REVERSIBLE MODIFICATIONS IN MOTOR OUTPUT FOLLOWING PURKINJE NEURON PHOTOSTIMULATION

by

DAVINA VERSYDA GUTIERREZ

Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Stefan Herlitze

Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY August 2011

2 COPYRIGHT NOTICE

Data presented in Chapter 2 and Figures 3 – 6 are from Gutierrez, DV et al., (2011) Optogenetic Control of Gi/o Signaling in Cerebellar Purkinje Cells

Alters Motor Coordination. Journal of Biological Chemistry, In press. These data do not require any copyright forms or permission.

3 TABLE OF CONTENTS

ABSTRACT 8 CHAPTER 1 10 INTRODUCTION 10 I. 10 A. Anatomical Divisions of the Cerebellum 10 1. Anatomical organization, landmarks and functional 10 neuroanatomy 2. Cortical or longitudinal zones 12 3. Biochemical characterization: Stripes 13 B. Cerebellar Histology 14 1. Cortical layers and intrinsic neurons 14 2. Cerebellar peduncles 18 3. 20 4. Vestibular nuclei 25 5. The olivo-cerebellar system 25 C. Normal Cerebellar Function 27 1. Motor and non-motor influence 27 2. Consequences of vermal injury on motor output 29 II. Purkinje Cells and Synaptic Plasticity 30 A. Firing properties & excitatory inputs 30 1. General firing properties 30 2. system 32 3. The system 33 B. Synaptic Plasticity 37 1. LTP, LTD and RP 37

4 III. G Coupled Receptors 39 A. GPCRS, G & effector proteins 39 B. GPCR mediated modulation 41

1. GABABR-mediated modulation in the cerebellum 42 C. G protein signaling cascade and the visual system 44 IV. & GPCRs 45 A. Chemically activated GPCRs: RASSLs, DREADDS & AlstR/AL 45 B. Light activated GPCRs: ChARGe, vRh & OptoXR 49

CHAPTER 2 56

OPTOGENETIC CONTROL OF Gi/o SIGNALLING BY VERTEBRATE 56 IN CEREBELLAR PURKINJE CELLS CHANGES MOTOR COORDINATION I. SUMMARY 56 II. INTRODUCTION 57 III. RESULTS 59 A. Creation of a new optogenetic mouse line vRh-GFP (TgflvRh-GFP) for the controlled expression of vRh in a -type specific manner 59

B. Gi/o pathway activation by vRh in vivo reduces the frequency of PC firing 60 C. In vitro and in vivo application of baclofen reduces the spontaneous firing rate of Purkinje neurons 61 D. Photostimulation of vRh in Purkinje cells alters motor behaviour 63 IV. DISCUSSION 65 A. vRh-GFP (TgflvRh-GFP) mouse for the cell-type selective control of Gi/o signalling 65 B. Modulation of simple spikes in the medial cerebellar regions leads to changes in motor behaviour 66 C. Considerations for controlling Gi/o signalling in vivo by light 67 V. MATERIALS AND METHODS 70

5 A. Generation and screening of transgenic mice 70

1. β-Galactosidase staining 71 2. Immunohistochemistry 72 3. NISSL staining 73 B. Stereotaxic surgeries and cannula placement 73 1. Fiber optics and photostimulation 75 2. Optrode contruction 75 C. Electrophysiological analysis 75 1. Brain slice recordings 75 2. In vivo recordings 76 3. Baclofen application 77 4. Lesion studies 77

D. Behavioral testing 78 1. Accelerating rotarod 78 2. Grip strength test 78 3. Pole test 79 4. Balance beam 79 CHAPTER 3 95 GENERAL DISCUSSION 95 I. Continued Application of Optogenetic Strategies 95 A. Designer GPCRs and 95 B. Vertebrate rhodopsin as an optogenetic tool 97 C. The connection between GPCR modulation and behavioral output 98 D. Future directions 100

6 LIST OF FIGURES Figure 1 Cerebellar landmarks 52 Figure 2 Cerebellar cortical organization 54 Figure 3 Cre recombinase-mediated expression of vRh 81 Figure 4 Photostimulation reduces PC firing rate in vRh-GFPPC mice 83 Figure 5 Application of baclofen decreases firing rate 85 Figure 6 Light activation of vRh induces changes in motor behavior 88 Figure 7 Cannula fixation during in vivo recordings 91 Figure 8 Optrode placement during in vivo recordings 93

7 Reversible Modifications in Motor Output Following Purkinje Neuron Photostimulation

Abstract by

DAVINA VERSYDA GUTIERREZ

A central goal in biology has focused on the ability to manipulate a defined population of cells within a model to determine the correlation between modulation and functional output. Ultimately, the technique utilized will ensure that the level of control will be precise, temporally realistic, genetically tangible and reversible. Devising a procedure that can modify a defined group of functionally related neurons both reversibly and rapidly creates a scenario where one can examine the effects of suppression or enhancement of neuro- and synaptic transmission and could establish a correlation between an electrophysiological outcome to a behavioral phenotype.

While numerous innovative approaches have attempted to establish a link between cellular activity and development, plasticity and behavior by modulating a specific group of neurons, studies were often confronted with a multitude of limitations that were intrinsic to the experimental approach. Classical methods faced obstacles in either kinetic, construct or pharmacological design that often rendered the targeted cellular population irreversibly or incompletely modified.

The discovery and utilization of light-sensitive opsins, coupled with traditional genetic approaches and innovation within the optogenetic field have revealed that the previously encountered spatial, temporal and reversibility restraints are essentially eliminated.

8 Taking this into consideration, we hypothesized that the targeted expression and light activation of vertebrate rhodopsin (vRh) within the cerebellum would not only alter the characteristic, cellular firing pattern but would also exert a visible and distinct change in motor behavior. In order to examine the physiological and behavioral effects of vRh expression and photoactivation in vivo, we had to first establish a transgenic mouse line whereby the expression of vRh was exclusively driven to Purkinje neurons. Subsequent steps included the development of a surgical procedure that would allow for the targeted delivery of light to the via an optrode as well as the in vivo electrophysiological and behavioral outcomes of light application in both transgenic and wild type littermates.

Our results revealed that vRh expression was restricted to the soma and proximal dendrites of Purkinje neurons in a punctate pattern. The in vivo data revealed that light activation of vRh in vermal Purkinje neurons significantly reduced the firing frequency in a manner similar to baclofen application.

Furthermore, we also discovered that a brief pulse of light (26 sec) sufficiently induced a loss of motor coordination and balance in positive transgenic mice.

9 CHAPTER 1

INTRODUCTION

I. CEREBELLUM

A. Divisions of the Cerebellum 1. Gross Anatomical Organization, Landmarks and Functional Neuroanatomy Upon initial examination, the most obvious and identifying morphological characteristic of the cerebellum is the presence of folds on the cortical surface.

While these folds form unique patterns within the anterior-posterior (1) axis that differ from the foliation configuration throughout the medial-lateral (ML) axis, they are ultimately organized into larger structures that establish a rather complex three-dimensional unit that is comprised of lobes, lobules and sublobules (2-4).

Specifically, the mammalian cerebellum is comprised of a medial structure, the vermis, and two wings that can be further subdivided into the paravermis (intermediate zone or pars intermedia) and the hemisphere (lateral cerebellum) (1, 5). Thus, the cerebellar cortex can be divided into three medio- lateral compartments: the vermis, paravermis and hemisphere (FIGURE 1) (1,

4). Aside from these divisions, two key fissures further categorize the lobes of the cerebellum from front to back. The primary fissure divides the cerebellum into anterior and posterior lobes and the posterolateral fissure separates the (comprised of the nodulus and ) from the posterior lobe; ultimately generating three lobes (4, 6). While these regions form the basis of the ML axis, they are not only morphologically distinct but also differ on both the molecular (i.e. gene expression) and functional levels.

10 Classic gross anatomical, organizational schemes have indicated that shallow fissures further subdivide the lobules into additional sublobules essentially creating a transverse-lobular design, comprised of four transverse zones. Specifically the transverse zones are labeled as follows, the anterior zone

(AZ, lobules I-V in the mouse), the central zone (CZ) again separated into the anterior CZa (lobule VI) and the posterior CZp (lobule VII), the posterior zone

(PZ, lobules VIII to dorsal IX) and the nodular zone (NZ, ventral lobule IX and lobule X) (1). Additionally, the vermis is also divided along the rostrocaudal axis to create and follow the transverse zone organizational scheme so that the anterior zone contains lobules I-V (lingula, and ), the central zone includes lobules VI-VII (declive, folium and tuber), the posterior zone consists of lobules VIII-IX (pyramis and uvula) and lastly the nodular zone is comprised of lobule X (nodulus) (4, 5, 7). Longitudinally, the vermis can be partitioned into several regions (A, AX, X, B and C) that are distinguished by the areas that correlate to the climbing fiber afferent input (via the inferior olive) and the destination of the output (in regards to the DCN). For this study, the main focus is on the A zone which extends over the entire vermis. More specifically, the A1 area receives input from the subnucleus b (subnuc b) caudal medial accessory olive (cMAO) and projects to the fastigial (medial cerebellar) nucleus

(1, 8). Additionally, there is a lateral strip of Purkinje cells that has been found to project to both the fastigial and vestibular nuclei (8). The overall vermal Purkinje neuron and circuitry and the influence on motor function will be explored in later sections.

11 2. Cortical or longitudinal zones Building upon the modular organization, each side of the cerebellum can be further arranged into eight mediolateral parallel longitudinal zones. Each half of the anterior lobe is divided into eight cortical zones (medial to lateral) and are named A, X, B, C1, C2, C3, D1 and D2 (9-12). The zones are thought to be electrophysiologically and functionally unique since each area receives a distinct climbing fiber input from a separate subnuclei of the inferior olive (10, 13). For example, the caudal medial accessory olive (MAO) sends climbing fibers to the a-zone, the b-zone acquires input from the caudal dorsal accessory olive (DAO), c-2 from the rostral MAO and d1 from the principal olive (PO) (10-12).

Because olivocerebellar projections are topographically organized, any overlap in their receptive fields within the longitudinal zones is defined as being a microzone (14, 15). A microzone is essentially a small structural unit that is a sagitally-oriented band of cortex, approximately 100-300 µm wide (1, 16). Within each microzone, Purkinje cell axons will project to either a cerebellar or vestibular nucleus after receiving input from mossy and climbing fibers. Studies have indicated that a microzone is essentially an amalgamation of excitatory and inhibitory inputs that functions to filter and correct error signals that ultimately grants the cerebellum some level of adaptation within the process of executing predictive and coordinated control that is based on long term depression (LTD)

(16). Furthermore, when spatially distinct microzones receive common climbing fiber input, they form a multizonal micro-complex structure that may be central to the processing and integration of information from multiple sources that in turn,

12 influences some aspect of movement (1, 9). It has been suggested that all cerebellar arrangements concerned with motor control are divisible into micro- complexes (14).

3. Biochemical characterization: Stripes Aside from the gross anatomical organization scheme of the lobe, lobule and sublobule patterns that define the AP coordinate system, the cerebellar cortex can be further classified into an intricate arrangement of parasagittal and transverse boundaries that can be immunohistochemically compartmentalized

(17). Specifically, the expression of several molecular markers organizes

Purkinje cells into stripe-shaped zones. The most studied of the markers, zebrin

II (aldolase C), has been found to be expressed in specific Purkinje neurons so that they form rostrocaudal orientated bands of positively expressing parasagittal bands that are dispersed amongst zebrin II nonimmunoreactive bands (17-19).

Several other substances follow the zebrin II pattern and are present in both

Purkinje cells and Bergmann glia and include, 5'-nucleotidase, protein kinase C delta, the glutamate transporter EAAT4, CDK5 P39 activator, the low affinity nerve growth factor protein and the metabotropic glutamate receptor mGluR1b (8, 20-24).

The zebrin regionalization of Purkinje neurons begins within the vermal posterior lobe on postnatal day 5 (P5) and is progressively distributed in an anterior manner and laterally first through the rest of the vermis and then throughout the lobules by P12; by P15 the adult parasagittal banding pattern is apparent and selective (1, 17, 25, 26). Studies of the rat cerebellum have

13 revealed 7 zebrin-positive bands (P1+ to P7+) and six zebrin-negative bands

(P1- to P6-); all being symmetrically arranged across the midline (1, 8, 26). The stripe configuration differs within transverse zones so that the AZ and PZ have alternating zebrin II +/- stripes, whereas the CZ and NZ are uniformly zebrin II positive (1).

A unique feature of the zebrin banding is the relationship with longitudinal zones. Specifically, there appears to be extensive co-localization between the longitudinal zones and the zebrin II/+ stripes (1, 8). Essentially, the overall architectural design of the cerebellum indicates that each division and subdivision do not function independently but most likely performs as a complex, interconnected configuration of hundreds of components collectively functioning towards normal motor function and coordination execution. This elaborate and organizational scheme will come into play when we consider the behavioral and electrophysiological downstream effects of manipulating the endogenous firing pattern of Purkinje neurons.

B. Cell types of the Cerebellar Cortex 1. Cortical layers and intrinsic neurons

Although the overall anatomical organization and divisions of the cerebellum are rather complex and intricate, the cerebellar cortex is actually quite homogeneous and divisible. The cortex itself is superimposed upon and three deep cerebellar nuclei (DCN). Historically, the four main types of neurons that have been described to comprise the cortex include granule cells,

Purkinje neurons, Golgi cells and stellate/basket cells. However studies have

14 indicated that while the cerebellar cortex is itself divided into three distinctive layers, the number of different cell types that encompass the cortex is actually closer to seven and includes candelabrum cells, Lugaro cells and unipolar brush cells (UBC).

Immediately above the white matter is the (GC) layer, which is described as containing 98% of the cerebellar neurons; most being granule cells

(27). Aside from granule cells, Lugaro cell interneurons, unipolar brush cells

(UBCs) and the somata of the are also localized within the granule cell layer (3, 28). Granule cells are glutamatergic, the most numerous cell-type in the cortex and are rather small with cell bodies ranging from 5-8 µm and dendritic arbors being approximately 50 µm in diameter (3, 6, 29). The terminals of mossy fibers directly contact the dendrites of multiple, excitatory granule cells in complex synapses (glomeruli) that in turn activates both AMPA and NMDA receptors (27, 30). Upon receiving an excitatory input from mossy fibers, the axons of granule cells (parallel fibers) ascend to the cerebellar cortex, bifurcate and excite Purkinje cells (via dendritic termination) and all other types of interneurons (4, 6).

Golgi cells co-localize for γ-aminobutyric acid (GABA) and glycine, are inhibitory interneurons that receive inputs from mossy and parallel fibers and have axonal projections to granule and UBCs (31). Morphologically, Golgi cells have a rather extensive distribution of ascending dendrites and meager spreading of descending dendrites (32). Feed-forward and feedback circuits are part of the Golgi signaling loop. Specifically, the feed-forward loop transmits

15 mossy fiber input signals to the distal dendrites of Golgi cells and the feedback circuit conveys signals from granule cells (via parallel fibers) to Golgi cells

(ascending dendrites) that will ultimately result in the inhibition of downstream cellular targets (4, 33, 34). Earlier studies suggested that a main function of

Golgi cells was to control the granule cell firing threshold which would ultimately result in fixing granule cell and parallel fiber activity at a constant rate (32).

