Intermittent hypoxia training after C2 hemisection modifies the expression of PTEN and mTOR.

In this study, we examined modulations in phosphatase and tensin homolog (PTEN) and mammalian target of rapamycin (mTOR) protein expression after a lateral C2 hemisection and subsequent intermittent hypoxia (IH) exposure and training, which initiates respiratory motor plasticity and recovery. PTEN and mTOR are significant molecules within a signaling pathway that directly influences dendritic sprouting, axonal plasticity, and regeneration. Expression levels of PTEN, mTOR and downstream effectors within this pathway were investigated, and it was found that following injury and IH exposure the expression of these molecules was significantly altered. This study directly demonstrates the implementation and feasibility of a non-invasive strategy to modulate the expression levels of intrinsic signaling molecules known to influence plasticity and regeneration in the CNS.

Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells.

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.

Delayed postnatal loss of P/Q-type calcium channels recapitulates the absence epilepsy, dyskinesia, and ataxia phenotypes of genomic Cacna1a mutations.

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 Ca(2+) 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 green fluorescent protein derivative Citrine into the first exon of Cacna1a and then crossed 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 caused by 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.

Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.

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 Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated Ca2+ 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.

Characterization of MPF and MAPK activities during meiotic maturation of Xenopus tropicalis oocytes.

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.