Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi͞o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltagegated Ca 2؉ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.A major challenge in understanding the relationship between neural activity and development and between neuronal circuit activity and specific behaviors is to be able to control the activity of large populations of neurons or regions of individual nerve cells simultaneously. Recently, it was demonstrated that neuronal circuits can be manipulated by expressing mutated ion channels or G protein-coupled receptors (GPCRs). For example, the regional expression of a genetically modified K ϩ channel in Drosophila was able to reduce the excitability of targeted cells (i.e., muscle, neurons, photoreceptors) (1). Silencing of cortical neurons was achieved by binding of the peptide allostatin to its exogenously expressed receptor (2). Recently, Zemelman et al. (3) elegantly demonstrated that light activation of the protein complex, encoded by the Drosophila photoreceptor genes (i.e., arrestin-2, rhodopsin, and G protein ␣ subunit), could induce action potential firing of hippocampal neurons. Activation and deactivation of neuronal firing could also be achieved when ligand-gated ion channels, such as the capsaicin receptor, menthol receptor, purinergic receptors, or lightcontrollable K ϩ channel blockers, were used to control firing in hippocampal neurons (4, 5). However, the application of these techniques to control neuronal function especially in neural circuits and living animals is limited by their relatively slow time course, the complex...

Inherited loss of P/Q-type calcium channel function causes human absence epilepsy, episodic dyskinesia, and ataxia, but the molecular ‘birthdate’ of the neurological syndrome and its dependence on prenatal pathophysiology is unknown. Since these channels mediate transmitter release at synapses throughout the brain and are expressed early in embryonic development, delineating the critical circuitry and onset underlying each of the emergent phenotypes requires targeted control of gene expression. To visualize P/Q-type Ca2+ channels and dissect their role in neuronal networks at distinct developmental stages, we created a novel conditional Cacna1a knock-in mouse by inserting the floxed GFP derivative Citrine into the first exon of Cacna1a, then crossing it with a postnatally expressing PCP2-Cre line for delayed Purkinje cell (PC) gene deletion within the cerebellum and sparsely in forebrain (purky). PCs in purky mice lacked P/Q-type calcium channel protein and currents within the first month after birth, displayed altered spontaneous firing, and showed impaired neurotransmission. Unexpectedly, adult purky mice exhibited the full spectrum of neurological deficits seen in mice with genomic Cacna1a ablation. Our results show that the ataxia, dyskinesia and absence epilepsy due to inherited disorders of the P/Q-type channel arise from signaling defects beginning in late infancy, revealing an early window of opportunity for therapeutic intervention.

The G protein-mediated signaling pathway provides a pivotal module for the adjustment of neuronal networks against physiological or behavioral tasks on a second to minute time scale (1). Among G proteins, the G i/o -mediated signaling pathway is the primary role in which GPCRs 2 mediate their inhibitory action on neuronal excitability (2). The processes and importance of such modulation in cellular and network functions has mainly been investigated with the application of drugs, activating or inhibiting more or less specifically a certain GPCR pathway. Recently, we demonstrated that light-activated vertebrate rhodopsin (vRh) is a suitable alternative to control ion conductances such as G protein-coupled inward rectifying K ϩ channel and voltage-gated Ca 2ϩ channels via pertussis toxin-sensitive G i/o protein-mediated signaling (3). Therefore, vRh may allow for the precise spatiotemporal control of G i/o -mediated pathway in vivo, leading to an investigation that focuses on the function of this pathway in animal behavior or brain functions such as motor coordination.The cerebellum plays a central role in overall motor coordination and motor learning. An extensive array of GPCRs is expressed throughout the brain and is believed to be involved in the modulation of network activity and synaptic plasticity. It has been recognized that the code for motor coordination and balance lies within the firing cadence and output pattern of cerebellar PCs, which are the sole-output neurons from the cerebellar cortex (4, 5). PCs integrate a range of cortical, vestibular, and sensory information via excitatory synaptic input from parallel and climbing fiber pathways and inhibitory synaptic input originating from neighboring interneurons. The PC firing pattern is determined by several factors that include the interplay between excitatory and inhibitory synaptic inputs, several ion channel conductances that support intrinsic firing properties, and modulation by postsynaptic GPCRs like the GABA B receptor (GABA B R) (6 -8). GABA B R activation by application of the selective agonist baclofen leads to a reduction in PC firing most likely due to membrane hyperpolarization induced by G protein-coupled inward rectifying K ϩ channel activation (9 -12). The exact mechanism in which G i/o -mediated GPCR modulation may occur within PCs and how such modulation may influence the single spike pattern and motor coordination has been difficult to address in vivo, as GABA B Rs and other G i/o -coupled receptors are expressed in various cell types in the cerebellum and can only be activated by slowly diffusing drugs.To overcome the kinetic and spatial issues that the pharmacological approach presents and to investigate the functional impact of G i/o protein-mediated modulation on cerebellar function via spike modulation in cerebellar PCs, we created an optogenetic mouse model for the cell type-specific expression of vRh and demonstrated that spike modulation of PCs affects motor coordination. EXPERIMENTAL PROCEDURES Generation and Screening of Tra...

Resumption of meiosis in oocytes of Xenopus tropicalis required translation but not transcription, and was marked by the appearance of a white spot and a dark ring, coincident with entry into metaphase I and the onset of anaphase I, respectively. Cyclin B(2)/p34(cdc2) activity increased prior to the first meiotic division, declined at the onset of anaphase I, and subsequently increased again. The capacity of egg cytoplasm to induce germinal vesicle breakdown (GVBD) was inhibited by cycloheximide, despite the fact that these oocytes contained cyclin B(2)/p34(cdc2) complexes. However, cycloheximide-treated oocytes underwent GVBD following injection of constitutively active mitogen-activated protein kinase (MAPK) kinase 2 (MEK2), p33(Ringo), or Delta 90 cyclin B. MAPK activity increased just prior to the first meiotic division and remained stable thereafter. Although injection of constitutively active MEK2 induced GVBD, treatment with the MEK inhibitors U0126 or anthrax lethal factor delayed GVBD and prevented spindle formation. Interestingly, the ability of egg cytoplasm to induce GVBD was unaffected by the inhibition of MEK activity. Our results indicate that the synthesis of a novel or short-lived protein(s) which acts in a MEK-independent fashion is required in order for egg cytoplasm to induce GVBD in X. tropicalis oocytes.

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