, n=4; Fig 2.15C).

Excitatory inputs from prefrontal cortex, hippocampus and thalamus target BLA principal neurons mainly through the lateral amygdala (LA) hence I examined evoked excitatory responses by placing the stimulating electrode in the center of LA (Fig 2.15D). Slices were incubated with PTX (100 µM) and CGP53432 (1 µM). In P21 neurons, a single pulse evoked EPSC showed multiple peaks (Fig 2.15E) which could only be inhibited by

100

CNQX suggesting the main component of eEPSCs are AMPA-mediated currents. D-AP5 had no effect on eEPSCs (data not shown). Application of BayK did not significantly alter eEPSCs (Fig 2.15 E and F) suggesting a lack of LTCC contribution towards excitatory synaptic strength (mean values eEPSC amplitude P7 Control: 59.6±15.2; P7 BayK: 59.2±11.2 pA, p=0.84, n=5; Fig 2.15F); and P21 Control: 94.3±34.1 pA; BayK: 104.3±28.4 pA, p=0.3, n=5; Fig 2.15F).

To examine short-term plasticity within the BLA circuit I employed paired-pulse stimulation with an increasing interpulse interval (IPI) from 20 ms to 1000 ms for both inhibitory and excitatory synaptic responses. However, my results showed no significant effect of BayK on the paired-pulse ratio observed for either P7 (Fig 2.16 A and B) or P21 neurons (Fig 2.16 C and D).

My results provided evidence that the mechanisms underlying spontaneous and evoked synaptic activity have different sensitivity to functional changes of LTCCs.

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Figure 2.15 BayK does not affect evoked post-synaptic responses in BLA neurons

Schematic diagram of a coronal section of basolateral amygdala showing the position of the stimulus (represented by a square pulse) and recording (pointed tip on the right) electrodes in panel A and D for eIPSC and eEPSC, respectively. Post-synaptic currents were evoked by electrical stimulation on the lateral cluster of intercalated GABAergic neurons (lPICs) for eIPSCs, and on the lateral amygdala (LA) for eEPSCs. Overlapped responses to 10 consecutive presynaptic pulses applied at 0.1 Hz are shown in gray; average control trace in black, BayK average trace in red, stimulation artifacts were 102 removed from the traces for clarity (B and E). For each experiment, the current amplitude was measured from the average response to 10 consecutive pulses at a frequency of 0.1 Hz. For eIPSCs, the membrane potential was held at 0 mV; for eEPSCs, the membrane potential was held at -60 mV. Neither excitatory nor inhibitory evoked current amplitudes were affected by BayK. Mean values are shown as bar graphs in panel C (P7, n=8; P21, n=6) and F (P7, n=5; P21, n=7). Data are presented as mean ± SEM.

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Figure 2.16 Paired-pulse ratio of synaptic responses are not affect by the application of (S)-Bay K8644 at both P7 and P21

Paired-pulse response was evoked by double stimuli at a series of intervals (20 ms, 40 ms, 60 ms, 100 ms, 200 ms, 500 ms, 1000 ms). The Paired-pulse ratio (PPR) was then calculated as the amplitude of the 2nd pulse divided by the 1st pulse, and plotted as a function of interpulse intervals. Representative traces on the left panels show the double

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IPSCs (A, traces are at 60 ms or 200 ms interval) and EPSCs (B, traces are at 60 ms or 200 ms interval) responses of P7 neurons, as well as the double IPSCs (C, traces are at 60 ms or 200 ms interval) and EPSCs (D, traces are at 40 ms or 100 ms interval) of P21 neurons. Plots in the right panels show that BayK application did not significantly alter the PPR of synaptic response for both P7 (A, IPSC, n=8; B, EPSC, n=5) and P21 (C, IPSC, n=4; D, EPSC, n=4) neurons. Data are presented as mean ± SEM.

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2.4 Discussion

The functional roles of neuronal LTCCs have largely focused on their regulation of postsynaptic immediate early gene expression, which in turn contributes to synaptic plasticity (Perrier et al., 2002a; Moosmang et al., 2005a; Yang et al., 2018; Kamijo et al., 2018a; Wild et al., 2019). However, LTCCs appear to also contribute towards regulating excitability in a subset of central and peripheral neurons (Baufreton et al., 2003; Perrier et al., 2002). Activation of LTCCs contributes to somatodendritic Ca2+ signals during tonic firing in vestibulocerebellar cells (Diana et al., 2007), to the slowing of sustained depolarization underlying burst firing in subthalamic nucleus neurons (Beurrier et al., 1999), and to mediating pacemaking activity in dopaminergic neurons (Liu et al., 2014). Thus, LTCCs might play a key role in regulating dynamic changes in neuronal firing modes. Notably, pathogenic alterations in the Cav1.2 and Cav1.3 LTCC genes have been correlated with neuropsychiatric and neurological disorders (Bhat et al., 2012; Berger & Bartsch, 2014; Kabir et al., 2017). In the case of elevated LTCC activity linked to pathological conditions such as epileptic disorders and sociability deficits associated with ASDs (Splawski et al., 2004b; Yan et al., 2007), it remains largely unknown as to the extent that increased LTCC activity affects neuronal excitability. In this study, I examined the contributions of LTCCs towards BLA principal neuron biophysical and synaptic characteristics at two stages of postnatal development and identified P7 neurons as being particularly sensitive to functionally increased LTCC activity.

For immature P7 BLA neurons, overall intrinsic excitability properties were significantly enhanced by application of BayK. The LTCC-mediated alterations included the frequency of repetitive firings above current threshold being significantly higher (Fig 2.3Aa) and a decreased rheobase current value (Fig 2.3Ab) albeit maintained at the same maximal steady-state firing frequency as in control resulting in a reduction in gain. Together, these imply that in the presence of LTCC agonist, P7 immature neurons tend to keep the same

106 high tone in response to different levels of input, as well as being more responsive to weaker input signals. In the presence of excitatory and inhibitory neurotransmission blockers, BayK produced a similar effect on gain, indicating that modulation of firing response properties by LTCCs mainly rely on the intrinsic properties of P7 BLA neurons.

Spike frequency adaptation (SFA) has been previously described in central neurons, including dopaminergic (Vandecasteele et al., 2011), hippocampal CA1 (Pedarzani et al., 2005) and pyramidal neurons from the lateral amygdala (Faber & Sah, 2002) wherein a gradual reduction in firing rate during prolonged depolarizations typical of SFA has been attributed to a calcium-dependent afterhyperpolarization (AHP) likely mediated by SK channels. In accordance with this model, increased intracellular concentration of Ca2+ would enhance AHP amplitude, in turn increasing SFA; therefore, Ca2+ influx during neuronal activity could significantly contribute to the activation of AHP (Andrade et al., 2012). However, contrary to that expected, current clamp recordings in P7 BLA neurons showed that the LTCC agonist BayK reduced SFA and AHP amplitude, similar to the impact of SK channel blockade (Fig 2.6). These results indicate that the SK channel- mediated AHP indeed contributes to the SFA observed in P7 BLA neurons, and that SK channels are likely involved in the effects of BayK on regulating spike frequency adaptation. However, the precise mechanisms relating LTCC function to SK-mediated regulation of SFA and AHP in BLA neurons requires further studies. Other yet-to-be identified voltage-gated or calcium-activated conductance(s) might be contributing to the modification of SFA by BayK (Rudy & McBain, 2001; Gu et al., 2007). Since SFA affects the dynamic range of frequency response (Benda et al., 2005), a reduced SFA in immature BLA neurons as a consequence of enhanced LTCC might have significant functional implications towards information processing in the developing amygdala circuit by limiting the ability to filter out input signals of lower frequency.

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A depolarizing plateau that outlasts a suprathreshold stimulus was another feature prominent across P7 neurons and was found to be significantly prolonged by BayK (Fig 2.7A). Further, in some P7 neurons that did not display a basal plateau potential, the application of BayK elicited a plateau potential (Fig 2.7B). Previous studies have shown a significant contribution of LTCCs to the generation and/or modulation of the plateau potential in spinal motoneurons (Perrier & Hounsgaard, 2003), nigral GABAergic neurons (Zhou & Lee, 2011) and prefrontal cortex pyramidal cells (Heng et al., 2011). Further, it has been suggested that LTCC modulation of plateau potentials results from the coupling of LTCC-mediated Ca2+ influx to calcium-dependent conductances such as non-specific cation channels (Lee & Tepper, 2007). I tested a candidate TRP channel by applying the inhibitor FFA known to contribute to plateau potential modulation (Zhang et al., 2011). However, FFA had no effect on either the decay time of plateau potential in P7 BLA neurons or on BayK-induced plateau potentials (Fig 2.7C). Previous studies have highlighted different possible mechanisms modulating plateau potential magnitude such as the activation of dopamine, serotonin and NMDA receptors (Perrier & Hounsgaard, 2003; Lee & Tepper, 2007b; Zhou & Lee, 2011). In my study, the ability of BayK to elicit plateau potentials in P7 BLA neurons was not affected by the presence of synaptic ionotropic receptor blockers CNQX, PTX and D-AP5 (data not shown). Thus, while the underlying mechanism of the LTCC-mediated plateau potential in immature BLA neurons is not fully understood, the sustained membrane depolarization and cation influx associated with the plateau are predicted to affect how BLA neurons integrate synaptic inputs from different sources (Zhang et al., 2011).

In P21 neurons, BayK did not modify the f-I relation, rather it promoted significant changes to burst firing: BayK increased the number of action potentials during the initial burst and enhanced rebound bursting firing (Figs 2.8 and 2.9). Phase plot analysis on initial burst was performed to examine the properties of the BayK evoked spikes

108 compared to the regular spike activity. The voltage dependence of either the depolarization or repolarization rate (dVm/dt) of the first action potentials of the burst did not show a significant difference; however, the additional spikes, initiated at more depolarized levels (Fig 2.8H), displayed a slow rate of change (Fig 2.8B, E, G). These slow action potentials might represent calcium spikes, perhaps elicited by membrane potential back-propagation from dendrites where LTCC expression is high (Markram et al., 1995; Christie et al., 1995). I speculate that the enhancement of initial burst firing might potentiate the reliability of signal transmission between neurons and contribute to the spike rhythmic within the network of the amygdala, as reported in supragranular cortical neurons and midbrain dopaminergic neurons (Brumberg et al., 2000; Anstrom & Woodward, 2005).

An enhancement of rebound firing by BayK was also observed in P21 neurons indicated as a significantly increased number of action potentials within the burst (Fig 2.9). Further as shown in Fig 2.10 and Fig 2.11, rebound action potential firing and rebound depolarization were both completely abolished by Ih inhibition and the effect of BayK was also occluded, indicating an important contribution of HCN channels to the LTCC- mediated regulation of rebound properties of BLA neurons. Further evidence for the contribution of Ih was the presence of hyperpolarization-activated depolarizing voltage Sag, a phenomenon mediated by the activation of HCN channel (Wahl-Schott et al., 2014). P21 recordings showed that rebound firing or depolarization always occurred in cells that display Sag at the hyperpolarization. In cerebellar neurons, Ih and IT both contribute to rebound characteristics (Perrier et al., 2002a; Molineux et al., 2006;

Sangrey & Jaeger, 2010). In the BLA IT did not appear to be a major contributor to the modifying effects of Bay K, although T-type calcium channels are expressed in BLA neurons and contribute to rebound spiking.

