Role of the Cav3-Kv4 Complex in Cerebellar Granule Cells

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2016 Role of the Cav3-Kv4 Complex in Cerebellar Granule Cells

Rizwan, Arsalan

Rizwan, A. (2016). Role of the Cav3-Kv4 Complex in Cerebellar Granule Cells (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27686 http://hdl.handle.net/11023/3228 doctoral thesis

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Role of the Cav3-Kv4 Complex in Cerebellar Granule Cells

Arsalan Rizwan

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN NEUROSCIENCE

CALGARY, ALBERTA

AUGUST, 2016

The cerebellum has a simple anatomy and a comparatively small number of neuronal classes connected by clearly defined excitatory and inhibitory projections. The cerebellum is thus an ideal area to study signal processing in neurons and how this behaviour contributes to overall circuit function. A key factor in how the postsynaptic machinery of a neuron converts the information it receives into an output is determined by their intrinsic level of excitability, which in turn determines the pattern or frequency of nerve impulse (spike) discharge. The intrinsic excitability of neurons is determined by the complement of ion channels expressed in the membrane. A low voltage-gated potassium channel of the Kv4 family that is important to regulating spike output in numerous CNS cells is highly expressed in granule cells. The Kv4 channels and low voltage-gated calcium channels of the Cav3 family are known to interact with each other in stellate cells of the cerebellum, whereby the influx of calcium ions enhances the efflux of potassium ions to reduce cell excitability. Thus, any change in the degree of this interaction can fine-tune the role of the Cav3-Kv4 complex in modifying membrane excitability and signal processing via Kv4 channels. This PhD project investigated the hypothesis that the differential expression of a Cav3-Kv4 channel complex and resulting Kv4 availability regulates granule cell excitability, spike output, and learning across the cerebellar lobules. This study used in vitro slices of rat cerebellum to conduct immunocytochemistry and electrophysiological voltage- and current-clamp recordings in the granule cells. Indeed, using immunocytochemistry, this study uncovered differential expression of the Cav3-Kv4 complex across the cerebellar lobules. The electrophysiology work also uncovered key differences in the postsynaptic expression and activity of the Cav3-Kv4 complex between anterior and posterior cerebellum that shapes the processing of mossy fibre input by granule cells. The synaptic plasticity data also

ii revealed a novel NMDAR-mGluR-ERK-Kv4 interplay in cerebellar granule cells that functions to regulate postsynaptic excitability and synaptic responses to mossy fiber input. Overall, this study advances our understanding of the ionic mechanisms that underlie differential signal processing across cerebellar lobules.

iii Acknowledgements

I would first like to thank Dr. Ray Turner for his incredible mentorship and guidance throughout my four years in the lab. I would also like to thank my committee members Drs. Gerald Zamponi and Patrick Whelan for providing invaluable input to my research project. During my time in the

Turner lab, I had the opportunity to be trained by some of the smartest people that I have ever met. These people include Colin Heath, Dr. Jordan Engbers and Dr. Theodore Bartoletti. I am also very grateful to Dr. Bartoletti in helping me setup my amazing electrophysiology rig. I was also very inspired by the Turner lab members who provided an amazing intellectual lab environment to work in. These Turner lab members include Brian King, Steven Dykstra, Dustin

Anderson, Hadhimulya Asmara, Mirna Kruskic, Jason de Mesa Miclat, Giriraj Sahu, Xiaoqin

Zhan and Brett Simms. This thesis was supported by operating grants from Canadian Institute for

Health Research (CIHR), and studentships from Alberta Innovates - Health Solutions (AIHS) and Queen Elizabeth II scholarship.

iv Dedication

I dedicate this thesis to my mother and father, the most valuable people (MVP) of my life. Thank you for sacrificing so much for the sake of my education.

v Table of Contents

Abstract ...... ii Acknowledgements ...... iv Dedication ...... v Table of Contents ...... vi List of Tables ...... ix List of Figures and Illustrations ...... x List of Symbols, Abbreviations and Nomenclature ...... xii Epigraph ...... xiii

CHAPTER ONE: INTRODUCTION ...... 1 1.1 General Introduction ...... 1 1.1.1 Anatomy and circuitry of the cerebellum: ...... 1 1.1.2 Granule cells: ...... 2 1.2 Ion channels in the granule cell layer: ...... 2 1.2.1 Kv4 Channels ...... 2 1.2.1.1 Kinase regulation of Kv4 channels ...... 4 1.2.2 Cav3 channels ...... 5 1.3 Calcium-dependent modulation of A-type currents: ...... 6 1.4 Other auxiliary subunits of Kv4: DPLP ...... 7 1.5 ERK and learning in cerebellum: ...... 8 1.6 Cerebellar timing: Role of Kv4 channels in granule cells ...... 11 1.7 Theories of signal processing in the granule cell layer: ...... 12 1.7.1 Diversity of microcircuitry in the granule cell layer: Specialized microcircuitry in the vestibulocerebellar lobules 9 and 10 ...... 15 1.7.2 Contribution of differential expression of ion channels to the diversity of granule cells: the Cav3-Kv4 complex ...... 16 1.8 Acknowledging Collaborators ...... 17

CHAPTER TWO: METHODS ...... 34 2.1 Electrophysiology ...... 34 2.1.1 Animal Care and Cerebellar Slice Preparation ...... 34 2.1.2 LTP Protocol ...... 34 2.1.3 Current-clamp recordings ...... 35 2.1.4 Voltage-clamp recordings ...... 35 2.1.5 Voltage-dependence of activation and inactivation ...... 36 2.1.6 Specific compounds ...... 37 2.2 Data analysis and Statistics ...... 37 2.3 Criteria of granule cell selection ...... 37 2.4 Tissue fixation ...... 38 2.4.1 Immunocytochemistry: ...... 38 2.4.2 Colocalisation analysis ...... 39 2.4.3 Primary antibodies ...... 39

CHAPTER THREE: EXPRESSION OF THE SUBUNITS OF THE CAV3-KV4 COMPLEX IN GRANULE CELLS ...... 41

vi 3.1 Introduction ...... 41 3.2 Methods ...... 42 3.3 Results ...... 42 3.3.1 Cav3.1 and Kv4.2 immunolabel in granule cells ...... 43 3.3.2 Cav3.1 and Kv4.3 immunolabel in granule cells ...... 44 3.3.3 Colocalization of Kv4.2 and Kv4.3 in granule cells ...... 44 3.3.4 Expression of Cav3.3 in granule cells ...... 45 3.3.5 Expression of KChIPs ...... 45 3.3.6 Expression of DDP6 in granule cells ...... 46 3.4 Discussion ...... 46

CHAPTER FOUR: A CAV3-KV4 COMPLEX DIFFERENTIALLY REGULATES SPIKE OUTPUT IN CEREBELLAR GRANULE CELLS ...... 69 4.1 Introduction ...... 69 4.2 Methods ...... 70 4.3 Results: ...... 70 4.3.1 Identification of granule cells ...... 70 4.3.2 Lobules 2 vs. 9: Resting membrane and spike properties of granule cells ...... 71 4.3.3 Passive membrane properties: ...... 71 4.3.4 Spike properties of granule cells ...... 71 4.3.5 Expression of Cav3 (T-type) calcium current in granule cells ...... 72 4.3.6 Biophysical properties of Kv4 channel-mediated A-type current in granule cells ...... 72 4.3.7 Blocking the Cav3-Kv4 channel interaction ...... 74 4.3.8 Gain of spike firing ...... 74 4.3.9 Role of the Cav3-Kv4 complex in regulating the granule cell response to physiological stimuli ...... 75 4.3.10 Presence of Cav3 T-type current and lack of rebound burst in cerebellar granule cells ...... 77 4.4 Discussion ...... 77

CHAPTER FIVE: LONG-TERM POTENTIATION AT THE MOSSY FIBER-GRANULE CELL RELAY INVOKES POSTSYNAPTIC SECOND MESSENGER REGULATION OF KV4 CHANNELS ...... 99 5.1 Introduction: ...... 99 5.2 Materials and Methods ...... 100 5.3 Results ...... 100 5.3.1 LTP increases the postsynaptic excitability of lobule 9 granule cells ...... 100 5.3.2 TBS stimulation reduces the availability of Kv4 channels ...... 101 5.3.3 The effects of LTP on Kv4 are mediated by select activation of NMDAR and mGluRs ...... 103 5.3.4 Blocking ERK phosphorylation prevents the postsynaptic effects of TBS stimulation...... 104 5.3.5 TBS-induced LTP is preserved in the presence of GABAergic circuitry .....105 5.3.6 Bursts of mossy fiber EPSPs uncover LTP of synaptic efficacy in the intact circuit ...... 105 5.4 Discussion ...... 107

vii 5.4.1 LTP at the mossy fiber-granule cell relay ...... 107 5.4.2 Role for kinase activation ...... 108 5.4.3 Functional role of Kv4 modulation of granule cell excitability ...... 109

CHAPTER SIX: DISCUSSION ...... 125 6.1 The Ca3-Kv4 channel complex in granule cells ...... 126 6.2 Role of the Cav3-Kv4 complex in regulating excitability and the response to mossy fiber input ...... 128 6.3 Activation of ERK pathway in granule cells by TBS of mossy fiber input ...... 130 6.4 Role of LTP in modifying synaptic properties at the input stage of the cerebellum.131 6.5 Conclusion ...... 133 6.5.1 Future directions: ...... 134 6.5.2 Role of retrograde messengers in regulating Kv4 channels: ...... 134 6.5.2.2 Endocannabinoids: ...... 134

APPENDIX A: INCLUSION OF WORK PUBLISHED OR SUBMITTED BY CANDIDATE ...... 136

REFERENCES ...... 137

viii List of Tables

Table 1: Immunoreactivity in granule cell layer of the Cav3-Kv4 macromolecular complex 50

Table 2: Properties of lobule 2 versus lobule 9 granule cells ...... 80

Table 3: NMDA and mGluR1,5 receptors collectively modulate Kv4 voltage dependence. 112

ix List of Figures and Illustrations

Figure 1-1: Trilayer neural circuitry of the cerebellum ...... 19

Figure 1-2: A cerebellar slice and the mossy fiber- granule cell synapse ...... 21

Figure 1-3: Classification, structure and expression of Kv4 ion channels ...... 23

Figure 1-4: Classification, structure and expression of Cav3 ion channels ...... 25

Figure 1-5: A functional complex between Cav3 and Kv4 channel in stellate cells of cerebellum ...... 27

Figure 1-6: The neuronal ERK/MAPK pathway ...... 29

Figure 1-7: Cerebellar lobules receive mossy fiber inputs from specific sources ...... 31

Figure 1-8: Graphical abstract of the hypothesis ...... 33

Figure 3-1: Cav3.1 and Kv4.2 coexpression in granule cells ...... 52

Figure 3-2: Cav3.1 and Kv4.3 coexpression in granule cells ...... 54

Figure 3-3: Kv4.2 and Kv4.3 coexpression in granule cells ...... 56

Figure 3-4: Cav3.3 expression in granule cells ...... 58

Figure 3-5: KChIP1 expression in granule cells ...... 60

Figure 3-6: KChIP2 expression in granule cells ...... 62

Figure 3-7: KChIP3 expression in granule cells ...... 64

Figure 3-8: KChIP4 expression in granule cells ...... 66

Figure 3-9: DPP6 expression in granule cells ...... 68

Figure 4-1: Firing behaviour of different classes of neurons in the granule cell layer ...... 82

Figure 4-2: Cav3 currents in cerebellar granule cells ...... 84

Figure 4-3: Pharmacology controls concerning the non-specific effects of Ni2+ and Cd2+ .....86

Figure 4-4: The Cav3 complex modifies Kv4 channel biophysical properties in lobule 9 .....88

Figure 4-5: Cav3-Kv4 complex regulates firing frequency in lobule 9 granule cells ...... 90

Figure 4-6: In vivo firing of granule cells ...... 92

x Figure 4-7: The lack of a Cav3-Kv4 complex in lobule 2 granule cells enables burst output in response to a presynaptic mossy fiber spike burst ...... 94

Figure 4-8: Granule cells in lobule 9 have greater frequency following capability for EPSCs delivered in a pattern simulating vestibular-like input...... 96

Figure 4-9: Lack of rebound depolarization in granule cells ...... 98

Figure 5-1: LTP at the mossy fiber- granule cell synapse increases postsynaptic excitability and properties of IA ...... 114

Figure 5-2: TBS-evoked effects on IA depend on activation of specific glutamate receptors ...... 116

Figure 5-3: TBS-evoked effects on IA are calcium- and ERK-dependent ...... 119

Figure 5-4: The effects of applying TBS on postsynaptic firing and IA with GABA circuitry intact ...... 122

Figure 5-5: LTP is expressed for burst-like input patterns in lobule 9 granule cells ...... 124

List of Symbols, Abbreviations and Nomenclature

Symbol Definition α-subunit pore-forming subunit aCSF artificial cerebrospinal fluid bAP backpropagating action potentials BAPTA 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid BAPTA-AM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid tetrakis(acetoxymethyl ester) CPCCPOEt 7-(Hydroxyimino)cyclopropa[b]chromen-1a- carboxylate ethyl ester DL-AP5 DL-2-Amino-5-phosphonopentanoic acid DNQX (6,7-dinitroquinoxaline-2,3-dione) Cav 3 T-type calcium channels DPLP dipeptidyl peptidase-like protein Ex Reversal potential for ionic species x EPSC Excitatory postsynaptic current EPSP Excitatory postsynaptic potential ERK extracellular signal–regulated kinase GABA γ-Aminobutyric acid HVA High voltage activated IA A-type current IT T-type current JNJ 16259685 (3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)- (cis-4-methoxycyclohexyl)-methanone KChIP voltage-gated potassium channel-interacting Kv4 A-type potassium channels LTD Long-term Depression LTP Long-term Potentiation LVA Low voltage activated MAPK Mitogen-activated protein kinase mGluR metabotropic glutamate receptors MPEP 2-Methyl-6-(phenylethynyl)pyridine NMDA N-methyl-D-aspartate PD98059 2-(2-Amino-3-methoxyphenyl)-4H-1- benzopyran-4-one PKA Protein Kinase A PKC Protein Kinase C TBS Theta-burst stimulus Va Half-activation potential Vh Half-inactivation potential

xii Epigraph

The hidden well-spring of your soul must needs rise and run murmuring to the sea;

And the treasure of your infinite depths would be revealed to your eyes.

But let there be no scales to weigh your unknown treasure;

And seek not the depths of your knowledge with staff or sounding line.

For self is a sea boundless and measureless.

Say not, "I have found the truth," but rather, "I have found a truth."

- Kahlil Gibran, The Prophet

xiii

Chapter One: Introduction

1.1 General Introduction

Classically, the cerebellum has been considered a motor structure because cerebellar damage causes severe neurological syndromes that involve a loss in coordination of movement (Holmes, 1917). However, there has been accumulating evidence that implicates the cerebellum in many higher cognitive functions (Steinlin, 2008; Stoodley and Schmahmann, 2010). For example, social and cognitive deficits in autistic children have been linked to cerebellar dysfunction (Wang et al., 2014).So, in addition to its motor function, the neural circuitry of cerebellum may play a role in producing the deficits experienced by at least autistic individuals. The addition of such a cognitive role to cerebellar function reveals that much more needs to be learned of the mechanisms of signal processing in cerebellar circuitry. The cerebellum functions by integrating sensory-motor information provided by the excitatory mossy fiber inputs that arise from the brainstem and spinal cord. Mossy fiber afferents project across 10 distinct lobules that divide the cerebellum over the anterior-posterior axis (Sillitoe and Joyner, 2007). Based on sensory information carried by mossy fiber inputs and related cerebellar outputs, a compartmentalization of function can be found, such that lobules 1-5 are related to limbs, lobules 6-8 to cognitive and oculomotor functions, and lobules 9-10 to vestibular functions (Robinson and Fuchs, 2001; Sillitoe and Joyner, 2007; Stoodley and Schmahmann, 2010; Barmack and Yakhnitsa, 2011; Glickstein et al., 2011; Stoodley, 2011). As this basic pattern of circuitry is repeated throughout the cerebellum, it raises the question as to how such widely different sources of sensory input can be processed, and suggests the existence of mechanisms to differentially process inputs across cerebellar lobules.

1.1.1 Anatomy and circuitry of the cerebellum:

Due to an almost crystalline-like morphology of the organization of the cerebellum, its structural aspects have received extensive study. The cerebellar cortex is organized into molecular, Purkinje and granule cell layers that are folded into 10 different lobules across the anterior-posterior axis (Figs. 1-1 and 1-2a). The Purkinje cell layer is the main output layer of the cerebellar cortex and the molecular layer consists of interneurons that relay information to the

Purkinje cell layer. The granule cell layer is the primary input layer of cerebellum in receiving excitatory mossy fiber inputs and conveying information to Purkinje cells. The granule cell layer also contains inhibitory interneurons called Golgi cells. Mossy fibers originate from multiple sources that include pontine nuclei, the spinal cord, the brainstem reticular formation, and the vestibular nuclei (Voogd and Glickstein, 1998; Glickstein et al., 2011). Thus, the flow of information within the cerebellar circuitry can be summarized as: mossy fibers à granule cells à Purkinje cells.

Given that the cerebellum contains a remarkably simple and homogeneous circuit across all lobules, it raises the key question: How can the uniform circuitry of cerebellum differentially process incoming information? My overarching hypothesis is that the postsynaptic protein machinery of granule cells, in addition to plasticity of mossy fiber inputs, contributes to a differential processing of inputs across cerebellar lobules.

1.1.2 Granule cells:

Granule cells are the most numerous neurons in the brain and outnumber the total number of neurons in the cereberal cortex. The importance of the granule cell layer lies in its strategic location within the cerebellar circuitry and its role as the main input layer of cerebellum. The mossy fiber-granule cell synapse is referred to as a glomerulus (Fig. 1-2B). The glomerulus is defined as a synapse where a mossy fiber terminal (rosette) contacts dendrites (claws) of ~ 20 granule cells (Eccles et al., 1967). Each granule cell gives rise to only four short dendritic branches that form a synapse with a mossy fiber axonal bouton. This synaptic arrangement is unique in that each dendrite projects into only one glomerulus where it contacts only one mossy fiber rosette (Eccles et al., 1967). The granule cell axon ascends through the Purkinje cell layer to bifurcate as a parallel fiber that converges onto the dendrites of Purkinje cells, the only output neuron of cerebellar cortex. 1.2 Ion channels in the granule cell layer:

1.2.1 Kv4 Channels

Active voltage-gated potassium (Kv) channels are formed by tetramerization of four α subunits (pore-forming subunits) (Birnbaum et al., 2004). Each α subunit consists of six

transmembrane domains. Based on this membrane topology they are differentiated from other groups of potassium channels. The first group has two transmembrane domains and generate inward-rectifying potassium channels (Kir). The second group has four transmembrane domains that comprise a class of a 2 pore domain “leak” potassium channels. The third group consists of voltage-gated and calcium-activated potassium channels and has six transmembrane domains. The fourth group has 7 transmembrane domains and consists of BK channels (Big Potassium) channels (Fig. 1-3A) (Birnbaum et al., 2004). Kv channels consist of 12 families (Kv1-12) encoded by approximately 40 genes (Fig. 1- 3A)(Gutman et al., 2005; Lai and Jan, 2006). Each member of a family is classified according to genes, as denoted by the number after the decimal point, such Kv1.1, Kv1.2, etc. The Kv1 channel was the first potassium channel to be cloned. Since earlier work in Kv channels was conducted in Drosophila, Kv1-4 channels were also referred to as Shaker (Kv1), Shab (Kv2), Shaw (Kv3) and Shal (Kv4) prior to reclassification by a numbering system (Birnbaum et al., 2004; Gutman et al., 2005). Structurally, Kv4 channels are divided into four parts: an N-terminal cytoplasmic domain, a tetramerization domain (T1), transmembrane domains (S1-S6) that include the voltage sensor (S4), an S5-pore loop-S6 region, which forms the pore domain, and a C-terminal cytoplasmic domain (Fig. 1-3B) (Birnbaum et al., 2004). Three different genes encode for Kv4 channels: Kv4.1, Kv4.2 and Kv4.3. Kv4 channels generate a transient outward current by exhibiting fast activation and fast inactivation in the subthreshold range of membrane potentials. Based on their kinetics, Kv4 current is also referred to as “A-type” current. These channels are also identified by their sensitivity to 4-aminopyridine. It should be noted that A-type current can also be generated by at least Kv1.4, Kv3.3 and Kv3.4 isoforms (Birnbaum et al., 2004). Connor and Stevens were the first to record A-type current in the neurons of molluscs (Connor and Stevens, 1971b). They were also the first to report the role of these currents in regulating the spiking behavior of molluscan neurons, especially in the middle and latter part of the interspike intervals (Connor and Stevens, 1971a). Kv4 channels are highly expressed in the rat’s brain, and intensive studies have investigated the expression of all the isoforms of Kv4 channels throughout the adult rat brain (Serodio and Rudy, 1998). The mRNA transcript distribution of all the three isoforms of Kv4 has been documented using in situ hybridization

(Fig. 1-3C). Kv4.1 is weakly expressed only in the olfactory bulb and hippocampus regions of the brain. On the other hand, Kv4.2 and Kv4.3 transcripts are abundantly expressed throughout the brain. In the case of cerebellum, Kv4.3 is highly expressed in the Purkinje cell layer and molecular layer (presumed interneurons), whereas Kv4.2 was absent from both of these layers. In the case of the granule cell layer, Kv4.2 and Kv4.3 isoforms are highly expressed. Interestingly, Kv4.2 expression dominates in granule cells of the anterior lobules (lobules 1-6), whereas Kv4.3 is strongly expressed in granule cells of posterior lobules (lobules 6-10) (Serodio and Rudy, 1998; Strassle et al., 2005; Amarillo et al., 2008). The reciprocal anterior-posterior gradient for expression of Kv4.2 and Kv4.3 is also seen for other molecular markers (Herrup and Kuemerle, 1997). Given these distinctions lobule 6 might represent the true boundary between anterior and posterior regions of cerebellum now suggested to mediate different functional roles (Stoodley, 2011). While no significant biophysical differences have been reported between the Kv4.2 and Kv4.3 isoforms (Birnbaum et al., 2004), the opposite gradients of Kv4 isoforms expressed in granule cells across the lobules again implies compartmentalization of function for these isoforms across cerebellum.

1.2.1.1 Kinase regulation of Kv4 channels

Kv4.2 and Kv4.3 contain amino acid sequences that can form putative phosphorylation sites for PKA, PKC, CaMKII and extracellular signal–regulated kinases (ERK) and mitogen-activated kinases (MAPK) (Birnbaum et al., 2004). Kinase mediated phosphorylation has been mainly studied for the Kv4.2 isoform since it is the isoform heavily expressed in dendrites of CA1 pyramidal neurons. The first evidence for regulation of Kv4 current in hippocampus came from Hoffman and Johnston, who found that application of 8-bromo-cAMP and forskolin (activators of PKA) led to a reduction of Kv4.2 current in CA1 pyramidal neurons (Hoffman and Johnston, 1998). This reduction was produced by a depolarizing shift in the activation curve of Kv4, which led to a decrease in the probability of opening Kv4 channels. Application of phorbol diacetate (a PKC activator) also reduced Kv4.2 current in CA1 neurons by invoking a depolarizing shift in the activation curve. A later study reported that both a PKA- and PKC-mediated reduction in Kv4 current reflects a convergence of these two pathways on ERK/MAPK (Yuan et al., 2002). ERK has 3 phosphorylation sites on the C-terminus of Kv4.2: T602, T607 and S616 (Schrader et

al., 2006). The effects of PKA or PKC on Kv4 were also blocked by the use of inhibitors of MEK, the upstream kinase whose only role is to activate ERK. Using phospho-specific antibodies, they found that PKA and PKC activators led to an increase in phosphorylated ERK (active ERK) and phosphorylated Kv4.2 (recognized by a phospho-antibody that recognizes all three ERK phosphorylation sites on Kv4.2). Another kinase that has received attention in the field of synaptic plasticity is calcium-calmodulin-dependent kinase type II (CaMKII). CamKII has been shown to be both necessary and sufficient for induction of long-term potentiation (LTP) at several synapses (Lisman et al., 2002). Interestingly, CaMKII has also been shown to regulate Kv4.2 channels through phosphorylation and to increase the surface expression of Kv4 channels in cultured CA1 pyramidal neurons (Varga et al., 2004). Co-expression of constitutively active CaMKII along with Kv4.2 in Xenopus oocytes showed no effect of CaMKII on the voltage dependence of activation or inactivation. Overall these studies reveal that direct phosphorylation of Kv4 channels is an important way through which these channels are regulated.

