Neuromodulation via Conditional Release of Endocannabinoids in the Spinal Locomotor Network

AbstractEndocannabinoids act as retrograde signals to modulate synaptic transmission. Little is known, however, about their significance in integrated network activity underlying motor behavior. We have examined the physiological effects of endocannabinoids in a neuronal network underlying locomotor behavior using the isolated lamprey spinal cord. Our results show that endocannabinoids are released during locomotor activity and participate in setting the baseline burst rate. They are released in response to mGluR1 activation and act as retrograde messengers. This conditional release of endocannabinoids can transform motoneurons and crossing interneurons into modulatory neurons by enabling them to regulate their inhibitory synaptic inputs and thus contribute to the modulation of the locomotor burst frequency. These results provide evidence that endocannabinoid retrograde signaling occurs within the locomotor network and contributes to motor pattern generation and regulation in the spinal cord

Neuromodulation via endocannabinoids and nitric oxide in the lamprey spinal cord

The overall objective of this thesis is to increase our understanding of neural networks generating locomotion. These networks called Central Pattern Generators (CPG), are localized in the spinal cord and can generate the basic locomotor pattern in the absence of sensory or supraspinal inputs. However, locomotor behaviour needs to be adapted to the changing environmental conditions, and this is proposed to be a result of neuromodulatory systems which can change the efficacy of synaptic transmission, and the intrinsic properties of CPG neurons. The work presented here focuses on the role of metabotropic glutamate receptors (mGluRs) in the spinal locomotor network. We show that a brief activation of postsynaptic mGluR1 results in a long-term potentiation of the locomotor frequency associated with a long-term depression of the mid-cycle inhibition and potentiation of the on-cycle excitation. These effects are blocked by a cannabinoid receptor 1 (CB1) antagonists and nitric oxide synthase (NOS) inhibitors, suggesting that endocannabinoids and nitric oxide (NO) are involved. Overall, endocannabinoids and NO can shift the levels of excitation and inhibition, in favor of excitation to induce the long-term potentiation of the locomotor frequency. Endocannabinoids are released on demand following activation of mGluR1 at the postsynaptic site and inhibit presynaptic glycinergic transmission. This de novo retrograde signaling via endocannabinoids enables network neurons to control their synaptic input and thus the level of their activation. We show that 2-Arachydonylglycerol (2-AG) is the primary endocannabinoid released by activation of mGluR1 and mediates the potentiation of the locomotor frequency and the associated depression of mid-cycle inhibition. In the lamprey spinal cord NOS is found in grey matter neurons and provides an intrinsic NO tone which enhances the locomotor frequency. NO increases the locomotor frequency by reducing mid-cycle inhibition via presynaptic mechanisms, and by increasing the excitatory drive via both pre-and postsynaptic mechanisms. Finally, endogenous activation of mGluR1, cannabinoid and NO signaling facilitates the excitatory drive underlying locomotion and thus contribute to the pattern generation within the spinal cord. The endogenous NO signaling is acting downstream CB1, while approximately 30% of the endocannabinoid tone is dependent on mGluR1 activation. In summary we propose novel modulatory signaling pathways within the spinal CPG and suggest that neuromodulation is a core process embedded within the CPG function that shapes the generation of locomotion

Parallel pathways from whisker and visual sensory cortices to distinct frontal regions of mouse neocortex

The spatial organization of mouse frontal cortex is poorly understood. Here, we used voltage-sensitive dye to image electrical activity in the dorsal cortex of awake head-restrained mice. Whisker-deflection evoked the earliest sensory response in a localized region of primary somatosensory cortex and visual stimulation evoked the earliest responses in a localized region of primary visual cortex. Over the next milliseconds, the initial sensory response spread within the respective primary sensory cortex and into the surrounding higher order sensory cortices. In addition, secondary hotspots in the frontal cortex were evoked by whisker and visual stimulation, with the frontal hotspot for whisker deflection being more anterior and lateral compared to the frontal hotspot evoked by visual stimulation. Investigating axonal projections, we found that the somatosensory whisker cortex and the visual cortex directly innervated frontal cortex, with visual cortex axons innervating a region medial and posterior to the innervation from somatosensory cortex, consistent with the location of sensory responses in frontal cortex. In turn, the axonal outputs of these two frontal cortical areas innervate distinct regions of striatum, superior colliculus, and brainstem. Sensory input, therefore, appears to map onto modality-specific regions of frontal cortex, perhaps participating in distinct sensorimotor transformations, and directing distinct motor outputs. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License