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The results are consistent with the previous characterization of the basal electrophysiological properties of BLA neurons at P7 and P21, and support the notion that at postnatal day 7 the functional characteristics of the BLA principal neurons correspond to an immature stage, with major changes occurring from P7 to P21 when the mature phenotype is established (Ehrlich et al., 2012). Age-dependent differences in BayK effects on intrinsic excitability might be related to the variation in the expression and function of various ion channels, transporters and membrane receptors associated to the developmental refinement of neuronal properties.

I was also interested in whether acute application of BayK could affect the spontaneous synaptic activity of BLA neurons as well as responses to the activation of specific synaptic inputs. Spontaneous neurotransmitter release occurring in the absence of action potentials accounts for only a small fraction of postsynaptic receptor activation compared to the evoked neurotransmitter release triggered by presynaptic action potentials, and its role in neuronal communication has been a subject of debate (Otsu & Murphy, 2003; Kaeser & Regehr, 2014). However, more recent studies indicate that miniature synaptic events participate in regulating neuronal excitability (Carter & Regehr, 2002), local dendritic protein synthesis (Sutton et al, 2006) and homeostatic neuronal signaling (Kavalali et al., 2011). My analysis of mPSCs show that BayK significantly increased the frequency of GABAergic mIPSCs at both P7 and P21 (Fig 2.13, Fig 2.14). mIPSC amplitude cumulative plot of P7 neurons also displayed a steeper initial phase (Kolmogorov test value: 0.18) corresponding to an increased number of events at around mean amplitude with no change in the average itself as shown in the inset bar graph (Fig 2.13B). I observed a noticeable presynaptic effect on mIPSCs at both postnatal ages, with a higher relative increase in mIPSCs frequency at P21 (Fig 2.14C). Since miniature synaptic events are at least partially regulated by Ca2+ (Schneggenburger & Rosenmund, 2015), and the spontaneous opening of presynaptic LTCCs can contribute to regulating

110 the spontaneous release (Goswami et al, 2012), one could speculate that a similar mechanism might occur in the BLA. A functional link between LTCCs and intracellular Ca2+ stores has been described in BLA (Power & Sah, 2005) but its role in spontaneous neurotransmitter release has not been explored. In contrast, the distinctive shift in the amplitude distribution of mEPSCs at P21 with no changes in frequency or time course of excitatory miniature events (Fig 2.14) indicates a postsynaptic mechanism in mature neurons, although the specific underlying mechanism remain to be described.

The principal neurons, which make up about 90% of BLA neurons, receive numerous sensory synaptic inputs from different brain regions and send glutamatergic outputs to various downstream areas (Olucha-Bordonau et al., 2015). For instance, the BLA receives glutamatergic inputs from the thalamus and prefrontal cortex along with strong feedforward inhibitory inputs from the GABAergic lateral intercalated cells (lPICs), and sends outputs to the central amygdala and ventral hippocampus (Rosenkranz & Grace, 2001; Felix-Ortiz & Tye, 2014; Strobel et al., 2015). Here, I examined the effects of an acute functional increase in LTCC activity on synaptic transmission onto the BLA by testing evoked post-synaptic inhibitory currents elicited by electrical stimulation of GABAergic neurons from the lateral intercalated nucleus, and also on excitatory postsynaptic currents evoked by stimulating glutamatergic projections entering the BLA through local connections from the lateral amygdala (Fig 2.15). Consistent with previous reports, I find that excitatory connections are not fully developed in the BLA at P7 (Bosch & Ehrlich, 2015; Arruda-Carvalho et al., 2017) when recorded evoked responses are small and variable in amplitude (Fig 2.15 E). At P21 I observed a complex EPSC waveform evoked by stimulation of the lateral amygdala, consisting of both CNQX- sensitive component and D-AP5-sensitive components, and indicating that at this postnatal stage the characteristics of mature glutamatergic transmission are already present. The characteristics of evoked IPSCs at both P7 and P21 were similar to those

111 reported previously (Ehrlich et al 2013). Interestingly, my results showed no noticeable effect of BayK on inhibitory or excitatory responses, and further that no changes were found using paired-pulse stimulation (data not shown). Taken together, functionally increasing LTCC activity did not alter the properties of synaptic strength or paired-pulse plasticity.

Collectively, I observed functionally relevant changes in intrinsic excitability of immature (P7) BLA pyramidal neurons associated with increased LTCC activity: neurons are more responsive to low magnitude depolarization and display a reduced ability to discriminate between different stimulus intensity at low firing frequencies. Also, they can sustain action potential firing for an extended period of stimulation due to reduced frequency adaptation, and exhibit a prolonged depolarizing plateau. In concert, these altered intrinsic electrophysiological properties could provoke a functional impairment in the network activity of the neonatal basolateral amygdala.

My findings for P21 principal BLA neurons suggest that at this neurodevelopmental stage the established functional complement of membrane conductances and the mature synaptic network are less vulnerable to dysregulation of L-type channels, and further that increasing of LTCC activity has limited effects on altering either neuronal excitability or synaptic activity. Compared to that for immature P7 neurons, LTCC-mediated alterations in P21 cells would be less likely to shift overall excitability and synaptic characteristics. Examination of the BLA for LTCC expression by quantitative RT-PCR showed no significant changes in the relative levels of Cav1.2 and Cav1.3 between the P7 and P21 stages (Fig 2.5). As such, the differential effects of BayK cannot be solely attributed to an age-dependent change in the expression of LTCCs during the postnatal periods examined. I cannot however, rule out the developmentally regulated distribution of LTCCs in neuronal membrane subregions and/or distinct modulatory mechanisms.

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Neuronal LTCCs consist of both Cav1.2 and Cav1.3 subtypes and it is difficult to dissect their individual contributions with pharmacological tools. Despite the lower sensitivity of

Cav1.3 channels to DHP antagonists (Bell et al., 2001), BayK has been shown to increase currents through recombinant Cav1.3 channels (Xu & Lipscombe, 2001b), impeding the identification of the subunit composition underlying BayK effects in my study. However, a previous study in transgenic mice has shown an increase in firing frequency in BLA neurons from adult Cav1.3 knockout animals (McKinney et al., 2009b). Also, the Cav1.3 channel activation threshold is more hyperpolarized that that of Cav1.2, arising the possibility that currents mediated by the two channel types operate in parallel in regulating distinct intrinsic membrane properties. Importantly, gain of function mutations in both Cav1.2 and Cav1.3 L-type isoforms are linked to autism spectrum disorders

(Splawski et al., 2004b; Pinggera et al., 2015b). Cav1.2 and Cav1.3 LTCCs are both expressed in BLA neurons across developmental stages and their contributions to overall excitability and synaptic signaling is likely critical towards mechanisms underlying neurodevelopmental disorders caused by LTCC gain-of-function mutations.

Overall, I describe novel contributions of LTCCs towards BLA intrinsic excitability and synaptic activity at critical neurodevelopmental stages. In order to determine whether the acute manipulation of LTCC activity translates to long-lasting effects on BLA function I am currently studying how the in vivo administration of BayK into the BLA at various developmental stages impacts BLA circuitry and certain behaviors. Together, these studies will further elucidate the roles of LTCCs in pathological states relevant to disorders such as ASD.

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3 CHAPTER Ⅱ: A pharmacological rat model of autism spectrum disorder: Behavioral and neuronal alterations caused by neonatal exposure of the basolateral amygdala to an L-type calcium channel agonist

3.1 Introduction

Early neurodevelopment involves multiple levels of cellular and molecular processes, such as neuron migration, axon and dendrite growth and pruning, programmed cell death and synaptic formation, all having to occur with precise timing and within specific brain areas (Bauman & Kemper, 2005). Calcium signaling through neuronal LTCCs Cav1.2 and

Cav1.3 are thought essential in multiple neurodevelopmental processes including sculpting of neurites, functional wiring, synaptogenesis and fine-tuning of growing networks (Oh et al., 2016). Many signaling molecules acting downstream of LTCCs such as calcium/CaMK and protein kinase C have been shown to play critical roles in morphological remodeling during early development (Takemoto-Kimura et al., 2007; Takemoto‐Kimura et al., 2010). In developing neurons, the genetic knockout LTCCs results in shorter neurites while introducing an LTCC gain-of-function mutation during perinatal development impairs cortical radial migration and that can be reversed postnatally to rescue normal corticogenesis (Kamijo et al., 2018b).

The crucial roles of LTCCs in regulating neuronal maturation that ultimately influences behaviour has been highlighted by the developmental brain dysfunction and impaired behaviors caused by point mutations identified in LTCC genes. For instance, Timothy Syndrome (TS), a severe disorder caused by gain-of-function mutations in LTCCs has been associated with the core symptoms of ASD. In addition, loss-of-function variants

114 identified in LTCC genes have been found correlated with schizophrenia (Anon, 2009). Although the exact neural mechanisms underlying impairments observed in ASD or schizophrenia patients remain unknown, it is not unreasonable to predict that altering the electrical properties and downstream calcium-signaling pathways normally contributed by LTCCs could drive underlying pathophysiologies associated with abnormal neurodevelopment.

As stated, the present study focuses on the BLA since functional alterations of the BLA neuronal network have been correlated with the impairment of social interaction abilities associated with ASD (Truitt et al., 2007; Sturm et al., 2013; Sosa-Díaz et al., 2014). Moreover, synaptic plasticity in the amygdala is believed to underlie various behavioral responses, such as the acquisition of fear, anxiety, addiction, and social activities (Rainnie et al., 2004; Truitt et al., 2007).

In Chapter I showed that a functional increase in LTCC activity caused increased excitability and altered spontaneous synaptic activities in BLA principal neurons, with more potent and broader effects in P7 compared to P21 neurons (Zhang et al., 2020). From these, I hypothesized that alterations in LTCC activity during critical periods of early neurodevelopment might lead to BLA circuit imbalances that predispose the circuitry to behavioral modifications resembling the LTCC gain-of-function mutations underlying certain genetic forms of ASD.

In part, our current poor understanding of the underlying mechanisms of how mutant LTCCs might trigger specific behavioral features are due to a lack of appropriate animal models that recapitulate predicted neurodevelopmental imbalances without affecting the cardiovascular, endocrine and immune systems. ASD is a complex condition characterized and diagnosed based on a triad of behavioral traits; decreased social

115 interactions and delayed communication skills and language development until later stages in infancy, with the strong prospect that the underlying neurogenesis processes emerge at much earlier stages (Stagni et al., 2015). As such, to investigate and dissect the underlying mechanisms of behavioral characteristics associated with ASD it is important to manipulate LTTCs activity at different stages during neural development.