The next layer is comprised of a monolayer of Purkinje cell (PC) bodies,

Bergmann glia cell bodies and candelabrum cells (3, 35). As a whole, cerebellum development transpires in four steps: 1) classification of cerebellar territory in the , 2) formation of two cellular proliferation zones that yield

Purkinje and granule cells, 3) cellular migration and 4) formation of structural units, circuitry and additional cellular differentiation (4, 36, 37). Purkinje cells are the first neurons born in the cerebellar cortex (E10-E13 in the mouse), are initially fusiform shaped and arranged into multiple layers/clusters until P10 whereby they form a monolayer of cells (1, 4, 38).

It has been estimated that the mouse cerebellar cortex contains approximately 160,000 Purkinje cells and that each Purkinje neuron is subjected to influence from inputs by climbing fibers (from the inferior olive) and mossy fibers (from a variety of brainstem and spinal cord sources) (1, 6). Purkinje cells are GABAergic neurons whose myelinated axons terminate onto neurons within deep cerebellar nuclei and brainstem nuclei. Overall, the dendrites of Purkinje cells are rather flat and perpendicularly orientated to the parallel fibers. Proximal to the soma, the dendrites are smooth and innervated by multiple synapses from

16 a single climbing fiber (39). Distal dendritic branchlets are covered with spines and serve as the termination sites for parallel fibers (6).

The most superficial layer is the molecular layer and is mostly comprised of Purkinje cell dendrites, Golgi cell dendrites and parallel fibers but also includes stellate cells, basket interneurons and palisades of Bergmann glia fibers (3, 6).

Basket and stellate cells are often classified as being variations of a single class of inhibitory interneuron, however a more thorough consideration of both cell types reveals that there are key morphological and physiological differences.

Despite the variations in morphology and physiology, one of the main objectives of these two cells types is to provide inhibitory innervation to Purkinje neurons, which is thought to modulate both the firing rate and pattern of Purkinje cells and ultimately the output of the cerebellar cortex (40-42).

The axons of basket cells horizontally pass through the lower molecular layer, which ultimately descend to surround the somata and axon hillock of

Purkinje cells, producing a basketlike formation (pinceau) (43-45). In addition to projecting descending collaterals to Purkinje neurons, the axons of basket cells also send beaded tendrils into the molecular, upper granule cell and Purkinje cell layers (44). Studies have indicated that more than one contributes an inhibitory (GABAergic) influence onto each pinceau (4, 44, 45). Dendrites of basket cells are rather long and extensive and are categorized as either being numerous and thick descending collaterals that project into the lower third of the molecular layer or as ascending collaterals (45).

17 Stellate cells are a group of polymorphous, inhibitory interneurons

(GABAergic) that reside within the outer two thirds of the molecular layer (4, 45).

One significant characteristic that differentiates stellate cells from basket interneurons is that their axonal arborization is localized to the dendritic shafts of

Purkinje neurons (4, 45). The outer third layer contains "superficial stellate cells" that have small somatas (5-9 µm diameter), irregularly contorted and profusely branched dendrites and a thin, short axon (40 µm long) (4, 45). The "deep long axon stellate cells" are defined as having cell bodies that are larger than their superficial counterparts, highly branched dendrites that radiate towards the molecular layer and very long axons (lengths up to 450 µm) (45). Figure 2 provides an example of the layers and cell types present throughout the cerebellar cortex (1).

2. Cerebellar peduncles

A general description of the cerebellar peduncles reveals three symmetrically paired groups of fiber tracts (entering or leaving) that connect the cerebellum to the brainstem (via the pons) and are as follows: the inferior (restiform body), the middle cerebellar peduncle (brachium pontis) and the superior cerebellar peduncle (brachium conjunctivum). Briefly, the superior cerebellar peduncles are connected to the midbrain and contain mostly afferent axons but also include some efferent fibers, the middle peduncles are linked to the pons and the inferior peduncles are connected to the medulla oblongata and possess both afferent and efferent axons (46).

18 The inferior cerebellar peduncles are situated on the posterolateral aspect of the superior medulla oblongata but eventually deviate from one another as they ascend into different cerebellar hemispheres (47). A majority of the fibers that comprise the inferior cerebellar penduncles are afferent and include: the dorsal (Clarke's nucleus to paleocerebellum), the cuneocerebellar tract (accessory cuneate nucleus to vermis), the (inferior olivary nuclei to neocerebellum cortex), the reticulocerebellar tract

(reticular formation to vermis) and the vestibulocerebellar tract (vestibular nuclei and vestibulocochlear nerve to archicerebellum) (46, 47). Fibers exiting the cerebellum via the inferior peduncles consist of: the cerebellovestibular fibers (to vestibular nuclei) and the cerebelloreticular fibers (to reticular formation in pons and medulla) (47).

Originating from the posterolateral pons and becoming part of the cerebellar white matter is the middle cerebellar penduncle (47). It is the largest of the all cerebellar penduncles and is shaped from transverse fibers of the corticopontiocerebellar pathway that initially arises from pontine nuclear neurons in one half of the pons, traverses the midline to the opposite middle cerebellar peduncle and eventually arrives at the neocerebellar cortex of the contralateral hemisphere (47).

The most medial peduncle situated between the stratum lemnisci (V- shaped structure; anterior cerebellar notch) and rostrally extends along the fourth ventricle is the superior cerebellar peduncle (47, 48). The afferent fibers associated with these peduncles are the ventral spinocerebellar tract (spinal

19 border cells to cerebellar cortex and globose and emboliform nuclei), rubrocerebellar fibers and tectocerebellar fibers (46, 47). The efferent fiber tracts include the globose-emboliform-rubral pathway that originates in both deep cerebellar nuclei, passes through the superior cerebellar peduncle, decussates to the opposite side and finally synapses with the contralateral red nucleus; ultimately crossing the midline twice and affecting motor output on the same side of the body (47). Also crossing through the superior cerebellar peduncle is the crosses the midline at the superior peduncle and terminates by synapsing with the ventrolateral thalamic nucleus (47). Additional details concerning these pathways will be discussed in an upcoming section.

3. Deep Cerebellar Nuclei (DCN)

The fastigial (medial), interposed (globose and emboliform) and dentate

(lateral) nuclei comprise the deep cerebellar nuclei system and are in essence, the sole output of the cerebellum. The cellular population within the DCN varies in size, origin of input, neurotransmitter consistency and innervation target.

Contained within each nuclei is a heterogeneous mixture of excitatory and inhibitory neurons and interneurons. It has been reported that excitatory neurons have a larger range of sizes (10 to 35 µm) and utilize glutamate and/or aspartate as their neurotransmitter; whereas inhibitory neurons are smaller and employ

GABA and/or glycine as their neurotransmitter (49-51). In general, excitatory projection neurons roughly represent 50-60% of the cellular DCN population and can innervate a range of targets that includes the thalamus, pontine and reticular nuclei, medulla oblongata, the superior colliculus and the spinal cord (51-53).

20 Inhibitory neurons comprise approximately 30-35% of DCN cells and predominately innervate the inferior olive (54). Inhibitory interneurons constitute the remainder of the group (10%), colocalize GABA and glycine and are intermingled with excitatory and inhibitory neurons (51, 52).

DCN neurons are the targets for all cerebellar input, pre-cerebellar nuclei and inferior olive, receive the foremost attention from Purkinje neurons (up to

75% of synaptic inputs are from PN) and are also recipients of serotonergic and cholinergic influence (51, 55, 56). Each DCN are chiefly influenced by two sources of input that includes a main contribution of GABA from Purkinje neurons and a moderately less glutamatergic influence from mossy and climbing fiber collaterals. While each nucleus is the recipient of identical transmitter input, the efferent projections and functional influence throughout the CNS varies considerably. Studies have indicated that Purkinje neurons can innervate multiple classes of post-synaptic projection neurons (GABAergic and non-

GABAergic) within the DCN and that a majority of GABAergic neurons contacted by Purkinje neurons would further project to the inferior olive (IO) (51). Excitatory and inhibitory efferent axons within the DCN exit the cerebellum via pathways that directly involve the superior or inferior cerebellar peduncles. All cerebellar nuclei send projections through the superior cerebellar peduncle whereby their projections bifurcate to generate ascending (upper brain stem termination) and descending (travels down to medulla) limbs (4). Conversely, inferior cerebellar peduncle fibers originate from the fastigial nucleus and move onto the vestibular nuclei and reticular formation (4). In addition to projecting through the cerebellar

21 peduncles, some cerebellar nuclear cells can directly traverse to the upper and lower brain stem and the spinal cord.

Studies have indicated that PN projections to the DCN are highly topographic and specifically organized in that each cerebellar cortical zone innervates a restricted portion within the deep nuclei. Additionally, each nucleus is uniquely organized in regards to anatomy within the nucleus and corresponding efferent targets. Specifically, anatomical tracing studies, single- unit recordings and electrical micro-stimulations in several experimental models have indicated that within each nucleus lies at least one map of the body and that inactivation of a nucleus will result in a motor deficit specific for that nucleus; fastigius induces impaired upright stance and gait, interpositus undergoes alternating agonist-antagonist muscle tremor on reach and the dentate experiences curved trajectory and overshoot on reach and a lack of finger coordination in grasping/pinching (57). Ultimately, signals entering the cerebellar cortex are first segregated into a particular zone/complex and then further project to a specific, definable area within the DCN.

For example, the (DN) is distinct in both the anatomical arrangement and functional motor and non-motor domains (58). Specifically, studies have revealed that instead of the dentate nucleus only being responsible for movement generation and control via projections to the M1 region of the cortex, innervation of various areas of the cerebral cortex are also found to occur from specific localized regions of the dentate called output channels that are concerned with cognition and visuospatial functions (58). Early investigations

22 have also suggested that the dentate can exert influence onto the activity of spinal neurons through pathways that do not include the sensorimotor and premotor cortices (59).

The interpositus nuclei (IN) are flanked by the fastigial and dentate nuclei and are dorsal to the superior and lateral vestibular nuclei (4). As is the case with the dentate nucleus, projections from the IN can be further differentiated as originating from either the anterior or posterior interpositus. While the exact location of efferent fibers differ within the nucleus, it is known that the IN are the sole output of the intermediate cerebellum and similar to the DN, are innervated by 2 main sources: massive GABA-mediated input from cortical Purkinje neurons and weaker glutamatergic-mediated input from mossy and climbing fiber collaterals (60). Functionally, the IN are essential contributors to the acquisition and performance of the classically conditioned eyeblink response (60, 61).

Studies have indicated that the lesioning or inactivation of the IN eliminates the possibility of learning new and previously acquired conditioned responses (60,

62, 63).

Amongst the DCN, the fastigial nucleus (FN) is phylogenetically the oldest and is approximated to be double the volume (3.5 x 10-4 cm3) in comparison to the dentate nucleus and also contains twice as many neurons (64). The medial nucleus lies close to the vermal midline, in the roof of the fourth ventricle with its long axis rostrocaudally orientated and contains both large (10-30 µm in diameter) cells and rather significant population (~75%) of small multipolar cells

(4-16 µm diameter) (64, 65). Anatomically, the FN is distinguished by a

23 dorsolateral protuberance and a ventral portion that can be further subdivided into middle and caudomedial areas (66). Early investigations suggested that under resting conditions, cells within the FN fire at a high and steady frequency

(~ 37 Hz) and that the firing rate will adjust depending on the type of input received (excitatory versus inhibitory) (67). Powerful, inhibition arises from vermal Purkinje neurons and excitatory innervation has been shown to originate from fiber collaterals in the olivo-cerebellar, cuneo-cerebellar and spino- cerebellar tracts.

Studies have indicated that the vermis is topographically connected to the anterior, ipsilateral two thirds of the FN (67). Additionally, the FN contains clusters of cells that are similar in regards to somatotopy and provide an excitatory input to downstream targets such as the vestibular nucleus. In regards to this study, the primary focus will be on the vermis-FN connection. Specifically because it is known that the vermis is chiefly involved with the amalgamation of static and dynamic posture components and movements related to locomotion and standing and that the primary output from the vermis is via fastigial cells, it is only probable that the FN serves as an integration station for receptors dispersed over the body (64, 67). Taking this idea one step further, correct quadripedal movements are dependent upon the incorporation of forelimb and hindlimb inputs as well as the integration of ipsilateral and contralateral hindlimb (68); so that any deviation within the intrinsic circuit pathway, whether it be in regards to the firing properties of PN or other synaptic inputs, could potentially produce a disruption in one or more aspect of motor control and output.

24 4. Vestibular nuclei

Four nuclei comprise the vestibular complex and include, the lateral vestibular nucleus (Deiter's; LVN), medial vestibular nucleus (MVN), superior vestibular nucleus (SVN) and the descending vestibular nucleus (spinal; DVN).

Vestibular nuclei are topographically connected via the commissural system and cells within the nuclei send ascending and descending contralateral projections.

Ascending fibers from the vestibular complex synapse with targets located in the brainstem, thalamus, cortex and several folia of the cerebellum (includes the anterior and posterior vermis and flocculus) (69). Targets of descending vestibular nuclei projections include the spinal cord and the solitary nucleus.

Functionally, the vestibular system contributes to a multitude of tasks that include gaze stabilization, balance and postural control, spatial navigation, spatial perception and memory, voluntary movement planning and autonomic function

(70). Impediments to the normal functions of the vestibular system can induce disorientation, impaired breathing and blood pressure adjustments during movement and posture changes, loss of postural control and balance, decreased visual acuity and perceptual distortions (70, 71).

5. The olivo-cerebellar system

Anatomically, the inferior olivary nucleus is located within the caudal medulla and is comprised of the principal olive (PO), the medial accessory olive

(MAO), the dorsal accessory olive (DAO) and a group of subnuclei that include the ventrolateral outgrowth, the dorsal cap of Kooy, Beta-nucleus and the dorsomedial cell column (72). Each nucleus can be partitioned into a set of

25 uninterrupted sheets that ultimately serves to distinguish anatomically distinct subdivisions of cell clusters that are called lamella. In rodents, the MAO is comprised of horizontal, vertical and rostral lamellae, whereas the DAO and PO are divided into dorsal and ventral lamellae (73). The cellular composition of the inferior olive is rather uniform in that it contains few interneurons (<0.1%) and two types of projection neurons that vary in soma shape and size and overall dendritic shape (72). These myelinated, projection neurons give rise to the climbing fibers that project to a specific parasagittal zones of the contralateral cerebellar cortex and extend throughout multiple lobules of the cerebellum (74).

Climbing fibers also send collateral branches to the central cerebellar nuclei before ascending through the GCL to directly synapse onto a single Purkinje cell, establishing multiple synaptic sites on the spines of the PC dendritic tree (74-76).