1.2.2 Cav3 channels

Voltage-gated calcium channels are divided into two major classes: high-voltage activated (HVA) channels and low-voltage activated (LVA) channels (Simms and Zamponi, 2014). Unlike HVA channels, LVA channels only consist of Cavα1 (pore-forming) subunits and no ancillary subunits (Catterall et al., 2005). All the Cavα1 subunits have the same membrane topology of four transmembrane domains, each consisting of six transmembrane helices (S1-S6). S4 is the charged helix responsible for voltage-activation and S5-S6 is the pore domain (Fig. 1-4B) (Catterall, 2010). Based on Cavα1 subunits, Cav channels are divided into three families: Cav1, Cav2 and Cav3 (Fig. 1-4A) (Simms and Zamponi, 2014). Cav1 channels consist of four members (Cav1.1-Cav1.4) that encode different isoforms of L-type channels. Cav2 channels consist of three members: Cav2.1 (P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type). Cav3 channels consist of three members that all encode for T-type channels. T-type channels are typically considered the only calcium channels fully activated from low voltages, and are distinguished from HVA channels by their sensitivity to Ni2+ and resistance to block by Cd2+ (Perez-Reyes, 2003a). Cav3 channels are termed “T-type” because of their “tiny” conductance (~5-7 pS) and “transient” activation. Both Cav3 and Kv4 channels are unique members of their

respective families by exhibiting a low voltage for activation, rapid activation and inactivation. In situ hybridization studies of mRNA transcript distribution reveal that all Cav3 channel isoforms are highly expressed in the rat’s brain (Talley et al., 1999). In the case of cerebellum, the Cav3.3 isoform is only expressed in the granule cell layer at a low level and this expression is uniform across the anterior-posterior axis of cerebellum (Fig. 1-4C). In contrast, Cav3.1 is expressed in all of molecular, Purkinje, and granule cell layers (Fig. 1-4C). In the granule cell layer, Cav3.1 exhibits a differential expression pattern across the anterior-posterior axis. Cav3.1 expression is high in the granule cells of the posterior lobules (lobules 6-10) and is below detectable levels in granule cells of anterior lobules 1-5 (Talley et al., 1999). While earlier electrophysiological studies indicated the presence of transient calcium currents in cultured granule cells, these were attributed entirely to the HVA class of R-type calcium channels (Randall and Tsien, 1995; Tottene et al., 1996; Zamponi et al., 1996). Less than 1% of the cultured granule cells were reported to express calcium currents consistent with T-type channels (Randall and Tsien, 1995). There is thus a separation of evidence for the expression of Cav3 channels and T-type current in granule cells.

1.3 Calcium-dependent modulation of A-type currents:

The α subunits of Kv4 channels are known to form a complex with a class of calcium sensor called potassium channel interacting proteins (KChIPs) (An et al., 2000). KChIPs are an auxiliary subunit of Kv4 channels, and act to increase the surface expression of Kv4 channels and remodel their biophysical properties (Ledo et al., 2000; Jerng and Pfaffinger, 2014). Like calmodulin, the classic calcium sensor protein, KChIPs have four EF-hands that contain a helix- loop-helix folding motif to bind calcium ions. Interestingly, the EF-hand 1 of KChIPs is unable to bind calcium ion and EF-hand 2 binds to Mg2+ ion. Only EF-hands 3 and 4 are able to bind calcium ions with an affinity of 1-10 µM (Craig et al., 2002). There are four genes that encode for KChIPs in the brain: KChIP1, KChIP2, KChIP3 and KChIP4 (Jerng and Pfaffinger, 2014). The Turner lab first reported a novel function for KChIPs as a calcium sensor in regulating Kv4 channels (Fig. 1-5) (Anderson et al., 2010b; Anderson et al., 2010a). They found that only the KChIP3 isoform was able impart calcium-sensitivity to Kv4 channels. KChIP3 is also

referred to as DREAM (downstream regulatory element antagonist modulator) for its role in calcium-dependent gene regulation, or alternately described as calsenilin for its interaction with presenilin, an Alzheimer’s protein (Buxbaum et al., 1998; Carrion et al., 1999). The Turner lab also identified Cav3 calcium influx as the calcium source that activates KChIP3 in cerebellar stellate cells, promoting a selective shift in the voltage-inactivation relationship (Vh) of Kv4 current toward hyperpolarized potentials (Fig. 1-5A, B)(Anderson et al., 2010b; Anderson et al., 2010a). Thus, Cav3 channels proved capable of linking to a pre-existing Kv4-KChIP3 complex at an undetermined site to increase the availability of Kv4 currents near resting potential. The resulting increase in A-type current in the region of spike threshold then exerts substantial control over the firing properties of stellate cells (Fig. 1-5C) (Anderson et al., 2010b; Anderson et al., 2010a).

1.4 Other auxiliary subunits of Kv4: DPLP

Another modulatory subunit of the Kv4 channel complex is dipeptidyl peptidase-like protein (DPLP) (Nadal et al., 2003; Maffie and Rudy, 2008b; Jerng and Pfaffinger, 2014) . These proteins belong to the serine protease family but lack the crucial serine residue at the catalytic site, making these enzymes inactive (Kin et al., 2001). The structure of DPLP consists of a short cytoplasmic N-terminal segment, a single transmembrane domain and a large extracellular domain (Bezerra et al., 2015). The transmembrane domain of DPLP interacts with the voltage sensor of Kv4 channels. The role of DPLP is to augment channel recruitment to the membrane, produce a hyperpolarizing shift in the steady-state inactivation and activation curves, accelerate the rate of Kv4 inactivation, and change its toxin sensitivity (Maffie et al., 2013; Jerng and Pfaffinger, 2014). The two DPLP genes are DPP6 and DPP10. DPP6 was found to be an auxiliary subunit of Kv4 channels when it was co-purified along with Kv4 α subunit from the rat brain (Nadal et al., 2003). Interestingly, DPP6 is the only DPLP that is expressed in the cerebellar granule cell layer with 5 known variants: DPP6a, DPP6K, DPP6L, DPP6D and DPP6S (Nadin and Pfaffinger, 2010a). However, only DPP6a and DPP6K are required along with KChIP3 to generate an A-type current consistent with native granule cells when coexpressed in heterologous systems (Jerng and Pfaffinger, 2012).

1.5 ERK and learning in cerebellum:

Changes at the synaptic level are believed to underlie learning and the formation of memory traces (Bliss and Collingridge, 1993). Canadian neuroscientist Donald Hebb was one of the first to suggest this idea (Brown and Milner, 2003; Sjostrom et al., 2008). According to Hebb, if presynaptic and postsynaptic neurons are active together then it will lead to a strengthening of their synapses. The mossy fiber –granule cell synaptic relay in cerebellum allows for the transmission of input frequencies up to 1 kHz (van Beugen et al., 2013). These high input frequencies have been shown to induce LTP at the mossy fiber synapse through a combination of pre- and postsynaptic mechanisms that shape the response to subsequent inputs (Rossi et al., 1996; Armano et al., 2000a; Sola et al., 2004a; Nieus et al., 2006). In particular, a study on mossy fiber-LTP reported a change in intrinsic excitability (Armano et al., 2000a), with an increase in the gain of firing of granule cells that is consistent with a reduction of Kv4 current. This is important, as Frick et al (2004) reported that LTP in CA1 pyramidal cells incorporates a selective hyperpolarizing shift in the voltage-inactivation relationship of Kv4 channels (Frick et al., 2004). The effective reduction in A-type potassium current near resting potential then leads to an increase in the backpropogation of action potentials into dendrites (Frick et al., 2004). Notably, there was no alteration in the voltage for activating Kv4 currents, much like our effects of blocking the Cav3- Kv4 interaction in cerebellar stellate cells (Anderson et al., 2010b; Anderson et al., 2010a).

Interestingly, the shift in Vh of Kv4 current in CA1 pyramidal cells was further attributed to the activation of extracellular signal–regulated kinases (ERKs) in dendrites (Rosenkranz et al., 2009). ERK is the most well-known member of the mitogen-activated protein kinase (MAPK) cascade and is recognized for its role in cell growth, differentiation and proliferation (Fig. 1-6) (Zhang and Liu, 2002; Thomas and Huganir, 2004). The ERK signaling cascade involves an increase in the levels of GTP bound G-protein Ras through an increase in the activity of guanyl nucleotide exchange factors (GEFs) and a simultaneous decrease in the activity of GTPase activating proteins (GAPs). GEFs promote the active form of Ras (GTP bound form); whereas GAPs promote the inactive form of Ras (GDP bound form). The GTP bound Ras then leads to the activation of protein kinase Raf that leads to phosphorylation of MEK kinase. MEKK then

subsequently activates ERK1 and ERK2 kinases, the two isoforms of ERK. ERK1 and 2 have 84% amino acid sequence similarity with activities that are indistinguishable in vitro; it is believed that their different expression levels account for their apparent biological in vivo differences (Lefloch et al., 2008). Therefore, we will refer to both isoforms only as ERK. Overall, this cascade is also known as the Ras-Raf-ERK signal transduction cascade. ERKs are serine/threonine protein kinases, and their substrates include factors involved in growth and differentiation of cells (Thomas and Huganir, 2004). ERK is expressed in cerebellar granule cells and has been shown to play a key role in their survival during development (Bonni et al., 1999). However, the role of ERK signaling in adult granule cells (Boulton et al., 1991) has never been investigated. The role of ERK in synaptic plasticity has been well characterized in the CA3-CA1 synapse of hippocampus (English and Sweatt, 1996, 1997; Thomas and Huganir, 2004). Since LTP at the CA3-CA1 synapse is NMDA receptor (NMDAR)-dependent, it suggests a role for glutamatergic signaling in activating a neuronal MAPK cascade (Luscher and Malenka, 2012) (Fig. 1-6). But it is not known how the rise in intracellular calcium concentration in neurons during LTP leads to the increase in levels of Ras-GTP (Thomas and Huganir, 2004). The hippocampus is part of the brain involved in spatial learning (Bannerman et al., 2014), with training in the watermaze (a test for spatial learning) activating ERK in CA1 neurons (Blum et al., 1999; Vorhees and Williams, 2006). Blum et al. (Blum et al., 1999) also showed that infusion of an ERK blocker (PD98059) into the hippocampus blocks the storage of long-term spatial memory in the watermaze test. Thus, the role of ERK in hippocampal plasticity has been well- established by both the behavioral and electrophysiological studies.

1.5.1 Plasticity at cerebellar synapses

Learning in cerebellum is widely studied through classical eyeblink conditioning (Christian and Thompson, 2003; Freeman and Steinmetz, 2011). This procedure involves the pairing of neutral stimuli (e.g. tone), called a conditioning stimulus (CS), with a noxious stimulus (e.g. airpuff), and called an unconditioned stimulus (US). The US elicits a reflex response which involves the closing of the eyelid. Multiple pairings of CS and US over time results in the ability of neutral stimuli (CS) to elicit closing of the eyelid. This new response elicited by CS is termed a conditioned response (CR). There is strong evidence that the circuitry of cerebellum is both 9

necessary and sufficient for eyeblink conditioning (Krupa et al., 1993; Christian and Thompson, 2003). The first evidence for the role of ERK in cerebellar learning was obtained using this approach, where eyeblink conditioning leads to activation of ERK in the anterior vermis of the cerebellum (Zhen et al., 2001). Classically, long-term depression (LTD) at the parallel fiber- Purkinje cell synapse was thought to underlie learning in the cerebellum (Ito, 2001). LTD is defined as a decrease in synaptic efficacy resulting in the depression of excitatory postsynaptic currents. LTD at this synapse is induced by simultaneous activation of parallel fibers and a climbing fiber which activates signaling cascades that promote endocytosis of AMPARs in Purkinje cells (Ito, 2001). Over time, both LTD and LTP have been shown to occur at other synapses in cerebellum including the mossy fiber – granule cell synapse (Hansel et al., 2001). LTP of mossy fiber input was shown by D’Angelo and colleagues in a series of papers (Rossi et al., 1996; D'Angelo et al., 1999; Armano et al., 2000a; Maffei et al., 2002a; Rossi et al., 2002; Maffei et al., 2003b; Sola et al., 2004b; Gall et al., 2005b; Roggeri et al., 2008; D'Errico et al., 2009). Through high frequency mossy fiber discharge, they revealed that mossy fiber EPSCs can undergo potentiation through a process that involves both pre- and postsynaptic elements. Thus, LTP induction requires coactivation of NMDAR and mGluRs on granule cells, as well as an elevation in intracellular calcium and activation of PKC. The increase in synaptic currents correlated with enhanced neurotransmitter release from the presynaptic mossy fiber terminals. An additional postsynaptic increase in intrinsic excitability involved at least an increase in the granule cell input resistance (Rin) and a reduction in spike threshold. In examining the nature of changes in intrinsic excitability it appears that an effective reduction in Kv4 currents could be a key contributing factor. LTD has also been demonstrated at the mossy fiber-granule cell synapse (Gall et al., 2005a; D'Errico et al., 2009). Depression of this synapse can be induced either by short (100 ms) 100 Hz bursts or low frequency (< 10 Hz) inputs (Gall et al., 2005b). Using calcium-imaging to investigate the relationship between [Ca]i and the sign of plasticity D’Angelo and colleagues found that the mossy fiber-granule cell synapse follows the Beininstock-Cooper-Munro (BCM) rule of plasticity (Gall et al., 2005a; D'Errico et al., 2009). According to this rule, a decrease in the concentration of calcium below a certain threshold leads to LTD, whereas an increase in

calcium concentration above that threshold leads to LTP. The BCM rule was first defined for visual cortical synapses where a certain threshold of calcium concentration can determine the sign of plasticity (Cooper and Bear, 2012). Hippocampal synapses have also been shown to follow the BCM rule (Dudek and Bear, 1993). In the mossy fiber-granule cell synapse, nicotine, a neuromodulator, can alter the BCM plasticity rule by sliding the threshold for the LTD/LTP switch (Prestori et al., 2013). The most conclusive results regarding plasticity at the mossy-fiber granule cell relay came from an in vivo study where tactile stimulation of the whisker pad induced LTP in the recorded local field potential recorded from the granule cell layer (Roggeri et al., 2008b). Recently, The D’Angelo group has emphasized the importance of both the elevation of postsynaptic internal calcium concentration that support presynaptic mechanisms of modulated transmitter release in both LTP and LTD at the mossy fiber-granule cell synapse (D'Errico et al., 2009). The reason for stressing presynaptic over postsynaptic mechanisms is suggested because of significant changes in the paired-pulse ratio (PPR), coefficient of variation (CV) and failures observed during plasticity at this synapse. Interestingly, it is suggested that the postsynaptic increase in the intracellular concentration of calcium in granule cells triggers a retrograde cascade (either nitric oxide or endocannabinoids) targeting the release sites of presynaptic mossy fibers (Isope, 2010) (Maffei et al., 2003b).

1.6 Cerebellar timing: Role of Kv4 channels in granule cells

Timing is one of the most important aspects of cerebellar function (Hansel et al., 2001; D'Angelo and De Zeeuw, 2009). In the case of eyeblink conditioning, it is the interstimulus interval between the CS and US that is thought to be stored in cerebellum because Purkinje neurons “learn” to pause firing before the onset of the US (thus, a conditioned response) (Freeman and Steinmetz, 2011). As first spike latency and firing frequency are among the properties regulated by Kv4, these channels may play an important role in the transmission of information through the granule cell layer.

1.7 Theories of signal processing in the granule cell layer:

The first theory of cerebellar function, known as the Marr theory, assumes that processing in the cerebellar granule cell layer is based on spatial pattern separation and redistribution of the incoming mossy fiber information (Marr, 1969). The spatial topography of afferents to cerebellum creates a fractured somatotopy that reflects in part the separation of mossy fiber axons carrying input across different cerebellar lobules. Separation and redistribution of mossy fibers is proposed to minimize interference between different incoming mossy fiber patterns (Booth et al., 2007). Based on the Marr theory, it is logical to assume that different lobules exist in the cerebellum to process specific information and to minimize interference between the incoming signals. Newer theories of cerebellar function also incorporate the role of Golgi cells positioned in the granule cell layer. Most of the Golgi cells are GABAergic but can also release glycine, even though there are no known glycinergic receptors on granule cells (80% co-release GABA and glycine, 15% only release GABA, and 5% are glycinergic) (Galliano et al., 2010). Each granule cell receives ~ 4 inhibitory synapses per dendrite (D'Angelo, 2008). Golgi cells provide both phasic and tonic inhibition to granule cells (Farrant and Nusser, 2005). Phasic inhibition provided by Golgi cells generates IPSCs in granule cells (Rossi et al. 2003). Tonic inhibition regulates the gain of transmission from mossy fibers by modulating granule cell’s input resistance (Rin) and spike threshold (Chadderton et al., 2004). Thus an increase in Golgi cell activity is predicted to reduce the gain of mossy fiber-granule cell transmission. By providing a shunting inhibition, Golgi cells also regulate a depolarization-induced unblock of NMDA receptors and the induction of plasticity at the mossy fiber–granule cell synapse (Galliano et al., 2010).The phasic inhibition provided by Golgi cells, and driven by mossy fibers, comes in two forms: feedforward inhibition (mossy fiberàGolgiàGranule cells) and feedback inhibition (mossy fiberàGranule cellsàGolgiàGranule cells). Compartmentalization: Mossy fibers arise from multiple sources and at least some of them can be identified by their presynaptic properties (according to PPR, CV and EPSC amplitude) (Chabrol et al., 2015). In vivo recordings from mossy fiber boutons indicate that they fire spontaneously (Powell et al., 2015). Mossy fiber inputs to granule cells are distributed across ten different lobules divided on a gross scale according to anterior, posterior, and vestibular

divisions (Fig. 1-7). An increasing body of evidence suggests a functional division between anterior and posterior lobules based on anatomical, imaging, and behavioural studies. Much of this reflects an initial segregation of mossy fibers that carry information for specific sensory inputs or cortical feedback projections to different cerebellar lobules. Anatomically, a primary fissure in the cerebellum divides anterior lobules 1-5 from posterior lobules, which consists of the region between lobule 6 to the dorsal portion of lobule 9 (Stoodley and Schmahmann, 2010). A posterolateral fissure divides vestibular (flocculonodular) from posterior lobules with the vestibular portion comprised of ventral lobule 9 and lobule 10 (Sillitoe and Joyner, 2007). Based on this data, Stoodley et al. proposed a functional division between the anterior and posterior cerebellum, with the anterior lobules primarily involved in primary sensory-motor processing and the posterior lobules in higher cognitive behaviours (Stoodley and Schmahmann, 2010) (Fig. 1-7). Interestingly, the developmental origins of anterior and posterior lobules also differ (Hawkes et al., 1999). Using lac Z as a lineage marker, Hawkes et al. found that different granule cell lineages define the anterior-posterior divide at lobule 6 (Hawkes et al., 1999), providing a clear distinction between granule cells in the anterior vs. posterior lobules. The different developmental history of granule cells across the anterior-posterior divide could account for the difference in ion channel expression in granule cells and affect processing of sensory inputs. What kind of inputs does a single granule cell receive? Despite the compartmentalization seen at the gross anatomical level, there are different theories regarding cerebellar processing at the granule cell level. The uniform circuitry across the lobules of cerebellar cortex led to the idea of a ‘cerebellar algorithm’ performed on the incoming information (Dean et al., 2010). David Marr posited one of the most influential theories of cerebellar processing in 1969 (Marr, 1969; Strata, 2009). Marr suggested that a sparse coding process takes place in the granule cell layer, such that there are only a small number of granule cells simultaneously activated in response to a given mossy fibre input. Sparse coding would allow the granule cell to present data to the downstream Purkinje cells in an easier format as it allows for better discrimination of patterns from the multitude of mossy fibre inputs. Jorntell and colleagues later suggested that the cerebellum acts as an adaptive filter (Dean et al., 2010). An adaptive filter is a linear process where inputs are transformed into outputs using adjustable

parameters, for example gain and decay time. In the context of cerebellum, mossy fiber signals are decomposed to different components by granule cells, sent as a parallel fiber output, weighed at the parallel fiber to Purkinje cell synapse, and further processed to form the Purkinje cell (filter) output. The cerebellar filter is adaptive because parallel fibers to Purkinje cell synapses are modifiable by climbing fiber input (error signal). Jorntell also relegated the role of granule cells to that of coincident detectors, i.e. two or more of its synaptic inputs are required to elicit granule cell firing (Dean et al., 2010). This was consistent with their in vivo experimental evidence from decerebrate cats that a single mossy fiber could not provide the EPSP summation required to evoke spike discharge in granule cells of the C3 region (Jorntell and Ekerot, 2006). In the same cerebellar C3 zone of cats, the granule cells respond to regional specific inputs, such that one granule cell receives inputs from the same location on the skin (Jorntell and Ekerot, 2006; Ekerot and Jorntell, 2008). Functional equivalency (same source) of the mossy fiber input is known as the unimodal hypothesis (Huang et al., 2013). The unimodal hypothesis further supported the idea that granule cells are coincident detectors that need multiple simultaneous mossy fiber inputs to drive their firing. All of this implies that differential information processing in the cerebellum is solely based on the nature of afferent mossy fiber information (Ramnani, 2006). An alternative hypothesis developed in other experimental systems posits that granule cells are more than mere integrators and relayors of functionally equivalent synaptic inputs. Multimodality is defined as the convergence of functionally distinct mossy fiber input on all four dendrites of granule cells (Huang et al., 2013). The first strong evidence for multimodality at the level of a single cerebellar neuron was provided with single Golgi cell activity in the lateral Crus I lobule in response to stimulation of whisker-related primary sensory afferents (vS1) or motor (vM1) cortical afferents (Proville et al., 2014). Remarkably, they found that these separate inputs remained segregated throughout the brain until reaching cerebellum. Thus, sensorimotor information converges at the level of single cells in the cerebellum, indicating a focal point for integration of incoming information from different modalities. Yet the question of multimodal integration at the granule cell level remained unanswered until recently. Anatomical tracing studies have recently established that there is a convergence of information from different modalities at the granule cell layer (Huang et al., 2013; Chabrol et al.,

2015). Indeed, the new in vitro and in vivo studies provide strong evidence for multimodal integration at the level of a single granule cell (Chabrol et al., 2015; Ishikawa et al., 2015; Powell et al., 2015). In vitro electrophysiology studies and tracing techniques show that a single granule cell can respond to inputs from at least two different modalities (vestibular and visual) (Chabrol et al., 2015). The authors of the study revealed that the mossy fiber-granule cell synaptic properties (measured as varying PPR, CV and EPSC amplitude) differed based on the mossy fiber inputs. Thus, varying properties of a synapse could be a biophysical signature for the mossy fiber input. Recent in vivo studies have also demonstrated that a single granule cell can respond to stimulation from at least three different modalities ( auditory, visual and somatosensory inputs) (Ishikawa et al., 2015). This information is received onto different synapses of the same granule cell (distinguished by evoked response properties). Costimulation of the modalities lead to approximately linear summation of evoked responses, enhancing the output of granule cells (albeit not in all granule cells, which further highlights the diversity of granule cells). All the new findings regarding multimodal integration occurring at the single granule cell level do not negate old findings regarding unimodal integration observed in decerebrate animals. More likely than not different granule cell populations utilize different modes of integration, with a subset of granule cells using unimodal integration, and another subset using multimodal integration.