Seminal studies on the functional postnatal maturation of BLA principal neurons (Ehrlich et al., 2009, 2013, 2013; Ehrlich & Rainnie, 2015) indicate that considerable changes in electrophysiological properties such as resting membrane potential, input resistance, firing patterns, and synaptic activities occur from P7 to P21 when thereafter the circuit reaches maturity (Fig 3.1). Result from Chapter 1 are consistent with these previous reports and provide further support for the notion that a critical vulnerable period in the BLA occurs in early postnatal development. As such, manipulations or interventions affecting the electrical properties of neurons at specific times during this period could potentially result in long-lasting changes in neural networks and behaviors.

Here, I performed stereotaxic bilateral injections of the LTCC agonist BayK into the BLA at P7, P14 or P21, and then investigated long-term effects on electrophysiological properties at ~P28 reflecting an age of amygdala maturity (Fig 3.2). Given that the defining criteria for ASD are behavioral (diagnostic criteria for ASD provided by the American Psychiatric Association in the DSM-5, http://www.dsm5.org/ProposedRevisions/Pages/proposedrevision.aspx?rid=94, January 2011) and that some core features are social impairment and repetitive activities, I also employed behavioral tests based on these phenotypes to attempt to correlate functionally increased LTCC activity in the BLA with ASD-associated behaviours.

The results show that the local enhancement of LTCC activity in the BLA circuitry by the application of agonist BayK at specific early neurodevelopmental stages (P7, P14) results 116 in long-lasting alterations in neuronal excitability, neuronal passive properties, long-term synaptic plasticity, and behavioral phenotypes.

Figure 3.1 Time course of the neurodevelopmental critical period in the rat BLA during the first four postnatal weeks

The schematic diagram shows the rate of morphological and functional changes of BLA neurons occurring at different times of the postnatal development of BLA. Briefly, there is an expansion of dendrites and increased dendritic spine density in principal neurons. Electrophysiological recordings show an increase in GABAergic currents and a reduction in membrane input resistance. The blue curve indicates that a maximal rate of change 117 occurs between P7 and P21. The diagram was elaborated to summarize findings from Ehrlich et al., 2013; Ehrlich & Josselyn, 2016; Ryan et al., 2016, as well as Chapter Ⅰ of my study.

Figure 3.2 Timeline diagram of the experimental procedures performed on experimental groups of rats at different ages

Bilateral injections of BayK or DMSO were performed at either P7, P14 or P21 for the corresponding group. From the age of P28 to P30, the animals were allocated to 3 experimental test groups: brain slice electrophysiological experiments were performed to investigate the neuronal excitability and synaptic plasticity of BLA neurons; tissue samples were collected to examine the mRNA expression of Ca2+ signaling related genes; and behavioral tests were performed to investigate potential alterations of behavioral phenotypes, including three chamber test, social interaction test, self-grooming and open field test.

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3.2 Methods

Animals All experimental protocols were approved by the University of British Columbia Animal Care Committee (UBC ACC Protocol A16-0127). Pregnant Sprague Dawley rats (purchased from Charles River Laboratories, 17 days of pregnancy on the day of arrival) were housed at room temperature (24-26oC) in a 12 hours day/night cycle at the Animal Resource Unit of University of British Columbia. Rodents are sensitive to environmental changes thus I followed the UBC SOP on handing of rat and mouse pups that undergo procedures (September 27, 2016) for habituation purpose. Rat pups were handled initially in the presence of the mother, and then separated from the mother (30 minutes per day) from day 0 to the day of surgery. Rat pups were weaned at postnatal day 21 (P21). Pups were randomly assigned to 3 experimental groups with each of the groups receiving a one-time BayK or DMSO bilateral injection at a specific age (P7, or P14, or P21) (Fig 3.2).

Stereotaxic Surgery The bilateral injection was performed on rat male pups from different litters at postnatal day 7, day 14, or day 21. Initially, rat pups were placed in an acrylic chamber and anesthetized with isoflurane using a VetEquip vaporizer and following UBC SOP ACC- 01-2017 Rodent Anesthesia. After induction, anesthesia was maintained throughout stereotaxic surgery using a nose-cone / face-mask located on the stereotactic frame (David Kopf Instruments, Model 900, 100-micron resolution). The depth of anesthesia was determined by respiration rate and foot/tail pinch response every 3-5 minutes during surgery. Once the surgical plane of anesthesia was reached, the skin over the cerebrum was incised by a 1 cm long median-sagittal cut with the eyes protected from drying out by using a corneal lubricant. The skull was cleaned with autoclaved water and dried by a

119 mini vacuum. Then two symmetrical holes were made using a dental drill on each side of the skull over the cerebrum (P7: bregma -2.4 mm anterior-posterior, ±3.4mm medial- lateral, 6.8 mm dorsal-ventral; P14: bregma -2.6 mm anterior-posterior, ±3.8 mm medial- lateral, 7 mm dorsal-ventral; P21: bregma -3.8 mm anterior-posterior, ±4.0 mm medial lateral, 7.2 mm dorsal-ventral). Bregma coordinates were established for each age considering the rapid change in the position of brain structures during the periods examined (Khazipov et al., 2015). A Hamilton Neuros micro-syringe was inserted into the two holes on each side, to deliver 0.5 μl BayK (1mM, 20 μg/kg) or the same volume of DMSO vehicle into the BLA regions. The injection duration was 5 minutes at a rate of 0.1 μl/min. After injection, the micro-syringe needle was left stationary for a further 3 minutes before retrieval. After completion of the procedures, the skin was sutured with subcuticular sutures (6-0 coated Vicryl). To attenuate surgical pain in pups, bupivacaine (diluted in saline solution to 0.25%) was dripped onto the skin when closing the incision at the end of surgery.

In order to verify the location of bilateral injection within the BLA, a set of naive rats received 0.2 μl Methylene Blue (20%) stereotaxic injection at either P7, P14, or P21, respectively. The animals were euthanized and brains were dissected after dye injection. Brain slices were collected at 300 μm with a vibratom and checked under a 10X surgical microscope. The area of BLA containing Methylene blue indicated the accuracy and consistency of the bilateral injection surgeries. In addtion, to verify the diffusion area of BayK injection in BLA, animals (n=4) were euthanized and brains were dissected 1 hour after Bay injection, slices were collected and visually examined under microscpe. The BLA area showed an round area with 1 mm diameter, which was stained by the yellow color of BayK.

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Acute Brain Slice Preparation At age P28, animals were anesthetized using isoflurane [5% in oxygen (vol/vol)] and decapitated using a rodent guillotine. The brain was quickly removed and transferred to ice-cold sucrose cutting solution: 214 mM sucrose, 26 mM NaHCO3, 1.25 NaH2PO4, 11 mM glucose, 2.5 mM KCl, 0.5 mM CaCl2, 6 mM MgCl2, bubbled constantly with 95%

O2/5% CO2. Trimmed brain tissue was glued to a cutting chamber of a vibrating microtome (VT 1200, Leica). Coronal brain slices containing amygdala were cut to 300 µm thickness, and then incubated at 32 oC in artificial cerebral spinal fluid (ACSF): 130 mM NaCl, 30 mM NaHCO3, 3.5 mM KCl, 1.1 mM KH2PO4, 1.3 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose, 0.4 mM Sodium Ascorbate, 0.8 mM Thiourea, 2 mM Sodium

Pyruvate, saturated with 95% O2/5% CO2.

Electrophysiological Recordings After one-hour incubation in 32oC ACSF, individual slices were transferred into the recording chamber mounted on the fixed stage of a ZEISS microscope, maintained at 32 oC and perfused with ACSF at a flow rate of 2 ml/min. The recording chamber was grounded with an Ag/AgCl pellet. BLA pyramidal neurons were visually identified using IR-DIC in combination with a 40X water immersion objective. Some neurons were filled with biocytin (0.3%) via an internal patch solution to confirm localization in the BLA. All electrophysiological recordings were performed using a Multiclamp 700B amplifier with pClamp software version 11 and a DigiData 1550B (Molecular Devices).

All patch-clamp recordings were performed in standard ACSF solution. A P-1000 micropipette puller (Sutter Instrument) was used to pull patch pipettes from thick wall borosilicate capillary glass with a final resistance of 3-5 MΩ. For neuronal action potential recordings in current clamp, pipettes were filled with the following internal solution: 130 mM K gluconate, 2 mM KCl, 10 mM HEPES, 3 mM MgCl2, 2 mM K-ATP,

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0.2 mM Na-GTP, 5 mM Phosphocreatine Tris, the pH was adjusted to 7.3 with KOH, and osmolality was adjusted to 290 mosmol/L with D-Mannitol. Cells with a resting membrane potential less than -55 mV, or access resistance greater than 30 MΩ, or action potentials exhibiting no overshoot were excluded. The access resistance and bridge balance values were checked throughout the recording and cells with greater than15% changes were not included in data analysis. The values in current-clamp data presented in this thesis were not subtracted by the liquid junction potential, which was calculated as 11.4 mV. For current-voltage (I-V) relationship analyses, steady-state voltage changes were measured and averaged in a 400 ms period at the end of a 1 s current square pulse, and the input resistance (Rin) calculated from the linear part of the I-V curve near the resting membrane potential. The steady-state frequency of action potentials was obtained from the last 400 ms period of the depolarizing pulses, and plotted as the function of normalized current injection for f-I relationship. Gain was measured as the slope of the initial linear part of f-I curve. The recordings were obtained and low-pass filtered at 10 kHz and digitized at 50 kHz.

The following internal solution was used to record evoked EPSCs: 134.63 mM Cs- Methanesulfonate, 5 mM CsCl, 5 mM TEA-Cl, 0.4 mM EGTA, 10 mM HEPES, 2.5 mM Mg-ATP, 2.5 mM Na-GTP, 5 mM Phosphocreatine Tris, the pH was adjusted to 7.3 with CsOH, and osmolality was adjusted to 290 mOsm/kg with D-Mannitol. To analyze evoked EPSCs, BLA cells were held at -60 mV and a concentric bipolar stimulating electrode (CBAPC100 from FHC Inc.) was placed on the lateral amygdala (LA). Single pulses were generated with an S48 Stimulator via a Stimulus Isolation Unit SIU5 (Grass Instruments) at 0.05 Hz. In order to induce long-term potentiation (LTP), high-frequency stimulation (HFS) was applied at 100 Hz for 1s, repeated 5 times with an interval of 10s, while the cells were held at a membrane potential of +30 mV. EPSCs were recorded in ACSF containing PTX (100μM) and CGP52432 (1μM). Data acquisition was sampled at

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2 kHz and filtered at 50 kHz.

Two different approaches were used to examine the effect of BayK on synaptic plasticity: in one group of animals the induction of LTP was tested subsequent to the behavioural tests; in a second group, LTP recordings were performed without animals having been exposed to any behavioural experience (see Section 3.3.4).