Additionally, climbing fibers can also innervated Golgi cells.

There is a reciprocal and topographic organizational scheme between the inferior olive, cerebellar nuclei and parasagittal Purkinje cell zones that form a functionally distinct cerebellar module. Extensive levels of functional and anatomical organization exists throughout the cerebellar modules so that a specific group of climbing fibers will project to a particular strip of Purkinje cells which then innervate a precise part of the cerebellar nuclei (CN) that ultimately close the loop by sending projections back to the inferior olive (IO). Additionally, there is a direct connection from the IO to the CN that most likely originates from a CF collateral and adds to the modular organization of olivocerebellar system

(77).

26 The level of complexity, organization and connectivity throughout the olivo-cerebellar system functions to control the timing, execution and precision of both simple and complex motor commands and tasks. The idea of having a multi-faceted arrangement of control comes into consideration when examining the effects of modulating one or more levels of the olivo-cerebellar system. In regards to this study, a 1 mm hole was drilled in the midline between Bregma points -5.68 to -6.36 which includes lobules II-V of the anterior lobe of the cerebellar vermis. As previously described, three transverse boundaries partition the vermis along the rostro-caudal axis to subdivide the vermis into 4 transverse zones – the anterior, central, posterior and nodular zones. Additionally, a longitudinal organizational scheme exists that from medial to lateral, includes the

A, AX, X, B and C projection zones. Each zone can be organized based upon anatomical location, electrophysiological properties, CF input, DCN projections and zebrin expression. The cortical area that was examined in this study has been shown to receive climbing fiber input from group B of the caudal medial accessory olive (cMAO) and is topographically related to the anterior 2/3 of the fastigial nucleus (8, 68, 77).

C. Normal Cerebellar Function 1. Motor and non-motor influence

Historically, the cerebellum has been accepted and recognized as being influential in motor coordination, timing and learning, posture and equilibrium.

While previous studies have mainly focused on the exact functional role of the cerebellum in motor output and the physiological outcomes of cerebellar

27 damage, more recent evidence has suggested that the cerebellum also contributes to a variety of non-motor functions. Projections from the DCN and cortical areas (both motor and non-motor) are part of a closed-loop circuit that ultimately provides the cerebellum with the anatomical capacity to influence the management of both movement and cognition (78).

Researchers have organized the cerebellum into several functional units that consist of the vestibulocerebellum, the spinocerebellum and the cerebrocerebellum. Each area represents a specific function, is comprised of different cerebellar structures and as expected, varies in both afferent and efferent projections. For example, the vestibulocerebellum is responsible for sustaining equilibrium and eye movement control, receives mostly vestibular afferents and consists of the flocculonodular lobe (74). The spinocerebellum contains the anterior lobe, vermis and paravermis and primarily influences posture, locomotion and proximal musculature control via projections with the brain stem and spinal cord. Lastly, the cerebrocerbellum (or pontocerebellum) predominantly receives input from the cerebral cortex (transmitted by the pontine nuclei) and then sends extensive ascending projections back to areas of the cerebral cortex (via the thalamus) that influence movement planning and coordination of voluntary limb movements (74). Additionally, certain functions of the body are related to the cerebellum and can be organized into three categories that include reflexes (somatic and autonomic), posture and locomotion and voluntary movements (4). These specific categories are comprised of the following tasks, the vestibulo-ocular reflex (VOR), eye saccades, smooth eye

28 pursuit, postural and stretch reflexes, inter-limb coordination, postural adjustments, locomotion, voluntary limb movement, eye blink conditioning, somatic and autonomic reflexes, cardiovascular and respiratory reflexes, and lastly acoustic and startle responses (4).

Aside from the involvement in motor output, the cerebellum is also considered to contribute to a wide range of non-motor tasks such as attention, verbal and working memory, cognition, emotion and behavior (79, 80).

Cerebellar activation during cognition was initially revealed during neuroimaging studies of language and was later demonstrated to occur during a multitude of non-motor functions that include sensory processing, anticipatory planning and prediction, mental imagery, memory, and visuospatial functions (79, 81).

Functional imaging studies of the vermis indicate that activation is present during panic, sadness, and grief (81). Furthermore, autonomic cardiovascular arousal

(as during exercise and mentally stressful tasks) elicits activation of both midline and lateral hemispheres in the posterior cerebellum; whereas different aspects of pain (experience versus anticipation) activate either the anterior or posterior regions respectively (79, 81).

2. Consequences of vermal injury on motor output

The role of the cerebellum in motor output has been determined through lesion studies or via the examination of degenerative diseases and is most often distinguished by impaired balance, posture and gait, ataxia, limb dysmetria, dysdiadochokinesis, loss of tone, dysarthria, and oculomotor disorders (81). As

29 previously described, the cerebellum can be divided into zones that are somatotopically, functionally and anatomically distinct. Essentially, the somatotopic representation within the cerebellar cortex and DCN ensure that any motor deficit is unique to each nucleus so that damage to the dentate induces curved trajectory and reach overshoot and lack of finger coordination during grasping/pinching, interpositus injury elicits agonist-antagonist muscle tremor during reaching and fastigial damage generates impaired upright stance and gait

(82).

Disorders in motor output also arise when damage is incurred in the cerebellar cortex. Lesion data has indicated that the medial cerebellar region contributes to regulating extensor tone, sustaining upright stance and dynamic equilibrium and modulating the cooperative actions of flexor and extensor muscle groups involved in locomotion and that destruction of the vermis (regardless if the fastigial nucleus was included) yields difficulties in balance, stance and gait (80,

83, 84). Furthermore, studies involving subjects with midline lesions and their ability to execute rhythmic tapping revealed that while the patients were able to precisely establish when to initiate a response, they were unable to implement a response at the appropriate time; demonstrating an association between the vermis and implementation of a motor response (85).

II. Purkinje Cells and Synaptic Plasticity

A. Firing properties & excitatory inputs

1. General firing properties

30 Due to the extensive branching of the dendritic tree, Purkinje cells are able to incorporate a vast array of excitatory and inhibitory inputs, providing the sole output of the cerebellar cortex that translates into inhibitory innervation of target cells within the DCN. Under normal conditions, Purkinje neurons fire in a characteristic, spontaneous manner; a direct result from the interplay and incorporation of a vast amount of cortical, vestibular and sensory information, excitatory inputs from parallel and climbing fibers, inhibitory inputs originating from cerebellar interneurons and intrinsic currents from activated potassium channels, P/Q-type calcium channels and a resurgent sodium channel that induce membrane depolarization, generation and membrane repolarization (86-88).

Furthermore, electrophysiological recordings from both cerebellar slices and in vivo paradigms have found that as the cerebellum matures a trimodal pattern of firing that is defined as an initial phase of tonic, spontaneous firing that increases until the PC bursts and is followed by an intermittent episode of silence

(89). Functionally, these trimodal oscillations ensure that the correct combination of augmentation and inhibition of agonist and antagonist muscle groups will ensue. Additionally, the cerebellum is able to achieve and execute motor coordination through the generation and maintenance of precise timing signals.

It has been suggested that these timing signals are encoded in the transient adjustments in the PC rate of firing (89). Because Purkinje cells are the exclusive neuronal outlets of the cerebellum, the postsynaptic amalgamation of excitatory and inhibitory inputs is critical for motor information processing (90). Because

31 Purkinje cells are GABAergic neurons, their activation results in the inhibition of their target neurons in the vestibular and other cerebellar nuclei (9).

Essentially, the output activity of Purkinje neurons has been categorized into spontaneous simple spikes and complex spikes. While both simple and complex spikes are produced ~15-20 mm from the Purkinje cell soma in the proximal axon, they differ in firing frequency, type of information transmitted and overall functional output (91). Additionally, the fibers that deliver simple or complex spikes vary in location of origin and will be discussed in the following sections.

2. Mossy fiber system

There are several nuclei within the brainstem that receive input from the cerebral cortex and proceed to project to the cerebellum, providing somatosensory input to the cerebellar cortex. These nuclei include, the inferior olive, pontine nuclei and the lateral reticular nucleus. While it is thought that the inferior olive supplies a majority of the climbing fibers, the source of mossy fibers arises from all other precerebellar relay nuclei. Mossy fibers originate from a multitude of sites that include the cerebral cortex, brainstem and spinal cord and travel via the dorsal spinocerebellar tract, cuneocerebellar tract, vestibulocerebellar tract, and the pontocerebellar tracts to synapse with the dendrites of granule cells (GCL). Out of all the possible areas of origin, the pontine and lateral reticular nuclei are regarded as the most important cerebrocerebellar relays due to the vast amount of fibers that terminate within each nuclei and eventually project onward to the cerebellum (92). Mossy fibers

32 that originate from the spinal cord ascend to the ipsilateral cerebellum whereas those coming down from the cerebral cortex cross the midline.

The mossy fibers are one of two sources of excitatory (glutamatergic) input to the cerebellar cortex and are essential for modulating Purkinje cell discharge (93). It is thought that each granule cell receives an average of four

MF inputs, which generate an 8-12 mV depolarization (94). Essentially, the MF contribution of rapid signal transduction is that of a graded response that is ultimately dependent upon the activity of a multitude of mossy fibers that are collectively and synchronously active. Studies have indicated that each mossy fiber is capable of innervating hundreds of granule cells whose axons ascend and bifurcate to form the parallel fiber system; approximately 200,000 parallel fibers cross the dendritic field of each Purkinje cell to establish a single synaptic connection (75, 92, 95). In response to cortical input via the MF system, granule cells generate an excitatory post synaptic potential (EPSP) in PC dendrites that then allows for the discharge of simple spikes. Purkinje cells can exhibit high frequency simple spike firing that can range anywhere from 10-100 Hz and is contingent upon the parallel fiber excitation (74, 91). An investigation by Rokni et al. suggested that granule cell axons (located directly under the PC targets) are more efficient at multi-synaptic, synchronous Purkinje cell innervation in comparison to parallel fibers (75). They further imply that the lateral arrangement between PFs and PCs may induce a rather weak activation of PCs because of the variable synaptic connections and conduction velocities of parallel fibers (75).

33 It should be noted that Golgi cells provide feedback inhibition to granule cells by receiving excitatory input (from both CF and MF) and synapsing with the dendrites of granule cells. Consequently, by inhibiting the MF induced excitation of granule cells, Golgi cells modulate the excitatory input to the cerebellar cortex and ultimately PC excitation. Additionally, it is thought that the feedback inhibition from Golgi cells induces synchronization of the rhythmic activity in the

GCL and appears to influence the strength and timing of PF excitation of Purkinje cells (74).

2. The climbing fiber system

In contrast to the MF-PC connection, each PC receives input from a single

CF that is quite robust and provides approximately 1500 synaptic contact locations to ultimately produce a burst of spikes known as a complex spike (76).

It should be noted that the response generated by climbing fibers is not graded, occurs at a lower frequency and is opposite to the effects produced by parallel fiber afferents. Aside from evoking a Purkinje cell response, the climbing fiber afferent system is thought to contribute to a variety of functional outputs. While numerous groups have attempted to establish some sort of commonly recognized viewpoint concerning the function(s) of climbing fibers, there is still a general lack of consensus. Extensive research has however, indicated that one critical function of CF input is to provide a signal that an unanticipated interruption has taken place during the intended movement (93). For example,

Gellman et al. indicated that olivary neurons almost exclusively carry sensory signals, that are either significantly reduced or absent during active movement

34 (96). So that when an unexpected somatic event takes places (i.e., one that interrupts movement part way through the trajectory), the subsequent firing of IO cells serves to indicate that a problem has arisen (96). While Mano et al. presented a scenario where complex spike activity increased during visually guided wrist tracking movements in the absence of any external disturbances,

Ojakangas and Ebner proposed that the climbing fiber system was directly involved in the adjustment of limb movements necessary to correct errors encountered during the proper placement of a cursor to a target box by making considerable, short-term modifications in the cerebellar cortical output (97, 98).

While the previous studies demonstrated several conditions in which climbing fibers were activated, it should be noted that the initial CF activation is not always solely dependent upon a mismatch during movement but most often seems to include the occurrence of an error signal sometime during the course of the testing period. Additional studies have indicated that activation may come about during other scenarios that include, sensory information encoding, tonic activity regulation, complex motor sequence generation and simple spike activity modulation (93). For example, in a study that utilized visual stimulation paradigms coupled with recordings from individual inferior olivary neurons, researchers were not only able to demonstrate the principle of zonal organization between activated climbing fiber inputs and specific, downstream cerebellar targets in the flocculus, but were also able to provide evidence that a changes in the stimulus pattern was actually registered as a retinal slip/error and serves as the basis for climbing fiber activation (99).

35 In regards to activity regulation, multiple investigations have reported that lesions of the inferior olive resulted in a marked increase in simple spikes that was accompanied with an almost complete elimination of complex spikes. The change in simple spike frequency was directly attributed to the suppression of olivocerebellar neuron activity and essentially diminished within several weeks

(100, 101). These results suggested that the function of the olivocerebellar system is to provide tonic inhibition onto Purkinje neurons, thereby controlling the excitability of downstream targets. Additionally, researchers have attempted to correlate the onset of movement with an increase in complex spike discharge.

This topic has however, generated conflicting resulting with one side proposing that subsets of IO neurons demonstrate periods of activity that are both rhythmic and temporally predictable during movement; whereas other investigations have suggested that while complex spike activity does increase at the onset of movement, the overall discharge rate is random (102, 103).

The last aspect of climbing fiber function pertains to the roles in both long term and short-term heterosynaptic action on Purkinje neurons. Briefly, while numerous studies have suggested that recurring and robust electrical stimulation of climbing fibers in conjunction with mossy fiber activation will induce long-term depression (LTD), the exact role of LTD in memory is still under investigation, whereas it is the best candidate for motor learning induction (104). The possibility that CF input influenced PC excitability was initially proposed in the

Marr-Albus hypothesis and further investigated by Ito (104, 105). The combination of theoretical and experimental work by these two groups suggested

36 that a memory trace was created upon the coincidental activation of Purkinje cells from both climbing and parallel fibers during a specific behavioral task; thereby creating a scenario where the Purkinje cells become less responsive to input from the same set of parallel fibers (93, 104, 105). While there is a general agreement that some sort of long-lasting change in Purkinje cell excitability is present during learning, a clear and unified idea of the relationship between LTD and memory is still lacking. Conversely, the short-term action of climbing fibers refers to a brief (10-20 ms) period of membrane inactivation and subsequent pause in simple spike activity following a complex spike (106). The modification of the short-term response of Purkinje neurons to parallel fibers can result in either an increase or decrease in the discharge rate following input from a spontaneous complex spike (93, 106).