1.7.1 Diversity of microcircuitry in the granule cell layer: Specialized microcircuitry in the vestibulocerebellar lobules 9 and 10

Unipolar brush cells (UBCs) are differentially distributed across the cerebellar lobules. They are highly enriched in the vestibulocerebellar lobules 9-10. They are also occasionally found in lobules 1-3 (Dino et al., 1999). UBCs are a class of glutamatergic interneurons found in the granule cell layer. The paintbrush like dendrioles of UBCs innervates the glomerulus, receiving inputs from mossy fibers. The axons of UBCs synapse onto granule cells in a fashion similar to mossy fibers. Due to their unique placement within the cerebellar circuitry they are referred to as “cortex-intrinsic mossy fibers” (Mugnaini et al., 2011). The presence of these excitatory interneurons leads to a specialized microcircuit within these lobules. The incoming mossy fiber information can be relayed through UBCs, expanding the

synaptic connectivity. The resulting circuit can be summarized as: mossy fiber àUBCs à granule cells à Purkinje cells. The location of UBCs within the circuitry allows them to shape the granule cell output in response to mossy fiber input. The presence of specialized microcircuitry in the vestibular lobules adds credence to diversity across the lobules at the granule cell layer.

1.7.2 Contribution of differential expression of ion channels to the diversity of granule cells: the Cav3-Kv4 complex

Granule cells like any other excitable cells express a complement of ion channels, and in particular, generate a high density of A-type current reflecting the expression of Kv4 potassium channels (Fig. 1-3) (Cull-Candy et al., 1989; Serodio and Rudy, 1998; Maffie and Rudy, 2008a). Recently, it was reported that calcium influx mediated by the LVA class of Cav3 calcium channels can increase the availability of Kv4 (Anderson et al., 2010b). The differential expression of Cav3 calcium channels in granule cells (Fig. 1-4) provides the potential to regulate the functional expression of the Cav3-Kv4 complex, and thus contribute to heterogeneity in granule cell output across cerebellar lobules. The postsynaptic changes in excitability following induction of LTP of mossy fiber inputs could further reflect the actions of synaptically triggered second messengers and regulation of subunits of the Cav3-Kv4 complex. Thus, the preliminary data in the field has led me to propose the following:

Hypothesis: The differential expression of a Cav3-Kv4 channel complex and resulting Kv4 availability regulates granule cell excitability, spike output, and response to mossy fiber input across cerebellar lobules.

Aim 1: Determine the expression of the Cav3-Kv4 complex in granule cells between the anterior and posterior lobes of cerebellum. Aim 2: Determine the role of the Cav3-Kv4 complex in regulating the excitability of granule cells and their postsynaptic processing of incoming mossy fiber inputs. Aim 3: Determine the potential for long term potentiation of mossy fiber inputs to induce a change in postsynaptic excitability by modifying the availability of IA in granule cells.

Methods: Patch clamp recordings and immunocytochemistry will be conducted on sagittal sections of rat cerebellum to examine the expression pattern and physiological function for a Cav3-Kv4 complex. For practical reasons recordings will be centered upon granule cells in lobules 2 and 9 given their position at either extreme of previously reported gradients of Kv4 expression, and ready access through tissue slice preparation.

Significance: This work will provide the first molecular insights into the differential processing of information across the the cerebellum, and which aspects of synaptic and intrinsic excitability are modulated in the course of establishing a change at the synapse believed to represent the underpinnings of learning.

1.8 Acknowledging Collaborators

The work presented in this thesis involved collaborations with N.C. Heath, M. Kruskic and

X. Zhan. Data that include their contributions will be indicated in the appropriate figure legends.

Figure 1-1: Trilayer neural circuitry of the cerebellum

The outermost layer is the molecular layer that consists of dendrites of Purkinje cells and the inhibitory interneurons, stellate and basket cells. Below the molecular layer is the Purkinje cell layer containing the cell bodies of Purkinje cells. The innermost layer is the granule cell layer comprised of granule cells and Golgi cell inhibitory interneurons. Adapted from Dean et al. (2010).

Figure 1-2: A cerebellar slice and the mossy fiber- granule cell synapse

A, Sagittal view of cerebellum demonstrating the 10 lobules that are spread across the anterior- posterior axis. B, The glomerulus comprised of the mossy fiber-granule cell synapse containing one mossy-fiber terminal (blue), the dendrites of ~ 50 granule cells (red), dendrites of Golgi cells (green), and axons of Golgi cells (yellow). Modified from Arenz et al. (2009).

Figure 1-3: Classification, structure and expression of Kv4 ion channels

A, Classification of potassium channels based on transmembrane (TM) topology. Voltage-gated potassium channels (Kv) belong to the 6TM family. Kv1-4 channels were initially referred to as Shaker, Shab, Shaw and Shal, respectively. B, Functional Kv4 channels are formed by the tetramerization of four α-subunits. An α-subunit (pore-forming) of Kv4 consists of an N-terminal domain, tetramerization domain (T1), 6 TM segments (S1-S6), S5-helix-S6 pore loop, and an N- terminal domain. The S4 helix is the voltage sensor that is lined with positively charged arginine residues. Two closely associated proteins of Kv4 channels are KChIPs and DPLPs. C, The distribution of mRNA transcripts of Kv4 isoforms in the granule cell layer of separate cerebellar lobules revealed through in situ hybridization. Kv4.2 and Kv4.3 mRNA is expressed in opposing anterior-posterior gradients in the granule cell layer, such that Kv4.2 expression is higher in anterior lobules (i.e. lobule 2) and lower in the posterior lobules (i.e. lobule 9), whereas Kv4.3 expression is higher in posterior lobules (lobule 9) and lower in the anterior lobules (lobule 2). Kv4.1 mRNA is not expressed in cerebellum. Adapted from Serodio and Rudy (1998).

Figure 1-4: Classification, structure and expression of Cav3 ion channels

A, Classification of voltage-gated calcium channels into high voltage-activated (HVA) channels and low voltage-activated (LVA) channels. B, Membrane topology of a calcium channel α- subunit (pore-forming) consisting of 4 domains (Domains I-IV) connected by cytoplasmic linkers. Each domain consists of 6 transmembrane helices and S5-helix-S6 pore loop. The positively charged fourth helix, S4, in each domain forms the voltage sensor. C, In situ hybridization to detect mRNA transcript distribution of the Cav3 isoforms in the granule cell layer of cerebellum. Cav3.3 is uniformly expressed in granule cells and Cav3.1 uniformly expressed in the Purkinje cell layer across cerebellar lobules. Cav3.1 is expressed in a distinctly different manner in the granule cell layer of anterior vs. posterior cerebellum, with high expression in the posterior lobules (i.e. lobule 9) and lower in the anterior lobules (i.e. lobule 2). The mRNA transcript for Cav3.2 is very lightly expressed in cerebellum. Adapted from Talley et al. (1999).

Figure 1-5: A functional complex between Cav3 and Kv4 channel in stellate cells of cerebellum

A, A schematic of the Cav3-Kv4 complex linked at the level of the C terminus and illustrating influx of calcium ions through Cav3 channels and efflux of K+ ions through Kv4 channels. KChiP3 is positioned beneath the Kv4 subunits to act as the calcium sensor for the Cav3-Kv4 complex. B, Calcium influx increases the efflux of K+ ions by inducing a depolarizing shift in the inactivation-voltage relationship of Kv4 channels to increase the availability of Kv4 channels near resting membrane potentials. C, The Cav3 channel-mediated increase in the availability of Kv4 channels promotes a reduction in the firing rate of stellate cells.

Figure 1-6: The neuronal ERK/MAPK pathway

ERK kinase is activated by influx of calcium ions through NMDA receptors and voltage-gated calcium channels (VGCC) that leads to an increase in the level of Ras-GTP. Ras-GTP activates Raf, a protein kinase, that activates mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which eventually activates ERK. ERK phosphorylates both nuclear and cytoplasmic proteins. Modified from (Thomas and Huganir, 2004)

Figure 1-7: Cerebellar lobules receive mossy fiber inputs from specific sources

Schematic illustration of the dorsal view of cerebellum indicating the distribution of mossy fiber inputs that arise from spinal regions (motor control), pontine nuclei (cortical input), and vestibular nuclei across the lobules. All indicated distributions are with respect to the midline. Shaded bars at right depict the general regions of cerebellar function defined by the distribution of inputs together with lesion and fMRI studies. Modified from: Sillitoe and Joyner (2007)

Figure 1-8: Graphical abstract of the hypothesis

The cerebellum processes input ranging from motor to vestibular signals carried by mossy fibers to granule cells across 10 cerebellar lobules. Motor-related mossy fiber input is conveyed by high frequency bursts and processed in anterior lobules. Vestibular inputs are conveyed as longer spike trains of varying frequencies to posterior lobules. The extent to which afferent input can be differentially processed across lobules is unknown, but a highly uniform trilayer circuit structure implies a role for postsynaptic signal processing. Our hypothesis is that the differential expression of the Cav3-Kv4 complex in the granule cell layer can thus support differential signal processing and learning across cerebellar lobules.

Chapter Two: Methods

2.1 Electrophysiology

2.1.1 Animal Care and Cerebellar Slice Preparation Sprague-Dawley rats were obtained from Charles River and maintained according to the Canadian Council of Animal Care and the University of Calgary Animal Care committee. Established protocols were used for tissue dissection and slice preparation (D'Angelo et al., 1999; Anderson et al., 2010b). Male pups between the ages of postnatal (P) 19 and 23 were used as granule cells older than P19 are considered representative of the adult condition (Rossi et al., 1994). All chemicals for slice preparation and recordings were obtained from Sigma unless otherwise specified. Rats were anesthetized by inhalation of isoflurane until unresponsive to tail pinch. The cerebellum was dissected out and harvested in ice-cold artificial cerebrospinal fluid (aCSF) composed of (mM): 125 NaCl, 3.25 KCl, 1.5 CaCl2, 1.5 MgCl2, 25 NaHCO3, and 25 D-glucose preoxygenated by carbogen (95% O2, 5% CO2) gas. Parasagittal (240-300 µm) slices from the cerebellar vermis were obtained in ice-cold aCSF gassed by carbogen (95% O2 / 5% CO2) using a Vibratome (Leica VT1000 S, Germany). Slices were then recovered in aCSF maintained at 37°C for 10-30 min before storing at room temperature. Electrophysiological recordings from slices were made in carbogen-gassed aCSF at 32-33°C on the stage of an Olympus BX51W1 microscope equipped with differential interference contrast optics and infrared light transmission (Olympus America INC., Center Valley, PA, USA).

2.1.2 LTP Protocol Mossy fibers were stimulated using a bipolar concentric tungsten electrode (Frederick Haer, Bowdoin, ME, USA) connected to a stimulus isolation unit (Digitimer, Welwyn Garden City, UK). We employed a quasi-physiological stimulus protocol used by Sola et al. (Sola et al., 2004a) to induce LTP of mossy fiber input to granule cells. Mossy fibers were activated using a “theta-burst stimulus” (TBS) pattern (8 bursts of 10 impulses at 100 Hz, 250 msec interburst interval) at a stimulus intensity that initially generated a submaximal EPSC or EPSP from a holding potential of -70 mV. For voltage clamp recordings of IA the synaptic stimulus was also

paired with a postsynaptic voltage step from -70 mV to -40 mV for the duration of the synaptic stimulus train (3 sec total), as used in (D'Angelo et al., 1999). Current-clamp recordings were maintained at -80 mV resting potential and in 14/17 cases employed only synaptic stimulation without a postsynaptic depolarization given adverse affects on spike amplitude when both were applied. A 5 min period was used to establish baseline synaptic amplitudes before presenting TBS and EPSCs/EPSPs then recorded every 7 min for 15-25 min to monitor the induction of LTP.

2.1.3 Current-clamp recordings Whole-cell current-clamp somatic recordings were made using an Axoclamp 700B amplifier and a Digidata 1322 A-D converter using a DC-10 kHz band-pass filter and pClamp software (Molecular Devices, California). Negative bias current of less than 10 pA was applied to maintain the resting potential of granule cells at ~-70 mV. When delivering either short trains of mossy fiber simEPSPs at 100 Hz or sinusoidally varying simEPSP frequency trains, negative bias current was applied to maintain the resting membrane potential of ~-62 mV to ensure that enough spikes were evoked during the simEPSP stimulus to conduct spike frequency analysis. Current-clamp recordings used an electrolyte of (in mM): 126 K-gluconate, 4 NaCl, 5

HEPES, 1 MgSO4, 0.15 BAPTA, 0.05 CaCl2, pH 7.25 via KOH, with 5 di-Tris-creatine phosphate, 2 Tris-ATP, and 0.5 Na-GTP added from fresh frozen stock each day. This internal nominally buffered the resting internal concentration of calcium ([Ca]i) to 100 nM (Gall et al., 2003; Sola et al., 2004a). Electrodes had a resistance of 6-8 MΩ and access resistance of 8-15 MΩ, with cells rejected for any drift in access resistance of >20%. A calculated junction potential of -10.7 mV was subtracted from current-clamp recordings.

2.1.4 Voltage-clamp recordings

Isolation of Kv4 current: External solution: The external aCSF used while conducting voltage-clamp recordings of Kv4 current contained (in mM): 125 NaCl, 3.25 KCl, 1.5 CaCl2, 1.5 MgCl2, 25 NaHCO3 and 25 d- glucose, preoxygenated by carbogen (95% O2, 5% CO2) gas. The aCSF further contained 50 µM picrotoxin, 1 µM CGP55845, 2 mM CsCl and 5 mM TEA.

Internal solution: Voltage-clamp recordings in Chapter 5 used an electrolyte of (in mM): 140

KCl, 10 HEPES, 2.5 MgCl2, 0.15 BAPTA, 0.05 CaCl, 5 TEA, 0.1 QX-314, pH 7.25 via KOH, with 5 di-Tris-creatine phosphate, 2 Tris-ATP, and 0.5 Na-GTP added from fresh frozen stock each day. This internal solution buffered the [Ca]i to 100 nM to establish an EK = -98 mV, ECl = 0 mV. No external Cd2+ was used to preserve HVA channels and avoid observed shifts in the voltage dependence of Kv4 channel activation. Importantly, this combination of blockers allowed for recording of Kv4 current while preserving intact mossy fiber-evoked EPSCs in granule cells in Chapter 5. Voltage-clamp recordings of Kv4 in Chapter 4 used an internal solution in mM: 140 KCl, 0.1 EGTA, 10 HEPES, and 2.5 MgCl2, pH adjusted to 7.3 with KOH. Synaptic transmission was blocked in all recordings in Chapter 4 with bath applied DL-AP5 (25 µM), DNQX (10 µM, Tocris), CGP-55845 (1 µM, Tocris), and picrotoxin (50 µM). Isolation of Cav3 current: External solution: The external aCSF used to isolate Cav3 current was similar to that for Kv4 current recordings, but it also contained 5 mM 4-aminopyridine (4-AP) and 30 µM Cd2+ to block HVA calcium channels, including R-type calcium channels that are known to be expressed in granule cells (Randall and Tsien, 1997; Heath et al., 2014). Internal solution: Voltage-clamp recordings used an electrolyte of (in mM): 100 CsCl, 10

EGTA, 10 HEPES, and 3 MgCl2 , pH 7.25 via KOH, with 5 di-Tris-creatine phosphate, 2 Tris- ATP, and 0.5 Na-GTP added from fresh frozen stock each day.

2.1.5 Voltage-dependence of activation and inactivation The steady-state voltage for inactivation and activation plots for Cav3 and Kv4 currents were constructed using a steady-state clamp protocol from a holding potential of -110 mV and applying successive 10 mV steps to initially activate and then inactivate Cav3 current over 1 sec, followed by a test potential to -30 mV (250 msec). Inactivation plots were constructed using Origin8.0 (OriginLab, Northampton, MA, USA). Inactivation curves were fitted according to the

Boltzmann equation: I = 1/(1 + exp((V−Vh)/k)), where Vh is the half-inactivation potential and k is the slope factor. Activation curves were fit according to the Boltzmann equation: I = 1/(1 + exp((Va−V)/k)), where Va is the half-activation potential and k is the slope factor. Activation

plots of Kv4 were constructed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA).

2.1.6 Specific compounds A mouse monoclonal antibody directed against all the KChIP isoforms (Pan-KChIP; NeuroMab, UC Davis) was included in the patch electrode at 1:100 dilution in specific experiments. The stocks of DNQX, DL-AP5, S-DHPG (Abcam, Cambridge), NMDA, glycine, and MPEP (Tocris, Burlington ON) were dissolved in water and frozen at -20oC before dilution in aCSF to the desired final concentration. The stocks of CGP55845 (Abcam), PD 98059, CPPCCOEt , JNJ 16259685 (Tocris) were dissolved in DMSO and frozen at -20 oC before being diluted in aCSF to the desired final concentration.

2.2 Data analysis and Statistics Data analyses were performed using a combination of pCLAMP 10 (Molecular Devices, California), Origin 8.0 (OriginLab, Northampton, MA), GraphPad Prism (GraphPad Software, La Jolla, CA, USA) softwares, and custom Matlab scripts. Average values are expressed as mean ± S.E.M where sample values represent the number of individual cell recordings. Statistical significance was set at p < 0.05 in all cases. The Shapiro-Wilk normality test was used to ensure that data was drawn from a normally distributed population and statistical significance determined using the paired Student’s t test unless otherwise specified (all within group comparisons for data obtained at baseline recording and 15 min after TBS). The regression slopes of F-I plots were compared using a two-tailed test in Graphpad Prism software. One-way repeated measures ANOVA was used for within group comparisons of Va and Vh over time, and two-way repeated measures ANOVA for between group comparison of Va and Vh over time, with Tukey’s post hoc analysis.

2.3 Criteria of granule cell selection Granule cell recordings were established using a set of criteria previously shown to distinguish between other cell types scattered within the dense layer of granule cells. Golgi cell recordings were distinguished on the basis of visual inspection (20 µm diameter for Golgi cells vs. 7-8 µm for granule cells), a larger membrane capacitance (40-50 pF Golgi cells vs. 4-9 pF

granule cells), and marked spike accommodation during current pulse injection (n = 3)

(Locatelli, 2012). Unipolar brush cells (UBCs) were distinguished by Rin (750-850 MΩ for UBC cells vs. > 1 GΩ for granule cells), membrane capacitance (10-12 pF UBC cells vs. 5-8 pF granule cells), and adaptation of spike firing in response to current injection (D'Angelo et al., 1998; Diana et al., 2007; Locatelli, 2012). Granule cells were accepted for recording if input resistance >1 GΩ, peak spike amplitude exceeded 0 mV, no spontaneous firing was encountered at rest, and cells exhibited an RMP between -50 to -60 mV in the absence of bias current injection (RMP, -60.4 ± 5.43, n = 8).

2.4 Tissue fixation Tissue slices for immunocytochemistry were obtained from male rats (P25-35) deeply anesthetized with isoflurane VSP by inhalation until unresponsive to ear pinch. Animals were perfused intracardially with 250 ml of 0.1 M phosphate-buffer (PB, pH 7.4) followed by 100 ml of 4% paraformaldehyde (PARA, pH 7.4) at room temperature. Brains were placed into 4% PARA at room temperature for 1 hr and left overnight at 4ºC in new 4% PARA. Cerebellar sections of 40-50 µm thickness were cut by vibratome (Leica VT1000 S, Germany) in the sagittal plane in PB.

2.4.1 Immunocytochemistry: Fixed tissue sections were reacted in a working solution consisting of 3% normal donkey serum, 0.2% dimethylsulphoxide (DMSO), and 0.1% TWEEN-20 in PB. Primary antibodies were included in the working solution for 24-72 hrs with gentle agitation on a rocker at 4° C. After thorough washing in working solution sections were exposed for 2-3 hrs at room temperature to AlexaFluor 488-conjugated goat anti-rabbit secondary IgG (1:1000) or AlexaFluor 594-conjugated donkey anti-mouse secondary IgG (1:1000) (Molecular Probes, OR). Sections were washed 3 x 20 min in PB, mounted on microscope slides in anti-fade medium (Fluoromount, Sigma-Aldrich), and stored at –20 ºC. Controls consisted of omitting the primary antibodies, and were included in all experimental tests to compare the relative labeling intensity between control and test conditions.

Fluorescent labeling was imaged on a Zeiss Axioimager microscope (Zeiss, Germany) equipped with Colibri LED illumination and optical sections obtained via Apotome grid illumination using a 0.27 µm section thickness. Post processing of images was restricted to whole image adjustments of levels in Adobe Photoshop CS3 and figures assembled in Adobe Illustrator CS3 (Adobe Systems Incorporated, California).

2.4.2 Colocalisation analysis

Quantitative analysis of colocalisation was measured by Pearson’s correlation coefficient (r),

(Zen2 software, Zeiss), which analyzes pixel by pixel signal intensities from red (X) and green

(Y) channels, plotted in a scatter plot.

, where X refers to the intensity values of the red channel and Y refers to the green channel intensity, respectively, of an individual pixel, and � and Y refer to the mean intensities of the red and green channels, respectively, across the entire image. r value is 1 for two images that are perfectly correlated . r value of −1 refers to two images whose fluorescence intensities are perfectly, but inversely, related to one another. r values near zero reflect distributions of probes that are random and not correlated with one another.

2.4.3 Primary antibodies Cav3.1 -Kv4.x: Kv4.2 and Kv4.3 primary antibodies used for labeling tissue sections in Fig. 3-1 and Fig. 3-2 were monoclonal mouse antibodies (1:500; Neuromab, UC Davis). The anti- Kv4.2 antibody recognizes the extracellular S1-S2 loop (residues 209-225) of Kv4.2, and the Kv4.3 antibody the cytoplasmic C-terminus region of the Kv4.3 isoform. Cav3.1 channel immunolabel was detected using a polyclonal rabbit antibody (1:500; Alomone Labs, Jerusalem) that recognizes the intracellular N-terminus of Cav3.1 (residues 1-22). Cav3.3: Antibody directed to Cav 3.3 was produced by using the Caulobactor expression system (Invitrogen) and corresponded to amino acids 1013–1115 (GenBank accession no.AF290214) found within the II-III linkers region of the Cav3.3 calcium channel. Purified

proteins from the expression system were then injected into rabbits, and the Cav3.3 polyclonal antibodies (1:500) were purified from rabbit’s blood plasma before use. See Molineux et al. (2006) for further details. Kv4.2-Kv4.3: The Kv4.2 primary antibody used for tissue sections was a monoclonal mouse antibody (1:500, Neuromab, UC Davis) and the Kv4.3 primary antibody a rabbit monoclonal antibody (1:500; Alomone Labs, Jerusalem); The Alomone antibody recognizes intracellular C- terminus (451-468) of Kv4.3 isoform. KChIP: Anti-KChIP1, 2, 3 and 4 antibodies used for tissue sections were monoclonal mouse antibodies obtained from Neuromab, UC Davis (1:250). DPP6: DPP6 primary antibody for tissue labeling was a polyclonal rabbit antibody that was obtained from Abcam and used at a dilution of 1:500.

