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.
Highlights
The functions of the cerebellum range from the control and coordination of movements to autonomic regulation [1]
These results show that the ChR2-EYFP expression induced by the sL7 promoter was highly specific to Purkinje cells (PCs)
We developed a method for manipulating PC activity using a lentivirus-based optogenetic technique, and applied it to the analysis of cerebellar cardiovascular control
Summary
The functions of the cerebellum range from the control and coordination of movements to autonomic regulation [1]. In past studies, lesioning, electrical stimulation, and chemical activation/deactivation have been used to investigate local functions of the cerebellar cortex [3,4,5,6] These manipulations affect Purkinje cells (PCs), the sole output neurons of the cerebellar cortex, and local excitatory, inhibitory, and modulatory cells, as well as non-local cells that send their axons to the cerebellar cortex. It is still unclear whether the effects of stimulation or lesions are due to the activation or absence (deactivation) of PCs. To elucidate the causal relationship between PC activity and a function of the cerebellum, a method for selective and rapidly reversible manipulation of PC activity is necessary
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