B. Synaptic Plasticity 1. LTP, LTD and RP The PF-PC synapse is home to several types of modification that includes pre- and postsynaptic long-term potentiation (LTP), long-term depression (LTD) and rebound potentiation (RP). It is thought that use-dependent changes in the intensity between PF-PC synaptic transmission is a fundamental component of particular types of motor learning that includes eye blink conditioning and vestibulo-ocular reflex adaptation (107). LTP occurs when the PF-PC synapse is strengthened with recurring PF stimulation at frequencies that range from 4-8 Hz

(presynaptic LTP) or at 1 Hz (postsynaptic LTP) (107, 108). Aside from the differences between frequency and site initiation, the two forms of LTP also vary

37 in the type of second messengers involved and physiological outcomes. For example, studies on presynaptic LTP induction have suggested the involvement of a rapid elevation in cAMP levels that aids in the generation of long-term enhancement of neurotransmitter release at the PF-PC synapse (108, 109).

Conversely, postsynaptic LTP initiation is dependent upon NO and not cAMP or cGMP and has been found to have a reciprocating relationship with LTD in that both act on AMPA receptors and can be undone by the other (108).

Another example of plasticity thought to influence motor learning and memory involves the repetitive, concurrent transmission between parallel and climbing fibers that results in LTD. Anatomically, LTD occurs at the parallel fiber-

Purkinje cell synapse and has functionally been found to not only adjust information flow but is also an essential component of motor learning. During

LTD induction, glutamate is released from PF terminals and binds to post- synaptic mGluR1 along the dendritic spines of PC, resulting in activation of the

Gq protein and the PLCβ4 cascade, the generation of DG and IP3 and the release of Ca2+ from internal stores (110). The activation of this signaling cascade in conjunction with PKC mediated phosphorylation of the AMPAR GluR2 subunit, induces the endocytosis of AMPAR at the PF-PC synapse (110, 111). It should be noted that there is also a high level GABABR expression localized on the dendritic spines were LTD transpires. It has been proposed that the

2+ activation of GABABR, augments the intracellular Ca response mediated by mGluR1 through the direct involvement of the Gi/o protein pathway (112).

Additionally, the colocalization of mGluR1 and GABABR suggests the possibility

38 of functional crosstalk between the two receptors which may function to influence

LTD induction (112). Numerous pharmacological investigations have demonstrated that the post-synaptic activation of GABABR facilitates mGluR1 mediated LTD (110-112). CF activity induces membrane depolarization within

PC dendrites, inducing the opening of voltage-gated calcium channels (VGCC)

2+ 2+ and subsequent Ca influx which also contributes to the rise in [Ca ]i (110).

Aside from the two types of synaptic modulation discussed thus far, rebound potentiation (RP) is an additional mode of plasticity that is also dependent upon the activity from an excitatory afferent. Specifically, RP is triggered through the activation of excitatory synaptic input at the CF-PC synapse that then initiates membrane depolarization and induces a transient rise in

2+ postsynaptic [Ca ]i that is followed by the potentiation of a GABAAR mediated

IPSC (33, 113). It is thought that RP is physiologically significant in that it contributes to the regulation of PC excitability and may possibly play a role in motor learning.

III. G Protein Coupled Receptors A. GPCRS, G proteins & effector proteins Approximately 80% of the signals transmitted across biological membranes are mediated by the largest group of cell surface proteins, the G protein coupled receptors (GPCRs) (114). While all GPCRs are structurally similar in that they consist of seven-transmembrane (TM) domains and an extracellular N-terminus and intracellular C-terminus and signal via a heterotrimeric guanyl nucleotide binding protein (G protein), they are functionally

39 unique, have been subclassified into 7 families (Class A Rhodopsin like, Class B

Secretin like, Class C Metabotropic glutamate/pheromone, Class D Fungal pheromone, Class E cAMP receptors, Frizzled/Smoothened and Orphans) and have been found to modulate various physiological processes, contribute to numerous disease states and are the targets of approximately 50% of all current therapeutics (114, 115).

The principal task of GPCRs is to transmit an extracellular stimulus into an intracellular signal. Numerous detectable, external cues such as ions, odorants, hormones, photons, peptides and neurotransmitters activate GPCRs, whereas those receptors with no known endogenous ligand are coined orphan GPCRs.

Upon agonist binding, the GPCR undergoes a conformational change that allows for activation of a G protein. Two classes of G proteins exist and are defined as being either heterotrimeric G proteins (α, β and γ subunits) that directly associate with GPCRs and signal transduction or small cytoplasmic G-proteins (116). In order to convey a signal from an activated receptor to an effector protein, the heterotrimeric G protein must undergo an activation-inactivation cycle that involves GDP to GTP exchange on the α-subunit and dissociation from the βγ complex; inactivation consists of hydrolysis of GTP back to GDP and sequestering the βγ complex by Gα-GDP (117).

The four major classes of G proteins, Gαi (Gtr, Gtc, Gg, Gi1-3, Go and Gz),

Gαs (Gs and Golf), Gαq (Gq, G11, G14 and G15/16) and Gα12, (G12 and G13) are defined by their a subunits, the receptors that each couples to and the specific biochemical pathway involved in signaling (116, 118). Once activated, G protein

40 subunits can act as secondary messengers or directly mediate cellular and physiological responses by interacting with effectors that include tubulins, adenylate cyclases, ion channels, protein kinases, phospholipases and phosphodiesterases (117, 119). For example, the Gi signaling pathway increases intracellular cAMP levels via adenylyl cyclase inhibition and has been found to modulate neurotransmission, slow the contraction rate of cardiac myocytes, and promote chemotaxis (118). Because this project examines the behavioral and electrophysiological outcomes of vertebrate rhodopsin activation in the cerebellum, focus will be given to GPCRs specifically localized to Purkinje cells.

B. GPCR mediated modulation GPCRs, G-proteins and downstream effector proteins are key contributors to cellular and neuronal circuit modulation that influences physiological or behavioral functions within seconds to minutes (120). Modulation can occur pre- or postsynaptically and includes mechanisms that affect the control of neurotransmitter release and synaptic transmission respectively. Inhibition of neurotransmitter release from presynaptic terminals can be mediated by a variety of GPCRs that couple to the Gi/o protein and include β, α1 and α2 adrenoreceptors, cannabinoid CB1 receptors and GABAB receptors and is thought to involve three possible mechanisms that consist of inhibition of voltage- gated calcium channels (VGCC), activation of voltage-gated potassium channels and through the direct action on the vesicular release machinery (121, 122).

Activation of the Gi/o protein pathway also modulates synaptic transmission

41 through the activation of G protein-coupled inward rectifying K+ channels

(GIRKs) and the inhibition of presynaptic VGCC, resulting in reduced AP firing and transmitter release.

1. GABABR-mediated modulation in the cerebellum

A number of transmitters couple to the Gi/o pathway via specific GPCRs throughout the brain but the focus of this section will be on GABABR expression and activity in the cerebellum. Briefly, the GABABR is a member of the class C

GPCRs and are composed of the 2 homologous subunits, GABAB1 where GABA binds and GABAB2 which is responsible for activation of the G protein (123).

Within the molecular layer of the cerebellum, GABABRs are presynaptically expressed at PF terminals and postynaptically at the PC annuli of dendritic spines (124). Activation of the Purkinje cell GABABR is dependent upon the synaptic release of GABA from innervating interneurons and GABA spillover (90).

Once activated, GABABRs exert typical presynaptic and postsynaptic modifications via the Gi/o protein. Specifically, the presynaptic event involves the inhibition of neurotransmitter release via modulation of pre-synaptic voltage- gated calcium channels (P/Q type) that ultimately decreases spontaneous firing and transmitter release (125). In regards to the postsynaptic effects, GABABR activation induces a long lasting K+ conductance by coupling to G-protein inward

+ rectifier K channels (GIRK) via the Gi/o protein to induce a hyperpolarized membrane potential and decreased firing probability – thereby mediating neuronal excitability (90, 126).

42 GABABRs have also been found to modulate VGCC and mGluR1 and

AMPAR mediated glutamate signaling in Gi/o protein dependent and independent methods (90, 111, 124-127). In regards to VGCC, GABABR mediated modulation acts in both a GIRK dependent and independent manner that ultimately functions to either downregulate glutamate release from P/Q type Ca2+ channels or decrease glutamate release via N-type Ca2+ channels, respectively

(127). Glutamate released from parallel fibers stimulates both AMPARs and mGluR1s; activation of a particular receptor is dependent on the level of activity.

Excitation of AMPARs elicits excitatory postsynaptic potentials (EPSPs), whereas stimulation of mGluR1 generates slow EPSPs through the Gq protein (128, 129).

Studies utilizing whole-cell patch clamping revealed that application of the

GABABR agonist baclofen, activated a GIRK current that resulted in hyperpolarization of both the resting Em and AMPA mediated EPSP levels (90,

128).

At the PF-PC synapse, GABABRs have been found to colocalize and functionally couple with another group C GPCR, type-1 metabotropic glutamate receptors (mGluR1s); numerous studies have suggested the existence of crosstalk between the two receptors (128, 130, 131). Data from these investigations propose that the extracellular domain (ECD) of the GABABR

2+ interacts with extracellular calcium (Ca o) to generate a constitutive increase in glutamate sensitivity, enhancement of mGluR1-mediated transmission and

2+ augmentation of [Ca ]i (130, 131). Studies have also suggested that the

43 cooperative interaction between GABABR and mGluR1 may possibly enhance

LTD (111, 128, 132).

C. G protein signaling cascade and the visual system As previously described, GPCRs act as signaling molecules in that they respond to a variety of environmental and physiological signals such as hormones, gustatory and olfactory stimuli, neurotransmitters and light and mediate the transmission of cellular responses into extracellular signals. Among this list of GPCR mediated signaling cascades, the pathway involving rhodopsin is the best characterized.

Contained within the outer segment of rod photoreceptor cells is the phototransduction machinery that allows for dim-light vision in vertebrates and includes rhodopsin and other transduction proteins. Rhodopsin is a 40kDa protein, member of the Class A group of GPCRs and is comprised of a protein, and the chromophore 11-cis retinal, that acts an inverse agonist to suppress constitutive activity from the opsin (133, 134). The onset of activation and the subsequent signaling cascade is initiated when a photon is absorbed by rhodopsin and is followed by the following steps: light-induced isomerization of

11-cis retinal, generation of the early intermediate bathorhodpsin (Batho) and all- trans retinal, thermal relaxation of the receptor through the intermediates lumirhodopsin (Lumi) and metarhodopsin I (Meta I) and proton transfer and transition into active metarhodopsin IIa (Meta IIa) whereby the configuration of rhodopsin allows for the binding and activation of the G protein, transducin (Gt) and transformation into Meta IIb (133-135). Transducin mediates signal

44 transmission between rhodopsin and the effector enzyme, cGMP specific phosphodiesterase (PDE), ultimately resulting in membrane hyperpolarization of rod photoreceptors, closure of VGCC and reduced glutamate release (134).

Hyperpolarization terminates the activation phase of the photoresponse and is followed by a recovery period that includes rhodopsin phosphorylation and subsequent arrestin binding, cascade shut-off that specifically involves transducin deactivation and cGMP resynthesis (134). At post-synaptic bipolar cells, mGluR6 mediated signaling is inhibited, membrane depolarization occurs and the retina transmits signals to visual centers within the brain (133).

Because the G protein transducin (Gt) is a member of the Gi/o family, it would be interesting to determine if rhodopsin could functionally couple to additional Gi/o-mediated pathways in other cellular populations besides the retina.

This idea was initially tested in cultured hippocampal neurons and results demonstrated that the photoactivation of vertebrate rhodopsin induced, membrane hyperpolarization and decreased neuronal firing (136). While these results demonstrate the feasibility of utilizing vertebrate rhodopsin to control

GIRK and VGCC conductances in vitro, the involvement of Gi/o mediated activation and signaling in vivo is major point of contention and a future project aim. A brief overview of the data obtained from the photoactivation of vertebrate rhodopsin in vermal Purkinje cells will be discussed in the next section.

IV. Optogenetics A. Chemically activated GPCRs: RASSLs, DREADDS & AlstR/AL

45 GPCRs are distinguished as being the largest superfamily in the human genome and the most common target for therapeutic drug discovery (137).

Despite the vast amount of work that has concentrated on the diversity of tissue localization, involvement of cellular signaling pathways and functional output following activation as well as the endogenous and exogenous ligands associated with certain GPCRs, direct modulation of the signaling pathways connected to specific GPCRs has presented a continuous challenge. The capacity to manipulate the stimulation of a particular GPCR in a single cellular population or specific tissue in vivo would contribute to gaining a more complete understanding of the functional and physiological consequences of G protein mediated signaling pathway activation. In order to circumvent some of the intrinsic problems associated with GPCR modulation in vivo, several innovative designer receptors have been created that not only aim to exclusively link one

GPCR to a ligand that does not activate any additional targets but also promotes expression in a specified group of cells.

The first attempt at creating a technology that aimed to control G protein signaling in vivo was reported by Strader et al. in 1991, and involved engineering a receptor so that it was insensitive to its endogenous ligand but was instead fully activated by synthetic, small-molecule drugs (138, 139). The original receptor activated solely by synthetic ligands (RASSLs) was genetically engineered by mutating the Asp113 residue of the Gi-coupled β2-adrenergic receptor and was activated by catechol esters and ketone ligands both which lacked the capacity to activate the wild-type receptor (138). Despite this technological breakthrough,

46 there was an overall deficiency in the potency of both synthetic ligands so that supplementary in vivo use would be futile.

The next approach at engineering a receptor that exclusively responds to a synthetic compound focused on the mutageneis of the Gi/o-coupled κ opioid receptor (KOR) and resulted in the development of Ro1 (RASSL based on opioid receptor, no. 1) and Ro2 (118). Between the two RASSLs, the greatest decrease in opioid peptide binding and signaling was evident with Ro1; both retained affinity for small molecule drugs that included bremazocine and spiradoline (118).

Because numerous investigations have studied and utilized Ro1 RASSSLs in both in vitro and in vivo paradigms, several derivatives of Ro1 have been created and include Rog, Rog-A, Rog-µ and Rog-µA (137, 140, 141). Additionally, multiple transgenic mouse lines have been generated that express modified Ro1

RASSLs in various tissues that include the heart and liver, taste cells, and osteoblasts; thereby allowing for the investigation and analysis of how the modulation of Gi/o-mediated signaling affects physiological processes and behaviors in vivo (118, 142-148).

Several noteworthy issue associated with the utilization of RASSLs have come to light and essentially involve the following, the presence of basal constitutive activity with endogenous G proteins, the potential activation of native, wild-type receptors via synthetic ligands, low potency with synthetic compounds for in vivo use and the possibility for the stimulation of additional proteins outside of the intended target (137). To circumvent these potential concerns, a new generation of RASSLs has been generated; these modified GPCRs are referred

47 to as DREADDs (designer receptors exclusively activated by designer drugs)

(149). The development of DREADDs involved multiple cycles of random mutagenesis of the human muscarinic acetylcholine receptor (mAChR) subtype 3

(hM3), resulting in a double mutant that was no longer constitutively active or had the ability to interact with acetylcholine (natural ligand) but was instead stimulated by the small molecule drug clozapine-N-oxide (CNO) (137). The major advantage of this system involves the notion that CNO is a pharmacologically inert molecule that possesses no possible binding affinity for off-target receptors (137, 150). This idea ultimately encouraged the construction of a family of DREADDs, hM1-5D, that are solely stimulated by CNO; thereby promoting the study of GPCR function and modulation and all G protein mediated signaling (Gi/o, Gq and Gs) in vitro and in vivo (137, 149, 151, 152).