Chapter Three: Expression of the subunits of the Cav3-Kv4 complex in granule cells

3.1 Introduction

Earlier studies on the Cav3-Kv4 complex in cerebellar stellate cells identified the subunits required to create a functional ion channel complex to allow Cav3 calcium influx to enhance Kv4 availability near resting potentials. Protein biochemistry and electrophysiology established that the Cav3-Kv4 complex can be comprised of subunits from any of the Cav3 or Kv4 isoforms, but has an absolute requirement for the expression of KChIP3 (Anderson et al., 2010b; Anderson et al., 2010a). None of KChIPs 1, 2 or 4 can substitute for KChIP3 to modify Kv4 current when coexpressed in tsA-201 cells (Anderson et al., 2010b). It is thus possible that the functional expression of a Cav3-Kv4 complex in different brain regions will depend on the coexpression pattern of all three elements of Cav3, Kv4, and KChIP3 subunits. Intense labeling has been reported for both Kv4.2 and Kv4.3 mRNA through in situ hybridization and immunocytochemistry, including rostro-caudal gradients for subunit expression in the granule cell layer (Serodio and Rudy, 1998; Strassle et al., 2005; Amarillo et al., 2008). Thus, Kv4.2 is strongly expressed in granule cells of anterior lobules whereas Kv4.3 is highly expressed in posterior lobules. By comparison, reports on the expression pattern of Cav3 channels and recording of T-type current in granule cells has varied. Early electrophysiology studies using cultured granule cells reported only R-type channel expression and little if any calcium current consistent with T-type channel expression (Randall and Tsien, 1995; Tottene et al., 1996). A continued lack of evidence for this current in intact tissue further delayed any study of the function of Cav3-mediated T-type current in granule cells (Rossi et al., 1994). The apparent lack of T-type current might also reflect variability in the cerebellar lobule targeted for recordings, an aspect of cerebellar physiology not considered extensively until recently. In this regard, in situ hyridization for Cav3 channel isoforms reported mRNA for Cav3.1 and Cav3.3 in cerebellar granule cells (Talley et al., 1999). The labeling for Cav3.1 mRNA was distinctive in exhibiting very high levels of expression in granule cells of all cerebellar lobules posterior to lobule 5, while labeling in anterior lobules was restricted to the Purkinje cell layer. The Cav3.3 isoform exhibited a light but uniform level of expression in granule cells across all lobules, with no reported label for Cav3.2 (Talley et al., 1999).

The expression of KChIP3 in granule cells as the third required subunit of the Cav3-Kv4 complex also suggests regional labeling. Immunocytochemistry detected the expression of KChIPs 1, 3, and 4 but not KChIP2, with a strong association between Kv4.2 and KChIP1 (Strassle et al., 2005). In situ hybridization studies on KChIP3 in granule cells reported an anterior-posterior gradient, with a variable density of punctate KChIP3 immunolabel in the granule cell layer (Xiong et al., 2004; Clark et al., 2008). Anderson et al. also showed that DPLPs form an integral part of the Cav3-Kv4 complex (Anderson et al., 2010b; Anderson et al., 2010a). DPP6, one of two family members of DPLPs, is highly expressed in the granule cell layer (Jerng and Pfaffinger, 2012). Clark et al. further reported that DPP6 exhibits an anterior-posterior gradient of expression similar to Kv4.2 (Clark et al., 2008). The possibility of T-type calcium current in regions of Cav3 expression, and the properties of A-type currents in anterior vs. posterior granule cells were thus unknown. The extent to which these channel subunits and KChIP3 are coexpressed in granule cells in these regions was also unknown. This is important given that differences in sensory innervations between anterior and posterior lobules may require that granule cells express the Cav3-Kv4 complex to manage signal processing requirements. As an initial part of assessing the role for the Cav3-Kv4 complex in granule cells the current chapter examines the relative distribution of Cav3, Kv4, and KChIP families and DPP6 across cerebellar lobules through immunocytochemistry. Selective attention is focused on granule cells in lobules 2 or lobule 9c as representative examples of reported differences in channel expression between anterior and posterior cerebellar lobules.

3.2 Methods

Methods for this section are described thoroughly in Chapter 2 of the thesis.

3.3 Results

The results of our immunohistochemistry analysis are summarized in Table 2. Since multiple variables can affect the fluorescent intensity of immunohistochemistry signals, these results should not be taken to infer absolute expression levels of different components of the Cav3-Kv4

complex. At best these results tell us whether a certain component of the Cav3-Kv4 complex is expressed in the granule cell layer and if the relative level of fluorescent intensity (loosely translates to expression) differs across the cerebellar lobules.

3.3.1 Cav3.1 and Kv4.2 immunolabel in granule cells

Cav3.1 immunolabel was detected throughout the cerebellar vermis (Fig. 3-1A). The intensity of Cav3.1 label was higher in the Purkinje cell and molecular layers compared to the granule cell layer. Interestingly, the granule cell layer showed the weakest intensity for Cav3.1 immunolabel in lobule 9 compared to other lobules (Fig. 3-1A; Table 1). Despite a lower relative intensity for Cav3.1 label in the lobule 9 granule cell layer, higher magnification at the single cell level revealed fluorescence for Cav3.1 as a diffuse label in the cytoplasm and presumed plasma membrane. Additional diffuse labeling was detected in structures surrounding granule cells in presumed synaptic glomeruli (Fig. 3-1D), a result supported in Chapter 4 in terms of the sensitivity of synaptic transmission to Cav3 channel blockers. The intensity of fluorescent signal for Kv4.2 immunolabel was highest in the anterior lobules and weakest in the posterior lobules (Fig. 3-1B; Table 1). This result confirms the previously reported gradient of expression of Kv4.2 immunolabel in the granule cell layer across cerebellar lobules (Serodio and Rudy, 1998). Using higher magnification in lobule 9c, we detected the presence of Kv4.2 as an intense label that excluded the nucleus and was distributed in the cytoplasmic and plasma membrane regions of granule cell somata (Fig. 3-1E). An initial estimate of the degree of colocalization of Cav3.1 and Kv4.2 was gained according to the yellow emission of the combined labels in Figure 3-1C, with strongest apparent colocalization in the anterior lobules and weakest in the posterior lobules. At the single granule cell level in lobule 9c, a weak colocalization of Cav3.1 and Kv4.2 was observed in granule cells that included apparent plasma membrane-like label of individual granule cells (Fig. 3-1F), with a colocalizaton analysis (Zen2 software, Zeiss) reporting a Pearson correlation coefficient, r, of 0.32 (Fig. 3.1-G).

3.3.2 Cav3.1 and Kv4.3 immunolabel in granule cells

Kv4.3 immunolabel in cerebellum showed a gradient of expression in the granule cell layer across lobules that were distinct from Kv4.2 immunolabel (Fig. 3-2B; Table 1). We detected the strongest Kv4.3 immunolabel in the granule cell layer in lobules 9c and 10 whereas other lobules had relatively weaker label (Fig. 3-2B). Using higher magnification, individual granule cells in lobule 9c exhibited pronounced Kv4.3 immunolabel in a plasma membrane-like distribution that contrasted with a lack of nuclear label (Fig. 3-2E). Potential colocalization of Cav3.1 and Kv4.3 as a yellow emission in merged images of dual labeled tissue was strongest for the granule cell layer in posterior lobule 9c (Fig. 3-2C). While the granule cell layer of lobule 10 also showed coexpression, it was not uniform throughout the lobule. At higher magnification in the granule cell layer of lobule 9c there was strong apparent Cav3-Kv4 colocalization in the plasma membrane of individual cells (Fig. 3-2F), with a colocalizaton analysis (Zen2 software, Zeiss) reporting a Pearson correlation coefficient, r, of 0.20 (Fig. 3.2-G).

3.3.3 Colocalization of Kv4.2 and Kv4.3 in granule cells

Given the immunohistochemical data supporting opposing gradients of expression of Kv4.2 and Kv4.3 isoforms in the granule cell layer across cerebellar lobules, we examined if Kv4.2 and Kv4.3 are coexpressed in single granule cells in the anterior and posterior lobules. Dual labeling for Kv4.2 and Kv4.3 showed coexpression of each channel isoform in individual cells of lobule 2 (Fig. 3-3A,B), with merged images reporting putative colocalization in a plasma membrane-like pattern in almost every cell (Fig. 3-3C) (Pearson correlation coefficient, r, = 0.40). Dual labeling for Kv4.2 and Kv4.3 also revealed coexpression of these isoforms in the vast majority of individual granule cells of lobule 9 (Fig. 3-3D, E) and a colocalization at the plasma membrane level (Fig. 3-3F) (Pearson correlation coefficient, r, = 0.29). Collectively, these data suggest that both Kv4.2 and Kv4.3 isoforms contribute to the A-type currents recorded in granule cells of both anterior and posterior lobules.

3.3.4 Expression of Cav3.3 in granule cells

Cav3.3 immunofluorescent label in granule cell layers varied from a diffuse to punctate pattern with no clear rostro-caudal level of intensity (Fig. 3-4A). Instead we detected clear Cav3.3 immunofluorescence in the Purkinje cell and molecular layers of all lobules (Fig. 3-4A), which differs from that Cav3.1. Using higher magnification, individual granule cells in lobules 2 and 9c exhibited Cav3.3 immunolabel as a combination of diffuse cytoplasmic label, with some evidence for a punctate plasma membrane-like distribution that contrasted with a lack of nuclear label (Fig. 3-4B,C).

3.3.5 Expression of KChIPs

Immunolabels for each of the KChIP1-4 isoforms were examined across cerebellar lobules. The most pronounced label with a distinct difference between anterior and posterior lobules was found for KChIP1 (Fig. 3-5A). In particular, KChIP1 labeling in the granule cell layer was very strong in the anterior lobules 1-5 but very weak in the posterior lobules (Fig. 3-5A; Table 1). This difference was punctuated by an abrupt transition in labeling intensity at lobule 5, where posterior lobules exhibited much lower labeling for KChIP1. Interestingly, this difference was mirrored in the label detected in the Purkinje cell layer, with brighter fluorescent label in posterior than anterior lobules, with a similar abrupt transition posterior to lobule 5 (Fig. 3-5A). In the anterior lobules, higher magnification revealed that KChIP1 immunolabel was distributed in a punctate manner in most granule cells and in others at much higher density as to label the cytoplasm and putative plasma membrane (Fig. 3-5B). Higher magnification analysis also revealed a punctate label for KChIP1 in lobule 9 granule cells but at a lower intensity than lobule 2 (Fig. 3-5C). Immunolabel for KChIP2 was of lower intensity in the granule cell layer throughout all cerebellar lobules compared to the Purkinje cell and molecular layers (Fig. 3-6A; Table 1). Higher magnification in the granule cell layers again indicated a punctate label for KChIP2 in individual cells in both lobule 2 and 9 (Fig. 3-6B,C). Of particular potential importance to functional expression of the Cav3-Kv4 complex is the distribution of KChIP3, the single KChIP isoform previously shown capable of supporting a functional modulation of Kv4 availability

(Anderson et al., 2010b). Yet no distinct pattern for KChIP3 immunolabel was apparent in the granule cell layer across lobules, with an apparent lack of label in the Purkinje cell layer of many lobules (Fig. 3-7A; Table 1). Higher magnification revealed one of the highest densities of punctate immunolabel for KChIP3 in individual granule cells in both lobule 2 and 9 (Fig. 3- 7B,C). Immunolabel for KChIP4 revealed relatively little apparent expression in Purkinje cell and molecular layers. Interestingly, the granule cell layer showed a variable level of intensity in each lobule, with some suggestion of an anterior-posterior gradient of expression. The highest intensity for KChIP4 immunolabel was in the deep sulci of lobules 1-3, some intermediate intensity again centered in the deep sulci region of lobules 6, 7, and 8, and the lightest level of KChiP4 immunofluorescence in lobules 9 and 10 (Fig. 3-8A; Table 1). Higher magnification images revealed a punctate, presumed membrane associated immunolabel for KChIP4 in individual granule cells in both lobule 2 and 9c (Fig. 3-8B, C).

3.3.6 Expression of DDP6 in granule cells

DPP6 immunolabel was apparent in the granule cell layer with distinct expression across the lobules, with higher labelling intensity in lobules 1-3 and 10 (Fig. 3-9A; Table 1). The cell bodies and large dendrites of Purkinje cells in the molecular layer also exhibited very strong labelling for DPP6. High magnification images of the granule cell layer revealed diffuse labelling of DPP6 in the presumed plasma membrane of granule cells of lobules 2 and 9c (Fig. 3- 9B, C).

3.4 Discussion

The immunohistochemistry experiments conducted here establish that all the principal components of the Cav3-Kv4 channel complex are expressed in granule cells across all lobules of cerebellum, but with a differential expression pattern for specific subunits. The intensity of fluorescent label clearly demonstrated an anterior-posterior gradient of apparent expression level for Kv4.2 in granule cells, strongest in the anterior lobules and weakest in posterior lobules, as previously reported (Fig. 3-1) (Serodio and Rudy, 1998). However, we found strong labeling for Kv4.3 expression only in granule cells of lobules 9c and 10, with lower

relative expression in all the other lobules. This pattern is different from the previously reported anterior-posterior gradient of Kv4.3 expression reported in the granule cell layer for both mRNA and immunolabel, where Kv4.3 expression was higher in posterior lobules (6-10) than the anterior lobules (1-5) (Serodio and Rudy, 1998; Amarillo et al., 2008). Previous in situ studies indicated that mRNA of Cav3.1 is strongly expressed in granule cells of posterior lobules (lobules 6-10) compared to anterior lobules (Talley et al., 1999). In contrast, we found that Cav3.1 immunolabel in the granule cell layer had a relatively uniform expression throughout all the lobules except for lobule 9, where there was relatively lower intensity of label for Cav3.1. Despite a low relative expression in lobule 9, Cav3.1 immunolabel was detectable at the level of single granule cells. On the other hand, Cav3.1 immunolabel did not show any noticeable regional differences across the anterior-posterior axis for either the Purkinje cell or molecular layers. We also detected Cav3.3 immunolabel intensity in the granule cell layer as being relatively uniform through all the lobules, in agreement with Tally et al. (Talley et al., 1999) Both the Talley et al. in situ study (Talley et al., 1999) and our immunohistochemical labeling was conducted in adult rats, so the age of animals cannot account for the discrepancies in pattern of Cav3.1 expression. The reason for an apparent discrepancy between Cav3.1 fluorescent signal intensity and the density of mRNA label in lobule 9 (Talley et al., 1999) is not fully known but could reflect the specific isoform of Cav3.1 detected by the antibody used here. It is also known that the intensity of mRNA labeling cannot be directly correlated to the level of protein translation or membrane expression. In summary, our Cav3 immunohistochemistry results would suggest that both Cav3.1 and Cav3.3 isoforms are expressed in granule cells across the cerebellar lobules, including lobule 9. We also found that Kv4.2 and Cav3.1 mainly coexpress in the anterior lobules, whereas Kv4.3 and Cav3.1 coexpress in lobules 9c and 10. Thus, the ion channels that are required to constitute a Cav3-Kv4 complex are found in granule cells across the cerebellum. The current set of experiments also provides evidence for the first time that two isoforms of Kv4, Kv4.2 and Kv4.3, are colocalized at the single granule cell level in cerebellum. Kv4.2 (69 KDa) and Kv4.3 (71 KDa) are highly homologous proteins, and there are no reported biophysical differences between the two isoforms, at least when expressed in heterologous systems in vitro (Hsu et al., 2003). Thus, we expect both Kv4.2 and Kv4.3 to contribute to A-type currents recorded in

granule cells. It is important to note that an active Cav3-Kv4 complex can consist of either Kv4.2 or Kv4.3 isoforms (Anderson et al., 2010b). Therefore, there is a distinct possibility that these two isoforms form heteromeric Kv4 channels in granule cells in which the Cav3-Kv4 complex is active. Kv4 channels are regulated by two distinct modulatory subunits: KChIPs and DPLPs. Thus, expression of these two subunits with Kv4 across cerebellum may be an important consideration to fully understand the differential activity of Cav3-Kv4 activity across cerebellum. Our immunohistochemistry analysis revealed that KChIP1 is highly expressed in granule cells of anterior lobules, with a sharp reduction in expression posterior to lobule 5. This is consistent with a previous study where a similar pattern of expression was observed for KChIP1 in the granule cell layer (Strassle et al., 2005) . We also found uniform expression of KChIP2 in the granule cell layer of cerebellum. This result is in contrast to previous studies where KChIP2 was reportedly not expressed in the granule cell layer (Strassle et al., 2005). In our studies, there was a uniform expression of KChIP3 and KChIP4 protein immunofluorescence throughout the granule cell layer, also consistent with a previous study (Strassle et al., 2005). The expression of KChIP3 throughout the granule cell layer of cerebellum is important because KChIP3 is absolutely required for a functional Cav3-Kv4 complex. We found KChIP3 at the level of single granule cell as a dense punctate label suggestive of localization within a channel complex at the level of plasma membrane. The uniform expression of DPP6 across cerebellar lobules also suggests that Cav3-Kv4 complex is active in granule cells throughout all the lobules. There are two genes in the DPLP family: DPP6 and DPP10. DPP6 is the only member of the DPLP family expressed in the cerebellar granule cell layer, with the DPP6 gene producing four different protein variants (DPP6a , DPP6K, DPP6L, DPP6S) (Jerng and Pfaffinger, 2012). In granule cells DPP6 sets the

V0.5 of steady state inactivation curve to ~ -70 mV, with co-expression of DPP6a, DPP6K, Kv4.2 and KChIP3 in xenopus oocytes producing A-type current that biophysically resembles native A- type current recorded in granule cells. Since DPP6L and DPP6K also have minor contributions to the native A-type current in granule cells, we used a DPP6 antibody that recognizes all the variants of DPP6. Our immunohistochemistry data shows that DPP6 is expressed in the granule cell layer, albeit with higher expression in lobules 1-3 and 10. Since we are interested mainly in

lobules 2 and 9, the DPP6 expression data suggests that there might be a biophysical difference between Kv4 A-type current recorded from these lobules, and will be explored in the next chapter. Overall, we established that all the components of a multi-protein Cav3-Kv4 ion channel complex along with its calcium sensor, KChIP3, and DPP6 are expressed in the granule cells in all the lobules of the cerebellum. While we know from protein biochemistry and Cav3 and Kv4 subunits can form a link at the molecular level (Anderson et al., 2010), a common expression pattern of the different subunits that contribute to this complex does not in itself define where a Cav3-Kv4 is functionally expressed.

Granule cell layer Anterior Posterior Gradient

Kv4.2 ++++ + Anterior > Posterior

Kv4.3 + ++++ (Lob. 9c) Posterior > Anterior KChIP1 ++++ + Anterior > Posterior

KChIP2 + + Uniform

KChIP3 ++ ++ Uniform

KChIP4 +++ (Lob. 1-3) + Anterior> Posterior

Cav3.1 + + Uniform except for low

expression in lobule 9

Cav3.3 + + Uniform

DPP6 +++ (Lobules 1-3) +++ (Lobules 10) High expression in

lobules 1-3 and 10

Table 1: Immunoreactivity in granule cell layer of the Cav3-Kv4 macromolecular complex

Summary of Kv4.2, Kv4.3, Cav3.1, Cav3.3, KChIP1, KChIP2, KChIP3, KChIP4 and DPP6 in granule cells across the anterior-posterior axis. -, absent; +, low; ++ moderate; +++, strong;

++++, high.

Figure 3-1: Cav3.1 and Kv4.2 coexpression in granule cells

Dual label immunocytochemistry to localize the expression pattern of Cav3.1 channels (green) and Kv4.2 channels (red) in granule cells across cerebellar lobules. A-C, Immunolabel distribution for Cav3.1 (A) and Kv4.2 (B) across cerebellar lobules reveals expression of both channel isoforms (C) in many regions of cerebellum. The location of anterior lobule 1, and posterior lobules 9c and 10 are indicated in (A), as are the granule cell (GCL) and molecular layers (mol). Note an anterior-posterior gradient in the intensity of Kv4.2 immunolabel in (B). D-F, Higher magnification image of granule cells in lobule 9c indicates Cav3.1 (D) and Kv4.2 (E) immunolabel in individual granule cells. The merged images in (C, F) reveal an apparent partial colocalization of labels in lobule 9c, with a Pearson correlation coefficient value of r = 0.32 measured through scatter plot in (G). Scale bars, A-C, 500 µm; D-F, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-2: Cav3.1 and Kv4.3 coexpression in granule cells

Dual label immunocytochemistry to localize the expression pattern of Cav3.1 channels (green) and Kv4.3 channels (red) in granule cells across cerebellar lobules. A-C, Immunolabel distribution for Cav3.1 (A) and Kv4.3 (B) across cerebellar lobules reveals expression of both channel isoforms (C) in many regions of cerebellum. The location of anterior lobule 1, and posterior lobules 9c and 10 are indicated, as are the granule cell (GCL) and molecular layers (mol). Note the high density of Kv4.3 in lobules 9c and 10 in (B). D-F, Higher magnification image of granule cells in lobule 9c indicates Cav3.1 (D) and Kv4.2 (E) immunolabel and colocalization of both channel isoforms in individual granule cells. The merged images in (C, F) reveal an apparent partial colocalization of labels in lobule 9c, with a Pearson correlation coefficient value of r = 0.20 measured through scatter plot in (G). Scale bars, A-C, 500 µm; D- F, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-3: Kv4.2 and Kv4.3 coexpression in granule cells

Dual immunolabel for Kv4.2 (red) and Kv4.3 (green) in granule cells of cerebellar lobules 2 and 9. Immunolabelling for Kv4.2 and Kv4.3 in lobules 2 (A-C) and 9 (D-F) reveals coexpression of both Kv4 channel isoforms within single granule cells. Merged images in (C, F) reveal plasma membrane-like colocalization for both labels in lobules 2 and 9, with a Pearson correlation coefficient value for colocalization of r = 0.40 and 0.29, respectively (G, H). Scale bars, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-4: Cav3.3 expression in granule cells

Cav3.3 immunofluorescence exhibits a uniform pattern across cerebellar lobules. A, Low power image reveals the strongest Cav3.3 immunolabel in the Purkinje cell and molecular layers across all lobules. The granule cell layer exhibits Cav3.3 immunolabel that varies from a diffuse to punctate pattern but no clear rostral-caudal gradient of intensity. The location of anterior lobule 2 and posterior lobules 9c are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals a combination of diffuse and punctate labeling most clearly associated with cytoplasmic regions but with some cases of plasma membrane-like punctate labeling. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-5: KChIP1 expression in granule cells

KChIP1 immunolabel is differentially expressed across cerebellar lobules. A, Low power image of immunolabelling for KChIP1 reveals a high intensity in granule cells in the anterior lobules, with a sharp reduction in label posterior to lobule 5 (arrow). The location of anterior lobule 2 and posterior lobules 9c and 10 are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals punctate plasma membrane-associated immunolabel and higher density labeling in lobule 2. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-6: KChIP2 expression in granule cells

KChIP2 immunolabel exhibits a uniform expression across cerebellar lobules. A, Low power image reveals a high intensity of KChIP2 immunolabel in the Purkinje cell layer and molecular layers, with lower intensity labeling in granule cell layers and no obvious gradient in the anterior-posterior axis. The location of anterior lobule 2 and posterior lobules 9c and 10 are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals punctate plasma membrane-associated immunolabel. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-7: KChIP3 expression in granule cells

KChIP3 immunolabel exhibits a uniform expression across cerebellar lobules. A, Low power image suggests a moderate intensity of KChIP2 immunolabel in the Purkinje cell and molecular layers, with lower labeling in granule cell layers and no obvious gradient in the anterior-posterior axis. The location of anterior lobule 1 and posterior lobules 9c and 10 are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals high density of punctate plasma membrane-like immunolabel. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-8: KChIP4 expression in granule cells

KChIP4 immunofluorescence exhibits a gradient of intensity between cerebellar lobules. A, Low power image reveals at most a low level of KChIP4 immunolabel in the Purkinje and molecular layers. Labeling in granule cell layers was variable and brightest in the deep sulci of anterior lobules 1-5, intermediate in lobules 6, 7, and 8, and some of the lightest immunolabel intensity in lobules 9 and 10. The location of anterior lobule 2 and posterior lobule 9c are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals a high density of punctate plasma membrane-like immunolabel. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Figure 3-9: DPP6 expression in granule cells

DPP6 immunolabel exhibits high expression in lobules 1-3 and lobule 10. A, Low power image reveals a strong DPP6 immunolabel in the Purkinje cell layer, with high labeling in granule cell layers in certain lobules. The location of anterior lobule 2 and posterior lobules 9c are indicated, as are the granule cell (GCL) and molecular layers (mol). B, C, Higher magnification view of granule cells of lobules 2 (B) and 9 (C) reveals diffused labeling of plasma membrane. Scale bars, A, 500 µm; B, C, 10 µm. Immunohistochemistry performed by M Kruskic.