Behavioral tests

All tests were performed during the dark period of the day/night cycle in a quiet symmetrically designed test room under dark conditions. Animal behavior was recorded with an overhead infrared camera (Amcrest ProHD 1080P camera; 1.5 m away above the test chamber). At the age of P28, the juvenile animals were examined in the following ASD related behavioral tests:

Three chamber test: assesses cognition in the form of general sociability and interest in social novelty in rodent models of CNS disorders; rodents normally prefer to spend more time with another rodent. Experimental animals were tested in a 0.89 m L x 0.48 m W x 0.4 m H Plexiglas box divided into three chambers (Malkova et al., 2012). Rats could freely move between chambers through a small opening (10 x 8 cm). At the beginning of the test, rats were placed in the center chamber for 10 minutes to explore the empty box in order to evaluate bias for either of the side chambers. Before the main part of the test, I confirmed that each animal had no evident side bias. At the end of the habituation session, a stranger rat of the same sex and close body weight was placed in a wire cage (12 cm height, 9 cm diameter) on one side chamber chosen randomly, and another empty cage was placed on the opposite side chamber as a non-social object. The time spent in each of the three chambers was measured, and the social preference was calculated as

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[timesocial chamber/(timesocial chamber + timenon-social chamber)] x 100 – 50. The test chambers were cleaned with 70% ethanol between tests.

Social interaction test: social interaction behaviors are employed in juvenile and adult rats for evaluating social motivation and non-play social behaviors (Vanderschuren et al., 2016; Pellis et al., 2018). Experimental animals were placed individually in the test box (0.89 m L x 0.48 m W x 0.4 m H Plexiglas box with an open-top) 10 minutes prior to the test for habituation. Then, an age-, sex-, and weight-matched stranger rat, which had not been housed together before the test, was placed in the test box. Social interaction behaviors of the two juvenile rats were recorded for 10 minutes. The frequency and duration of the following behavioral patterns from experimental animals were measured by a blind-to-treatment observer with a chronometer using the video recording: a) chasing/sniffing – the experimental rat follows the stranger rat and sniffs its back and genital area; this parameter evaluates social exploration. b) climbing - a dominant posture in which the experimental rat stands over the back or the exposed ventral area of the stranger rat and with its front paws presses the shoulder of the opponent against the floor; this parameter evaluates the ability to engage in active social interaction with conspecifics.

Self-grooming test: self-grooming can provide an index of repetitive/stereotypic behavior in rodents (Silverman et al., 2010). I tested individual rats in a glass beaker for 10 minutes as it is known that a restricted environment induces repetitive behavior (Lewis et al., 2007). The self-grooming behavior was evaluated in a 10 cm diameter x 15 cm tall glass beaker covered with a filter top. After 10 minutes habituation in the beaker, the number and duration of self-grooming were recorded for a further 10 minutes. All beakers were thoroughly cleaned with 70% ethanol between tests.

Open field test: was originally developed as a test for emotionality or locomotor activity

124 in rodents; and more recently it has been widely used for testing the level of anxiety. The animal was placed into the open field (0.4 m L x 0.32 m W x 0.3 m H Plexiglas box) and behaviors recorded over 10 minutes. Rodents typically prefer not to be in the center, and tend to walk close to the walls since the chamber is a novel, presumably stressful, environment to the animal. A decreased amount of time spent in the center area (0.18 m L x 0.14 m W) of the open field may indicate a higher anxiety level.

Data Analysis Electrophysiological data analysis was performed using Clampfit version 11 (Molecular Devices) and Origin version 2019 (OriginLab). For current-voltage (I-V) relationship analysis, voltages were measured and averaged at the end of the 400 ms current square pulse, and the input resistance (Rin) was calculated from the linear part of the I-V curve around the resting membrane potential. The steady-state frequency was obtained from the last 400 ms period of the current pulse. The amplitude of EPSCs was calculated as the peak current in the first 50 ms following the stimulating artifact. Data are reported as mean ± SEM. Statistical comparison was performed with paired Student’s t-test, one-way ANOVA, or non-parametric Kolmogorov- Smirnov test, as appropriate. Student’s t-test was used to analyze the significance between BayK and DMSO treatment groups in behavioral tests. Data from behavioral tests were compared between BayK and DMSO treatment groups with the same age and gender, and were analyzed by student’s t-test.

Drugs (S)-Bay K8644, nifedipine, D-AP5, CNQX, CGP52432, and biocytin were purchased from Tocris Bioscience. Picrotoxin (PTX) was purchased from Sigma Aldrich. (S)-Bay K8644, nifedipine, CNQX were dissolved in DMSO and stored at -20 oC before use.

Other drugs were dissolved in nanopure H2O. All drugs were applied by perfusion in the bath solution.

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3.3 Results

3.3.1 Bilateral injection of BayK at early post-natal ages produced long-lasting alterations in BLA that manifest in juvenile behavioral patterns. Several behavioral tests are commonly used in animal models of ASD, including the three-chamber test, social interaction and self-grooming test. The three-chamber paradigm, also known as Crawley’s sociability and preference for social novelty, has been successfully employed to study social affiliation in rodents (Kaidanovich-Beilin et al., 2011). As such, for testing whether BayK injection into the BLA affected behaviours in yound adult rats, I performed three-chamber test on P28 animals. I measured the ratio of the total time a subject rat spends in the chamber of a stranger rat over the time in chamber of the novel object. At P28, animals that had been treated with BayK at P14 showed an decreased ratio compared to DMSO treatment (Fig 3.3 B; middle bars, DMSO: 5.27±0.94, n=9; BayK: 2.48±0.55, n=11; p=0.022), whereas no significant differences were observed between BayK and DMSO treatment in either the P7 experimental group (DMSO: 3.49±0.36, n=9; BayK: 3.59±0.52, n=10, p=0.76) or the P21group (DMSO: 3.37±1.12, n=8; BayK: 3.71±0.61, n=8, p=0.67). Next, by measuring the time a subject rat spent in the chamber of stranger rat, there is a significant decrease induced by BayK treatment at P14 compared to DMSO treatment (Fig 3.3 C; middle bars, DMSO: 425.85±20.7, n=9, BayK: 338.27±27.7, n=11; p=0.13), no significant difference between BayK and DMSO treatment in the experimental group of P7 (DMSO: 400.2±19.1, n=9, BayK: 356.33±44.4, n=10; p=0.19) or P21(DMSO: 374±37.4, n=8, BayK: 393.46±36.9, n=8; p=0.72). Similarly for animals that received BayK treatment at P14, a corresponding increase was observed in the time spent in the chamber of the novel object (Fig 3.3D, DMSO: 102.9±15.3, n=9, BayK: 178.2±27.4, n=11; p=0.037). Again, no significant differences were found for the P7 (DMSO: 126.3±9.9, n=9, BayK: 131.8±15.2, n=10; p=0.76) and P21 groups (DMSO: 152±31.7, n=8, BayK: 133.7±24.5, n=8; p=0.66). The results indicate that at P14, but not P7 or P21, the injection of BayK 126 into the BLA results in impaired social motivation towards stranger rats and an increased interest in novel objects at an older age. The altered behaviours at P14 have been precviously suggested to resemble certain phenotypes observed in ASD patients.

Figure 3.3 BayK injection at P14 selectively produced long-term social affiliation impairment at P28

Schematics in panel A show the three-chamber apparatus with the white rat indicating the experimental subject and the yellow rat indicating the stranger. The social behaviours of the rats were measured by the time the experimental rats spent around the stranger, and/or the novel object (empty cap), as well as the time ratio. Bar graphs show that the animals treated with BayK at P14 have significantly lower stranger/object ratio (B, middle; DMSO n=9, BayK n=11) compared to DMSO treated rats. These animals spent less time exploring the stranger rat (C, middle), and more time around the object (D, middle). 127

Whereas animals that received BayK injection at P7 (B, C, C, left; DMSO n=9, BayK n=10) or P21 (B, C, D, right; DMSO n=8, BayK n=8) showed no significant differences in these parameters compared to DMSO. Data are presented as mean ± SEM. * P<0.05.

I next examined whether BayK injection during ealy development affected P28 rats with regard to multiple aspects of social interaction, including juvenile play and non-play social exploratory behavior using a social exploratory test. I measured the total duration a subject rat spent on chasing/sniffing a stranger rat, a typical pattern of social exploration. Also measured was the frequency of climbing up on the back of a stranger rat as an index of active social interaction (values are shown in Table 3.1). No significant differences were found between BayK and DMSO treatment in any of the experimental groups (Fig 3.4 and Table 3.1)

Table 3.1 Parameters in the exploratory social behavior test

The occurrence of repetitive behaviour was also examined (Fig 3.5 A). Animals treated with BayK at P7 showed a significant increase in self-grooming duration at P28 compared to DMSO treated animals (DMSO: 107.1±35.6 ms, n=9, BayK: 182.5±25.7 ms, n=11, p=0.046), indicating a higher repetitive behavior level. No differences were found in P14 or P21 experimental groups (P14, DMSO: 129.6±30.1 ms, n=9, BayK: 128

147.2±31.3 ms, n=10, p=0.35; P21, DMSO: 133.9.9±36.1 ms, n=6, BayK: 137.1±25.4 ms, n=7, p=0.77).

Taking into consideration that both human and animal studies indicate important roles for the amygdala in mediating fear and anxiety, and in the manifestation of anxiety disorders (Gina Forster, 2012), I examined the effects of BayK injection on long-term anxiety levels in juvenile rats (Fig 3.5 B). The results show that animals treated with BayK at P7 (DMSO: 62.5±12.3 ms, n=9, BayK: 35.9±5.1 ms, n=11; p=0.03) exhinit a higher level of anxiety at P28 compared to DMSO treatment. Whereas no significant differences were found in animals that received treatment at either P14 (DMSO: 61.9±19.5 ms, n=9, BayK: 44.8±8.5 ms, n=10; p=0.36), or P21 (DMSO: 42.1±14.6 ms, n=8, BayK: 33.9±6.9 ms, n=8, p=0.62).

Taking together, the results from behavioral studies at P28 following bilateral injection of BayK in rat pups showed distinct long-term alterations: BayK at P7 produced higher anxiety levels and repetitive behaviors, while BayK at P14 induced an impaired ability for social affiliation, together suggesting the involvement of LTCC activity in BLA circuits underlying these behavioral phenotypes. Interestingly, BayK injection into the BLA had no significant behavioural affects at P28 on any of the behavours examined. The changes observed for the P7 and P14 experimental groups suggest the existence of a vulnerable period during early development, while the lack of observable effects at P21 may reflect that neural circuits underlying these behaviouras have reached a stage where they are not sensitive to changes in LTCC activity.

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Figure 3.4 Exploratory social behaviors in an open field test were not affected by BayK treatment

Schematics show the test apparatus (A); after freely exploring the box for 10 min, the experimental rat (black) was allowed to interact with a stranger rat (green) for another 10 min. Bar graphs show that, relative to DMSO treatment, BayK treatment did not induce significant differences in any age group on sniffing/chasing behavioral duration (B), nor the duration of climbing on stranger’s back (C). Black bars indicate DMSO groups, while red bars represent BayK groups. Data are presented as mean ± SEM.