The need for a genetic method that promotes the modulation of a particular cellular population and permits the quick and reversible manipulation of neural activity, while using GPCRs, led to the development of AlstR/AL receptor/ligand system (153). This approach utilized the Drosophila allatostatin receptor (AlstR), a GPCR that is activated by the insect peptide allatostatin (AL) and has no capacity for activation by ligands of related mammalian receptors

(153, 154). Studies revealed that while AlstR has the ability to activate mammalian GIRK channels via Gi/o proteins in Xenopus oocytes, can rapidly and reversibly silence ferret cortical neurons in vitro, and reduces neural activity in cortical and thalamic neurons of rats, ferrets and monkeys in vivo, there are

48 kinetic and pharmacological restraints that make this method less than ideal

(153-155).

The innovation and technical allowances associated with RASSLs,

DREADDs and the AlstR/AL system have promoted the generation of additional tools that examine the physiological and behavioral consequences of GPCR activation in vivo that also aim to avoid the spatiotemporal and kinetic difficulties encountered with these designer GPCRs.

B. Light activated GPCRs: ChARGe, vRh & OptoXR Emerging technologies are still focusing on the direct functional analyses of neuronal circuits by developing methods that not only ensure groups of genetically specified neurons can be stimulated but also avoids the use of pharmacological agents that are most often associated with limitations in both application and wash-out. The first approach that sought to stimulate a functionally related neuronal population without applying synthetic compounds utilized light to control GPCR pathways and was termed the chARGe system

(156). The chARGe method was developed by coexpressing the Drosophila photoreceptor genes that encode for arrestin-2, rhodopsin NinaE and the α subunit of the Gq protein (156). Activation in Xenopus oocytes evoked a current when an initial dose of retinal was applied and photostimulation in hippocampal neurons consistently elicited action potentials (156). While this novel approach was successful in vitro, there have been no reports on the feasibility of in vivo use and most likely, the requirement of expressing three individual genes has hindered this endeavor.

49 Building upon the momentum of the chARGe system and the idea that light and photosensitive GPCRs could be utilized to study G-protein mediated signaling in vitro, researchers targeted vertebrate rhodopsin as a potential source for neuronal modulation. To review, vertebrate rhodopsin couples to the G protein transducin, a member of the Gi/o family, with activation modulating presynaptic VGCC and postsynaptic GIRK channels. The expression of vertebrate rhodopsin with either GIRK or P/Q type calcium channels and subsequent photoactivation (475 nm light application) in HEK293 cells resulted in an increase of the K+ current or inhibition of Ca2+ currents, respectively (136).

Activation in hippocampal neuron cultures induced membrane hyperpolarization and decreased the overall firing rate via the Gi/o pathway, whereas expression and illumination in the chick spinal cord elicited synchronization of network activity. These initial studies reveal the true potential that vertebrate rhodopsin has in regards to controlling pre- and post-synaptic functions and downstream targets on a millisecond time scale through the Gi/o protein.

Subsequent work focusing on the next generation of G-protein coupled light-activated receptors led to the creation of a chimeric receptor that was constructed by replacing the cytoplasmic domains of rhodopsin with those of the

β2-adrenergic receptor (β2-AR) (157). Photoactivation of the chimera resulted in an increase in [cAMP]i that was mediated by the Gs protein and ultimately confirmed the thought that GPCRs have a universal activation mechanism (157).

By employing the same chimeric design, an additional family of optical tools

(optoXRs) were produced that replaced the intracellular loops of rhodopsin with

50 those of either the Gq-coupled human α1a-adrenergic receptor (opto-α1AR) or the

Gs-coupled hamster β2-adrenergic receptor (opto-β2AR) and a fluorescent tag

(158). Characterization of the two optoXRs indicated that light application selectively activated distinct, signaling pathways in HEK cells, that either

2+ enhanced [Ca ]i via the Gq protein (opto-α1AR) or recruited cAMP via Gs (opto-

β2AR) (158). Sterotaxic injection of either optoXR into the nucleus accumbens also revealed opposite effects on firing so that opto-α1AR enhanced spike firing in slices, whereas opto-β2AR reduced network activity. The place preference assay revealed that the light activation of opto-α1AR in the nucleus accumbens was sufficient to modify the behavior in freely moving mice with the aid of an optrode.

The engineering of chimeric receptors demonstrates another facet of

GPCR modulation that utilizes photostimulation to activate G protein signaling pathways in targeted cellular populations and essentially presents an enhanced opportunity to modulate and control behavioral output in behaving animals.

51

52 Figure 1: Schematic diagram demonstrating the general organizational scheme of the cerebellar cortex. Major landmarks, such as the primary fissure, separate the cortex into the anterior and posterior lobes. In the mouse, the cortex is structurally divided in the vermis, paravermis and the lateral hemisphere.

53

54 Figure 2: Cellular arrangement of the cerebellar cortex. Three separate layers divide the cortex and include the granule cell layer (GCL), Purkinje cell layer (PCL) and the superficial molecular later (ML). Excitatory inputs (+) arise from mossy and climbing fibers; the sole output of the cerebellum is mediated by the Purkinje cells and is inhibitory (-). Interneurons have been excluded.

55 CHAPTER 2

OPTOGENETIC CONTROL OF Gi/o SIGNALLING BY VERTEBRATE RHODOPSIN IN CEREBELLAR PURKINJE CELLS CHANGES MOTOR COORDINATION

I. SUMMARY

In this study, we sought to examine the effects of Gi/o pathway modulation on the firing pattern of cerebellar neurons and determine how this modification relates to the functional output of the cerebellum. In order to investigate the effects of cell-type specific Gi/o pathway activation, we generated the vRh-

GFP(TgflvRh-GFP) transgenic mouse line that exclusively expressed vRh in

Purkinje neurons and restricted the activation of this GPCR to a subset of cerebellar vermal cells by light. We demonstrate that the in vivo light activation of vRh decreased simple spike firing in vermal Purkinje neurons to a level similar to the reduced rate visualized after the activation of Gi/o-coupled GABAB receptors in the same area. Additionally, light applied to the cerebellar vermis of freely moving mice resulted in a loss of motor coordination. We reasoned that the defects in motor output were possibly due to the functional coupling between vRh-mediated Gi/o pathway photoactivation and the activation of GABABR downstream targets.

Therefore, this investigation not only provides an example of how the modulation of Gi/o-mediated signaling can affect spike firing and motor coordination, but also presents a novel technique to examine the physiological and behavioral role of GPCR-mediated signaling in a specific cellular population.

56 II. INTRODUCTION The G-protein mediated signaling pathway is a key component in the pursuit of obtaining a more thorough understanding of how neuronal network modulation influences physiological and behavioral tasks on a second to minute time scale (120). Amongst G proteins, activation of the inhibitory Gi/o-mediated signaling pathway is the principal method in which GPCRs mediate neuronal excitability (159). Thus far, studies aimed at investigating the modulation of cellular and/or network function have mainly relied on the application of pharmacological agents that essentially activate or inhibit a GPCR pathway in a nonspecific manner. Recent advancements in the area of optogenetics in conjunction with the exploitation of opsins have provided alternative means to examine the physiological effects of GPCR-mediated signaling within a specified cellular population and have circumvented previously encountered kinetic and pharmacological obstacles. For example, it has been demonstrated that the photoactivation of vRh effectively controls both G protein-coupled inward rectifying K+ channel and voltage-gated Ca2+ channel conductances via pertussis toxin-sensitive Gi/o-mediated signaling in vitro (136). These findings suggest that the expression and light activation of vRh in a genetically designated cellular population may grant the opportunity to not only spatially control the Gi/o pathway in vivo, but may also aid in determining the correlation between Gi/o pathway modulation, animal behavior and brain functions that include motor coordination.

It has been investigated and recognized that within the firing cadence and pattern of cerebellar Purkinje neurons lies the code for motor coordination,

57 learning and balance (84). Purkinje cells incorporate a vast amount of cortical, vestibular and sensory information, excitatory inputs from parallel and climbing fibers and inhibitory inputs originating from cerebellar interneurons. Under normal conditions, Purkinje neurons fire in a characteristic, spontaneous manner; a direct result from the interplay of excitatory and inhibitory inputs, intrinsic ion conductances from Ca2+ activated K+ channels, P/Q type channels and a resurgent Na+ channel, as well as modulation from postsynaptic GPCRs that includes the GABABR (86, 88, 89). Activation of the Purkinje cell GABABR, is dependent upon the synaptic release of GABA from innervating interneurons and neighbor cell GABA spillover (90). Once activated, GABABRs exert typical presynaptic and postsynaptic modifications that includes the inhibition of neurotransmitter release and decreased spontaneous activity and induction of a

+ long lasting K current via GIRK channel coupling through the Gi/o protein that promotes membrane hyperpolarization, respectively (90, 124-126). Additionally, in vitro studies that apply baclofen reveal that GABABR activation causes decreased AP firing that is presumably a result of somatodendritic GIRK channel activation and diminished presynaptic transmitter release through inhibition of

VGCCs (90, 160-162).

The details concerning the manner in which the Gi/o protein mediates the modulation of GPCRs in vivo, and especially within PCs, has been problematic because of the dependence on pharmacological agents that pose difficulties in application, localization, specificity and washout. With the intention of gaining a more comprehensive understanding of how Gi/o modulation may affect the spike

58 pattern of PCs and the functional output of the cerebellum in vivo, while avoiding the spatial and kinetic setbacks encountered with the use of pharmacological compounds, we generated an optogenetic mouse model that exclusively expressed vRh in PCs. The in vivo photoactivation of vRh in the cerebellar vermis revealed a reduction in the single spike pattern of PCs that ultimately influenced motor coordination and emphasized the significance of the Gi/o signaling pathway in regards to the influence that PC firing has on motor control.

III. RESULTS

A. Creation of a new optogenetic mouse line vRh-GFP(TgflvRh-GFP) for the controlled expression of vRh in a cell-type specific manner

To investigate the cell-type specific function of Gi/o pathway activation within neuronal networks in vivo and to analyze the functional impact of pathway activation on mouse behavior, we created transgenic mice to specifically activate the Gi/o coupled, light activated GPCR vRh by Cre recombinases. We first identified positive pCZW-fl-Lac-Z-vRh-GFP transgenic founders by genotypic analysis and examination of b-galactosidase expression (Figure 1A). In this construct, Lac-Z is flanked by loxP sites and followed by the vRh-GFP. The expression of Lac-Z and vRh-GFP is under the control of the ubiquitous chicken

β-actin promoter-cytomegalovirus enhancer. The vRh-GFP is only expressed when Lac-Z is excised by Cre recombinases, while LacZ is present throughout the central nervous system (CNS) when Cre is not expressed (Figure 1A) (163).

By performing b-galactosidase staining of both coronal and sagittal sections, we were able to visualize abundant LacZ expression throughout the

59 CNS. Staining was especially robust in the cerebellum, hippocampus and caudate putamen (Figure 1A) and was also detected in other tissues outside the

CNS such as gut, pancreas and stomach (data not shown). To demonstrate that vRh-GFP expression can be induced cell-type specifically, we crossed mice that expressed Cre recombinase under the PCP2/L7 promoter with pCZW-fl-LacZ- vRh-GFP mice (Figure 1B) for the selective expression of vRh-GFP in cerebellar

PCs (164). We call this mouse line vRh-GFPPC. Immunohistochemical staining with GFP and calbindin antibodies verified that vRh expression was exclusive to

PCs in mice that had undergone site-specific recombination (Figure 1B). Upon closer examination, we detected vRh in the PC soma in a punctate pattern and in the proximal dendrites. Thus, the vRh-GFP(TgflvRh-GFP) mouse allows for the for cell-type selective, Cre recombinase mediated expression of vRh-GFP.

B. Gi/o pathway activation by vRh in vivo reduces the frequency of PC firing

We first examined if activation of vRh by light would modulate PC firing as would be expected from GPCRs coupling to the Gi/o pathway. To test our hypothesis, an optrode coupled to a delivered a 26 second 473 nm light pulse to vermal PCs in vivo. Throughout the experiments PCs were selected by their characteristic regular spiking pattern and by the occurrence of complex and simple spikes. Additionally, we confirmed the location of the in vivo recording site by an electrolytic lesion at the end of the experiments (Figure 3A). The recording paradigm consisted of an initial 10 sec recording of simple and complex spikes followed by a 26 sec light pulse and a post-light recording of 30 sec. vRh-GFPPC

60 mice exhibited an 30.8 ± 4.5 % (n=9) reduction in the spontaneous firing rate in comparison to a 10.9 ± 6.3 % (n=10) increase in firing in control mice when the

26 sec light pulse was applied (Figure 3B and 3C). No change in the CV was observed before and after light stimulation (control before and after light, 0.46 ±

0.03 and 0.47 ± 0.03 (n=10); vRh-GFPPC before and after light, 0.57 ± 0.06 and

0.61 ± 0.07 (n=9), Figure 3D). Post-light recordings indicated that reduction of firing persisted for at least 30 sec after light was switched off (-28.4 ± 7.6% (n=9),

Figure 3D). Thus, our data show that light-activation of vRh, selectively expressed in PC, reduce the firing frequency of PCs in vivo.

C. In vitro and in vivo application of baclofen reduces the spontaneous firing rate of Purkinje neurons

We next investigated if the activation of the Gi/o pathway within PC by an endogenously expressed Gi/o coupled GPCR such as GABABR would induce comparable modulation of PC firing as observed for the light activation of vRh.

Because we are interested in comparing the effects of GABAB-R mediated Gi/o activation to vRh, we first compared the expression between vRh and GABAB1-R in cerebellar PCs. Immunohistochemical staining of sagittal cerebellar sections revealed that GABAB1-R expression is present in both granule and Purkinje cells and can be detected in cell bodies, dendrites and spines of PCs (Figure 4A)

(124, 162). Overlay studies revealed colocalization between GFP and GABAB1-R expression in the soma and proximal dendrites in PCs (Figure 4A), suggesting the possibility that in these subcellular PC regions, Gi/o pathways activation by

61 light could potentially activate GABAB-R downstream targets but this idea needs to be further investigated.

In order to investigate how GABABR activation influences the firing properties of PCs in vivo, we iontophoretically applied the GABABR agonist baclofen in 3 month old mice. A 26s lasting iontophoretic application of 1 mM baclofen led to a reduction in the firing frequency by 33.6 ± 12.3% (n=8), which was significantly different from the 5.3 ± 4.1% (n=10) reduction in firing frequency when saline was applied (Figure 5B and 5C). No change in the coefficient of variation (CV)) was detected before and after application of baclofen or saline

(Figure 5D; CV before and after saline application, 0.49 ± 0.06 and 0.52 ± 0.06

(n=10); CV before and after 1 mM baclofen application, 0.62 ± 0.07 and 0.69 ±

0.08 (n=8)). A 22.0 ± 11.9 % (n=8) reduction in firing frequency was still observed

30 sec after baclofen wash out (Figure 5B). In conclusion, GABABR activation by baclofen in the PC layer of anaesthetized mice caused a reduction in the firing rate of PCs.

