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. Cerebellum 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. Deep cerebellar nuclei 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. Mossy fiber system 32 3. The climbing fiber system 33 B. Synaptic Plasticity 37 1. LTP, LTD and RP 37 4 III. G Protein Coupled Receptors 39 A. GPCRS, G proteins & 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. Optogenetics & 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 RHODOPSIN 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 cell-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 opsins 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 Purkinje cell 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 cerebellar vermis 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 flocculonodular lobe (comprised of the nodulus and flocculus) 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, central lobule and culmen), 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,