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Figure 3.5 Bilateral injection of BayK at P7 induced increased anxiety and enhanced repetitive behavior at an older age

Repetitive behavior was measured as the duration of self-grooming in the restricted space, as shown in the schematic (A). Bar graphs show that animals receiving BayK injection at P7 exhibited an increase in self-grooming behavior duration when test at P28 (A, left bars), whereas no differences were found in the P14 and P21 experimental groups (A, P14, middle bars; P21, right bars). Levels of anxiety were tested in an open field test and measured as the time spent in the centre area (B, upper panel). Animals with BayK treatment at P7 showed less time spent in the centre, indicating an increased anxiety level compared to the DMSO group (B, left bars). No differences were found in the other P14 and P21 experimental groups (B, P14, middle bars; P21, right bars). Data are presented as mean ± SEM. *p<0.05.

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3.3.2 BayK differentially affects long-term potentiation in BLA neurons at different postnatal stages. Long-term potentiation (LTP) of synaptic activity is the most widely studied form of synaptic plasticity and it is thought to play an important role in learning, memory formation, fear, stress and social activities (Nicoll, 2017; Jung et al., 2013; Leuner & Shors, 2013). Here, I examined whether BayK injection into the BLA at P7, P14 and P21 mediated changes at P28 in synaptic plasticity for excitatory inputs from the lateral amygdala into the BLA.

In the presence of GABA receptor blockers PTX and CGP52432, single pulse stimulation at low frequency, applied in the area of LA, elicited EPSC responses in BLA neurons were recorded as basal synaptic activity during a 5 min period (Fig 3.6 Left representative traces). In giving high-frequency stimulation (HFS: a 1-sec train of repetitive pulses at 100 Hz, repeated 5 times at 0.1 Hz), paired with membrane potential depolarization to +30 mV, an increase in EPSC amplitude was observed in BLA neurons from animals treated with BayK at P7 and P14. In contrast, LTP was not induced by the paired HFS in neurons from DMSO treated animals at either P7 or P14. The enhancement in synaptic responses lasted more than 30 min after the HFS. BLA neurons following BayK treatment at P7 showed a significant increase in EPSC amplitude over the approximate 30 min recording (Fig 3.6 A, normalized EPSC amplitude at 25 minutes: DMSO 0.94±0.31, n=3, BayK 1.70±0.34, n=5, p=0.025). Similar LTP was also induced in neurons from animals treated with BayK-P14 (Fig 3.6 B, normalized EPSC amplitude at 25 minutes: DMSO 1.04±0.29, n=5, BayK 1.92±0.27, n=5, p=0.019). No LTP was found to occur in neurons from either the DMSO or BayK P21 groups (Fig 3.6 C, normalized EPSC amplitude at 25 min: DMSO 0.96±0.25, n=4, BayK 1.12±0.26, n=4, p=0.35).

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Figure 3.6 Bilateral BayK injection at P7 and P14 induce LTP in BLA neurons at P28

Schematic (left) shows the recording strategy for measuring LTP in BLA neurons. The stimulating electrode was placed in the LA while the whole-cell patch clamp recoding pipette was on a BLA neuron. Single EPSC responses were elicited by giving stimulations at 0.05 Hz; a high-frequency stimulation (HFS) at 100 Hz (1s duration, repeat 5 times at 0.1 Hz, membrane potential was holding at +30 mV) was used to induce LTP. The representative traces in each panel show single EPSCs before (left) and 25 min after (right) HFS. Black traces represent recordings in DMSO groups; red traces represent recordings in BayK groups. LTP was induced in the animals that received BayK treatment at P7 (A) or P14 (B) by approximately 2-fold compared to their DMSO counterparts. Notably, no LTP was observed from animals treated with BayK at P21. Data are presented as mean ± SEM.

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3.3.3 Bilateral injection of BayK at P14 modified passive electrophysiological properties of BLA neurons recorded at P28 I next examined whether postnatal BayK injection resulted in any long-lasting effects on the basal electrophysiological properties of BLA principal neurons. Resting membrane potential and input resistance are essential factors contributing to tuning neuronal excitability. Here, I utilized whole-cell patch-clamp techniques to examine the passive properties of BLA neurons at P28. The current-voltage (I-V) relation (Fig 3.7B upper panel), an important characteristic of the cell membrane for encoding stimuli, shows that when BayK treatment was applied at P14, that P28 BLA neurons display a significant increase in steady-state voltage responses towards hyperpolarizing currents, indicating a reduction in the inward rectification in the I-V relationship, together with a trend towards increased input resistance although not statistically significant (Fig 3.7B,lower panel, left; DMSO: 77.1±21.3 MΩ, n=5; BayK: 128.9±40.6, n=10, p=0.12; see Table 3.2). These data suggest that 14 days post-BayK injection BLA neurons would tend to exhibit larger membrane potential responses compared to DMSO treatment when receiving a given current stimulus. Also following BayK injection at P7, the resting membrane potential was found to be more significantly depolarized (Fig 3.7 B, lower panel, right; DMSO: - 59.7±0.4 mV, n=5; BayK: -55.1±1.3, n=10, p=0.037; Table 3.2), making it closer to the action potential threshold. In contrast, animals that received BayK treatment at either P7 or P21 did not show significant differences in BLA neuron passive properties at P28 compared to the DMSO group. For animals treated at P7 and recorded at P28, the input resistance = 94.5±10.6 MΩ (n=5) in the DMSO group, and 130.0±37.5 MΩ (n=7) in the BayK group (Fig 3.7A, table 3.2, p=0.51). The resting membrane potential showed no significant difference between groups (DMSO: -58.4±1.1 mV, n=5; BayK: 53.8±2.1; n=7, p=0.15). Similarly, rats that received injections at P21 showed no significant difference between BayK and DMSO treatment groups in input resistance (Fig 3.7A, Table 3.2, DMSO: 80.67±8.8 MΩ, n=6, BayK: 84.8±8.1 MΩ, n=6, p=0.92) or resting membrane potential (DMSO: -55.9±0.8 mV, n=6, BayK: -55.5±0.8 mV, n=6, p=0.68).

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Table 3.2 Parameters of the passive properties of BLA neurons

Data are presented as mean ± SEM. *p<0.05.

Figure 3.7 Bilateral injection of BayK at P14 produced long-lasting alterations in the I-V relationship and resting membrane potential recorded at P28

Current-voltage (I-V) relationship plots corresponding to each experimental group are shown in the upper panels. (A, P7; B, P14; C, P21). I-V curves were analyzed to calculate

135 input resistance (Ro); representative traces in the middle panel show voltage responses to hyperpolarizing current pulses; mean values for each experimental group are shown as bar graphs in the lower panels (left). The averaged values of resting membrane potentials are shown on the right of each lower panel. The group treated with BayK at P14 displayed a significantly depolarized resting membrane potential (B, lower panel right) with no significant change in the input resistance (B, lower panel left) compared to DMSO control group (Panel B). No obvious changes were found in the I-V relationship, input resistance, or resting membrane potential in groups treated with BayK at P7 (A) or P21 (C). Data are presented as mean ± SEM. * P<0.05.

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3.3.3 Neuronal excitability increased in animals that received BayK injection at P7 To examine the effect of postnatal BayK injection on neuronal excitability, I analyzed the f-I relation (steady-state firing frequency versus current injection) in BLA neurons at P28. Compared to DMSO, in animals treated with BayK at P7 the BLA neuron steady-state maximal frequency was higher across the entire range of current injection tested (Fig 3.8 A) without showing a difference in gain (DMSO: 0.16±0.08 Hz/pA, n=10; BayK: 0.18±0.1; n=10, p=0.53) which indicates that neuronal sensitivity in response to electrical stimuli is not affected. The rheobase was not significantly changed (DMSO 65±12.6 pA, BayK 66.7±24 pA; p=0.95). In contrast, animals that received BayK treatment at P14 or P21 did not show significant differences in the f-I relation (Fig 3.8 B, C), gain values (P14 group, DMSO: 0.20±0.09 Hz/pA, n=8; BayK: 0.18±0.08 Hz/pA, n=6, p=0.68; P21 group, DMSO: 0.15±0.09, n=4, BayK: 0.17±0.07; n=4, p=0.26), or rheobase (P14 group, DMSO: 83.3±14.7 pA, n=8; BayK: 75.6±15.9, n=6, p=0.74; P21 group, DMSO: 76.6±24.9 pA, n=4; BayK: 79.2±19.3 pA, n=4, p=0.69), compared to corresponding DMSO control group. These results indicate that the firing frequency response of P28 BLA neurons is selectively sensitive to BayK treatment at P7.

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Figure 3.8 BayK injection at P7 produced a long-lasting increase in the firing response of P28 BLA neurons as indicated by the f-I relation

The frequency-current (f-I) relationship was analyzed as the steady-state firing frequency as a function of current injection (upper panels). Representative traces in lower panels

138 show a train of action potentials elicited by depolarizing square pulses of different intensity. Only animals with BayK treatment at P7 show an increased steady-frequency across all current injection range, compared to the DMSO control group (A). No obvious changes were found in the animal groups with BayK treatment at P14 (B) or P21 (C). Data are presented as mean ± SEM.

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3.3.4 BayK-associated induction of LTP in the LA to BLA synapse is enhanced but not determined by prior behavioural experience. It is known that environmental novelty and/or the exposure to behavioral tests may impact the subsequent induction of LTP (Roman et al., 1999; Moncada & Viola, 2007; Eckert & Abraham, 2010). In a previous study on the valproate rat model of autism, the induction of LTP at thalamic inputs into the lateral amygdala was reported to occur after behavioral experiments measuring fear conditioning and extinction (Lin et al., 2013), however similar results were observed in the absence of any preceding behavioural tests. Here, I asked whether the LTP observed in animals treated with BayK could have occured as a consequence of the behavioural experience itself during assessment of behavioral traits, and also if treatment itself could cause permissive functional changes for LTP to occur in the intra-amygdala circuit. I performed patch-clamp recordings on P28 animals that received BayK or DMSO injections at P14 without any behavioral tests or environmental disturbance prior to the recordings. While the data show some quantitative differences, overall, they indicate that BayK treatment alone is sufficient to produce functional changes in LTP. Compared to the DMSO treatment group, BLA neurons from animals treated with BayK displayed reduced inward rectification on the I-V curve (Fig 3.9 A) and an upward shift in the f-I relationship (Fig 3.9 B) but with no change in input resistance (Fig 3.9 C). The data also indicate that without performing behavioral tests, the effects of BayK on LTP induction (Fig 3.9E, normalized EPSC amplitude at 25 minutes: DMSO 0.85±0.1, n=4, BayK 1.54±0.18, n=4, p=0.027) and depolarizing the resting membrane potential (Fig 3.9 D, -57.8±2.1 mV in DMSO vs. -55.4±0.9 mV in BayK) were more moderate compared to the results following behavioral testing (Fig 3.6 B, normalized EPSC amplitude at 25 minutes: DMSO 1.04±0.29, n=5, BayK 1.92±0.27, n=5, p=0.019; Fig 3.7, -59.7±0.4 mV in DMSO vs. -55.1±1.3 mV in BayK), which is consistent with previous findings that the behavioral experiences can modify synaptic plasticity. My data could also serve as additional evidence that LTCC-driven activity changes within BLA circuit are also sufficient to induce LTP.