In order to investigate if the reduction in firing frequency is caused by intrinsic or extrinsic PC modulation, we performed extracellular recordings of PC firing in cerebellar slices from 4 week old mice and blocked the inhibitory as well as excitatory inputs into PCs with 10 µM CNQX and 100 µM picrotoxin. We concentrated on tonically firing PCs, and excluded PCs demonstrating a trimodal spiking activity. Application of 10 µM baclofen reduced the AP firing by 21.9 ±

4.1% (n=5) (Figure 5F and 5G). Again, no change in the CV was detected before

62 and after baclofen application (Figure 5H; CV before and after baclofen application, 0.09 ± 0.014 and 0.09 ± 0.013 (n=5)).

Thus, GABAB-R activation by baclofen in PC in vivo induced a reduction in firing frequency as observed by light activation of vRh, suggesting that vRh and

GABAB-R activate a similar intracellular signalling pathway to modulate PC firing.

D. Photostimulation of vRh in Purkinje cells alters motor behavior In order to investigate the functional consequence of Gi/o-mediated modulation of PC firing, we implanted a laser guide positioned on top the cerebellum to illuminate the cerebellar cortex (Figure 6A). We chose the anterior vermis as the specific illumination area because it is known to be involved in balance, equilibrium and motor execution (1, 83, 85, 165). In all motor tests administered, a significant difference was detectable between wild type and transgenic vRh-GFPPC adult mice after a 26s long light stimulus was applied to the vermis. Specifically, vRh positive mice either fell off the pole after light delivery (scored as 120 sec) or took at least twice as long to descend to the bottom of the pole (Figure 6B, wild type pre-pulse 17.51 + 1.67 seconds; post- pulse 11.63 + 0.87 seconds; vRh-GFPPC pre-pulse 18.85 + 2.87 seconds; post- pulse 102.1 + 12.4 seconds; n= 10, ANOVA ***p<0.0001). The accelerating rotarod test was administered by delivering a pulse of light at the beginning of the experiment, followed by a performance evaluation without light application. This behavioral paradigm was designed this way to control for the possibility that the duration of time spent on the rotarod would increase because of the acquisition of motor skill learning, regardless of transgenic expression, and could potentially

63 mask any effects that light activation of vRh may have on firing and behavioral output (166). Accelerating rotarod testing revealed that the vRh-GFPPC mice stay on the accelerating rod for a shorter amount of time after light application in comparison to wild type mice (Figure 6C, wild type 93.25 + 9.63 seconds versus vRh-GFPPC mice 72.65 + 13.7 seconds; n=10, ANOVA **p<0.001). There was no significant difference in the time spent on the rotarod without any light application between the two groups of mice (wild type 109.99 +10.57 seconds versus vRh-

GFPPC mice 101.61 + 14.05 seconds n=10). Beam walk testing (Figure 6D) also revealed that the modulation on motor behavior was dependent on light (wild type pre-pulse 13.27 + 2.14 seconds; post-pulse 10.72 + 1.38 seconds; vRh-

GFPPC pre-pulse 7.43 + 0.53 seconds; post-pulse 17.36 + 4.37 seconds; n = 10,

ANOVA *p<0.05).

As an additional control for each behavioral test, grip strength for both hind and front paws was analyzed before and after light treatment. These tests were performed to demonstrate that any significant differences between wild type and positive transgenic mice detected throughout the behavioral tests were attributable to the photoactivation of vRh and are not a result of insufficient strength or muscle ability. Measurements of front grip strength revealed no significant difference between the two groups both before and after light application (Figure 6E; wild type pre-pulse 73.0 + 4.99 g; post-pulse 54.9 + 4.55 g; vRh-GFPPC pre-pulse 73.0 + 3.14 g; post-pulse 51.9 + 1.76 g; n=10 ANOVA n.s.). There were also no indications of changes in hind grip strength before and after light application (Figure 6F; wild type pre-pulse 24.5 + 1.9 g; post-pulse 22.0

64 + 1.26 g; vRh-GFPPC pre-pulse 23.67 + 2.82 g; post-pulse 21.1 + 2.31 g; n=10

ANOVA n.s.). Thus our results indicate that Gi/o-mediated modulation of PC firing is sufficient to alter motor coordination in behaving mice.

IV. DISCUSSION A. vRh-GFP(TgflvRh-GFP) mouse for the cell-type selective control of Gi/o signalling The pursuit to gain a more thorough understanding of the physiological roles of cell-type specific GPCR signalling in vivo and in vitro has resulted in the development of two new approaches that circumvent the use of traditional receptor-specific agonists and antagonists. The first consists of a chemical approach that utilizes engineered GPCRs such as DREADDs, which are activated by inert chemical compound (137, 149). The second technique is a physical scheme that employs light-activated proteins to evoke intracellular signalling pathways, like PTX-sensitive Gi/o-coupled vRh in neurons (136, 167,

168). The advantage of using light-activated proteins is the guaranteed precise temporal control, which cannot be achieved with application of chemical compounds. To further develop and utilize this tool for cell-type specific applications, we created mice whose expression of vRh-GFP was dependent upon the use of cell-type specific expression of Cre recombinase. The vRh-GFP

(TgflvRh-GFP) mice were crossed with PCP2/L7-Cre recombinase (TgPcp2-cre) mice for selective expression of vRh-GFP in PCs (164). The vRh expression was induced one week after birth following Cre-expression and was restricted to PCs of vRh-GFPPC mice (Figure 1B). In order to visualize vRh-GFP after 1-3 months of age, an antibody against GFP had to be used, suggesting that the vRh-GPF

65 concentration within PCs is low. Despite the potential lower expression levels, light stimulation of vRh in vivo led to a significant reduction of AP firing in PCs that was comparable to the effects induced by application of GABABR agonist, baclofen. As shown by the intense lacZ staining, especially within hippocampus and basal ganglia (Figure 1A), the vRh-GFP (TgflvRh-GFP) mouse line is a promising tool that could be used in the investigation of Gi/o signalling in other neuronal populations. According to our studies in PCs, vRh-GFP (TgflvRh-GFP) mice provide a new optogenetic tool for the analysis of in vivo function of

GPCRs.

B. Modulation of simple spikes in the medial cerebellar regions leads to changes in motor behavior One of the surprising findings of our study was that a 20-30% reduction in vermal PC firing was sufficient to cause motor deficits in freely behaving mice.

This finding was especially remarkable because the expression level of vRh appeared to be relatively low and limited throughout cerebellar PCs. While no quantitative measurement was taken of expression levels, vRh was only visible with antibody application. The seemingly restricted vRh concentration in vermal

PCs not only exhibits the necessity to create an alternative and optimized method for in vivo expression but also highlights the magnitude of influence that the Gi/o pathway has on motor control and the endogenous firing properties of

PCs. Numerous examinations of the cerebellum and specifically the medial cerebellar region have indicated that this area plays a pivotal role in regulating extensor tone, sustaining upright stance and dynamic balance control (83, 165,

169). It is thought that the cerebellum employs anticipatory and feedback

66 mechanisms to maintain balance during locomotion and that failure in these systems induce an ataxic-like phenotype (83, 170, 171). Behavioral testing revealed that the photostimulation of positive vermal PCs in vRh-GFPPC mice induced changes in motor output. Specifically, an overall lack of balance, coordination and performance was quite apparent with positive transgenic mice that significantly differed from control littermates. These results are consistent with prior studies that have examined the correlation between vermal lesions and gait ataxia, postural defects and motor coordination difficulties and highlight the importance of Gi/o modulation of PC firing for motor control. As a side note, an early examination of light delivery to positively expressing vRh PCs indicated that the optimal length of activation was around 20 seconds. Similar behavioural responses could be elicited with longer light pulses but was ultimately found to be unnecessary. Furthermore, brief pulses of light were unable to reliably evoke changes in the intrinsic firing properties of vermal PCs.

C. Considerations for controlling Gi/o signalling in vivo by light

While we have provided an effective means to modulate the activity of a single neuronal population and network, there are several concerns associated with this study that may be influenced by the overall methods utilized. These potential issues include the extent and range of light penetration within the cerebellum and the presence of any plausible variables related to light delivery that may influence the in vivo behavioral and/or electrophysiological testing.

Previous studies investigating the feasibility of controlling neuronal excitability in a noninvasive and light-dependent manner revealed that vertebrate

67 rhodopsin promoted the modulation of GIRK and P/Q type Ca2+ channels via a functional coupling to the pertussis toxin-sensitive, Gi/o protein pathway (136).

Because vertebrate rhodopsin couples to the G protein transducin, whereby the

α subunit belongs to the Gi subfamily, these findings offer supporting evidence that mammalian are capable of coupling to other Gi/o family members in vitro. In order to examine the possibility that vRh may also promote the precise spatio-temporal control of the Gi/o pathway in vivo, we established an investigation that focused on the function of this pathway in animal behavior and system coordination such as motor control.

Activation of the Gi/o pathway in a membrane delimited way is the main inhibitory action of GPCRs on neuronal excitability (159). Many different transmitters, such as glutamate, acetylcholine (Ach), (5-HT) or GABA couple via specific GPCRs to the Gi/o pathway, which are expressed throughout the brain. Among them, the GABAB receptor (GABABR) is widely distributed throughout the brain including the cerebellum (172) and is located in the granule cell, PC and molecular layer. Within the molecular layer GABABRs are found at the presynaptic terminals of parallel fibers and at the PC dendrites and spines

(124, 173, 174). Taking all of this into consideration, our in vivo data seem to suggest that the photoactivation of vRh in vermal Purkinje cells acts via the Gi/o mediated signalling pathway in general. Up to this point, we have not detected the activation of other G protein pathways using vRh such as Gq or Gs in cellular or neuronal culture systems.

68 Our attempt to control endogenous Gi/o-signalling by exogenously expressed vRh still remains to be further developed. There are several matters to consider that may influence the feasibility of controlling Gi/o signalling and include the following: Firstly, Gi/o-coupled GPCRs have a variety of downstream signalling targets and have a binding preference to each of their respective targets. Secondly, more than one type of Gi/o-coupled GPCR is expressed in a single neuron and spreads in a specific distributing pattern. Lastly, some Gi/o- coupled receptors can form heterodimers with other types of GPCRs. Although we recently demonstrated the ability to target and modify GPCRs by tagging vRh with the C-terminal signalling domain of a specific GPCR and were able to control 5-HT1A/Gi/o specific signalling properties of neurons (168), further ingenuity is required to overcome the intrinsic issues presented thus far. An additional point of contention surrounds the idea that GABABRs in dissociated

PCs have been suggested to inhibit P/Q-type Ca2+ channels (175), establish a heterodimeric functional coupling with mGluR1 at postsynaptic sites of the PF-PC synapse (90, 130) and are thought to be involved in synaptic plasticity (176, 177).

Therefore, future studies should be focused on investigating which downstream signalling pathway is activated and whether discrete motor learning tasks can be modulated by the photoactivation of vRh.

An additional concern focuses on the delivery of a maximal but specific and controlled amount of light to the brain tissue. To achieve this goal, a stripped, multimode optical fiber (200 µm diameter) was coupled to a blue laser light (20 mW of power at 473 nm) and affixed above the cerebellar vermis. It is

69 understood that the light scattering properties within the brain are influenced by species and age, incident wavelength, and physiological characteristics of the tissue (178-181).

Specifically, the blue laser light utilized (473 nm) for this study has been described as having a high propensity for scattering within the brain and is also weakly absorbed (179-181). The specifics of this optrode have been previously characterized in detail and it has been estimated that the fiber tip produces a total tissue volume experiencing > 1 mW mm-2 light intensity to be ~0.5 mm3

(179). These fiber optic specifics correlate with our data in that the most significant decrease in the firing rate of vermal Purkinje cells was elicited in neurons located at more superficial tissue depths; thereby supporting the notion that increase tissue depth corresponds to a lower level of light intensity.

In summary, we generated a new mouse line that allows for the cell-type specific activation and modulation of the Gi/o pathway through vRh, and demonstrated the feasibility of modifying the firing properties of a single neuronal population through the utilization of light. Thus for the first time, our experimental results revealed that the in vivo modulation of the Gi/o protein pathway in PCs has a significant functional influence on motor control and coordination.

V. MATERIALS AND METHODS A. Generation and screening of transgenic mice In order to generate a colony of vRh-GFPPC transgenic mice, homozygous transgenic Purkinje cell specific CRE (TgPcp2-cre) mice were crossed with heterozygous vRh-GFP (TgflvRh-GFP) mice (164). Routine screening of all

70 transgenic mice was accomplished by adding either tail or toe tissue to 0.3 ml of lysis buffer containing 100 mM Tris (pH 8.5), 5 mM EDTA (disodium salt), 0.2%

SDS and 200 mM NaCl. Twenty microliters of proteinase K (20 mg/ml, Roche

Diagnostics) was added to the lysis buffer and the mixture was shaken overnight at 55oC. Following tissue dissolution, the mixture was heated to 99oC for 10 minutes and then cooled to room temperature. A PCR master mix contained either of the following oligos: vRh-GFP (5’ CATGCTCACCACCGTCTGCT and 5’

AAGATGGTGCGCTCCTGGAC) or Cre-Recombinase (5’

TCTCACGTACTGACGGTGG and 5’ ACCAGCTTGCATGATCTCC). The 50 ml final PCR reaction contained 1 ml gDNA, 1 ml of each primer, 1 ml dNTP mix (10 mM each of dATP, dTTP, dCTP, dGTP; New England Biolabs (NEB)), 5 ml 10X

Thermopol II Reaction Buffer (NEB), 5 ml dimethyl sulfoxide, 0.5 ml Taq

Polymerase (NEB) and 35.5 ml dH20. PCR reactions were run on an Eppendorf thermocycler, using the following conditions: 92oC for 30 s, 60oC for 45 s and

72oC for 1 min run for 40 cycles or 95oC for 30 s, 55oC for 1 min and 72oC for 1 min 30 sec for 40 cycles to detect vRh-GFP or Cre-Recombinase respectively.

PCR products were analyzed on a 1% agarose gel utilizing standard electrophoresis conditions. Positively identified PCP2-vRh-GFP mice expressed both the vRh and Cre recombinase genes. Wild type littermates were distinguished as being negative for either vRh or Cre recombinase or both.

1. β-Galactosidase staining Animals were deeply anaesthetized with 0.2cc/g Avertin (tribromoethanol;

Sigma) and transcardially perfused with 1X PBS followed by a neutral buffered

71 formalin solution (4% paraformaldehyde). Upon complete perfusion, brains were isolated and post-fixed in the same paraformaldehyde solution for 15 minutes.