Chapter Four: A Cav3-Kv4 complex differentially regulates spike output in cerebellar granule cells

4.1 Introduction

The fast inactivating Kv4 channels are highly expressed in cerebellar granule cells (Serodio and Rudy, 1998; Maffie and Rudy, 2008a). The A-type current (IA) carried by Kv4 channels is known to regulate multiple aspects of spike discharge in neurons, including first spike latency

(FSL), spike threshold, and frequency (Jerng et al., 2004; Molineux et al., 2005). Since IA is most available at hyperpolarized membrane potentials that relieve steady-state inactivation, one would expect a direct relation between FSL and membrane potential. However, in a recent study, our lab demonstrated that there is a non-monotonic relation between FSL and membrane voltage in cerebellar stellate cells, with FSL being the longest at the intermediate level of hyperpolarization (-74 to -70 mV) (Molineux et al., 2005). Latency to first spike was found to be shorter in the preceding hyperpolarizing or depolarizing range. Cav3 calcium channel blockers reduced the long FSL in the intermediate voltage range, thus eliminating the non-monotonic relation between

FSL and membrane voltage. Stellate neurons express Cav3 current (IT) and IA, and it was the coexpression of these two conductances that produced the novel spike latency profile of stellate cell firing. In a later study, our lab showed that Cav3 channels form a complex with Kv4 channels, whereby the influx of calcium ions enhances the efflux of potassium ions to reduce stellate cell excitability (Anderson et al., 2010b). Thus, Cav3 blockers reduce the availability of IA, shifting its inactivation-voltage relationship in the hyperpolarizing direction. KChIP3, an auxiliary subunit of Kv4 channels, was found to be the calcium sensor of the Cav3-Kv4 complex. Infusion of antibodies against KChIP3 proved to have a similar effect as blockers of Cav3 channels, i.e. a shift of the inactivation-voltage relation in the hyperpolarizing direction. Anderson et al. also established that the Cav3-Kv4 complex can be comprised of any of the Kv4 channel isoforms and any of the three Cav3 channel isoforms (Anderson et al., 2010b; Anderson et al., 2010a) Intriguingly, the mRNA levels of the constituents of the Cav3-Kv4 complex in granule cells exhibit an anterior-posterior gradient across cerebellar lobules. Talley el al. (1999) showed that mRNA for the Cav3.1 isoform is strongly expressed in the posterior half of cerebellum (lobules 6-10) and only weakly expressed in the anterior lobules (lobules 1-5). Similarly, for Kv4 channels, the Kv4.2 mRNA levels are highest in the anterior half of cerebellum, whereas Kv4.3

mRNA levels are highest in the posterior half (Serodio and Rudy, 1998). Since the Cav3-Kv4 channel complex can include any of the isoforms of these channels, it is plausible that the variable expression pattern of Cav3 channels may influence the functional expression of the Cav3-Kv4 interaction across the different lobules. Thus, the experiments described in this chapter test how a differential expression of the subunits of the Cav3-Kv4 complex in granule cells across cerebellum regulates the excitability and postsynaptic processing of incoming mossy fiber inputs.

4.2 Methods Methods for this section are described thoroughly in Chapter 2 of the thesis.

4.3 Results:

4.3.1 Identification of granule cells

The granule cell layer consists of a wide variety of neurons that include granule cells, unipolar brush cells (UBCs), Golgi cells, Lugaro cells and other poorly defined large interneurons (Mugnaini et al., 2011). Since granule cells are the smallest neurons within the granule cell layer (7-8 µm), the cells were initially identified based on visual inspection. We also used electrophysiological parameters to ensure that the recordings were conducted from granule cells. One of the distinguishing properties of granule cells during current-clamp recordings is tonic firing in response to current injection without spike adaptation (Fig. 4-1) (D'Angelo et al., 1998). However, a class of UBCs not unlike granule cells in somatic diameter also do not exhibit spike adaptation (Kim et al., 2012). Thus, UBCs were distinguished from granule cells based on their exhibiting a membrane capacitance that is 4-6 times larger than granule cells (Mugnaini et al., 2011). This agrees with our measurements in finding that granule cells exhibit a membrane capacitance in the range of 5–8 pF (Heath et al., 2014). Capacitance was also helpful as a distinguishing parameter when granule cells were recorded in the voltage-clamp mode. Therefore, granule cell recordings were only accepted if the measured membrane capacitance value was less than 10 pF.

4.3.2 Lobules 2 vs. 9: Resting membrane and spike properties of granule cells

The intrinsic properties of granule cells were studied using the whole-cell current-clamp configuration in the presence of excitatory and inhibitory synaptic blockers, which allowed us to study the intrinsic postsynaptic properties of granule cells independent of synaptic influences.

4.3.3 Passive membrane properties:

We measured input resistance (Rin), cell capacitance (Cm), and resting membrane potential

(RMP) of lobule 2 and 9 granule cells. Rin is an important membrane parameter that can affect the excitability of neurons, and is dependent upon cell size and the density of ion channels open at rest. Rin is defined as the change in membrane voltage evoked in relation to current injection and can be measured as the slope of current-voltage (I-V) plots. A high Rin implies high excitability of a neuron in that small current injections will lead to a larger membrane voltage response, whereas low Rin implies the opposite. RMP is a property that is set by resting ion channels. Cm is a property of a cell’s surface area and affects the time constant, a measure of how quickly the neuron’s membrane potential responds to synaptic inputs. Differences in RMP of two populations of neurons are an indirect way to identify differences in ion channel composition or activation. Based on our hypothesis that there is a differential expression of Cav3 and Kv4 subunits across cerebellum, we tested for a difference in the intrinsic properties between granule cells of lobules 2 and 9.

We found a significant difference between the mean Rin values (lobule 2 Rin = 1.6 ± 0.18 GΩ, n = 10; lobule 9 Rin = 2.4 ± 0.15 GΩ, n = 15; p < 0.05) but no significant differences between the Cm and RMP values under conditions of no applied bias current (Table 2).

4.3.4 Spike properties of granule cells We found no significant differences in the spike properties between lobule 2 and 9 granule cells, except for the voltage threshold of action potential discharge, defined as the first point in the rising phase of an action potential. Lobule 2 granule cells had a spike threshold of -35.60 ± 1.07 mV (n = 17), which was significantly more depolarized than lobule 9 granule cells that had a voltage threshold of -43.12 ± 1.22 mV (n = 19; p < 0.05) (Table 2).

4.3.5 Expression of Cav3 (T-type) calcium current in granule cells

According to in situ hybridization data, the Cav3.1 isoform shows strong labelling in granule cells of lobules 6-10 and weak labelling in granule cells of lobules 1-5 (Talley et al., 1999). By comparison, Cav3.3 isoform labelling is uniform throughout the granule cell layer of all the lobules. In this set of experiments, we tested if there is a differential expression of Cav3 current between anterior lobule 2 vs. posterior lobule 9. T-type calcium current was recorded by perfusing 30 µm Cd2+ to block HVA and R-type calcium channels (Randall and Tsien, 1995; Randall and Tsien, 1997; Engbers et al., 2012). 2+ Cav3 current was also identified by its sensitivity to 300 µm Ni , a concentration within the IC50 for Cav3.1 and Cav3.3 isoforms (Lee et al., 1999). The external recording solution also contained blockers of Kv4 potassium (5 mm 4-AP), sodium (1 µm TTX), hyperpolarization- activated cyclic nucleotide-gated (HCN) (2 mm CsCl), KCa2.x (100 nm apamin), KCa1.1 channels (5 mm TEA), and excitatory and inhibitory synaptic blockers. Under these conditions, we found Ni2+-sensitive LVA transient inward current in both lobule 2 and lobule 9 granule cells. The current activated near approximately −75 mV and peaked at approximately −30 mV in both lobules on current–voltage (I–V) plots (Fig. 4-2) and was fast activating and inactivating (Fig. 4- 2b). The reversal for the isolated Ca2+ current was in the range of 0–20 mV on I–V plots (Fig. 4- 2A) (Iftinca et al., 2006). The density of Cav3 current was smaller in lobule 2 cells (at −30 mV), with approximately four times greater current density in lobule 9 granule cells (Fig. 4-2C). Due to a small amplitude Cav3 current in lobule 2, we recorded conductance and inactivation properties only in lobule 9. Voltage-conductance plots for Cav3 current in lobule 9 revealed a Va

−46.7 ± 4.61 mV, and voltage-activation plots for Cav3 current in lobule 9 a Vh −78.8 ± 1.75 mV. Overall, these recordings revealed a higher density of Cav3 current in lobule 9 than lobule 2 granule cells.

4.3.6 Biophysical properties of Kv4 channel-mediated A-type current in granule cells

We used a whole-cell voltage clamp configuration to record IA in granule cells. 2+ IA was recorded under conditions where all the calcium current were preserved (no external Cd to avoid the effects on Kv4 activation voltage) (Fig. 4-3). Under these conditions, we were able to record large amplitude IA (> 1 nA) in both lobules 2 and 9 granule cells (Fig. 4-4A). We found

that IA in lobule 9 granule cells had a Vh of −72.3 ± 1.47 mV (n = 11). The IA Vh in lobule 2 granule cells was significantly left-shifted by ~ 5 mV to −77.7 ± 1.48 (n = 10; p < 0.05). There was no significant difference in IA Va between the two lobules (lobule 9 Va = 6.3 ± 6.9, n = 11; lobule 2 Va = -1.6 ± 4.59, n = 10; p > 0.05) (Fig. 4-4B). In stellate cells, a class of interneuron found in the molecular layer of the cerebellum, it was shown that blocking Cav3 channel calcium influx induced a ~ 10 mV hyperpolarizing shift in IA 2+ Vh (Anderson et al., 2010b). Thus, we applied 300 µM Ni as a blocker of Cav3 channels to investigate if the IA in granule cells is modulated by Cav3-mediated calcium influx. This concentration of Ni2+ was found to have no effect on Kv4 current expressed in tsA-201 cells (Fig. 2+ 4-3B). Application of 300 µM Ni led to no significant change in IA Vh of lobule 2 granule cells 2+ (Control Vh = −77.7 ± 1.48; Ni Vh = 82.7 ± 1.77 mV, n = 10; p > 0.05) (Fig. 4-4C). However, 2+ when we applied Ni to granule cells in lobule 9, there was a significant leftward shift in IA Vh from −72.3 ± 1.47 to −82.8 ± 2.53 mV (n = 11; p < 0.001) ( Fig. 4-4C). Moreover, after Ni2+ application IA Vh in lobule 9 granule cells became similar to that of lobule 2 cells (p > 0.05) (Fig. 4-4C). Overall this data suggests that Cav3 calcium channels in lobule 9 cells normally produce a rightward shift in IA Vh but not to any significant extent in lobule 2 granule cells.

Next, we compared the current density of IA (pA/pF) between the two lobules to determine if the reduction in IA amplitude was entirely voltage-dependent or could also reflect a change in channel density. The reason we conducted this comparison is because the shift in the Vh of IA determined in Figure 4-4C predict that there should be no change in IA availability when tested from −110 mV (for a step to −30 mV). Given the range of voltages over which IA Vh shifts when Cav3 channels are blocked (Fig. 4-4), we would predict that Ni2+ application would reveal no change in the availability of IA (tested at -30 mV) from a holding potential of -110 mV but a 2+ reduction in IA when tested from a holding potential of -80 mV. We found that Ni application did not affect the density of IA when tested from a holding potential of -110 mV in either lobule 2 or 9 (lobule 2, p > 0.05; lobule 9, n = 10; p > 0.05) (Fig. 4-4D). However, when the density of IA was tested from -80 mV, Ni2+ application produced a significant reduction in both lobules 2 (p < 0.01) and lobule 9 (p < 0.01) (Fig. 4-4E). Overall, this set of data reveals that a Cav3 calcium- mediated modulation of IA occurs in both lobules, but is greater lobule 9 granule cells.

4.3.7 Blocking the Cav3-Kv4 channel interaction

Anderson et al. (2010) found that calcium influx through Cav3 channels modulates IA in cerebellar stellate cells by interacting with the calcium sensor protein KChIP3. Since there is no known blocker of KChIP3, Anderson et al. used specific antibodies against the protein to interfere with the Cav3-Kv4 interaction. Specifically, they found that a broad spectrum antibody against all KChIP isoforms (PanKChIP), disrupted the Cav3-Kv4 complex when applied through internal dialysis through the patch electrode (1:100 dilution). By interfering with the complex they found a leftward shift in the IA Vh that reduced the availability of IA in the region of resting potentials. This approach also avoided the confounding effects that can arise from direct Cav3 channel blockade, such as secondary effects on calcium-activated potassium current (Fig. 4-3C). I thus interrupted KChIP function in granule cells through internal perfusion of a PanKChIP antibody. We found that anti-PanKChIP infusion had no significant effect on several resting and spiking properties of granule cells, including Rin (Table 2). This is particularly important for interpreting results on firing properties as not reflecting a baseline change in membrane excitability and only reflecting an interruption of the Cav3-Kv4 complex.

4.3.8 Gain of spike firing Granule cells can be driven to fire repetitively by depolarizing current pulse injections of sufficient intensity, with the frequency of firing remaining stable over a 1 sec depolarizing pulse (D’Angelo 1998). The aim of this set of experiments was to test for any differences in the gain of spike firing (Hz/pA) of lobule 2 vs. 9 granule cells. Since interruption of the Cav3-Kv4 complex reduces the availability of IA, we hypothesized that the complex would regulate the frequency of firing of granule cells, at least as indicated by bath perfusion of 1 mM 4-AP (D’Angelo et al. 1998). We found a significant difference in the level of firing rate gain between lobule 2 and lobule 9 granule cells under control conditions (lobule 2 gain = 6.7 ± 0.32 Hz/pA, n = 5; lobule 9 gain = 5.3 ± 0.49 Hz/pA, n = 6; p < 0.05) (Fig. 4-5). This result could be explained by the differential expression of the Cav3-Kv4 complex: the higher availability of IA modulated by a Cav3-Kv4 complex in lobule 9 cells would lower the gain of firing compared to lobule 2 granule

cells. Internal infusion of a PanKChIP antibody significantly increased the gain of lobule 9 granule cells to 8.8 ± 0.57 Hz/pA (n = 6; p < 0.05) but did not affect the gain of lobule 2 granule cells (p > 0.05) (Fig. 4-5). Thus, these data suggest that the Cav3-Kv4 complex is more functional in lobule 9 granule cells and contributes to a lowering of cell excitability, as measured in the gain of firing. We note that we cannot provide the key companion data for coimmunoprecipitation between Cav3 and Kv4 channels in tissue isolated specifically from lobule 9 as evidence for a specific link between these channels. However, there is strong correspondence between

PanKChiP antibody modulation of IA in cerebellar stellate cells, coimmunoprecipitation between Cav3 and Kv4 from cerebellum, and other parameters including nanodomain interactions between Cav3 and Kv4 channels (Anderson et al., 2010a,b). For this reason we will interpret the effects of lead us to interpret the effects of PanKChIP antibody modulation of IA as evidence for a functional Cav3-Kv4 complex.

4.3.9 Role of the Cav3-Kv4 complex in regulating the granule cell response to physiological stimuli

We next investigated the role of the Cav3-Kv4 complex in regulating the response to physiologically relevant synaptic input patterns. The activity of cerebellar granule cells has been measured in vivo in response to sensory input (van Beugen et al., 2013). Whisker or perioral stimulation in rats resulted in short, high frequency bursts in Crus I and 2a granule cells (rostral regions) while vestibular stimulation results in continuous, frequency modulated patterns in granule cells of the flocculus (caudal region) in rabbits (Fig. 4-6). Based on reports of this nature, we hypothesized that the Cav3-Kv4 complex in granule cells has a role in differential processing of incoming mossy fiber signals. The high expression of Cav3.1 channels in lobule 9 (Figs. 1-4 and 4-2) and their modulation of Kv4 availability would serve to more effectively repolarise the membrane and allow for the processing of continuous vestibular mossy fiber inputs. A lower functional role for the Cav3-Kv4 complex in lobule 2 may be optimal to allow granule cells to process high frequency bursts of short duration. We tested this hypothesis by measuring the response of granule cells to postsynaptic stimulation mimicking the physiological input (Fig. 3-4). Physiologically relevant mossy fiber inputs were simulated by

the postsynaptic injection of 4 simEPSCs at 100 Hz (Fig. 3-4). As predicted from the low functional expression of the Cav3-Kv4 complex in lobule 2 granule cells, this 4 simEPSP input was almost directly converted into a 1:1 EPSP-spike output (3.3 ± 0.33 spikes, n=6) (Fig. 4-7A, B). The higher excitability of lobule 2 granule cells was also reflected in a short latency to first spike (14 ± 3 ms, n=6) (Fig. 4-7C). By comparison, subsequent infusion of PanKChIP antibody into the electrode had no significant affect on these parameters in lobule 2 granule cells (p > 0.05) (Fig. 4-7). Under control conditions, lobule 9 granule cells had a lower conversion of EPSP input to spikes (1.3 ± 0.21 spikes, n=6) (Fig. 4-7A, B) and a longer latency to first spike (27 ± 2 ms, n=6) (Fig. 4-7B). The differences in these parameters between lobule 2 and 9 cells were statistically significant under control conditions (Fig. 4-7). However, infusion of the PanKChIP antibody increased the number of spikes and reduced the latency to first spike only in lobule 9 cells (post anti-PanKChIP = 4.2 ± 0.58 spikes; p < 0.001 and latency to first spike = 8 ± 3 ms; p < 0.001) (Fig. 4-7B,C). To test the ability of a granule cell to follow a vestibular-like mossy fiber input, we injected the simEPSC stimulus using a stimulus pattern that smoothly varied from 40 Hz to 80 Hz over two periods over 4 sec. An analysis of spike frequency revealed that the Cav3-Kv4 interaction underlies a difference in frequency-following capability between lobule 2 and 9 cells. Under control conditions lobule 2 cells exhibited a rapid increase in frequency to a mean of 67 ± 14.4 Hz (n = 5) by the peak of the first input cycle of simEPSCs, and a decrease to peak frequency of 54 ± 8.1 Hz on the second cycle (Fig. 4-8B). Lobule 9 cells instead produced a graded response in spike discharge as simEPSCs gradually increased spike output to 46 ± 7.0 Hz (n = 6) by the peak of the first oscillatory phase (Fig. 4-8C). Spike output in lobule 9 cells then responded in an equivalent manner during the second phase of the oscillatory input and reached a similar peak frequency as the first oscillation (Fig. 4-8C, D). Infusing PanKChIP antibody to block the Cav3-Kv4 interaction had more of an effect on lobule 2 cells than the short burst stimulus in enhancing the initial increase and peak in firing frequency and the lower limit of output frequency when EPSC stimuli returned to 40 Hz (Fig. 4- 8B). In lobule 9 cells infusion of PanKChIP antibody essentially converted the pattern of spike output to that encountered in lobule 2 cells, with a rapid increase in firing to a higher peak frequency on the first phase of the oscillatory input and a pronounced reduction in frequency-

following capability on the second phase of the oscillation (Fig. 4-8C,D). Thus, the Cav3-Kv4 complex regulates the gain of granule cell firing and how they respond to physiological relevant inputs in lobule 9.

4.3.10 Presence of Cav3 T-type current and lack of rebound burst in cerebellar granule cells

We used whole-cell patch-clamp recordings to investigate if granule cells can generate rebound bursts. Cells were held at -70 mV and then given a hyperpolarizing step (100-200 msec long) to activate all the T-type channels. Surprisingly, our recordings (n=7) revealed that granule cells do not generate rebound bursts (Fig. 3-7). The granule cell layer also contains another class of inhibitory interneurons called Golgi cells that also express the Cav3.1 isoform (Molineux et al., 2006). In contrast to granule cells, Golgi cells do generate rebound burst (n = 2) (Fig. 4-9). Thus, granule cells in do no generate rebound bursts despite the expression of Cav3 T-type current.

4.4 Discussion Previously, our lab has shown the role of the Cav3-Kv4 complex in regulating the excitability and spike output of stellate cells (Anderson et al., 2010b; Anderson et al., 2010a). The data in this chapter establish several key findings that update and expand our understanding of the ion channels involved in regulating granule cell excitability and processing of sensory-like inputs. Our study is the first to identify the presence of T-type Cav3 current in granule cells. Moreover, there was a pronounced regional difference in expression, with substantially more Cav3 current in lobule 9 vs. lobule 2 granule cells. These results stand in contrast to a previous study that negated the presence of Cav3 current in granule cells (Randall and Tsien, 1995). However, this study was limited because it recorded current from only cultured granule cells that were dissociated from P2-P5 rats. We also obtained results in support of the additional expression of R-type calcium channels in granule cells, in agreement with Randall and Tsien (see Heath et al.) (Heath et al., 2014). In this regard another study reported that HVA channels are highly expressed in granule cells of younger animals and subsequently down-regulated as they age to P-18 (Rossi et al., 1994). Our recordings by comparison were all obtained over the age of P20 to focus on presumed adult equivalent membrane properties. Thus, different

developmental profiles of voltage-gated calcium channels at the younger age could account for the small amount of LVA current recorded by the Tsien’s group studies in cultured granule cells. A recent expression study also negated the presence of the Cav3.1 isoform in cerebellar granule cells (Hildebrand et al., 2009). Given the contrasting in situ hybridization data of Talley et al. (Talley et al., 1999) and our immunocytochemical data (Chapter 3) this difference presumably reflects the expression patterns of different alternative splice isoforms of Cav3.1. Our study is also the first to report the lack of rebound depolarization in granule cells despite the presence of Cav3 T-type current. These results are consistent with the interpretation that

Cav3 channels in granule cells largely form a complex with Kv4 channels to modulate IA, thus supporting an important role for the Cav3-Kv4 complex in granule cells.

Both lobule 2 and 9 cells expressed substantial levels of IA, with similar baseline biophysical properties between the lobules. Thus, the opposing gradients of Kv4.2 and Kv4.3 immunolabel expression across cerebellar lobules and their colocalization in individual cells (Chapter 3) did not become readily apparent in terms of differences in voltage for activation or inactivation. However, testing for the functional modulation of Kv4 current by applying Ni2+ as a Cav3 channel blocker revealed a much greater role for the Cav3-Kv4 complex in lobule 9 cells, where 2+ Ni shifted the Vh in the order of -10 mV. By comparison, no significant shift was detected in lobule 2 cells upon applying Ni2+. This difference was also reflected in the extent to which Ni2+ altered the firing rate gain of granule cells in current-clamp recordings, with more effect in lobule 9 cells. The higher effective function of the Cav3-Kv4 complex in lobule 9 cells presumably reflects the higher density of Cav3.1 channel mRNA reported by Tally et al. in posterior lobules. The lower level of IT in lobule 2 cells is expected to reflect the activity of Cav3.3 channels reported in anterior lobule granule cells (Talley et al., 1999). Cav3.3 channels can also form a functional complex with Kv4 to modulate Vh (Anderson et al., 2013), although this was established in tsA-201 cells likely overexpressing all subunits. Given the readily detectable difference in IT density between lobule 9 and 2 the data suggest that the expression density of at least the Cav3.1 subunit is an important determinant of Cav3-Kv4 complex function. The role of the Cav3-Kv4 complex in granule cells is thus similar to its role in stellate cells where it regulates excitability in terms of the gain of spike output. Since the granule cell layer is

the main input layer of the cerebellum, it receives a myriad of inputs from incoming mossy fibers. The current study examined the potential for the Cav3-Kv4 complex to modulate the processing of mossy fiber inputs designed to mimic physiologically relevant signals in the cerebellum. Specifically, the Cav3-Kv4 complex allowed lobule 9 granule cells to respond to long oscillatory vestibular-like inputs with reproducible spike output and little spike failure. Conversely, the relative lack of Cav3-Kv4 function in granule cells in lobule 2 allows them to respond to high frequency, short bursts of mossy fiber input. It is worth noting that most of the data presented in this chapter was published in Heath et al. (Heath et al., 2014). This study was based on the prevailing data at that time that there is a clear cut compartmentalization between anterior and posterior lobules in terms of the inputs that they receive. For this reason we hypothesized that anterior lobules exclusively receive burst-like high frequency mossy fiber inputs, whereas the posterior lobules receive sinusoidal vestibular inputs (Arenz et al., 2009; Valle et al., 2012; Chadderton et al., 2014). Future studies on the role of the Cav3-Kv4 complex in regulating the processing of other sensory inputs may then extend these findings to those beyond the vestibular-like inputs tested here.