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Figure 3.9 Changes in synaptic strength induced after BayK treatment without preceding behavioral tests

Data from P28 animals treated with BayK or DMSO at P14. The I-V relationship shows a downward shift in BayK group compared to DMSO (A). Panel B shows no significant change in the f-I curve between BayK and DMSO treatment. A similar result is shown in panel C for input resistance. Input resistance was measured at the linear part of the I-V curve near the resting membrane potential. BayK slightly depolarized the resting membrane (Fig 3.9 D). High frequency stimulation paried with postsynaptic depolarization induced LTP that was significantlt different between BayK and DMSO control groups (Fig 3.9 E). Data are presented as mean ± SEM. *p<0.05, ‡p<0.01.

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3.3.5 BayK injection does not produce long-term change in CREB and BDNF expression. The results from Chapter Ⅰ indicate that the acute effects of BayK treatment on BLA neurons are not dependent on relative different levels of calcium channel expression at the different postnatal stages. Here, I examined long-term effects of BayK injection on gene expression. Samples from coronal brain slices containing the BLA were collected using micro-punches from P28 rats that had previously received BayK or DMSO treatments. mRNA was extracted and reverse transcribed into cDNA for real-time quantitative PCR analysis. Cav1.2 and Cav1.3 mRNA expression was calculated relative to GAPDH expression. The results show no difference between DMSO and BayK experimental groups in animals treated at P7 (Fig 3.10A and B) (Cav1.2 mRNA expression level: DMSO 0.0016±0.0002, BayK 0.0014±0.0002, n=6, p=0.16; Cav1.3 mRNA expression level: DMSO 0.0027±0.0002, BayK 0.0023±0.0003, n=6, p=0.32),

P14 (Cav1.2 mRNA expression level: DMSO 0.0011±0.0002, BayK 0.0011±0.0004, n=5, p=0.53; Cav1.3 mRNA expression level: DMSO 0.0023±0.0002, BayK 0.0021±0.00004, n=5, p=46), or P21 (Cav1.2 mRNA expression level: DMSO 0.0013±0.0002, BayK

0.0015±0.0001, n=4, p=0.36; Cav1.3 mRNA expression level: DMSO 0.0026±0.0002, BayK 0.0023±0.0001, n=4, p=41).

Cav1.2 and Cav1.3 LTCCs are responsible for the regulation of gene expression through various intracellular signalling pathways, including cAMP response element-binding protein (CREB) (Dolmetsch et al., 2001). Here, CREB mRNA expression in the BLA (Fig 3.10 C) showed a tendency to increase in animals treated with BayK at P7 (DMSO: 0.0013±0.00007, BayK: 0.0017±0.0001, n=6, p=0.09) and P14 (DMSO: 0.0016±0.0003, BayK: 0.0024±0.0003, n=5, p=0.06) compared to DMSO treatment; however, the differences were not statistically significant. The trend was not observed at P21 (BayK (0.0012±0.0001, n=4), DMSO (0.0013±0.0002, n=4; p=0.38).

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Brain-derived neurotrophic factor (BDNF) is a major downstream signaling component of CREB-dependent pathways, with an important role in neurodevelopment and responsible for the regulation of long-term synaptic plasticity (Miranda et al., 2019). The results show that BDNF mRNA expression levels (Fig 3.10 D) were not statistically different between animals treated with BayK at P14 compared to DMSO treatment (DMSO: 0.0036±0.0005, BayK: 0.0047±0.0002; n=5, p=0.07). In animals treated at P7 and P21, the differences between BayK and DMSO group were subtle yet also not statistically different (P7, DMSO: 0.0035±0.0005, BayK: 0.004±0.0005; n=6, p=0.27; P21, DMSO: 0.0046±0.0002, BayK: 0.0041±0.0003, n=4, p=0.64).

Figure 3.10 BayK injection did not induce long-term alterations in the mRNA expression of LTCCs, CREB and BDNF

143 mRNA expression levels of the genes were analyzed as values relative to GAPDH. Bar graphs show that BayK injection at P7 (n=6), P14 (n=5), or P21 (n=4) did not induce any significant changes in the mRNA expression levels of Cav1.2 (A), Cav1.3 (B), CREB (C), or BDNF (D) genes. Data are presented as mean ± SEM.

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3.4 Discussion

It has been suggested that early disruption in the development of medial temporal lobe structures, including the amygdaloid complex, could affect neuroanatomical and neurochemical organizations as well as the functioning of subcortical and cortical areas, such as the neostriatum, basal forebrain, and prefrontal and parietal cortices, which share strong anatomical connections with medial temporal lobe structures (Bachevalier, 1994; Hermann et al., 2002). Anatomical studies have revealed the disruption of the normal neurodevelopmental pattern in patients with ASD (Courchesne, 1997). On the other hand, ASD is mainly characterized by deficits in communication and the understanding of others’ minds involving emotional expressions thought to be related to the atypical function and structure of the amygdala. The performances of people with ASD on emotional recognition tasks resemble those seen in people with acquired amygdala damage in early life (Baron-Cohen et al., 2000a; Howard et al., 2000). In addition in rats, prenatal stress has been shown to alter the development of socioemotional behavior and amygdala neuron excitability (Ehrlich & Rainnie, 2015). Together, these studies support my hypothesis that disturbance of the amygdala during the early neurodevelopment could produce long-lasting alterations in amygdala-related physiological processes, and might help towards understanding the contributions of the amygdala in the pathological mechanisms of ASD.

To examine electrophysiological development of the amygdala, Erlich and colleagues used whole-cell patch clamp techniques to study changes in excitability and passive properties of BLA neurons in rats during postnatal development. According to their results, BLA neurons are at an immature stage from birth until ~ P7 wherein a rapid developing period around P14 occurs and is followed by maturation around P21 (see Fig 3.1). These studies provide for the notion that a suitable critical time window to test for the role of LTTCs in the amygdala circuit and ASD-relevant behaviours fall between P7

145 and P21. The results of Chapter Ⅰ here indicate that a functional increase of LTCC activity induced by acute application of BayK at P7 leads to multiple different alterations of BLA neuronal properties compared to that observed for BayK-treated P21 neurons (Zhang et al., 2020), reinforcing the idea of a vulnerable period of BLA neurons early during development. This hypothesis is also supported by the valproic acid rat model, which involves injecting valproic acid into pregnant rat uterus at gestation day 12.5 to interfere with the central neural tube formation and causing autistic behaviors in her offsprings (Ingram et al., 2000). In order to test the functional consequences of altering the BLA development by manipulating the function of LTCCs, I chose three time points: two turning points at P7 & P21 and the fastest developing period, P14.

Studies have also shown that the amygdala may contribute to psychopathological diseases such as schizophrenia. Several animal models have been established for the investigation of these mood disorders. For instance, ibotenic acid-induced amygdala lesions at P7 resulted in a variety of behavioral impairments later in life, whereas no alterations were found in animals that received lesions at P21 (Wolterink et al., 2001), indicating P7 serves as a potentially critical time point for amygdala-related developmental processes and for studying neurodevelopmental disorders.

Although my data here show that acute application of BayK around the critical period of development produced differential changes in neuronal excitability and synaptic transmission depending upon age, the excitability and synaptic ability alterations in the amygdala related behaviors in vivo has not been explored. As such, it is important to examine the contributions of LTCCs towards excitability and neurotransmission around the critical period in a neurodevelopment disorder-related animal model. In establishing the pharmacological bilateral BLA injection rat model at early developmental stages, my

146 results showed multiple long-term alterations in BLA neuronal excitability, synaptic plasticity, as well as behavioral phenotypes (discussed below).

First, investigations at the behavioral level showed that for the three-chamber test, animals receiving bilateral BLA injection at P14 exhibited a significant decrease in time spent with a stranger rat, and increased time with a novel object, together indicating impairment in social affiliation abilities. In contrast, animals receiving BayK treatment at P7 or P21 did not show any significant differences compared to DMSO control groups. Therefore, P14 appears to serve as a crucial time point for the development of social behaviors within BLA circuits.

The social interaction test investigates social interest and exploratory behaviors from the analysis of chasing/sniffing, while climbing is an indication of inviting for play(Vanderschuren et al., 2016; Pellis et al., 2018). The results did not reveal any long- term effects of BayK treatment at any postnatal age compared to DMSO controls. Further behavioral paradigms are likely required for the determination of any involvement of the amygdala circuit concerning these social behaviors. Pinning, defined as pressing the recipient animal against its ventral bodyside, has also been seen in a small fraction of experimental animals in my study, but the sample size was not sufficient for proper analysis. Repetitive behavior is one of the main phenotypes of ASD patients. I evaluated the self-grooming behavior of rats when confined in a restricted space. The data showed that animals receiving bilateral BLA injection at P7 exhibited increased self-grooming duration over the 10 min recording period compared to its control group. No significant differences were found in the other groups suggesting that BLA circuitry is involved in the development of repetitive grooming behaviors, and that P7 is a vulnerable time point

147 towards this development. The amygdala is traditionally thought to play a role in regulating anxiety thus I performed an open field test for the investigation of anxiety levels. Results indicated that rats with BayK treatment at P7 showed a decreased time spent in the centre area of the open field compared to the DMSO control group, indicating an increased anxiety level, and suggesting the involvement of BLA in the neuronal circuits of anxiety around P7. Again, no significant differences were found for the other groups. Taking together, the results from the behavioral tests support the hypothesis that the functional increase in LTCC activity at certain postnatal critical periods during early neurodevelopment can induce long-term changes in behaviors previously shown related to ASD phenotypes.

To date, the cellular mechanisms underlying many animal behaviors, including learning and memory formation, emotional expression and response, remain some of the most intriguing unknowns. What are the long-term consequences of the functional increase in LTCC activity at critical developing periods and how might these relate to behavioral phenotypes? Numerous studies have suggested that long-term synaptic plasticity at excitatory synapses is the most likely mechanism that underlies many behaviors (Kolb & Whishaw, 1998; Di Filippo et al., 2009). In some instances, LTP is shown induced in the pathway transmitting sensory inputs from the thalamus to the amygdala during fear conditioning (Li et al., 1998; Huang & Kandel, 1998). Interestingly, this thalamo- amydala LTP was found independent of NMDA receptors and instead dependent upon LTCCs (Weisskopf et al., 1999b). Blockade of LTCCs during the conditioning stimulus completely abolished LTP of the TH response to elevated KCl, suggesting that LTCCs play a crucial role in the activity-dependent plasticity of transmitter receptor expression in sensory neurons. Further, exposure to depolarizing stimuli during early development may alter neuronal response properties at later ages (Brosenitsch et al., 1998). From these studies, I hypothesized that a functional increase in LTCC activity in the BLA at an early

148 postnatal stage could affect the induction of LTP which in turn underlie behavior alterations.