Frozen, embedded brains (OCT, Tissue TEK) were cut into 25-30 micron section on a rotary microtome, mounted onto Superfrost/Plus Microscope Slides (Fisher), allowed to dry at room temperature for 1 hour and permeabilized with PBST

(0.2% Triton X-100) for 15 minutes. Slices were incubated overnight with 1mg/ml

X-gal staining solution (200 mM ferricyanate; Sigma, 200 mM ferrocyanate;

Sigma, X-gal (40 mg/ml in DMSO); Sigma, 1 M MgCl2; Sigma, 0.02% NP40;

Sigma, and 1X PBS) at 37oC in a humid chamber.

2. Immunohistochemistry Animals were deeply anaesthetized with 0.2cc/g Avertin (tribromoethanol;

Sigma) and transcardially perfused with 1X PBS followed by a neutral buffered formalin solution (4% paraformaldehyde). Upon complete perfusion, brains were isolated and post-fixed in the same paraformaldehyde solution for 1 hour followed by a 30% sucrose solution for 24-48 hours. Frozen, embedded brains

(OCT, Tissue TEK) were cut into 25-30 micron section on a rotary microtome, mounted onto Superfrost/Plus Microscope Slides (Fisher) and allowed to dry at room temperature for 1 hour. Sections were washed with 1X PBST for 15 minutes and blocked with 2% goat serum (1X PBST, 2 ml goat serum, Invitrogen) for 1 hour at room temperature. Primary antibodies (1:200 Anti-GFP, Synaptic

Systems and 1:200 Anti-Calbindin, Swant or 1:200 Anti-GFP, Millipore and 1:200

Anti-GABAB1 R, Novus Biologicals) were incubated on the sections overnight at

4oC, followed by three washes in 1X PBST for 15 minutes per wash. Anti-

72 species specific secondary antibodies (anti-mouse Alexa 546 and anti-rabbit

Alexa 488 or anti-rabbit Alexa 546 and anti-mouse Alexa 488, Invitrogen) were incubated on the sections for 2 hours at room temperature, followed by three rinses in 1X PBST for 15 minutes per wash. Images were taken utilizing standard epifluorescence microscopy and processed with Volocity software.

3. NISSL staining Sagittal sections (30 µm) of transcardially, perfused brains were mounted onto

Superfrost/Plus Microsoft slides and allowed to air dry for 24 hours. In order to stain and remove the lipids and residual fixation solutions from the tissue, slices were placed into a 1:1 chloroform/ethanol solution for 45 minutes, 5% cresyl violet acetate for 3 minutes and 50% ethanol/acetic acid solution (approximately

4 drops) for 3 minutes with each step followed with a distilled water wash.

Following the initial stain, slices were dehydrated by placing them into a 70% ethanol solution for 3 minutes, 96% ethanol for 3 minutes, two isopropanol washes for 3 minutes each. Two, 5 minute changes of xylene made any unstained parts of the tissue transparent. Finally, coverslips were mounted onto the slides with DePeX mounting medium and allowed to dry overnight. Images were taken on a Zeiss Axiophot equipped with a CCD camera (SensiCam, PCO,

Kelheim, Germany).

B. Stereotaxic surgeries and cannula placement Three to six-month old male vRh-GFPPC and wild type littermates were the subjects of these experiments. All surgeries were performed under aseptic conditions. Rodents were anaesthetized using isoflurane for one hour or less.

73 Sedation was verified by using the gentle toe pinch withdraw reflex. A lubricating ophthalmic ointment was applied to prevent corneal drying during surgery. Mice were mounted into the stereotactic frame (Narishige Group, Model SR-6M) by placing non-rupture ear bars into the ear canals and gently tightened into place.

Confirmation of correct ear bar placement was dependent upon complete lateral immobilization of the head. The rodent’s mouth was secured by using the incisor adapter on the anterior mount of the apparatus. The nose was placed into the nose clamp and the head was checked for a level position (in regards to the apparatus). Fur from the top of the top of the head was removed and cleaned with 70% ethanol and 10% povidone-iodine. A midline incision was made and all soft tissue from the skull surface was removed. One 1 mm wide hole was drilled through the skull with a battery-operated drill at Bregma points -5.88mm to -

6.24mm and two additional 1.5 mm, anterior-lateral holes were drilled for mounting screws (Figure 7) (182). The dura was manually removed. The modified, flanged cannula guide (Plastics One) and skull screws (Plastics One) were cleaned in ethanol and saline and vertically lowered into their correct coordinates. The flanged cannula guide was kept in place by two 2.4 mm long,

1.57 mm wide mounting screws. A cap of dental cement (3M, Rely-X luting) was applied on top of the head and surrounded the cannula guide. The wound was permanently closed by applying a thin layer of Vetbond tissue adhesive (3M) to each side of the scalp. Animals were subcutaneously administered 1 ml of sterile saline and recovered on a heating pad in individual cages.

74 In regards to the in vivo electrophysiology, data were obtained by performing the above surgical procedure on three to six-month old vRh-GFPPC, vRh-GFP (TgflvRh-GFP) and C57/B6 mice. Surgeries were identical except that only one hole was drilled at Bregma points, -5.88mm to -6.24mm. Upon completion of the recording session, mice were euthanized by cervical displacement.

1. Fiber optics and photostimulation For blue-light photostimulation through the modified cannula guide, a diode pumped crystal laser (20 mW, 473 nm, CrystaLaser, Reno, NV, BCL-473-

020) was coupled into a multimode hard polymer-clad fiber (200 µm core diameter, 0.37 numerical aperture, Thorlabs BFL37-200). The animal behavior photostimulation protocol involved applying a 26 second light pulse to the cerebellar region located directly under the cannula opening. Protocols for the in vivo recordings included a 26 second light pulse applied 10-20 seconds into each sweep (total sweep time approximately 1 minute).

2. Optrode construction

A cleaved multimode glass optical fiber (50 µm core diameter, 0.37 numerical aperture, Thorlabs AFS50/125Y) was stripped of the outer polymer jacket and a glass coated tungsten microelectrode (Impedance 1-2.5MOhm) was attached to the stripped end of the optical fiber with epoxy. The optrode was coupled to a blue laser (Crystal Laser BCL-473-020). Triggering of the laser was controlled by a custom made Matlab program and a corresponding D/A card.

C. Electrophysiological analysis 1. Brain slice recordings

75 Sagittal sections (250 µm thick) were cut from the cerebellum of P21,

C57/B6 mice. Mice were anaesthetized with isoflurane and decapitated. The cerebellum was dissected out, cooled and sliced in an ice-cold solution containing 87 mM NaCl, 75 mM sucrose, 2.5 mM KCl, 0.5 mM CaCl2, 7 mM

MgCl2, 1.25 mM NaH2PO4, 25 mM NaHCO3, and 20 mM glucose bubbled with

95% O2 and 5% CO2 with a vibratome (VT1000S, Leica). Slices were kept for at least 1 hour at room temperature in a recording artificial cerebral spinal fluid composed of 124 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.23 mM

NaH2PO4, 26 mM NaHCO3, and 10 mM glucose bubbled with 95% O2 and 5%

CO2. Slices were continuously perfused with an external solution containing

10 µM CNQX and 100 µM picrotoxin. Extracellular recordings from Purkinje cells were made at (temperature) with 10 µM baclofen being perfused during steady state firing. Patch pipettes (2-4 megaohms) were filled with an internal solution comprised of 140 mM potassium methyl sulfate, 4 mM NaCl, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg-ATP, 0.3 mM Na-GTP, and 10 mM Tris-phosphocreatine, pH 7.3 (KOH). Membrane voltages were recorded with an EPC10/2 amplifier

(HEKA). PatchMaster software (HEKA) was utilized to control voltage and data acquisition. Data was further analyzed with Igor Pro 6.0 software (Wavemetrics).

2. In vivo recordings Extracellular recordings were taken from vermal Purkinje cell layers of adult vRh-GFPPC and wild type littermates that underwent stereotactic surgery.

Recordings from actual Purkinje cells were confirmed by the presence of both complex and simple spikes. The custom optrode was lowered into the vermis

76 and recordings were taken from Purkinje cells ranging in depths from 1100 to

3200 mm below the surface (Figure 8). Activity was amplified and filtered

(bandpass 0.5 to 9 kHz) with a multi-channel spike sorter (Plexon Inc., Austin,

TX) and stored on a computer disk with a sampling rate of 32 kHz. During off-line analysis, simple spikes (SS) and complex spikes (CS) were discriminated using custom made software implemented in Matlab (MathWorks, Natick, MA). Single cell spike activity was used to calculate mean firing rates and inter spike intervals. The coefficient of variation (CV) of the SS interspike intervals was calculated to quantify the variability in spike activity.

3. Baclofen application A Union-40 iontophoresis pump (Kation Scientific) was used for the extracellular delivery of 1 mM baclofen (dissolved in 150 mM NaCl, pH = 3.5) or saline through a Carbostar-3 (Kation scientific) carbon electrode, which includes

2 barrels for microiontophoresis. Baclofen was delivered by + 50nA ejection pulses and retaining currents were -20nA. Baclofen or saline was applied for 26 seconds to the Purkinje cells that had both simple and complex spikes.

4. Lesion studies In order to confirm the position of the recording electrodes electrical microlesions were created at different sites following the completion of the in vivo recordings. Lesion areas were at least 700 µm apart. The designated regions received a 10 µA anodic current for 1 minute via the recording electrode by using an A365 stimulus isolator (World Precision Instruments). Following lesions, mice immediately underwent a paraformaldehyde perfusion.

77 D. Behavioral testing 1. Accelerating rotarod Mice were placed on a 3.0 cm x 9.5 cm rotating drum of an accelerating rotarod (Columbus Instruments, Rotamex-5 Rotarod). The rod was elevated

44.5 cm above the floor of the apparatus. While the mice were allowed to acclimate to the rotarod for 1 minute before beginning the experimental protocol, no formal prior training was introduced into the testing paradigm. Testing conditions included application of either no light or a 26 second light pulse. Upon receiving photostimulation, mice were promptly placed onto the rotarod where the duration and speed of the run were recorded. Constant acceleration of 40 rotations/min was applied until the mouse fell from the rod and activated the infrared beam. The running duration and rotarod speed at time of fall were recorded. The runs were consecutively measured, three times with a 5 min rest period between each run. In the case of no light pulse, animals were allowed to rest for 1 min in between each run. If mice were unable to stay on the rotarod, they were assigned a baseline value of 5 seconds. The latency to fall and speed were recorded for each mouse. Data was averaged over 3 trials per mouse.

2. Grip strength test The muscle strength of wild type and transgenic mice was assessed utilizing the Chatillon DFE Series Digital Force Gauge (AMETEK TCI Division -

Chatillon Force Measurement Systems, Largo, Fla). The instrument measures both fore- and hindlimb grip strength in laboratory rodents by employing an electronic digital force gauge that directly calculates the animal’s peak force value exerted upon a pull bar. To measure forelimb grip strength, animals were

78 held by the tail base and lowered at an angle onto the flat wire mesh of the pull bar so that the forelimbs would be exclusively examined. The mouse was slowly pulled away from the bar at approximately 2.5 cm/sec until release whereby the force gauge recorded the peak tension. Hindlimb grip strength was determined by similar means except that the hindlimbs were solely in contact with the pull bar. Measurements were averaged over 5 trials per mouse with and without light pulses and are recorded as the peak tension (g) and is calculated from the force applied to the bar when grasp is released.

3. Pole test Balance and motor coordination were examined by calculating the capacity of the mice to navigate an angled pole. Mice were held by the tail and lowered, head-upward onto the top of a vertical rough-surfaced pole (diameter 8 mm; height 55 cm). The time required for descent to the base of the apparatus was recorded with a maximal duration of 120 seconds. If the mouse was unable to descend completely and fell off the pole, a maximal default time of 120 sec was assigned to the animal. Experimental conditions included no light application and a 26 sec light pulse so that each animal was measured twice.

4. Balance beam In order to assess fine motor coordination and balance abilities, the capability to cross a narrow beam onto an enclosed platform was analyzed for each mouse. The horizontally placed, 70 cm long beam was 7 mm in diameter and situated 50 cm above the table surface. One end of the beam was mounted to a small, illuminated supportive area while the other end was fastened to an

79 enclosed (20 cm2) box. Mice underwent training on the beam for three days (3 trials a day) before data collection. Briefly, the mouse was placed at an illuminated end of the beam and the time required to traverse the beam to the safety platform was recorded. In addition to recording the latency, hind feet slips were also noted. Measurements were taken both with and without light pulses.

Data was averaged over 3 trials per mouse.

80

81 Figure 3: Cell-type specific Cre recombinase-mediated expression of vertebrate rhodopsin in cerebellar Purkinje cells. (A) Schematic description of the construct used to create the transgenic animals expressing floxed vRh. vRh was cloned into the pCZW vector, which contains a CMV enhancer and b- actin promoter and a lacZ expression cassette, flanked by two loxP sequences.

X-gal staining of sagittal brain slices from the vRh-GFP(TgflvRh-GFP) mouse line shows b-galactosidase expression throughout the brain with robust expression localized in the cerebellum (left) and the hippocampus (middle) and caudate putamen (right). (B) Diagram revealing the results of Cre-mediated recombination events indicates an excision of the lacZ expression cassette and cell type specific expression of vRh-GFP driven in PCs. Cre recombinase- mediated induction of vRh-GFP expression in PCs was accomplished by crossing vRh-GFP(TgflvRh-GFP) mice with Purkinje cell specific CRE (TgPcp2-cre) mice. PC specific expression of vRh was verified by immunohistochemical staining for GFP (middle) and calbindin (a calcium binding protein associated with Purkinje cells, left). (Right) Three-dimensional reconstruction of confocal z- stack images revealing colocalization of vRh-GFP and calbindin in the soma and proximal dendrites of PCs. Scale bars (left and middle) 25 µm, (right) 10 µm.

82

83 Figure 4: In vivo photostimulation of the cerebellar vermis of vRh-GFPPC induces a reduction in the firing rate of Purkinje cells. (A) NISSL stain of sagittal cerebellum slices after electrolytic lesions indicate that the in vivo recordings and the application of baclofen (see Figure 3) were directed to the

Purkinje cell layer. (B) PC firing rate recorded and calculated as percent change in firing before and after light application. The vRh-GFPPC transgenic line demonstrated a significant reduction in firing during the light pulse that persisted after 30 second once light was switched off. (C) Representative firing rates (Hz) of individual PCs from control and vRh-GFPPC mice with and without illumination reveal that only neurons from the vRh-GFPPC line reveal decrease in the firing rate after light treatment. (D) Analysis of the coefficient of variation (CV) after light application indicates no significant difference between wild type littermates and vRh-GFPPC mice. (E) Raw traces of control littermates and vRh-GFPPC mice before and during the 473 nm light pulse reveals a reduction in the PC firing rate only in vRh-GFP positive transgenic mice. The presence of both simple and complex spikes as well as a comparative analysis of depth with a standard mouse brain atlas confirmed that recordings were taken from Purkinje cells.