Lobule 2 Lobule 9 Lobule 2 Lobule 9 Lob 2 Lob 9

Control Control p PanKChIP PanKChIP p p Con p Con vs. Pan vs. Pan

Cm (pF) 7.5 ± 0.51 6.3 ± 0.37 0.06 5.74 ± 0.65 5.0 ± 0.46 0.93 0.07 0.13 (20) (24) (7) (5)

RMP (I = 0) -68.2 ± 2.32 -60.4 ± 5.43 0.19 -63 ± 6.66 -62 ± 8.62 0.93 0.36 0.88 (9) (8) (3) (3)

Rin (GΩ) 1.6 ± 0.18 2.4 ± 0.15 * 0.02 1.35 ± 0.05 2.15 ± 0.22 * 0.02 0.48 0.63 (10) (15) (3) (3)

Spike height (mV) 66.22 ± 1.94 70.1 ± 1.86 0.16 67.4 ± 3.46 69.2 ± 5.03 0.76 0.76 0.84 (18) (18) (7) (5)

Spike height (mV) 52.5 – 86 61 – 89 54 – 76 50 - 75.93

Spike half-width (ms) 0.83 ± 0.04 0.84 ± 0.05 0.92 0.75 ± 0.04 0.60 ± 0.05 * 0.04 0.26 0.025 (18) (18) (7) (5)

Spike threshold (mV) -35.6 ± 1.07 -40.2 ± 0.97 0.003 -43.1 ± 2.07 -48.5 ± 2.92 0.15 0.002 0.002 (17) ** (19) (7) (5)

Table 2: Properties of lobule 2 versus lobule 9 granule cells

Resting membrane and spike properties of lobule 2 vs. 9 granule cells in control conditions and following internal infusion of a PanKChIP antibody (1:100 dilution). Con, control; Pan, antiPanKChIP. Sample values are shown in brackets, mean ± SEM. * p < 0.05, ** p < 0.01 Student’s t-test.

Figure 4-1: Firing behaviour of different classes of neurons in the granule cell layer

Capacitance and spiking activity of other neurons (capacitance of greater than 10 pF) within the granule cell layer of the cerebellum. A, Example of current-clamped interneurons, which are not identified as granule cells, found in the granule cell layer. B, Example of a current-clamped granule cell (capacitance of less than 10 pF).

Figure 4-2: Cav3 currents in cerebellar granule cells

A, I-V plots comparing Cav3 calcium current activation and peak amplitude between granule cells recorded in lobules 2 and 9. B, Representative T-type currents recorded in granule cells of lobules 2 and 9. C, Mean T-type current density in granule cells of lobules 2 and 9 evoked by a step from −110 to −30 mV. D, Steady-state conductance and voltage-inactivation plots of Cav3- mediated T-type calcium currents in lobule 9. Sample numbers for mean values are shown in parentheses. Cav3 current was isolated by blocking HVA channels (30 µm Cd2+), Kv4 potassium (5 mm 4-AP), sodium (1 µm TTX), hyperpolarization-activated cyclic nucleotide-gated (HCN) (2 mm CsCl), KCa2.x (100 nm apamin), KCa1.1 channels (5 mm TEA), and excitatory and inhibitory synaptic blockers in the bathing medium.

Data collected by: N.C. Heath (Heath et al., 2014)

Figure 4-3: Pharmacology controls concerning the non-specific effects of Ni2+ and Cd2+

A, B Application of 30 µM Cd2+ to Kv4.2 channels expressed in isolation in tsA-201 cells produced a shift in the voltage-inactivation curve of Kv4 current. Application of 300 µM Ni2+ to Kv4.2 channels expressed in isolation in tsA-201 had no effect on the voltage-inactivation of Kv4.2 channels expressed in isolation in tsA-201 cells. C, Effect of 300 µM Ni2+ on conductances evoked by a ramp. Recordings were conducted in a lobule 9 granule cell in the presence of internal 4-AP (2 mm) and external TTX (1 µm) and CsCl (2 mm) to block Kv4, sodium, and HCN channels. Perfusing 300 µm Ni2+ reveals an inward LVA calcium current and outward calcium-activated potassium current (arrow). Data collected by: N.C. Heath and A.P. Rizwan (Heath et al., 2014)

Figure 4-4: The Cav3 complex modifies Kv4 channel biophysical properties in lobule 9

A, A representative whole-cell recording of IA in a lobule 9 cerebellar granule cell in response to steady-state command pulses. B, Whole-cell steady-state activation and inactivation plots of IA 2+ recorded in lobules 2 and 9. C, Effect of 300 µM Ni on the IA voltage-inactivation relationship recorded in lobule 2 and 9 granule cells. D, Bar plots of IA density evoked by a step to -30 mV from two different holding potentials that are beyond (-110 mV) or within (-80 mV) the range of 2+ Ni -sensitive shifts in IA Vh. E, Bar plots of IA density evoked by a test potential to −30 mV from either −110 or −80 mV before and after the application of 300 µm Ni2+. Data collected by: N.C. Heath (Heath et al., 2014)

Figure 4-5: Cav3-Kv4 complex regulates firing frequency in lobule 9 granule cells

Representative recordings from granule cells in lobules 2 or 9 in response to 1 sec current step injections under control conditions and after internally perfusing anti-PanKChIP (1:100 dilution). B, Average F-I plots for granule cells in lobules 2 and 9 over a range of 1 pA current steps. C, Bar plots of the average gain (slope) of spike firing from F-I plots of granule cells in lobule 2 vs. 9 in control and anti-PanKChIP conditions. Data collected by N.C. Heath, A.P. Rizwan. (Heath et al., 2014)

Figure 4-6: In vivo firing of granule cells

Shown are representative recordings of granule cell firing patterns when recorded from rostral vs. caudal cerebellar lobules. A, An extracellular recording of granule cell firing of an awake rabbit when recorded from the floccular region (caudal lobule) that receives vestibular input. Granule cells in the floccular lobe exhibit high frequency rhythmic bursts in response to contralateral movement of the head. B, An extracellular recording of a granule cell from the crus I (rostral lobule 5) region of an awake mouse. Granule cells exhibit a single high frequency burst after the onset of a whisker deflection stimulus. Modified from Van Beugen et al. (2013).

Figure 4-7: The lack of a Cav3-Kv4 complex in lobule 2 granule cells enables burst output in response to a presynaptic mossy fiber spike burst

A, Representative whole cell current-clamp traces of responses to a 100 Hz 5 pulse input in control and following internal infusion of a PanKChIP antibody (1:100). B, Bar plots of the number of spikes evoked by a 100 Hz EPSP train. Lobule 2 granule cells respond with a greater number of spikes than those in lobule 9. Pan KChIP antibody infusion causes an increased number of spikes in lobule 9. C, The latency to the first spike is longer in granule cells in lobule 9 as compared to lobule 2. Pan KChIP antibody greatly reduces the latency to first spike in lobule 9 selectively. **p < 0.01; ***p < 0.001, Student's unpaired t test. Data collected by: N.C. Heath, A.P. Rizwan (Heath et al., 2014).

Figure 4-8: Granule cells in lobule 9 have greater frequency following capability for EPSCs delivered in a pattern simulating vestibular-like input. A, Example of the train of MF simEPSCs evoked by postsynaptic injection with a pattern shifting in an oscillatory manner between 40-80 Hz. Single EPSCs are shown expanded in the lower panel. B-D, Average spike frequency plots over time of lobule 2 and 9 granule cells in response to the input shown in (A), with responses superimposed in (D). Left panels show that lobule 9 granule cells respond in a more graded manner and can better follow oscillatory simEPSC inputs compared to lobule 2 cells. Right panels show that dialyzing anti PanKChIP (1:100 dilution) in the electrode to block the Cav3-Kv4 interaction converts the response of lobule 9 cells (C) to one closely resembling lobule 2 cells (B). Shaded areas in (B, C) reflect SEM.

Data collected by: N.C. Heath, A.P. Rizwan (Heath et al., 2014)

Figure 4-9: Lack of rebound depolarization in granule cells

Shown are representative recordings of tests for the ability for T-type calcium channels to evoke a rebound depolarization following a current-evoked membrane hyperpolarization in current clamp recordings. A, Golgi cells, a class of inhibitory interneurons of the granule cell layer, exhibit rebound burst depolarization and a burst of spike discharge. B, Granule cells exhibit no evidence for a rebound depolarization or spike burst despite expressing Cav3 T-type calcium channels.

Chapter Five: Long-term potentiation at the mossy fiber-granule cell relay invokes postsynaptic second messenger regulation of Kv4 channels

5.1 Introduction:

Granule cells receive excitatory mossy fibers from both sensory afferents and pontine nuclei to form the primary synaptic relay into cerebellum. Any factors that regulate IA in granule cells are thus in a privileged position to influence signal processing across cerebellar lobules. Mossy fiber input to granule cells can exhibit a long-term potentiation (LTP) that depends on both NMDA and metabotropic glutamatergic (mGlu) receptor activation (Rossi et al., 1996; D'Angelo et al., 1999; Hansel et al., 2001; Gall et al., 2005a; Andreescu et al., 2011). LTP at this synapse can further include a postsynaptic increase in granule cell excitability and probability of spike output (Armano et al., 2000a), which on inspection, exhibits characteristics that are consistent with a reduction in Kv4 channel activity. In this regard, LTP of Schaeffer collateral input to CA1 pyramidal cells invokes a hyperpolarizing shift in IA voltage for inactivation that reduces IA availability (Frick et al., 2004). While the molecular mechanism for this finding was not determined, Kv4 channel biophysics can be regulated by the actions of protein kinase A (PKA), protein kinase C (PKC), and extracellular signal-related kinase (ERK) (Watanabe et al., 2002; Yuan et al., 2002; Schrader et al., 2006; Hu et al., 2007; Rosenkranz et al., 2009). NMDAR activation is further capable of reducing Kv4 channel membrane density in hippocampal cells through PKA-mediated phosphorylation (Kim et al., 2007; Hammond et al., 2008). However, the mechanism by which mossy fiber input triggers a long-term change in intrinsic excitability of cerebellar granule cells has not been determined. Here we report that LTP at the mossy fiber-granule cell synapse leads to a 50% reduction in the availability of Kv4 current in lobule 9 granule cells by shifting channel voltage dependence and decreasing membrane current density. Postsynaptic modification of granule cell excitability involves an ERK-mediated phosphorylation process activated in a glutamate receptor-specific manner. LTP of synaptically evoked spike output was most evident for short bursts of mossy fiber input, allowing potentiation to enhance the response to signals characteristic of sensory input. The data thus reveal a key role for a postsynaptic signaling cascade that modifies Kv4 channel properties to regulate granule cell excitability and LTP at the mossy fiber-granule cell relay.

5.2 Materials and Methods

Methods for this section are described thoroughly in Chapter 2 of the thesis.

5.3 Results

5.3.1 LTP increases the postsynaptic excitability of lobule 9 granule cells

Subgroups of cerebellar granule cells can receive mossy fiber input from multiple sensory sources that converge to allow integration of sensory modalities (Azizi and Woodward, 1990; Arenz et al., 2009; Ishikawa et al., 2015). Many of these inputs arrive as high frequency, short duration bursts of mossy fiber discharge (Chadderton et al., 2004; Rancz et al., 2007; Powell et al., 2015). The vermal region of lobule 9 corresponds to the uvula that receives vestibular, somatosensory, and corticopontine mossy fiber inputs involved in mediating aspects of optokinetic and postural responses (Voogd et al., 2012). In lobule 9 bursts of mossy fiber input can range in frequency up to 105 Hz (Arenz et al., 2008). This is important as a theta burst stimulus (TBS) pattern using a 100 Hz intraburst frequency is known to induce LTP at the mossy fiber-granule cell relay (D'Angelo et al., 2005). We thus used a theta burst stimulus protocol consisting of 8 bursts of 10 impulses at 100 Hz (250 msec interburst interval) to study LTP in granule cells in lobule 9, with recordings centered primarily near the border of the granule cell layer with the white matter in lobule 9c. Mossy fiber LTP induced by TBS is expressed as an increase in EPSP amplitude and an increase in spike output from granule cells (Armano et al., 2000; Sola et al., 2004; Nieus et al., 2006). Postsynaptic increases in intrinsic excitability can be evoked at even low levels of excitation during the TBS (Armano et al., 2000), but is often considered to have a secondary role compared to presynaptic mechanisms in mossy fiber LTP (Maffei et al., 2002; Sola et al., 2004; D'Angelo et al., 2005; Nieus et al., 2006). We previously reported differences in granule cell excitability according to the expression pattern of a Cav3-Kv4 channel complex in the vermis of lobules 2 and 9 (Heath et al., 2014). Here we specifically examined the effects of delivering TBS to mossy fiber inputs on the contributions of Kv4 A-type current to postsynaptic excitability. Spike firing preceding TBS was assessed by constructing an F-I plot (1 sec pulse) to establish the threshold for firing and instantaneous firing frequency. The TBS protocol was then delivered and

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F-I measurements repeated every 7 min thereafter until spike amplitude characteristics dropped below acceptable levels (up to 25 min post TBS). Unless indicated all recordings were conducted in the presence of 50 µM picrotoxin and 1 µM CGP55845 to block GABA-A and GABA-B responses, respectively. We found that TBS to mossy fibers induced an increase in the intrinsic excitability of granule cells within 10-15 min, apparent as a substantial increase (up to 3 fold) in the instantaneous firing frequency for a given level of current injection (Fig. 5-1A, B). The F-I plots for granule cells after TBS revealed a marked reduction (~75%) in the current threshold to evoke firing and a 2-3 fold increase in the gain of firing (Baseline gain = 2.3 ± 0.3 Hz/pA; Post TBS gain = 5.7 ± 0.2 Hz/pA, n = 8, p = 0.001) (Fig. 5-1B). In many cases the increase in excitability was sufficiently pronounced to cause progressive spike inactivation at current injection levels that were originally capable of evoking stable firing (n = 7/12). In 4 cells spike accommodation was prominent enough to restrict output to only 1-2 spikes during current injection. Data from these cells were not included in F-I calculations but were deemed healthy in retaining the ability to discharge a normal spike in response to a synaptic stimulus. These TBS-induced changes in spike frequency were also accompanied by a substantial reduction in the latency to discharge the first spike from the onset of current injection (Fig. 5-1C). The increase in excitability was not associated with any significant change in input resistance (Baseline Rin = 1.8 ± 0.3 GΩ; Post

TBS Rin = 2.4 ± 0.4 GΩ, n = 8, p = 0.15). However, the absolute voltage threshold for spike discharge was substantially reduced by ~10-15 mV for all levels of current injection 15 min following TBS (Fig. 5-1D), helping account for the leftward shift in current threshold to discharge a spike on the F-I plot (Fig. 5-1B).

5.3.2 TBS stimulation reduces the availability of Kv4 channels

We have previously shown that Cav3 calcium channels allow calcium influx to interact with an associated potassium channel interacting protein 3 (KChIP3) subunit to produce a depolarizing shift in the half inactivation voltage (Vh) of Kv4 (Anderson et al., 2010a,b, 2013; Heath et al., 2014). Reducing calcium influx or interfering with the Cav3-Kv4 interaction produces a select leftward shift in Vh that reduces IA availability. We thus recorded IA in lobule 9 granule cells under conditions that left postsynaptic calcium influx intact to identify any role for

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the Cav3-Kv4 complex. IA was isolated by including 2 mM CsCl (HCN channels) and 5 mM TEA (KCa1.1, Kv1.x, Kv3.x) in the bathing medium (Coetzee et al., 1999; Gutman et al., 2005), with 100 µM QX-314 (Nav1.x) and 5 mM TEA in the internal solution. Together these conditions isolated IA postsynaptically but produced no significant change in the amplitude of the synaptically evoked EPSC (n = 5, p = 0.54).

Under baseline conditions IA Vh was -71.3 ± 0.8 mV (n = 5) and the half activation voltage (Va) was -18.9 mV ± 3.2 (n = 5) (Fig. 5-1E, F). Delivering the TBS protocol produced a significant hyperpolarizing shift in IA Vh of ~-11 mV (15 min Post TBS Vh = -82.1 ± 1.4 mV, n = 5, p = 0.008). The shift in Vh was apparent within 5 min of delivering the TBS protocol and continued to increase up to 15 min post TBS (two-way repeated measures ANOVA, Vh, (F(1, 4) = 11.7, n = 5, p = 0.03) (Fig. 5-1G). TBS also induced ~-12 mV hyperpolarizing shift in Va (15 min Post TBS Va = -30.7 ± 1.5 mV, n = 5, p = 0.003) that reached statistical significance at 15 min post baseline recording (two-way repeated measures ANOVA, Va, (F(1, 4) = 10.18, n = 5, p = 0.03) (Fig. 5-1G). Time-matched controls revealed no significant change in either Vh or Va over time without TBS (Fig. 5-1G) (one-way repeated measure ANOVA: Vh, F (2,3) = 1.88, p = 0.29; Va, F (2,3) = 2.55, n = 5, p = 0.22). Delivering only the postsynaptic voltage step to -40 mV also did not cause a shift in IA Vh (Baseline Vh = -74.4 ± 1.2 mV; 15 min Post voltage step Vh = -76.5 ± 1.1 mV, n = 3, p = 0.39) or Va (Baseline Va = -18.6 ± 2.9 mV; 15 min Post voltage step Va = -20.2 ± 2.1 mV, n = 3, p = 0.46). NMDAR activation has been shown capable of reducing Kv4 channel membrane density in hippocampal cells (Kim et al., 2007; Hammond et al., 2008). We tested the potential effects of

TBS on IA density (pA/pF) in granule cells but found no reliable shift over time compared to a set of equivalent time controls (two-way repeated measures ANOVA, Va, (F(1, 4) = 0.47, n = 4, p = 0.53). Collectively the changes in Kv4 Vh and Va induced more than a 50% reduction in the availability of Kv4 current at -70 mV (the nominal resting membrane potential of granule cells) after TBS (n = 5, p = 0.0001) (Fig. 5-1H). These results are consistent with the role for Kv4 channels in modulating spike voltage threshold and gain of firing in granule cells (Heath et al., 2014). Since the Cav3-Kv4 interaction invokes a selective shift in Kv4 Vh (Anderson et al., 2010b; Heath et al., 2014) the TBS-induced hyperpolarizing shift of both Vh and Va indicates an action

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on Kv4 function that could be separate or in addition to that mediated by Cav3 channels. We have established that internal perfusion of a PanKChIP antibody blocks the Cav3-mediated effect on Kv4 Vh (Anderson et al., 2010b; Heath et al., 2014). However, including the PanKChIP antibody in the electrode (1:100 dilution) did not impede the synaptically-induced hyperpolarizing shift in Vh or Va (Baseline Vh = -72.7 ± 3.2 mV; Post TBS Vh = -79.9 ± 3.8 mV, n = 6, p = 0.046; Baseline Va = -21.9 ± 3.9 mV; Post TBS Va = -32.5 ± 4.7 mV, n = 4, p = 0.004). Thus, the ability for TBS to induce hyperpolarizing shifts in Vh and Va under conditions when the Cav3-Kv4 interaction is blocked would suggest that the effect of TBS on IA occurs through mechanisms beyond a disruption of the Cav3-Kv4 interaction.

5.3.3 The effects of LTP on Kv4 are mediated by select activation of NMDAR and mGluRs

It was shown that LTP at the mossy fiber-granule cell synapse requires coactivation of NMDAR and mGluRs (Rossi et al., 1996). We thus tested the contribution of these classes of glutamate receptors to the long term changes in Kv4 properties. For this we applied the NMDAR blocker DL-AP5 (25 µM) or a combination of CPCCOEt (10 µM) and JNJ 16259685 (1.5 µM) that target the mGluR1 isoform, and MPEP (1 µM) that targets the mGluR5 isoform. Application of either NMDAR or mGluR blockers did not entirely eliminate mossy fiber-evoked EPSCs, with the additional postsynaptic step command to -40 mV during TBS ensuring a minimal level of postsynaptic depolarization. We found that applying NMDAR or mGluR blockers alone or in combination entirely prevented the TBS-induced shift in IA Vh and Va (Fig. 5-2A-C, Table 3). We next tested if direct agonist stimulation of these glutamate receptor subtypes was sufficient to induce a shift in IA properties. For these tests TBS was substituted with a 5 min perfusion of an agonist followed by 15-20 min washout. Voltage for inactivation and activation were tested before and 15-20 min following the end of agonist perfusion. Application of 50 µM S-DHPG as a group I mGluR agonist slightly left shifted Vh (~-5 mV) but not Va (Fig. 5-2D, Table 3). Conversely, perfusion of 100 µM NMDA had no effect on Kv4 Vh but did left shift Va (Fig. 5-2E, Table 3). Finally, simultaneous application of NMDA and S-DHPG produced large hyperpolarizing shifts in both Vh (~-18 mV) and Va (~-30 mV) (Fig. 5-2F, Table 3). In summary, NMDAR and mGluR agonists were able to induce select but relatively minor shifts in

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IA voltage-dependent properties, whereas large effects on both Vh and Va were encountered upon co-activating NMDAR and group I mGluRs.

To test the calcium-dependence of ligand-gated shifts in Kv4 properties, we recorded IA Vh and Va in the presence of 10 µM BAPTA-AM in the bath. These tests revealed that in the presence of BAPTA-AM the shift in IA Vh induced by direct co-application of NMDA and S- DHPG was blocked (Vh, n = 4, p = 0.83) (Fig. 5-3A) (Table 3). In contrast, NMDA and S- DHPG coapplication in the presence of BAPTA-AM caused a hyperpolarizing shift in Va (n = 4; p = 0.01) (Fig. 5-3A) (Table 3).

5.3.4 Blocking ERK phosphorylation prevents the postsynaptic effects of TBS stimulation

Co-activation of NMDAR and mGluRs has been shown to have synergistic effects on the activation of ERK (Yang et al., 2004). ERK is part of a signaling cascade activated by an increase in internal calcium that elevates the level of Ras-GTP. Ras-GTP then activates RAF, a protein kinase that activates Mitogen-activated protein kinase kinase (MEK). MEK in turn phosphorylates ERK to target Ser/Thr phosphorylation sites, three of which have been reported on Kv4.2 channels (Thomas and Huganir, 2004; Schrader et al., 2006). We therefore examined the effects of 20 µM PD 98059, a cell-permeable inhibitor of MEK (Hu et al., 2006). Bath application of PD 98059 prior to mossy fiber stimulation had no effect on IA Vh or Va (Vh, n = 5, p = 0.96; Va, n = 5, p = 0.33). In the presence of PD 98059 TBS delivered to mossy fibers failed to induce a hyperpolarizing shift in IA Vh, but did produce a hyperpolarizing shift in IA Va (Fig. 5-3B; Table 3). We further noted that TBS-induced no shift in Vh in the presence of the alternate ERK blocker Selumetinib (10 µM) in the electrode (n = 3, p = 0.93). Next, we recorded in current-clamp mode and delivered TBS in the presence of PD 98059 to determine the role of ERK in modulating firing frequency. Inhibition of MEK by PD 98059 prevented the TBS- induced increase in granule cell firing evoked by current injection (Fig. 5-3C). Similarly, direct application of 100 µM NMDA and 50 µM S-DHPG in the presence of 10 µM BAPTA-AM to reduce calcium increases blocked the agonist-induced increase in postsynaptic firing (Fig. 5-3D). Altogether the data suggest that the increase in intrinsic excitability following TBS reflects primarily a hyperpolarizing shift in IA Vh that involves a calcium-dependent process and

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activation of an ERK signaling cascade. An additional hyperpolarizing shift in IA Va appears different in reflecting a process that develops with a slower time course (Fig. 5-1G), can be at least partially evoked by NMDA receptors (Fig. 5-2E) but is calcium-independent (Fig. 5-3A) and relatively insensitive to an ERK blocker (Fig. 5-3B). While a hyperpolarizing shift in Va should increase IA availability, the overall reduction in IA following TBS (Fig. 5-1H) suggests that the effects of TBS on IA Vh has a greater net influence to increase postsynaptic excitability.