Previous studies have shown that induction of LTP in BLA neurons ranges from 30% to 50% (Li et al., 1998; DeBock et al., 2003). Many of these studies were performed under ACSF conditions without the GABA receptor blocker PTX and which has proven to enhance the excitatory synaptic strength as well as LTP induction (Gustafsson et al., 1987; Ko et al., 2014). In my recordings with PTX present, I found that animals with BayK treatment at P7 or P14 showed a roughly 2-fold increase in EPSC amplitude after the HFS compared to the DMSO control group, and that both of the HFS-induced amplitude increase lasting longer than 30 minutes. In animals that received bilateral BLA injection at P21 LTP could not be induced at P28. These results indicate that increased LTCC activity at P7 and P14, but not P21, produced a long-lasting effect on the long-term synaptic plasticity at excitatory synapses in BLA, and that correlated with the behavioral alterations that found in these two experimental groups.

In various animal models of developmental diseases researchers have demonstrated potential contributions of neuronal excitability towards the underlying mechanisms of neurodevelopment. For instance, in mouse models of ASD: the Mecp2 models of Rett syndrome and the Met-knockout model, both alterations in intrinsic neuronal excitability and changes in synaptic strength were demonstrated (Shepherd & Katz, 2011). Similarly, in my bilateral BLA BayK injection model, significant changes in multiple neuronal electrophysiological properties were observed in the animals receiving BayK treatment at P7 or P14. These include the input-output relationship (f-I curve), resting membrane potential, as well as the I-V relationship. These alterations in neuronal properties and excitability are likely to affect the development and maturation of BLA circuits, which in turn might account for the observed abnormalities in LTP induction and behavioral 149 phenotypes. It is possible that the distinct long-term effects displayed in the different experimental groups (P7 vs. P14 group) might be associated with specific contributions of LTCCs towards the underlying behavioral phenotypes examined.

Gene expression analyses showed no significant difference in the mRNA expression levels of LTCCs in the BLA between BayK and DMSO treatment groups, suggesting that the long-term effects of BayK were a direct result of changes in overall LTCC expression. Similarly, no significant differences in the mRNA expression levels of CREB and BDNF in BayK treated versus DMSO control animals at either P7 or P14 were observed. However, these data cannot rule out any potential BayK-driven changes in LTCC protein expression or in alternative splicing. Long-term functional alterations affected by BayK such as phosphorylation levels could also affect neuronal excitability and synaptic transmission.

I lastly examined whether the observed effects of BayK treatment on synaptic strength and neuronal excitability themselves affected by environmental stimuli and behavioral testing. It has been shown in animal behavioral studies that a weak stimulus, such as a brief behavioral training, can induce a transient form of short-term memory (Moncada et al., 2011). Further, when this weak stimulus is associated with exposure to a novel environment, it can lead to the formation of longer-term memory. This process is known as behavioral tagging and its underlying mechanisms involve the synthesis of specific synaptic plasticity-related proteins important for LTP (Moncada & Viola, 2007; Moncada et al., 2011). Here, I tested groups of animals that were not previously exposed to environmental novelty or behavioral testing prior to the electrophysiological testing and found that animals that received bilateral BLA BayK treatment successfully elicited LTP with a statistically significant amplitude increase (~156%). The magnitude of LTP was however smaller compared to the groups that also underwent behavioral tests. The data

150 provide evidence that, 1) BayK administered to the BLA indeed produces long-term change in synaptic plasticity, and 2) the behavioral tagging phenomenon triggered by behavioral tests prior to the electrophysiological recordings can impact the induction of LTP.

Overall, my results support the idea that manipulations affecting LTCC activity at sensitive time points (P7 and P14) within the critical period of postnatal BLA development can produce long-lasting effects in adolescent life. The data also demonstrate the potential contribution of LTCCs to the development and maturation of BLA circuitry, as well as the functional involvement of the BLA circuits in multiple social and non-social behavioral phenotypes.

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4 Conclusion

4.1 Summary and discussion of research findings The ideas of this dissertation were initially inspired by the observation of the similarities between the gain-of-function TS mutations and the effects of BayK on the biophysical properties of LTCC currents. Mutations in LTCCs are relatively rare compared to other

VGCC families, partially due to the high expression and functions of Cav1.2 and Cav1.3 in the cardiovascular system where mutations affect crucial heart functions. The discovery of the Cav1.2 G406R mutations that result in Timothy Syndrome opened the door for investigating the functional contributions of LTCCs in neurological disorders, including early neurodevelopmental disorders such as ASD.

According to studies over the past couple of decades several brain regions are implicated in ASD, including the prefrontal cortex, thalamus, cerebellum and the amygdala (see Fig 1.4) (Amaral et al., 2008). These brain regions display multiple morphological and/or functional alterations across human patient and animal models. Amongst these, the amygdala complex, specifically the basolateral amygdala is a relatively new hotspot to be considered as a core of ASD-related neural networks that underlie social behaviors (Baron-Cohen et al., 2000a; Amaral et al., 2003). Furthermore, the structure, efferent/afferent connections, as well as the neuronal composition in BLA area, make it relatively straightforward to examine compared to some other brain regions. Together with the known endogenous expression of LTCCs (Karst et al., 2002b), the BLA was chosen to investigate the roles of LTCCs in network functioning during development.

To start, I utilized whole-cell patch clamp techniques to test the effects of the LTCC agonist BayK on the electrophysiological properties of BLA principal neurons. To investigate the roles of LTCCs during different developmental stages, I focused the initial 152 study on P7 and P21 BLA neurons, as they display distinct excitability features and represent different stages during the maturation (Ehrlich et al., 2009, 2013, 2013; Ehrlich & Rainnie, 2015). I found that BayK not only increased the excitability of P7 neurons by altering firing frequency and gain, but also induced various distinct effects on P7 neurons such as plateau potential and firing frequency adaptation. Burst firings were primarily observed in P21 neurons and were enhanced by BayK. These results suggest that: 1) a functional increase of LTCC activity enhances BLA neuronal excitability, 2) changes in neuronal excitability induced by BayK could potentially alter how BLA neurons integrate synaptic activity and subsequently, shift the input/output balance within the amygdala circuitry. For instance, the BayK-induced high firing frequency response at lower stimulation range, reduced gain, as well as the loss of firing frequency adaptation, together with reduced the ability of P7 BLA neurons to differentiate between input stimuli of different intensities. However, BayK increased the sensitivity of BLA neurons in response to low frequency or “weak” excitatory input. This could result in an attenuation of filtering ability allowing increased excitatory signals to pass through the BLA, 3) in both P7 and P21 neurons the increase in the frequency of spontaneous inhibitory GABAergic neurotransmission was induced by BayK. Since the amygdala is considered as a gatekeeper to filter somatosensory input in brain circuitry, one might hypothesize that enhanced inhibition in the amygdala could suppress its filtering ability resulting in an increased overall brain synaptic activity, and 4) the contribution of LTCCs to neuronal excitability is different and versatile in immature neurons compared that in more mature neurons, indicating that LTCC could serve as a potential target in early developmental period to manifest the maturation and plasticity of BLA circuitry (see Chapter Ⅱ of this study).

Upon demonstation of involvement of LTCCs on BLA neuronal excitability, I next asked whether a onetime induction of these gain-of-function effects at specific early stages

153 could produce long-lasting alterations. And if so, whether any long-term alterations led to behavioral changes. This hypothesis was indirectly supported by the classic valproic acid rat model, which involves onetime pharmacological treatment at a specific time point during the embryonic stage (Bringas et al., 2013), and also the schizophrenia rat model which involves ventral hippocampal lesion at P6 (Tseng et al., 2009). The offspring of these modeled animals display altered social interaction behaviors, anxious behaviors and learning and memory impairment. To achieve my goal, the approach of a single stereotaxic injection of BayK into BLA area was employed. The injection was performed bilaterally to evade the issue of amygdala lateralization. One critical aspect of this model is the injection time point. According to previous studies on BLA morphology and electrophysiological property development, I chose to perform BayK treatment at either P7, the immature stage and the starting point of BLA development, or P14, the rapid developing period, and P21, which close to the mature stage (Ehrlich et al., 2009, 2013, 2013; Ehrlich & Rainnie, 2015). After bilateral injection and recovery, juvenile rats at P28 were used to perform a series of tests, including multiple behavioral tests, gene transcription level examination and electrophysiological recordings on BLA slices. In order to correlate the results of behavioral tests to gene expression and BLA neurons functions, as well as to reduce animal usage, each experimental animal underwent behavioral tests one or two days prior to the subsequent experiments.

Animals treated with BayK displayed an upward shift in the f-I relationship (P7 group) compared to DMSO control, indicating higher neuronal excitability and suggesting that the conductances involved in generating and shaping action potentials, such as voltage- gated ion channels, calcium-activated SK or BK channels, are likely affected by BayK injection. For the P14 group BayK induced a reduction inward rectification compared to control, suggesting that the performance of membrane conductances underlying inward rectification, such as IH and/or rectifier potassium channel (IKIR) (Tanaka et al., 2003), are

154 possibly modified by BayK treatment. BayK is unlikely to directly affect these targets and it is more likely that BayK injection affects gene expression to accomplish the long- lasting changes in firing frequency response and the I-V properties. Homeostasis is a mechanism that regulates and maintains neuronal overall excitability and prevents neural activity from being driven towards hypo- or hyperactive states. One potential homeostatic mechanism is the adjustment of synaptic strength to maintain balanced excitability. LTCCs are critical for the expression of neuronal homeostasis plasticity. For instance, chronic treatment of hippocampal cultures with the LTCC antagonist nifedipine reduces GABAergic synaptic transmission (Saliba et al., 2009). Whereas long time excited CA1 pyramidal neurons by optogenetic techniques result in a homeostatic down-regulation of both NMDAR and AMPAR-mediated responses in a process that can be occluded by nifedipine (Goold & Nicoll, 2010). I hypothesize that the BayK-induced long-term changes in neuronal excitability might involve a shift or impairment in homeostasis plasticity during early development. Together with the fact that the application of BayK onto BLA neurons changes the spontaneous synaptic activity (Chapter I), I speculate that the functional increase of LTCC activity at critical periods leads to profound alterations in neuronal homeostatic plasticity which in turn impairs the maintenance of normal physiological balance. The impairment of homeostasis in early development likely drives to long-lasting pathological changes observed. As neuronal homeostasis is thought to be counteracted by synaptic plasticity (Turrigiano & Nelson, 2000, 2004), such as LTP, this impairment of homeostasis might be further affected by the BayK-induced enhancement in LTP.

Neuronal activities can lead to long-term synaptic plasticity changes, such as LTP and LTD, to destabilize the activity of neural circuits and form a new homeostatic balance (Turrigiano & Nelson, 2000, 2004). LTP in the lateral amygdala has been suggested to serve an important role in regulating fear memories. In my study, BayK-injection

155 experimental groups spanning the critical period (P7 and P21) displayed LTP with a 2- fold increase in EPSC amplitude. In the amygdala LTCCs are known to contribute to LTP. For instance, LTP induced from a high-frequency tetanization or pairing of presynaptic and postsynaptic inputs require LTCCs (Magee and Johnston, 1997). In the LA, calcium influx through LTCCs is necessary for LTP at thalamic input synapses (Weisskopf et al., 1999b). Similarly, in my study the functional increase of LTCC activity might play an important role concerning the induction of LTP in BLA neurons and affect the development and/or maintenance of balanced of synaptic plasticity.