Statistical significance was evaluated with ANOVA. (** P < 0.01). Given values are mean ± S.E.M.

84

85 Figure 5: In vivo and in vitro application of baclofen decreases the firing rate of cerebellar Purkinje cells. (A-E) In vivo recordings of cerebellar PCs before and after baclofen application. (A) Comparative immunohistochemical

PC staining of GABAB1R (red) and vRh-GFP (green) reveals the expression of

PC GABAB1R in both PCs and cerebellar cortical neurons. vRh-GFP expression is restricted to PCs and colocalized partly (yellow) with GABAB1Rs. Scale bar 25

µm. (B) Percentage change in number of spikes for a 10 sec time interval during

(t = 16-26 sec) and after (t = 20-30 sec) 1 mM baclofen application compared to control (saline application). Bar graphs indicate a significant decrease in the firing of Purkinje cells with baclofen application. (C) Spike frequency (Hz) before and after saline or baclofen application for each recorded PC demonstrates an overall reduction in firing that corresponds to baclofen administration. (D)

Calculated values for the coefficient of variation (CV) between PCs treated with saline or baclofen reveals no significant difference in data dispersion between the two treatment groups. (E) Raw traces of PCs before and during either saline or baclofen treatment indicates reduced firing in the present of 1 mM baclofen. The presence of both simple and complex spikes as well as a comparative analysis of depth with a standard mouse brain atlas confirmed that recordings were taken from Purkinje cells. (F-I) Cerebellar slice recordings of PCs before and after baclofen application. (F) Percentage change in spike number during the 10 mM baclofen bath application displays reduced firing. (G) Modifications in the overall firing rate (Hz) for the recorded PCs during baclofen application. (H) Calculated

CV values for extracellular PC recordings indicate no significant difference before

86 and during baclofen treatment. (I) Raw data traces demonstrating a decrease in

PC firing with baclofen. Statistical significance was evaluated with ANOVA. (* P <

0.05). All values are mean ± S.E.M

87

88 Figure 6: Light activation of vertebrate rhodopsin expressed in Purkinje cells of the cerebellum induces changes in motor behavior. (A) Photograph demonstrating the permanent placement of the cannula light guide used for behavioral testing. (B) Pole test performance of control and vRh-GFPPC mice

(n=10), before and after a 26 sec light pulse. The light activation of vRh in vRh-

GFPPC mice results in either a fall (scored as 120 sec) or an increase in the time required to descend from the pole. Light application to control littermates did not initiate any significant difference in the time required to descend the pole. (C)

Rotarod performance of wild type littermates (n=10) and vRh-GFPPC mice, before and after a 26 sec light pulse. Light activation of vRh in vRh-GFPPC mice produces a significant decrease in rotarod performance when compared to wild type littermates. Performance between the two groups when no light pulse is applied reveals no significant difference. (D) Beam walk analysis demonstrates an increase in the time required to successfully cross the length of the beam after vRh activation in vRh-GFPPC mice. Conversely, the time needed to cross the beam decreases in control littermates, regardless of the light pulse. Falls were assigned a value of 120 sec. Additionally, a measurement of the number of paw slips reveals a significant increase after light application for the left side of the vRh-GFPPC mice; whereas control littermates experienced no significant increase in slips post-light application. (E) Grip strength assessment of wild type and vRh-

GFPPC mice, before and after a 26 sec light illumination. No significant differences were observed for the grip strength of the front and hind paws between wild type littermates and vRh-GFPPC mice before and after light

89 application. Statistical significance in all behavior experiments was evaluated with ANOVA. (* P < 0.05; ** P < 0.01). Shown values are mean ± S.E.M.

90

91 Figure 7: Schematic diagram of region targeted for cannula fixation and in vivo recordings. The specific midline location corresponds to the anterior vermis of the cerebellum and the area receiving baclofen application and light stimulation during behavioral and in vivo experiments.

92

93 Figure 8: Example illustration of optrode placement during in vivo recordings. In vivo electrophysiologal recordings were restricted to Purkinje cells located within the anterior, medial vermis. Recordings were collected from variable depths that ranged from 1100 to 3200 mm. As described in the methods section, a glass coated tungsten microelectrode was attached to the stripped end of the optical fiber with epoxy.

94 CHAPTER 3 DISCUSSION

I. Continued Application of Optogenetic Strategies A. Designer GPCRs and opsins As previously described, the basis of optogenetic techniques centers on the idea that neurons expressing light-sensitive proteins can undergo photo- mediated modulation, which can alter cellular activity and behavioral output.

Overall, this system has proven to be advantageous in that it circumvents the use of pharmacological compounds; thereby avoiding associated kinetic and application restraints, is reversible, offers a higher level of resolution and can be utilized in freely moving animals or in vitro. Additionally, this method presents the opportunity to not only reveal extensive details about the circuitry involved in a particular behavior but also allows for the possibility to directly link neuronal events (membrane hyper- or depolarization, action potential generation, neurotransmitter release, etc.) or activation of a certain receptor and subsequent associated downstream signaling with complex behaviors (183).

Amidst the vast amount of methods developed thus far, two primary modes of technology have emerge as the primary means to regulate neuronal signaling: designer GPCRs (RASSLs, DREADDs and AlsR/AL) and light-gated microbial opsins (vertebrate rhodopsin, channelrhodopsin and halorhodopsin)

(183). In general, the development and utilization of designer GPCRs have granted the opportunity to examine the relationship between the manipulation of

G-protein receptor signaling pathways and/or modifications in membrane

95 excitability to and ensuing behavioral and/or functional outputs in a tissue specific manner that is reversible and exclusively responsive to non-endogenous compounds. Additionally, these receptors have the potential to induce both short- and long-term changes in activity and involve relatively simple administration methods of ligands via feeding or peripheral injection (184).

Despite the numerous advantages that designer GPCRs present, the approach is not ideal due to several intrinsic shortcomings. Specifically, the time required for receptor activation is within the tens of minutes range, ligands are often degraded and washout is relatively impossible (183, 185). In essence, the spatiotemporal constraints associated with this system are rather unfavorable for in vivo use and require additional investigation and manipulation in order to circumvent these issues.

In contrast to the issues surrounding the use of designer GPCRs, the advantages of employing opsins to modulate neuronal activity have definitely outweighed potential drawbacks. Moreover, opsin utilization appears to have driven the application of optogenetic techniques within numerous systems in vivo. The most obvious benefit of opsins and optogenetics centers on the swiftness of circuit modification. Numerous additional benefits are associated with the utilization of opsins and include, targeting specificity, elimination of exogenous ligand addition, exploitation of the endogenous agonist retinal, and the reversibility factor. It should be noted that photostimulation is also associated with several issues that are mostly associated with the implementation and integration of opsin stimulation and recording techniques. For example, if light is

96 applied for an extensive amount of time or at an extremely high intensity, tissue damage, abnormal neural activity or excitotoxicity may ensue (186). Additionally, recording from deeper structures is problematic due to a loss of light intensity that likely influences the volume of tissue activated. Other specific obstacles associated with the combination of light activation and neural recordings are the findings that optical stimulation can induce photoelectric or temperature- dependent effects on the conduction features of the electrode. Despite these potential concerns, solutions do exist that include using opaque glass electrodes or thin wire stereotrodes and minimizing exposed metal (183). To overcome light delivery issues, various light sources (LED or diode ) can be examined and optimized for use with a specific optical fiber.

B. Vertebrate rhodopsin (vRh) as an optogenetic tool Several projects within the lab have examined the ability to substitute opsins for native mammalian receptors. The first study revealed that when expressed in cultured hippocampal neurons and intact embryonic chick spinal neurons, vRh couples to the Gi/o, pertussis toxin-sensitive pathway to hyperpolarize the somato-dendritic membrane, decrease neuronal firing, modulate presynaptic transmitter release and paired-pulse facilitation and postsynaptic GIRK activity (136). The second approach involved constructing a chimeric receptor by tagging vRh with the C-terminus of the 5-HT1AR (Rh-CT (5-

HT1A)); expression and photoactivation in hippocampal neurons induced membrane hyperpolarization and was shown to traffic and signal similar to the native 5-HT1A receptor (183, 187). Taking these ideas one-step further, we

97 decided to establish a combined optical and GPCR-mediated approach to modulate neuronal signaling. Developing this system would not only be beneficial from an optogenetic method standpoint, but would also clarify a few questions about the correlation between the precise timing, firing pattern and intracellular second messenger pathways involved with accurate system functioning.

Specifically, our early in vitro work and current in vivo data suggest some form of functional coupling between vRh expression and the Gi/o signaling pathway. In essence, the data seemed to indicate that the photoactivation of vRh reversibly interrupted the firing pattern of Purkinje neurons by overriding the intrinsic Gi/o signaling pathway. Additionally, the data demonstrate the importance of uninterrupted Purkinje cell firing in accurate motor output. Taken together, it seems that key components of this project directly contribute to the advancement of the optogenetic arena and include: high spatiotemporal resolution through the combination of fiber optic and optrode use, genetic manipulation and lack of pharmacological agents.

C. Gi/o modulation, PC firing and motor output The discovery that the in vivo photostimulation of vRh in vermal PCs could induce a significant decrease in AP firing to a level comparable to the reduction in firing elicited during baclofen application is significant because it suggests that the mode of action between the two GPCRs is similar. Specifically, both vRh and

GABRAB are coupled to the PTX-sensitive Gi/o protein; whereby activation of such GPCRs is known to exert both pre- and postsynaptic effects. Of particular

98 importance to this project is the notion that activation of this specific type of

GPCR not only inhibits adenylate cyclase and voltage-gated calcium channels but ultimately activates GIRK channels, resulting in both membrane hyperpolarization and an overall reduction in neuronal excitability. The combination of our current in vivo data and previously obtained in vitro findings support the idea that the change in Purkinje cell firing is induced when vRh is photostimulated and the endogenous Gi/o signaling pathway is modulated. If vRh activation and the subsequent downstream signaling events are identical to those seen post GABABR activation, then GIRK channel activation most likely contributes to the diminished PC firing rates.

Building upon the electrophysiological data, the next uncertainty in vRh activation was determining if the alteration in PC firing would translate into a behavioral phenotype. By focusing on Purkinje neurons located within the anterior vermis, we anticipated that the reduction in simple spikes would trigger some sort of motor deficit because this region has been found to contribute to the regulation of extensor tone, sustaining upright stance and dynamic balance control (83, 165, 169). Furthermore, studies have indicated that vermal lesions are often associated with gait ataxia, postural defects and decreased motor coordination. Overall, our behavioral testing revealed that vRh activation lessened balance, coordination and motor performance. If we continue with the proposal that both the electrophysiological and behavioral effects exhibited after vRh activation in vermal PCs were due to the modulation of the intrinsic Gi/o signaling pathway, then it might also be inferred that GIRK channel activity is

99 potentially affected. Studies focusing on the patho-physiological roles of GIRK channels in several disease states have indicated that an overall loss of GIRK function can elicit excessive neuronal activity, whereas increased GIRK function significantly reduces neuronal excitability (188). Taken together, it would be interesting to not only determine the mechanism of vRh activation but to also establish which G protein signaling pathway is involved in vivo.

D. Future directions While our approach presents numerous advantages to traditional methods, there are several areas that need improvement. Because GPCRs amplify and transmit encoded information from extracellular sources into intracellular signaling, the time required for signal transduction is slower when compared to signaling via ion channels. Specifically, we found that the optimal time for complete photoactivation of vRh to be approximately 20 seconds in length. Clearly, this prolonged activation period is not the most ideal parameter for behavioral or in vivo investigations and it is possible that the increased time requirement is due to the low expression level of vRh. Specifically, because the amount of vRh expression seems to be limited within vermal Purkinje cells, it is plausible that the extended activation time is necessary to activate a sufficient volume of vRh receptors to elicit a significant response. When comparing the current in vivo data to previously acquired in vitro data, it is evident that a shorter light pulse (<1.5 sec) was required to elicit a significant hyperpolarizing response in both HEK cells and cultured hippocampal neurons that could possibly be

100 attributed to the higher level of vRh expression in approximately 70-80% of synaptic sites (136).

A potential approach that may circumvent this problem would be to employ a viral mode of targeted vRh expression within a specific region(s) of the cerebellum. This method would not only eliminate the time requirement and potential issues associated with creation and expansion of transgenic mouse lines but would also ensure that the expression is both limited and significant

(dependent on titer concentration). For example, a study on nonhuman primates was able to demonstrate the feasibility of establishing a system that was able to optically activate excitatory neurons on a millisecond-timescale by using lentiviral delivery of channelrhodopsin-2 (189). An additional advantage of this approach is that a single viral injection may label an area approximately 1 mm3, a value closely related to the volume of tissue potentially illuminated by single optical fibers; thereby posing the idea that several different viruses and optical fibers may be cooperatively used to examine the effects on cellular, behavioral and circuit function (189). Building upon this idea, it would be interesting to generate and perform a unilateral injection of a vRh viral construct (LV or AAV) into a specified, predetermined area of the cerebellum and determine how photostimulation influences motor output and the intrinsic cellular firing properties of vermal PCs.

Besides utilizing a viral system to generate targeted, concentrated vRh expression, our overall approach could possibly be improved by examining alternative modes of in vivo light delivery and recording methods. Ideally, a

101 motor behavioral analysis would be performed on freely moving mice that are not inhibited by an attached fiber. This proposal was investigated by designing a small, wireless and head-mountable light emitting diode (LED) module that consisted of a transmitter and receiver (190). The creation and use of such a device would promote a more thorough understanding of the consequences that

Gi/o protein pathway modulation has on motor output during an actual behavioral test (as opposed to light application before the test). Specifically, we could expand our behavioral analysis to include a thorough catwalk analysis that focuses on any changes in inter-limb coordination and gait that may be induced during the photoactivation of vRh in vermal PCs.

Another potential investigation focuses on the downstream effects of vRh photostimulation in vermal PCs. For this current study, we decided to focus on

PCs located within the A1 area of the cerebellar vermis, a zone that is known to project to the fastigial nucleus; together disruptions or lesions that involve the anterior vermis or FN result in impaired balance, gait and stance. It would be interesting to examine the electrophysiological relationship between vermal PCs and FN neurons when vRh is photoactivated. This proposal could be accomplished by utilizing the already established transgenic mouse line in combination with our optogenetic system that would specifically involve placing an optrode within the A1 region of the vermis, an additional optrode in the FN and varying the photostimulation protocol.

In conclusion, the data obtained from optogenetically targeting vRh exclusively to PCs may be added to an already rapidly progressing optogenetic

102 toolbox. The future of this area will likely involve the advancement of probes (for recording, light delivery and sensing), discovery of new opsins and improvements in gene delivery. Regardless of what transpires next in this field, optogenetics grants the opportunity to elucidate a more comprehensive understanding of the connection between circuitry, intrinsic (neuronal) firing properties and/or signaling pathways and the execution and regulation of a specific behavioral output that is characteristic of a certain structure or function.

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