Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

Abstract

Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum.

Central Autonomic System

“…Subsequently, both pressor and depressor pathways were reported to originate from different parts of the vermis in rabbits Spyer, 1989, 1990). More recently, specific optogenetic stimulation of cerebellar Purkinje cells in the uvula (lobule IX of the posterior vermis), was also shown to decrease blood pressure in anesthetized rats, whereas optogenetic inhibition of the same area led to increases in blood pressure (Tsubota et al, 2011). These responses are thought to be related to the role of the vermis in maintaining blood pressure during upright posture (Holmes et al, 2002;Tsubota et al, 2012).…”

A glass-coated tungsten microelectrode enclosing optical fibers for optogenetic exploration in primate deep brain structures

“…ChR2-EYFP was expressed by lentiviral vector-mediated gene delivery techniques Tsubota et al, 2011Tsubota et al, , 2012.…”

“…For the generation of the transfer vector pCL20c-CMV-hChR2-EYFP-WPRE (Lenti-CMVChR2-EYFP-WPRE), EcoRI/AgeI-digested CMV promoter from CS-CDF-CG-PRE (kindly provided by Dr. H. Miyoshi) was inserted into MluI/EcoRI-digested pCL20c-MSCV-hChR2-EYFP-WPRE by bluntend ligation to replace the MSCV promoter. With cultured HEK293T cells, two lentiviral vectors were prepared as described previously Tsubota et al, 2011). For the titration of viral stocks, cultured HEK293T cells were transduced, transfection efficiency was estimated by counting the EYFP-positive cells using an EPICS XL flow cytometer with EXPO32 software (Beckman Coulter), and the functional titer was calculated from a linear range.…”

“…For the histological analysis of actual EYFP expression, the rat was perfusion fixed as described previously Tsubota et al, 2011), and coronal brain sections of 40 m thickness were observed (BZ-9000, Keyence). By analyzing the lesion marks in the Hoechst-stained sections (Hoechst 33258, 1 mg mL −1 ; MolecularProbes), the recording tracks and the depths were reconstructed.…”

“…With optogenetic methodology, the causal relationship between the activity and function of a specific neuronal circuit is elucidated with high temporal and cell-type specificity (Baier and Scott, 2009;Goard and Dan, 2009;Miesenbock, 2009;Johansen et al, 2010;Kaneda et al, 2011;Tsubota et al, 2011;Tsunematsu et al, 2011;Yizhar et al, 2011a). Pioneering works have successfully applied the technique to primates in the dorsal areas of neocortex (Han et al, 2009;Diester et al, 2011).…”