Several studies have shown that amygdala-related long-term memory and fear conditioning are associated with LTP and that LTCC blockade selectively impairs the behavioral conditioning thus indicating a role of LTCC-dependent LTP in shaping animal behavioral phenotypes (Bauer et al., 2002; Davis & Bauer, 2012b). Indeed, Chapter II of my study shows that when animals are treated with BayK at P7 and tested in a series of behavioral trials at P28, they display increased anxiety and repetitive behavior compared to control vehicle treatment groups. The data support the notion that LTCCs likely contribute to regulating the formation of neural circuits underlying anxious and repetitive behaviors during a critical developmental period. These data also suggest the circuits underlying these behaviors have already emerged and entered a vulnerable period around P7.

Animals that received BayK at P14 did not show any significant effect on anxiety or repetitive behaviors compared to the DMSO control group. Rather, they displayed reduced social interest towards a stranger rat in the three-chamber test. This suggests an emerging and critical developmental period of the circuits underlying social affiliation behaviors around P14. The developmental time windows of different brain nuclei and neural circuits that underlie different behavioral phenotypes are suggested to follow a

156 specific sequence. For example, in the immature cortex, early network oscillations of a large calcium wave occur between P0 and P7 (Garaschuk et al., 2000). These oscillations are primarily driven by NMDA/AMPA receptors, and then from P7 recurrent patterns of large synaptic activity known as giant depolarizing potentials are driven by GABAA- mediated conductances. These synaptic activities during the first two postnatal weeks are essential for morphological and functional maturation of neurons and the establishment of networks, which are thought to begin to both encode sensory and motor behaviors and for subsequent integrative behaviors such as social behavior. LTCCs have been suggested to be involved in neurogenesis in early neurodevelopment. For example, LTCCs were shown to regulate the conversion of adult hippocampal neural precursors into immature neurons and to subsequently contribute to different forms of hippocampus-dependent learning and memory (Deng et al., 2010; Moon et al., 2018). I hypothesize that the contribution of LTCCs in neurogenesis in different stages of the developmental sequence might partially explain the distinct long-lasting effects of BayK on the multiple behavioral phenotypes when injected at different critical time points.

Animals treated with BayK at P21 did not show any significant differences compared to the vehicle control group in terms of behavior, BLA cell excitability, or long-term synaptic plasticity. It is known that sensorimotor behaviors, learning capability, spatial memory, as well as communication via ultrasonic vocalizations in rat pups develop and matures during the first two weeks after birth (Wood et al., 2003). As such, the results here confirm my hypothesis that if a disruption from normal does not occur during the critical period of development then a disturbance of cellular plasticity is not sufficient to produce long-lasting alterations in the system.

4.2 Research significance and potential limitations Neuronal LTCCs have largely been thought to contribute to the regulation of synaptic plasticity-related to calcium-dependent gene transcription (Perrier et al., 2002b). 157

However, accumulating evidence suggests additional roles concerning LTCC involvement in regulating excitability. For instance, BayK was found to induce a plateau potential in rat substantia nigra GABAergic neurons (Lee & Tepper, 2007a), and to significantly increase the slow AHP amplitude and a reduction in γ–frequency oscillation in mice hippocampal neurons (Driver et al., 2007). In chapter 2 I employed brain slices to investigate the functional roles of LTCCs in BLA neuronal properties. My investigations revealed the contribution of LTCCs towards multiple neuronal electrophysiological properties, including firing frequency adaptation, burst-firing, spontaneous firing, and spontaneous neurotransmission. Together, these data provide evidence that LTCCs play important roles in gating the mechanisms of neuronal excitability at neuronal level during early BLA development.

Previous single-channel recordings showed that BayK promotes Cav1 channel switching from gating mode 1, characterized by low open probability and short open time, to gating mode 2, with a higher open probability and longer open times (Hess et al., 1984). The significance of using BayK to mimic the impact TS mutations on Cav1 channels was further supported by a recent study in which the cryo-EM structure of the Cav1.2 channel was modeled to investigate the impact of TS mutations, G402S and G406R, on the structure and conformational changes of the channel (Korkosh et al., 2019). The study reveals that the α1-interaction domain (AID)-linked IS6 would bend at the flexible G402 and G406 residues, facilitating the activation gate closure. Modelling predcited that the TS mutations stabilize the open state and resisting pore closure, generally equivalent to the action of BayK application.

Contributing to our current poor understanding of the underlying mechanisms of ASD is the lack of animal models that mimic specific behavioral features of clinical ASD. Current pharmacological models such as valporic acid rodent model also affect the

158 endocrine and immune systems, while genetic rat models that integrate extraneous cassettes into LTCC genes potentially affect their expression in vivo. In the BLA bilateral injection rat model of my study, LTCC functional manipulation was restricted in the BLA area, and to a single injection at specific ages. Alterations in social interaction, repetitive behaviors and anxiety observed in a series of behavioral tests provide evidence that direct modulation of LTCC activity can mimic at least some behaviours associated with SD.

The current pharmacological model is still in the process of being more fully validated for ASD. However, that the defining criteria for ASD are behavioral, with no biological markers, it remains important we develop as many models and research tools as possible towards the study of the pathological mechanisms underlying ASD. Current models largely reflect a comprehensive set of assays for social interaction, social communication and repetitive behaviors in mice (Tordjman et al., 2007; Crawley, 2012; Mabunga et al., 2015). Other behavioral tests such as olfactory communication, ultrasonic vocalizations, motor stereotypies are also used for phenotyping rodent models of ASD. In addition, standardized tests for developmental milestones are used for identifying associated ASD symptoms such as body length, eye-opening, and both the size of the brain and different brain nuclei, EEG recordings, and anxiety levels. While the current model displays multiple behavioral features of ASD, such as impaired social preference, a high level of anxiety and repetitive behaviors, other important aspects of the model await future investigations.

In the BayK rat model, I first demonstrated that a single treatment with LTCC agonist at specific early ages (P7 & P14) leads to long-lasting effects in neuronal excitability and synaptic plasticity. Further investigations revealed distinct long-term effects of BayK injection concerning neuronal excitability, synaptic plasticity and behavioral traits between P7 and P14 experimental groups, supporting the hypothesis that P7 and P14

159 represent a critical developmental period of BLA neural circuits and underlying behaviors.

My results also support the notion that the amygdala circuitry is prone to functional manipulations of LTCC activity within a specific critical period, and provide evidence that the LTCCs play an important role in regulating the development and maturation of BLA neuronal functions and circuits, which in turn contribute to the emergence and development of complex behaviors. While highly interesting overall, further studies are required to shed light on our understanding of the underlying mechanisms of the neuropathology in developmental disorders such as ASD.

The research possesses some potential limitations towards examining the roles of LTCCs in BLA neuronal circuit functions. For example, as mentioned above, GABAergic activity is depolarizing at P7 thus native GABA currents should be tested around the resting membrane potential. However, in order to correlate and compare to GABAergic responses of P7 neurons with P21 neurons, I performed all the recordings when holding at 0 mV for inhibitory neurotransmissions. This still reflects the contribution of a functional increase of LTCCs to the synaptic strength, yet the underlying mechanism or the conductances involved in the process could be somehow different. For instance, the driving force of chloride mainly depends on the concentration gradient of chloride across the membrane, but would be altered under different holding membrane potentials. Thus, when holding at 0 mV, the amplitude of spontaneous IPSCs and evoked IPSCs may not reflect accurately the GABAergic synaptic strength.

Here, I examined the effects of BayK on gene expression post 7, 14 or 21 days after injection on neuronal LTCCs in the BLA, as well as CREB and BNDF, which are the downstream signaling factors for the regulation of gene transcription. The results showed

160 no significant difference in their mRNA expression between BayK and DMSO treatment groups. However, we cannot rule out potential expression changes induced immediately after BayK injection. In addition, other genes of synaptic plasticity related singling factors, such as activity-regulated cytoskeleton-associated protein (Arc), and Homer1, might be involved in the long-term changes.

The open field social interaction test is appropriate for evaluating exploratory social behaviors given that the stranger rat does not go through the habituation process in the open field or meet the experimental rat. However, this setup might suppress the social response of the stranger rat and amplify the exploratory behavior of the experimental animal. Some behavioral traits, such as chasing, sniffing, climbing, are also present in juvenile play or sexual behaviors. However, as the stranger/recipient is not responding to the experimental animal, these observed behaviors are not considered as social play which are important parameters for the evaluation of an ASD animal model. Further, the behavioral test parameters, such as frequency and duration, were measured by a blind-to- treatment observer with a chronometer and from a video recording. Optimally, tracking animal behavior would also involve travel distance and patterns measured using a video tracking system which are important factors for the evaluation of anxiety level and locomotor ability.

4.3 Future directions An important future study will be to identify downstream signaling pathway genes that are involved in regulating the long-term synaptic plasticity induced by BayK. These genes include Arc and Homer1 as mentioned. In addition, the glutamatergic receptors, such as AMPAR, NMDA2A and NMDA2B, are associated with LTP, and their expression has been found to change drastically during the early development. The switch of the expression of these genes may play an important role in regulating BLA circuit 161 development and maturation. Therefore, it will be interesting to examine if this switch was disrupted by BayK injection and which might account for the observed long-term alterations in synaptic plasticity.

In addition, an important future investigation is to examine the long-term consequence of BayK injection using juvenile play behavioral tests wherein a different format of play could be applied where both animals are habituated prior to testing. This approach would allow assessment of whether the treated animals are able to engage in a sequence of patterns typical of juvenile play. As mentioned, socio-emotional maturation occurs throughout the entire developmental period and that the neurobiological processes that underlie the “social brain” are comprised of a complex network of brain regions including the amygdala. Juvenile play has been identified as one of the key mechanisms involved in the healthy development of the social brain (Pellis et al., 2010). The functions involved in the juvenile play include assessing and interacting with social partners, understanding social hierarchies and emotional communication, as well as increased behavioral flexibility, therefore, analyses of these behaviors would provide other important parameters for analyzing social interactions in the rat BayK model.

While Chapter II of my study reveals the effects of LTCC functional manipulation at P28, two or three weeks after the BayK injection, the true extent of longer-lasting effects of BayK injection remains to be defined. Further, the gene expression levels of neuronal excitability related membrane conductances and synaptic plasticity-related intracellular signaling factors are going through drastic changes during the early development. I am very interested in investigating the progressive trajectory of the long-term changes in synaptic plasticity and homeostatic plasticity induced by BayK. Future directions include examining the effects of BayK on excitability, synaptic plasticity as well as behavioral traits at different time points after the injection, including 1 day, 3 days, and 7 days later.

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