5.3.5 TBS-induced LTP is preserved in the presence of GABAergic circuitry

The excitability of granule cells is closely regulated by Golgi cells, a class of GABAergic interneuron located within the granule cell layer (Mapelli and D'Angelo, 2007; Duguid et al., 2012). Previous reports indicated that GABAergic inhibition is robust enough to prevent induction of LTP of the mossy fiber EPSP and reduce the extent of spatial region expressing LTP (Armano et al., 2000; Mapelli and D'Angelo, 2007). To determine the ability to evoke LTP of postsynaptic excitability when GABAergic systems are intact we repeated TBS in the absence of picrotoxin and CGP55845. Here we found that TBS still evoked a long-term increase in the rate of current-evoked firing, along with a reduction in the spike voltage threshold (Baseline Vthresh = -38.7 ± 2.5 mV; Post TBS Vthresh = -52.6 ± 3.5 mV, n = 7, p = 0.001) and increase in gain of firing on F-I plots (Baseline gain = 2.3 ± 0.2 Hz/pA; Post TBS gain = 4.1 ± 0.6 Hz/pA, n = 7, p = 0.01) (Fig. 5-4A, B).

To again consider the physiological relevance of these changes to modulation of IA, we recorded the effects of inducing LTP on IA in medium lacking picrotoxin or CGP55845. TBS to mossy fibers again induced a ~-10 mV hyperpolarizing shift in IA Vh (Baseline Vh = -70.5 ± 2.1 mV; Post TBS Vh = -79.9 ± 3.2 mV, n = 7, p = 0.0002) and Va (Baseline Va = -16.9 ± 4.9 mV; Post TBS Va = -29.1 ± 4.6 mV, n = 7, p = 0.002) (Fig. 5-4C). These tests are important in demonstrating that a TBS-evoked increase in postsynaptic excitability of lobule 9 granule cells occurs in the presence or absence of GABAergic inhibition.

5.3.6 Bursts of mossy fiber EPSPs uncover LTP of synaptic efficacy in the intact circuit

It was important to determine the effective contribution of an increase in postsynaptic excitability in spike firing to LTP of the synaptically evoked response. As noted, previous work

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testing single evoked EPSPs concluded that LTP of the mossy fiber EPSP was only reliably detected when GABAergic circuits were blocked (Armano et al., 2000), raising questions as to the functional role of LTP in the intact system. However, recent work in vivo indicates that the physiological pattern of input for mossy fibers that evokes a response in granule cells is in the form of short bursts (Chadderton et al., 2004; Rancz et al., 2007; Duguid et al., 2012; Powell et al., 2015). We thus retested the ability to record LTP of the synaptic response and the effects of a TBS-induced increase in postsynaptic firing in the presence or absence of GABAergic inhibition. We first compared the ability to detect LTP of synaptic transmission using single evoked EPSPs in the manner traditionally used in studies of long-term plasticity. The intensity of synaptic stimulation was adjusted to the minimal level required to evoke a subthreshold EPSP, with a stable baseline EPSC or EPSP amplitude confirmed by stimulating 1 / min for 5 min prior to delivering TBS. Testing the effects of TBS on a single evoked EPSP in the presence of 50 µM picrotoxin and 1 µM CGP55485 to block GABAergic inhibition consistently revealed a potentiation of the EPSP and an increase in the probability of discharging a spike. Thus a single EPSP originally subthreshold for spike firing was increased after TBS to the point of reliably triggering a single spike per stimulus, a potentiated state that persisted for at least 15 min (n = 6/6). We also found a potentiation of EPSP amplitude in 5/5 cells after TBS in the presence of the ERK blocker PD 98059, with 3/5 cells exhibiting an increase in synaptically evoked spike discharge. However, when the same tests were conducted without GABA receptor blockers present TBS failed to evoke a long term increase in the amplitude of a single evoked EPSP or spike firing in 7/7 cells (p = 0.20), confirming previous reports on the inability to evoke LTP of single evoked EPSPs when GABA circuits are intact (Armano et al., 2000). We next examined the effects of evoking a short 4 pulse train of mossy fiber EPSPs (10, 20, 50 and 100 Hz) in the absence of GABA receptor blockers to retain GABAergic circuitry. Here we could record step-wise (quantal) changes in EPSP amplitude and intermittent failures from pulse to pulse, as expected for activation of individual mossy fiber axons (Sola et al., 2004; Nieus et al., 2006) along with a temporal summation of the EPSP (Fig. 5-5A). Applying TBS in the absence of GABA receptor blockers now revealed LTP of synaptic efficacy and increased spike output in response to the mossy fiber burst input in 5/5 cases (Fig. 5-5B). Thus, a short train of EPSPs exhibiting temporal summation but below threshold for spike discharge was

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converted by TBS to an enhanced EPSP amplitude and summation process that increased spike firing up to 8 spikes during a 4 pulse train (Fig. 5-5B). A TBS-induced increase in postsynaptic firing was further verified in a subset of cells using direct current injection (n = 2), confirming that the potentiated synaptic response was accompanied by an increase in postsynaptic excitability. By comparison, single evoked EPSPs tested in this group still failed to potentiate compared to the train of EPSPs (n = 3; p = 0.61). Interestingly, the degree of potentiation of spike output in response to burst input showed evidence for a frequency-dependent influence, such that the greatest degree of synaptically evoked potentiation occurred in the 20 - 50 Hz range (Fig. 5-5B). These data are important in establishing that the effects of TBS-induced LTP of synaptic transmission and postsynaptic excitability in granule cells are apparent in preparations with intact GABAergic circuits.

5.4 Discussion

The current study reveals that LTP at the mossy fiber-granule cell synaptic relay includes a modulation of postsynaptic Kv4 channels that dramatically increases the excitability of lobule 9 granule cells. A TBS-evoked shift in IA voltage dependence involves NMDA and mGlu receptors that invoke the signaling cascade leading to ERK activation. The final result is a long term reduction in postsynaptic IA that contributes to LTP of mossy fiber inputs that arrive in a burst- like pattern characteristic of physiologically relevant inputs.

5.4.1 LTP at the mossy fiber-granule cell relay

It was known that the mossy fiber-granule cell synapse is capable of exhibiting LTP following theta burst patterns of input both in vitro (D'Angelo et al., 1999; Mapelli and D'Angelo, 2007) and in vivo (Roggeri et al., 2008). LTP depends on mossy fiber activation elevating postsynaptic calcium concentration following coactivation of NMDA and mGlu receptors (Rossi et al., 1996; D'Angelo et al., 1999; Gall et al., 2005). The source of LTP has been largely attributed to presynaptic mechanisms (Maffei et al., 2002; Maffei et al., 2003; Sola et al., 2004). A second contributing factor is a postsynaptic increase in granule cell excitability, as reflected in a lower spike threshold and increased probability for spike firing (Armano et al., 2000; Nieus et al., 2006).

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In lobule 9 TBS evoked a decrease in the threshold for granule cell firing that supported a 2-3 fold increase in spike frequency and a higher probability of firing in response to synaptic input. Although a decrease in the threshold to trigger a spike has been reported (Armano et al., 2000), the TBS-evoked increase in excitability detected for lobule 9 granule cells was much more pronounced. Voltage-clamp analysis revealed a rapid and enduring hyperpolarizing shift in IA Vh following TBS and a hyperpolarizing shift of Va with a slightly later onset. The balance of these factors decreased IA availability, a result consistent with the change in spike threshold and rate of firing after TBS. Moreover, the change in IA Vh was maximal upon coactivation of NMDARs / mGluRs, with an increase in postsynaptic excitability apparent in the presence or absence of GABAergic inhibition. The current results are thus important in identifying modulation of Kv4 channels as a contributing factor to a marked TBS-induced increase of intrinsic excitability in lobule 9 granule cells in relation to physiologically relevant patterns of mossy fiber input.

5.4.2 Role for kinase activation

The molecular mechanisms underlying a change in postsynaptic excitability in granule cells after

LTP had not been identified. Our ability to block the TBS-induced hyperpolarizing shift in IA Vh, the decrease in Kv4 current density, and the increase in spike output with a MEK blocker strongly implicates an ERK-mediated phosphorylation process. While there are three known sites for ERK phosphorylation on Kv4.2 (Schrader et al., 2006) we cannot predict if IA results from homomeric or heteromeric combinations of Kv4.2 and Kv4.3 subunits. We thus do not know the site(s) on Kv4 subunits potentially targeted for ERK phosphorylation. We also cannot rule out the possibility that ERK may target other subunits of the Cav3-Kv4 complex including KChIP and dipeptidyl peptidase-like subunits. Cav3 channels can also link to a Kv4 complex containing

KChIP3 to produce a depolarizing shift in Kv4 Vh to augment IA (Anderson et al., 2010b;

Anderson et al., 2010a; Heath et al., 2014). The TBS-induced hyperpolarizing shift in Vh could then conceivably reflect a reversal of the Cav3-mediated effect on Kv4 current. The finding that infusion of an anti-PanKChIP antibody did not prevent the TBS-induced hyperpolarizing shift in

Vh suggests that the effects of TBS may be independent of the Cav3-Kv4 complex. On the other hand, the T607 phosphorylation site on Kv4.2 at which ERK can induce a rightward shift in Va in an expression system depends on KChIP3 coexpression (Schrader et al., 2006). The exact role

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for a Cav3-Kv4 complex in these results thus remains to be identified, as do the site(s) at which

ERK acts to modulate IA in granule cells. Some of our findings at least resemble Kv4 modulation following LTP induction in CA1 hippocampal pyramidal cells. A theta burst LTP-inducing stimulus to Schaffer collaterals induced a select hyperpolarizing shift in Kv4 Vh that reduced IA availability and increased the amplitude of dendritic spikes (Frick et al., 2004; Rosenkranz et al., 2009). The increase of dendritic spike amplitude was traced to a PKA-MAPK cascade (Rosenkranz et al., 2009), but the relationship of this kinase activity to the shift in IA Vh was not established. Instead, phosphorylation by PKA, PKC, or ERK results in a depolarizing shift in Kv4 Va with no effects on Vh (Johnston et al., 1999; Watanabe et al., 2002; Yuan et al., 2002). By comparison, the hyperpolarizing shift in IA Va seen here after TBS or direct glutamate receptor activation reflects a non-ERK mediated process that remains to be identified. The second messengers triggered by

LTP to modify IA in hippocampus thus differ from granule cells where ERK mediates a hyperpolarizing shift in IA Vh. An NMDAR-dependent internalization of Kv4.2 channels has also been reported in CA1 pyramidal cells that depend on phosphorylation at a specific PKA site on the C terminus (Kim et al., 2007; Hammond et al., 2008). In granule cells we found a reduction in Kv4 current density following TBS or by direct activation of NMDARs but not mGluRs. As the decrease in Kv4 density was prevented by the MEK blocker PD98059, it implicates kinase activity downstream from the direct PKA phosphorylation site relevant to CA1 pyramidal cells. The mechanism for reducing Kv4 channel density in relation to LTP in granule cells is thus again distinct from that in hippocampal pyramidal cells.

5.4.3 Functional role of Kv4 modulation of granule cell excitability

The ability to induce LTP and modulate Kv4 channels with stimuli centered on 100 Hz input is important given that a TBS-like pattern of tactile stimuli can induce LTP in granule cells in vivo (Roggeri et al., 2008). We view the postsynaptic contributions by Kv4 channels to mossy fiber LTP as an important complement to the established role of presynaptic factors underlying LTP at this synapse. It is known that mossy fiber LTP requires an increase in postsynaptic calcium (Gall et al., 2005). Our results now identify NMDA and mGlu receptor activation as a source for

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calcium to modulate Kv4 voltage-dependence and IA availability in lobule 9 cells. A key result of

IA modulation is a decrease in spike threshold that will support a shorter first spike latency of granule cell firing, as detected here in response to current injection (Fig. 1C,D). We also document an increase in the probability and number of spikes generated by granule cells following TBS, with a short burst of subthreshold EPSPs transformed into responses capable of triggering up to 8 spikes per EPSP. These data are important to the potential functional significance of LTP of mossy fiber input. Direct recordings in vivo establish that granule cells exhibit little response to spontaneous mossy fiber discharge and require burst input to reliably drive spike firing (Chadderton et al., 2004; Rancz et al., 2007; Powell et al., 2015). It is thus important that we found an effect of TBS specifically on the granule cell response to bursts of input as compared to single EPSPs. This would indicate that postsynaptic Kv4 channels can contribute to blocking a response to background spontaneous input (Jorntell and Ekerot, 2006; Powell et al., 2015) by lowering granule cell firing probability until a TBS-induced decrease in IA increases postsynaptic excitability. The number of spikes generated also showed a relation to the frequency of repetitive mossy fiber bursts, suggesting a frequency-filter effect presumably mediated by the combination of EPSP summation, Golgi cell inhibition, and Kv4 regulation of postsynaptic excitability. Interestingly, a frequency filter for input has been reported in Purkinje cells to restrict a response to high frequency bursts of parallel fibers. In that case the filter is established by a Cav3-KCa3.1 channel complex that suppresses temporal summation of EPSPs at low frequencies (Engbers et al., 2012). As a result Purkinje cells respond most effectively to short bursts of parallel fiber input from granule cells. It is known that cerebellar circuits can shape motor responses with a resolution of < 10 ms (D'Angelo and De Zeeuw, 2009). It has been proposed that the combined actions of a burst of mossy fiber excitation and feedforward inhibition by Golgi cells establish a time-window of opportunity for a granule cell to respond with a short latency first spike and/or burst (D'Angelo and De Zeeuw, 2009; Chadderton et al., 2014). The occurrence of LTP or LTD at this synapse was then proposed to modulate the timing of evoked spikes in relation to ~5 ms window for granule cells to respond to sensory inputs. Interestingly, using current injections we found that first spike latency was reduced by ~2/3 following TBS (Fig. 5-1C). Control over the timing of

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granule cell output through LTP could then be another means to influence temporal coding strategies by establishing time-delays between populations of granule cells activated by different sensory inputs (adding to that proposed for presynaptic mechanisms of plasticity) (Chabrol et al., 2015). Alternatively, LTP-induced shifts in intrinsic excitability could reduce the number of mossy fiber inputs of a specific modality needed to reach threshold for firing (Jorntell and Ekerot, 2006). The LTP-induced modulation of Kv4 control over granule cell excitability would thus appear to dovetail with other mechanisms in cerebellum that effectively preset the system to respond to bursts of sensory input conveyed by mossy fibers (Roggeri et al., 2008; D'Angelo and De Zeeuw, 2009; Chabrol et al., 2015; Ramakrishnan et al., 2016).

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Blocked Baseline Vh Post TBS Vh p Baseline Va Post TBS Va p

(mV) (mV) (Vh) (mV) (mV) (Va)

mGluR1,5 -73.0 ± 3.0 (4) -68.3 ± 1.4(4) 0.26 -13.61 ± 1.50 (4) -13.32 ± 2.37 (4) 0.51

NMDAR -72.72 ± 1.70 (4) -74.39 ± 2.18 (4) 0.41 -23.03 ± 2.45 (4) -27.12 ± 1.80 (4) 0.30

mGluR+NMDAR -71.10 ± 1.98 (4) -71.50 ± 2.49 (4) 0.19 -17.65 ± 5.67 (4) -23.63 ± 5.20 (4) 0.10

[Ca]i (BAPTA-AM) -71.6 ± 3.7 (4) -72.3 ± 3.0 (4) 0.83 -21.4 ± 3.9 (4) -25.8 ± 3.32 (4) * 0.01

ERK (PD98059) -73.0 ± 2.7 (5) -72.0 ± 2.1 (5) 0.83 -25.5 ± 3.7 (5) -36.5 ± 2.2 (5) * 0.01

Agonists Baseline Vh Post Agonist Vh Baseline Va Post Agonist Va

(mV) (mV) (mV) (mV)

DHPG -71.2 ± 0.3 (6) -76.7 ± 2.2 (6) * 0.04 -17.6± 4.0 (6) -21.6 ± 3.4 (6) 0.16

NMDA -69.4 ± 2.0 (6) -72.5 ± 3.2 (6) 0.4 -19.63 ± 2.64 (6) -31.50 ± 2.11 (6) * 0.01

DHPG+NMDA -71.73 ± 1.99 (5) -89.50 ± 4.62 (5) * 0.01 -18.10 ± 5.26 (5) -48.20 ± 7.90 (5) * 0.03

Table 3: NMDA and mGluR1,5 receptors collectively modulate Kv4 voltage dependence.

Receptor blockers were bath applied throughout recordings and prior to TBS of mossy fiber afferents. Receptor agonists were bath applied following control recordings in normal medium for 5 min in place of TBS. Receptor blockers: NMDA, DL-AP5 25 µM; mGlur1,5, CPCCOEt 10 µM, JNJ 16259685 1.5 µM, MPEP 1 µM. Agonists: NMDAR, NMDA, 100 µM; mGluR, S- DHPG 50 µM. BAPTA-AM, 10 µM. PD98059, 20 µM. Sample values represent number of individual cell recordings. Average values are mean ± SEM with sample values indicated in brackets. *, p < 0.05. Student’s paired t-test.

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Figure 5-1: LTP at the mossy fiber- granule cell synapse increases postsynaptic excitability and properties of IA

A, B, The results of delivering TBS stimulation of mossy fiber input on postsynaptic excitability. Shown is spike firing evoked by two levels of current injection (A) and mean F-I plots of the current-evoked instantaneous firing rate before and after TBS stimulation (B). Arrows indicate the current level to reach spike firing threshold. C, D, Whole-cell steady-state voltage- inactivation and conductance plots for IA isolated in lobule 9c granule cells before and after delivering the TBS stimulus. Tests included 2 mM CsCl and 5 mM TEA in the medium, and 0.1 mM QX-314 and 5 mM TEA in the electrode to isolate IA while retaining synaptic responses for

TBS. IA exhibits a significant hyperpolarizing shift in Vh (C) and Va (D) and a decrease in membrane current density (E) following TBS. Data in (E) are normalized to the peak current in control conditions. F, A step command from a holding potential of -70 mV (nominal resting potential) to -30 mV reveals a net decrease in IA availability post TBS. Asterisks in (C, D) refer to significant shifts in Vh (C) and Va (D) (Table 3). F-I plots were measured before and 15 min following TBS and all recordings conducted in the 50 µM picrotoxin and 1 µM CGP55485 to block GABAergic inhibition. Average values are mean ± SEM. *, p < 0.05; **, p < 0.01.

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Figure 5-2: TBS-evoked effects on IA depend on activation of specific glutamate receptors

A-C, Tests on the effects of applying TBS of mossy fiber input in the presence of mGluR or NMDAR blockers, with schematics of the stimulation protocol. TBS fails to induce a leftward shift in IA Vh or Va when delivered in the presence of mGluR1,5 blockers (10 µM CPCCOEt, 1.5 µM JNJ 16259685, 1 µM MPEP) or an NMDAR blocker (25 µM DL-AP5) applied either alone (A, B) or together (C). D-F, The effects of applying agonists to mGluRs (50 µM S-DHPG) or NMDARs (100 µM NMDA) on IA Vh and Va. All recordings were conducted in 50 µM picrotoxin and 1 µM CGP 55845 to block GABAergic inhibition. Sample values represent number of individual cell recordings. Average values are mean ± SEM. *, p < 0.05; **, p < 0.01. Student’s paired t-test. X. Zhan contributed to the collection of data in (D)

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Figure 5-3: TBS-evoked effects on IA are calcium- and ERK-dependent

A, B, The effects of applying agonists of mGluR (50 µM S-DHPG) and NMDAR (100 µM

NMDA) on IA Vh (A) and IA density (B) are blocked in the presence of 10 µM BAPTA-AM while IA Va is still exhibits a hyperpolarizing shift in the presence of BAPTA-AM (A). C, D, In the presence of the ERK blocker PD98059 (20 µM) the effects of TBS on IA Vh and membrane density are blocked (C, D) while the leftward shift in Va remains (C). E, The TBS-evoked increase in postsynaptic spike firing is blocked in the presence of the ERK blocker PD98059. F, The increase in spike firing induced by direct application of 100 µM NMDA and 50 µM S- DHPG is blocked in the presence of BAPTA-AM to reduce calcium increases. Average values are mean ± SEM. Sample values in (C) are shown in brackets. X. Zhan contributed to the collection of data in (D)

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Figure 5-4: The effects of applying TBS on postsynaptic firing and IA with GABA circuitry intact

A, B, The results of TBS of mossy fiber input on postsynaptic firing when no GABA blockers are present. Representative spike firing to current injection (A) with mean F-I plots of instantaneous frequency (B) before and after TBS. F-I plots were measured before and 15 min following TBS. Arrows indicate the current level to reach spike firing threshold. C, D, Voltage- inactivation and conductance plots for IA before and after TBS. IA was isolated while retaining synaptic responses for TBS using 2 mM CsCl and 5 mM TEA in the medium, and 0.1 mM QX-

314 and 5 mM TEA in the electrode. IA exhibits a significant hyperpolarizing shift in Vh and Va (C) but no change in current density (D) following TBS. Data in (D) are normalized to the peak current in control conditions. Average values are mean ± SEM.

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Figure 5-5: LTP is expressed for burst-like input patterns in lobule 9 granule cells

A-C, The results of TBS on single evoked mossy fiber EPSPs in the presence of 50 µM picrotoxin and 1 µM CGP55485 to block GABAergic inhibition (A, B), in the additional presence of 20 µM PD 98059 (B), or when GABA circuits are left intact (C). TBS stimulation reliably potentiates the EPSP and promotes a single evoked spike when GABA receptors are blocked (A, B) but not when GABA circuits are intact (C). The dashed line indicates the average value of spike amplitude in granule cells. D, E, The results of TBS on a 4 pulse burst of mossy fiber EPSPs at the indicated frequencies when GABA circuits are left intact. EPSP amplitude was initially set subthreshold for spike discharge. Shown are the number of spikes evoked during the burst input delivered at the indicated frequencies for 5 different cells before and after TBS. Average values are mean ± SEM. *, p < 0.05; **, p < 0.01. Student’s paired t-test.