Membrane potential correlates of sensory perception in mouse barrel cortex

Neocortical activity can evoke sensory percepts, but the cellular mechanisms remain poorly understood. We trained mice to detect single brief whisker stimuli and report perceived stimuli by licking to obtain a reward. Pharmacological inactivation and optogenetic stimulation demonstrated a causal role for the primary somatosensory barrel cortex. Whole-cell recordings from barrel cortex neurons revealed membrane potential correlates of sensory perception. Sensory responses depended strongly on prestimulus cortical state, but both slow-wave and desynchronized cortical states were compatible with task performance. Whisker deflection evoked an early (<50 ms) reliable sensory response that was encoded through cell-specific reversal potentials. A secondary late (50-400 ms) depolarization was enhanced on hit trials compared to misses. Optogenetic inactivation revealed a causal role for late excitation. Our data reveal dynamic processing in the sensory cortex during task performance, with an early sensory response reliably encoding the stimulus and later secondary activity contributing to driving the subjective percept

Voltage-sensitive dye imaging of mouse neocortex during a whisker detection task

Sensorimotor processing occurs in a highly distributed manner in the mammalian neocortex. The spatiotemporal dynamics of electrical activity in the dorsal mouse neocortex can be imaged using voltage-sensitive dyes (VSDs) with near-millisecond temporal resolution and similar to 100-mu m spatial resolution. Here, we trained mice to lick a water reward spout after a 1-ms deflection of the C2 whisker, and we imaged cortical dynamics during task execution with VSD RH1691. Responses to whisker deflection were highly dynamic and spatially highly distributed, exhibiting high variability from trial to trial in amplitude and spatiotemporal dynamics. We differentiated trials based on licking and whisking behavior. Hit trials, in which the mouse licked after the whisker stimulus, were accompanied by overall greater depolarization compared to miss trials, with the strongest hit versus miss differences being found in frontal cortex. Prestimulus whisking decreased behavioral performance by increasing the fraction of miss trials, and these miss trials had attenuated cortical sensorimotor responses. Our data suggest that the spatiotemporal dynamics of depolarization in mouse sensorimotor cortex evoked by a single brief whisker deflection are subject to important behavioral modulation during the execution of a simple, learned, goal-directed sensorimotor transformation. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License

Separate signalling mechanisms underlie mGluR1 modulation of leak channels and NMDA receptors in the network underlying locomotion

Metabotropic glutamate receptor subtype 1 (mGluR1) contributes importantly to the activity of the spinal locomotor network. For example, it potentiates NMDA current and inhibits leak conductance in lamprey spinal cord neurons. In this study we examined the signalling pathways underlying the mGluR1 modulation of NMDA receptors and leak channels, respectively. Our results show that mGluR1-induced potentiation of NMDA current required activation of phospholipase C (PLC) and was independent of the increase in the intracellular Ca2+ concentration because it was unaffected by the Ca2+ chelator BAPTA and by depletion of the internal Ca2+ stores with thapsigargin. We also show that the mGluR1-mediated inhibition of leak channels is mediated by activation of G-proteins. Finally, we show that blockade of protein kinase C (PKC) abolished the mGluR1-induced inhibition of leak current without affecting the potentiation of NMDA receptors. The contribution of mGluR1-mediated modulation of leak channels to the potentiation of the locomotor cycle frequency was assessed during fictive locomotion. Blockade of PKC significantly decreased the short-term potentiation of locomotor cycle frequency by mGluR1. These results show that the effects of mGluR1 activation on the two cellular targets, the NMDA receptor and leak channels, are mediated through separate signalling pathways