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Chapter Six: Discussion

This thesis examined the role of the Cav3-Kv4 complex in regulating excitability and postsynaptic processing in cerebellar granule cells, with each Chapter studying a different aspect of neurophysiological concepts for regulating neuronal excitability. The excitability of a neuron is determined by the complement of ion channels expressed. Therefore, in Chapter 3, I established that all the molecular components of the Cav3-Kv4 ion channel complex are expressed in the granule cell layer of cerebellum. These components include different Cav3 and Kv4 channel isoforms, the calcium sensor KChIP3, and a single-pass membrane protein, DPP6. Postsynaptic mechanisms, in addition to presynaptic mechanisms, contribute to synaptic processing capabilities of a neuron. Therefore, in Chapter 4, I studied the role of the Cav3-Kv4 complex in regulating the response of granule cells to mossy fiber input in lobules 2 and 9. I found that the Cav3-Kv4 complex is more active in lobule 9 compared to lobule 2, and that this leads to Cav3-dependent regulation of Kv4 Vh in lobule 9. Thus, a higher functional expression of the Cav3-Kv4 complex results in differences in excitability between granule cells, such that lobule 2 granule cells respond more effectively to a tactile stimulus-like burst input and lobule 9 granule cells to slow shifts in input frequency characteristic of vestibular input. Changes in ion channel properties contribute to changes in synaptic efficacy. Therefore, in Chapter 5, I studied the role of LTP in modifying postsynaptic excitability via modulation of Kv4s. I showed that TBS activation of mossy fiber input activates an ERK-dependent pathway in granule cells that leads to hyperpolarizing shifts in Kv4 Vh and Va, and a reduction in Kv4 membrane current density. This novel pathway involves the co-activation of NMDAR and mGluRs, and their effects on Kv4 channel properties are mediated by the activation of the ERK kinase signaling cascade. The TBS mediated effects on Kv4 channel properties leads to lowering of the current threshold to spike and an increase in the gain of spike firing by 2-3 folds. Given new reports indicating that granule cells filter out incoming mossy fiber inputs during the resting state, we provide new evidence regarding the role of plasticity in increasing granule cell responsiveness to incoming mossy fiber inputs. The one end point the work did not resolve was whether the changes in Kv4 properties following TBS specifically involved the Cav3-Kv4 complex. The cerebellum has a comparatively small number of neuronal classes connected by clearly defined excitatory and inhibitory projections, allowing for comparisons of synaptic processing 125

strategies in different regions of the cerebellum. The cerebellar granule cell layer is an ideal area to study signal processing in neurons and how this behavior contributes to overall circuit function. The findings presented here will thus enhance our understanding of the processing and learning that occurs at the primary input layer, i.e. granule cell layer, of the cerebellum. The data presented in this thesis advances the argument that postsynaptic mechanisms in the granule cell can fine tune cell responsiveness to particular features of a stimulus. I also provide the first evidence for the physiological role of an ERK signaling pathway in adult granule cells. Thus, this thesis explored novel mechanisms by which ion channels and their associated intracellular signaling pathways come together to control synaptic processes and responses. In this chapter, I will discuss how these molecular findings relate to the role of ion channels in the broader function of the cerebellum.

6.1 The Ca3-Kv4 channel complex in granule cells

Kv4.2 and Kv4.3 isoforms that mediate A-type potassium current are highly expressed in the brain (Serodio and Rudy, 1998). There have been reports since the 1980s that voltage-gated A- type currents might be sensitive to calcium influx (Macdermott and Weight, 1982; Zbicz and Weight, 1985; Chen and Wong, 1991). But it was not until the early 2000s that a physical link between Kv4 channels and KChIPs, a calcium sensor molecule with EF-hands, was discovered (An et al., 2000). Later, DPLPs were found to be another auxiliary subunit of Kv4 channels, with effects on the properties of Kv4 (Nadal et al., 2006; Nadin and Pfaffinger, 2010b; Pongs and Schwarz, 2010). The source of calcium for the modulation of Kv4 current was discovered by our lab to be Cav3 (T-type) calcium channels (Anderson et al., 2010c; Anderson et al., 2010a). Specifically, calcium influx through Cav3 channels modulates Kv4 current by activating a KChIP3 isoform bound to the N-terminus of Kv4 channel subunits. The functional interaction of all the components of the Cav3-Kv4 channel complex occurs at the level of a nanodomain (Anderson et al., 2010c). Cav3 and Kv4 channels share a number of similarities such as low voltage for activation, fast inactivation, near complete inactivation at resting potential, and an availability that is governed by preceding membrane hyperpolarization (Perez-Reyes, 2003b; Jerng et al., 2004). Our previous work in stellate cells and the current work in granule cells show that these similarities are not a mere coincidence, but rather reflect a close 126

synchrony between Cav3 and Kv4 channels that allow them to work together as an ion channel complex. We do note that we have no biochemical evidence for actual complex in lobule 9 but we do observe a functional expression/interaction. Our study was the first to address the relative expression patterns of all the subunits that make up the Cav3-Kv4 complex in the granule cell layer across cerebellar lobules. We confirmed the presence of all Cav3-Kv4 complex subunits expressed across cerebellar lobules, and in many cases with a regionally different pattern of expression across the anterior-posterior axis. While our immunocytochemistry confirmed Kv4.2 expressed as a gradient, our results differed in other ways from previous reports for Kv4.3, Cav3.1, DPP6 and KChIP3 expression. Kv4.3 is highly expressed in lobule 9c and 10, with relatively low expression in all the other lobules. We also find an apparent uniform label for Cav3.1 except for low labeling in lobule 9, where many of the electrophysiologal recordings were conducted. In partial agreement with previous literature (Clark et al., 2008), we find that DPP6 expression is higher in lobules 1-3 and 10 compared to other lobules. Clark et al. reported an anterior- posterior gradient for the expression of DPP6, with higher expression in lobules 1-5 and lower expression in lobules 6-10. We note that the Clark et al. study was carried out in mice, whereas we studied the expression of DPP6 in rats. These differences could then be due to different expression patterns in mice vs. rats. Compared to previous literature (Xiong et al., 2004), we also find that KChIP3 is uniformly expressed throughout all the cerebellar lobules. As discussed, KChIP3 is the only member of the KChIP family that imparts calcium sensitivity to Kv4 channels. Except for this property, all the KChIPs modulate Kv4 channels by increasing Kv4 surface expression, slowing the rate of inactivation and increasing the rate of recovery from inactivation (Maffei et al., 2003a; Jerng et al., 2004; Covarrubias et al., 2008). Interestingly, none of these parameters are calcium- dependent (Anderson et al., 2010c). Thus, KChIP3 is unique among KChIP members of the family in causing calcium-dependent changes in the voltage-inactivation relationship of Kv4 channels. To extend our analysis we also examined the expression pattern of all KChIP isoforms. We found that both KChIP2 and 3 are uniformly expressed in the granule cell layer, and only KChIP1 and 4 exhibits unique gradients (Table 1). Furthermore, we provide the first evidence regarding the co-expression of both Kv4.2 and Kv4.3 isoforms in a single granule cells. Thus,

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our results raise multiple questions as to how these different isoforms of KChIPs and Cav3s along with DPP6 come together in a granule cell to modulate IA in granule cells.

6.2 Role of the Cav3-Kv4 complex in regulating excitability and the response to mossy fiber input

Previous work from our lab in stellate cells has shown that the Cav3-Kv4 complex IA by right shifting the Vh to increase window current and reduce excitability and spike output of stellate cells. Yet, the Cav3-Kv4 complex was not known to be functionally expressed in granule cells, or how reported gradients of subunit expression might regulate Cav3-Kv4 function. In this thesis I provide the first evidence for the functional presence of a Cav3-Kv4 complex in granule cells. Both lobules 2 and 9 express IA but without obvious biophysical differences until considered under conditions that maintained calcium influx (no HVA or LVA blockers).

With calcium influx preserved Kv4 Vh in lobule 9 granule cells was more right shifted than in lobule 2 cells. As Cav3 channels modify the voltage dependence of Kv4 channel inactivation, we found that lobule 9 granule cells express T-type currents with a substantially higher density than lobule 2 cells. Thus, we provide the first evidence for T type currents in cerebellar granule cells. However, there had been no reported evidence for T type current in granule cells despite strong apparent evidence in in situ data showing a prominent difference between anterior and posterior lobules (Talley et al., 1999), providing conflicting data. Immature granule cells exhibit calcium spikes that are mediated by L-, N-, P-, and putative R-type HVA calcium channels (D'Angelo et al., 1997). At approximately P20, these HVA channels are downregulated as granule cells start to exhibit Na+-dependent action potentials (D'Angelo et al., 1998). Interestingly, our immunohistochemistry contrasts with the in situ hybridization data of Talley et al. (Talley et al., 1999), who reported a very strong expression in the posterior lobules and no expression of Cav3.1 in the anterior lobules. One possible reason for this disagreement between our immunohistochemistry and electrophysiological data could be an inability of Cav3.1 immunofluorescence to report an expression difference for Cav3 across lobules. Whole-cell voltage-clamp analysis is a more direct and sensitive technique to measure T-type currents, finding 4 times higher Cav3 current in lobule 9 granule cells compared to lobule 2 granule cells (Fig. 4-2). 128

The classical electrophysiological signature of Cav3 expressing neurons is their ability to generate rebound burst discharge. Here, we provide the first evidence for no rebound depolarization in granule cells despite the presence of T type currents in granule cells. A previous study from our lab showed that Cav3.2 and Cav3.3 expressing cells do not generate rebound burst discharge, and that these isoforms instead formed a complex with Kv4 channels in stellate cells (Molineux et al., 2006). We show here that granule cells, which express Cav3.1 in addition to Cav3.3, were also unable to generate rebound bursts (Fig. 4-9). Thus, a key function for Cav3 channels in granule cells is to form a complex with Kv4 channels. I also demonstrated in this thesis that perfusion of a PanKChIP antibody into cerebellar granule cells by dialysis through the electrode rapidly promoted a leftward shift of Kv4 Vh only in lobule 9 but not lobule 2 granule cells. This suggests a higher functional expression of the Cav3-Kv4 complex in granule cells in lobule 9 than lobule 2. However, using immunocytochemistry in chapter 3, we detected uniform expression of KChIP3 in granule cells across lobules. One possible explanation for these discrepancies could be that another component of the Cav3-Kv4 complex governs the activity of this complex. One such candidate is DPP6, which is highly expressed in lobule 2 granule cells but not in lobule 9 cells. DPP6 is known to induce a leftward shift in Vh of Kv4, and Kv4 Vh in lobule 2 granule cells is left shifted compared to lobule 9 granule cells (Jerng et al., 2004). Thus, the lack of effects of PanKChIP antibody in lobule 2 granule cells may reflect a high expression of DPP6 in that lobule with functions not fully tested at this time. A significant weakness of the use of a PanKChIP antibody to interfere with Cav3- Kv4 function is also that its specific site of action has not been identified. A more targeted approach to interrupt the KChIP3-Kv4 complex would be to use specific TAT-peptides against N-terminal regions of the Kv4 channel α subunit that are necessary to interact with KChIP proteins. We also found that the differential expression of the Cav3-Kv4 complex shapes the granule cell response to widely different forms of mossy fiber input. In vivo recordings from lobules 1-2 and lobules 9-10 indicate two general patterns of mossy fiber input and granule cell output (Arenz et al., 2009). Granule cells in lobule 1-2 generate high frequency bursts in response to high frequency input (4-5 mossy fiber spikes) from sensory brainstem nuclei (Arenz et al., 2009). On the other hand, granule cells in lobule 9-10 must respond to more tonic gradual increases or

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decreases in the frequency of mossy fiber input from vestibular nuclei (Barmack and Yakhnitsa, 2011). However, these distinctions are not as absolute as they were thought before. Mossy fiber input to anterior cerebellum is now known to include both tonic and bursting components (Witter and De Zeeuw, 2015). Moreover, recent studies have shown that even a single granule cell can receive inputs from multiple sources (Huang et al., 2013; Chabrol et al., 2015). For example, Chabrol et al. showed that vestibular inputs and visual inputs innervate single granule cells in lobule 10 of cerebellum (Chabrol et al., 2015). Interestingly, they also found the visual input to be burst-like and low frequency in nature. Even though we provided evidence for the Cav3-Kv4 complex to contribute to tuning of granule cell responses to different inputs in different lobules, we do not know how a Cav3-Kv4 complex would shape the response of granule cells to different incoming mossy fiber inputs on their individual dendrites.

6.3 Activation of ERK pathway in granule cells by TBS of mossy fiber input

Coactivation of NMDAR and mGluRs has been suggested as a mechanism for LTP induction at the mossy fiber – granule cell synapse. Interestingly, the coactivation of these two receptors has been shown to synergistically activate ERK (Yang et al., 2004). ERK phosphorylates Ser/Thr sites, and is known to have numerous substrate proteins involved in synaptic plasticity (Thomas and Huganir, 2004). One of the targets for ERK phosphorylation is Kv4 channels, which contain at least three known sites on the C- terminus: T602, T607, and S616 (Thomas and Huganir, 2004; Schrader et al., 2006). There are also numerous predicted ERK phosphorylation sites for Kv4.3 (Group-based Prediction System, ver 2.0), however the effects of ERK on Kv4.3 have not been reported yet. Since granule cells in lobule 9 express both Kv4.2 and Kv4.3 subunits, we cannot predict if a functional Kv4 channel results from homomeric or heteromeric combinations of Kv4.2 and Kv4.3 subunits. We thus do not currently know if the effects seen in our LTP experiments are mediated due to direct ERK phosphorylation of Kv4 or the associated auxiliary protein, i.e. KChIPs and DPLPs. Interestingly, our results suggests that the effects of TBS may be independent of the Cav3-Kv4 complex as infusion of PanKChIP antibody to disrupt Cav3- Kv4 complex did not occlude the TBS-mediated changes in Kv4. It has also been reported that

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Cav3-mediated calcium influx can activate ERK (Chen et al., 2010). Thus, it is conceivable that Cav3 channels regulate Kv4 function though the activation of ERK. In this study, we identified for the first time an ERK-mediated reduction in the availability and density of Kv4 current, accounting for the reported need to coactivate two forms of glutamatergic receptors. At first, our results seem to be similar to LTP-mediated effects on Kv4 channels reported in CA1 pyramidal neurons. Frick et al. reported that LTP in the CA1 pyramidal layer causes a shift in the inactivation curve of Kv4, enhancing backpropagating action potentials (bAPs) in the dendrites of CA1 pyramidal cells (Frick et al., 2004). However, they did not observe a change in either the activation curve or current density. Moreover, in CA1 neurons, there is a basal level of ERK activity that right shifts the Kv4 Va so that there is effectively less IA to reduce the amplitude of bAPs in the dendrites of CA1. Thus, application of

PD 98059 by itself produces a hyperpolarizing shift in Kv4 Va so that there is more IA to decrease the amplitude of bAPs.. Our results also show that the shift in Kv4 Va is calcium- independent. Thus, TBS of mossy fiber could be activating another calcium-independent pathway to increase the Kv4 current to counteract the reduction in Kv4 availability and density. However, this suggestion is only speculative and should be explored by future studies. Thus, our results only allow us to claim that a leftward shift in the Kv4 Va induced by TBS mossy fiber is independent of the ERK pathway.

6.4 Role of LTP in modifying synaptic properties at the input stage of the cerebellum.

Another form of plasticity recognized at the mossy fiber synapse is a long-term depression (LTD) induced by short bursts (< 250 ms) or low frequency (< 10 Hz) inputs that lead to only small increases in intracellular calcium (Gall et al., 2005b). The cellular pathways underlying LTD at mossy fiber – granule cell synapse have not been elucidated yet. Since we only activated the mossy fibers a TBS protocol, which consists of 8 bursts of 10 impulses at 100 Hz delivered at 250 msec interburst intervals, we mainly observed LTP in our recordings. It was known that LTP at the mossy fiber – granule cell relay involves both presynaptic and postsynaptic mechanisms, but the cellular mechanisms by which postsynaptic changes occur had not been identified (Armano et al., 2000b; Maffei et al., 2002b; D'Errico et al., 2009). Instead, D’Angelo and colleagues have mainly focused on elucidating the molecular pathways involved 131

in enhancing the presynaptic elements that support a subsequent long term increase in neurotransmitter release (Maffei et al., 2003a; Sola et al., 2004b; D'Errico et al., 2009). Furthermore, given the inability to apparently potentiate EPSPs in the absence of a block of GABAergic inhibition (Armano et al., 2000a), the physiological relevance of LTP at the mossy fiber –granule cell synapse was discounted by many. In this thesis, we uncovered the cellular pathways involved in mediating postsynaptic changes of synaptic potentiation at the mossy fiber – granule cell synapse. Postsynaptic mechanisms are important because they determine the input-output relationship of a neuron. One of the ways where postsynaptic contributions to input-output relationship are studied is by measuring the gain of firing by constructing F–I plots of spike output. The synaptic contribution to granule cell gain of firing was studied by the Silver lab using dynamic clamp (Rothman et al., 2009). Since synaptic transmission at the mossy fiber – granule cell synapse exhibits short-term synaptic depression (STD), Rothman et al. found that STD at this synapse contributes to a lowering of gain of granule cell firing. They also found that the tonic inhibitory GABAergic conductance in the granule cell layer lowers the gain of granule cell firing, something previously shown by Chadderton et al (Chadderton et al., 2004). Interestingly, Rothman et al. found that tonic GABAergic conductance modulated the gain of granule cells in an additive manner (a shift along the x-axis of an F-I plot), whereas STD modified gain in a multiplicative manner (modulation of the slope of an F-I plot) (Rothman et al., 2009). In this thesis, we provide the first evidence for the role of postsynaptic ion channels in regulating the gain of granule cell firing. In Chapter 4, we highlight the role of Cav3–Kv4 complex in modulating the gain of firing in only lobule 9 granule cells. In Chapter 5, we provide the first evidence of how granule cells can utilize synaptic plasticity to modulate the gain of spike output. Importantly, we also discovered that LTP at the mossy fiber-granule cell synapse in lobule 9 increases the gain of granule cell firing both in the presence and absence of GABAergic inhibition. Furthermore, we related the change in gain of firing to the burst-like nature of mossy fiber input received by granule cells. Mossy fiber bursts are better suited to lead to the activation of downstream Purkinje cells based on the synaptic arrangement of a single mossy fiber contacting a substantial number of neighboring granule cells (Xu-Friedman and Regehr, 2003). Thus, mossy fiber bursts can lead to

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activation of a population of granule cells, which can drive the activation of Purkinje cells. Moreover, based on new reports from in vivo recordings that granule cells are relatively quiescent at resting state despite a high rate of mossy fiber activity (Powell et al., 2015), we decided to investigate if granule cells responsiveness to mossy fiber input can be modulated by LTP. With GABAergic inhibition intact, LTP at the mossy fiber – granule cell synapse caused granule cells to respond to mossy fiber bursts of different frequencies but not to spontaneous mossy fiber inputs, i.e. a single pulse. A lack of granule cell response to single mossy fiber stimuli had been previously demonstrated by the Hausser group, who showed that mossy fiber bursts are necessary to drive granule cell spike output (Rancz et al., 2007). Thus, a lack of potentiation of single EPSPs when GABAergic inhibition is intact (Armano et al., 2000a) now follows in light of the known burst-like nature of mossy fiber inputs. Moreover, our data also suggests that granule cells are more tuned to respond to medium range intraburst frequencies (25-50 Hz) than high frequencies (100 Hz). The reason for this is unknown, but could even depend on the lobule of recording should granule cells in different regions require specific frequency-tuning to particular sensory modalities. However, given that only a relatively small number of cells could be tested for this property further work will be required to examine the extent to which this is a general property of cells in lobule 9.

6.5 Conclusion

Cerebellar granule cells are the most abundant neurons in the brain and live in a very dynamic environment, where they receive multiple inputs that range from motor control to cognition. A neuron’s excitability, determined by the expression and properties of ion channels, regulates the processing capabilities of a circuit. My work provides the first molecular basis for differential information processing in different regions of the cerebellum. I have also shown two different ways in which Kv4 channels can be modulated to regulate cell output: (1) the expression of a

Cav3-Kv4 complex that shifts the Kv4 Vh to depolarizing potentials, and (2) an LTP-induced downregulation of channel density and availability. Thus, a single class of channel can be differentially regulated according to association with other channels or by second messengers that are activated by entirely different inputs. This work thus provides the first molecular insights into differential processing of information by granule cells across the cerebellum, and aspects of 133

intrinsic excitability that are modulated in relation to synaptic plasticity, the cellular basis for learning.

6.5.1 Future directions:

6.5.2 Role of retrograde messengers in regulating Kv4 channels:

6.5.2.1.1 Nitric oxide: Nitric oxide (NO) has been shown to be a retrograde messenger in the granule cell layer with a significant role in producing LTP at the mossy fiber – granule cell synapse (Maffei et al., 2003a). Using an electrochemical probe positioned in the granule cell layer, Maffei et al. showed that NO is produced by TBS of mossy fibers and that NO scavengers present during TBS prevented LTP. Conversely, NO donors, such as DEA-NO, are enough to induce LTP at the mossy fiber – granule cell synapse. These findings are consistent with the high expression of nitric oxide synthase (NOS) and NMDARs in granule cells (Baader and Schilling, 1996). Indeed, NO is produced by the activation of NMDARs (Garthwaite et al., 1988). Even though Maffei and colleagues (Maffei et al., 2003a) focused on the role of NO in increasing the presynaptic components contributing to LTP, they did not rule out the possible postsynaptic effects of NO. In cardiac myocytes, NO affects both inward calcium and outward potassium conductances (Gomez et al., 2008). Gomez et al. showed that the application of NO donors, such as DEANO, leads to a reduction in the density of Kv4.3 current that is responsible for mediating transient outward potassium current (Ito1) (Gomez et al., 2008). They also showed that NO donors produce a hyperpolarizing shift in the Vh of Kv4.3, an action that will reduce Ito1 availability. Since our data suggests a strong role for ERK in reducing Kv4 current and availability to mediate the postsynaptic effects of potentiation at the mossy fiber – granule cell synapse, future studies should explore the connection between NO regulation of Kv4 channels in granule cells.

6.5.2.2 Endocannabinoids:

Endocannabinoids (ECs) are lipid signaling molecules that can act as retrograde signaling molecules in the brain. Two main endocannabinoids found in the central nervous system are

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anandamide (AEA) and 2-arachidonoylglycerol (2-AG) (Amoros et al., 2010). These ligands are produced in post-synaptic neurons and travel retrograde to presynaptic neurons to modulate neurotransmitter release (Diana et al., 2007). ECs act through the activation of G-protein-coupled receptors CB1 and CB2 (Amoros et al., 2010). However, some effects of ECs are “receptor- independent” (Amoros et al., 2010). One such receptor-independent effect of ECs is their inhibition of Kv4.3 channels in the cardiac myocytes (Poling et al., 1996; Amoros et al., 2010). Amaros et al. showed that the application of either AEA or 2-AG leads to a reduction of Kv4.3 current by shifting the Vh of Kv4.3 to more negative potentials (Amoros et al., 2010). These effects of ECs are very similar to the effects of NO on Kv4.3 (Gomez et al., 2008). However, ECs act as direct inhibitors of Kv4 channels, whereas NO triggers a signaling cascade to mediate its effects on Kv4 channels. It is interesting to note that AEA and 2-AG are derived from arachidonic acid, which is also known to block Kv4 channels and cause a hyperpolarizing shift in the Vh (Ramakers and Storm, 2002). CB1 and CB2 receptors are highly expressed in the cerebellum, with CB2 receptors preferentially located in mossy fibers (Suarez et al., 2008). The molecular machinery for the synthesis of endocannabinoids is also expressed in the cell bodies of granule cells (Suarez et al., 2008). Thus, the issue for future studies will be to understand if any of these retrograde signaling molecules have any role in regulating LTP mediated effects on Kv4 channels in granule cells.

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Appendix A: INCLUSION OF WORK PUBLISHED OR SUBMITTED BY CANDIDATE

Chapter 4:

Heath, N. C., Rizwan, A. P., Engbers, J. D., Anderson, D., Zamponi, G. W., & Turner, R. W.

(2014). The expression pattern of a Cav3–Kv4 complex differentially regulates spike output in cerebellar granule cells. The Journal of Neuroscience, 34(26), 8800-8812. [ A.P. Rizwan designed, performed and analyzed experiments. A.P. Rizwan also contributed to writing of the paper]

Chapter 5:

Rizwan, A. P., Zhan, X., Zamponi, G. W., & Turner, R. W. Long-term potentiation at the mossy fiber-granule cell relay invokes postsynaptic second messenger regulation of Kv4 channels. The

Journal of Neuroscience. (In review). . [ A.P. Rizwan designed, performed and analyzed experiments. A.P. Rizwan also contributed to writing of the paper]

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