Neuronal damage in the somatosensory system causes intractable chronic neuropathic pain. Plastic changes in sensory neuron excitability are considered the cellular basis of persistent pain. Non-coding microRNAs modulate specific gene translation to impact on diverse cellular functions and their dysregulation causes various diseases. However, their significance in adult neuronal functions and disorders is still poorly understood. Here, we show that miR-7a is a key functional RNA sustaining the late phase of neuropathic pain through regulation of neuronal excitability in rats. In the late phase of neuropathic pain, microarray analysis identified miR-7a as the most robustly decreased microRNA in the injured dorsal root ganglion. Moreover, local induction of miR-7a, using an adeno-associated virus vector, in sensory neurons of injured dorsal root ganglion, suppressed established neuropathic pain. In contrast, miR-7a overexpression had no effect on acute physiological or inflammatory pain. Furthermore, miR-7a downregulation was sufficient to cause pain-related behaviours in intact rats. miR-7a targeted the ?2 subunit of the voltage-gated sodium channel, and decreased miR-7a associated with neuropathic pain caused increased ?2 subunit protein expression, independent of messenger RNA levels. Consistently, miR-7a overexpression in primary sensory neurons of injured dorsal root ganglion suppressed increased ?2 subunit expression and normalized long-lasting hyperexcitability of nociceptive neurons. These findings demonstrate miR-7a downregulation is causally involved in maintenance of neuropathic pain through regulation of neuronal excitability, and miR-7a replenishment offers a novel therapeutic strategy specific for chronic neuropathic pain.
Several forms of depolarization-induced plasticity in inhibitory transmission have been reported to occur in cerebellar Purkinje cells (PCs), namely depolarization-induced suppression of inhibition (DSI), depolarization-induced potentiation of inhibition (DPI), and rebound potentiation (RP). Here, we describe another form of synaptic plasticity for gamma-amino butyric acid (GABA)ergic transmission in PCs. Immediately following depolarization trains in a PC, evoked inhibitory postsynaptic currents (eIPSCs) changed their direction from outward to inward currents under a recording condition in which eIPSCs were elicited as an outward current. Subsequently, the eIPSC amplitude remained depressed (depolarization-induced depression of inhibition [DDI]) for more than 20 min under the blockade of cannabinoid and N-methyl-D-aspartic acid (NMDA) receptor-mediated DSI and DPI, respectively. This DDI was completely abolished by intracellular infusion of the fast Ca(2+)-chelating agent BAPTA and by inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, DDI was strongly suppressed by calcium-activated chloride channel (CaCC) blockers, while an inhibitor of cation-chloride cotransporters (CCCs) partially blocked DDI during the early phase. Exogenous GABA-induced inhibition of spontaneous spike activity was attenuated in ?50% of the PCs by climbing fiber stimulation-induced depolarization. These results suggest that activation of both CaCCs and CCCs was necessary for alteration of [Cl(-)]i after activation of CaMKII following elevation of [Ca(2+)]i in PCs. DDI may provide another mechanism for regulation of inhibitory inputs to PCs within the neuronal networks of the cerebellar cortex.
Cerebellar outputs from the deep cerebellar nuclei (DCN) are critical for generating and controlling movement. DCN neuronal activity is primarily controlled by GABAergic inhibitory transmission by Purkinje cells in the cerebellar cortex and is also modulated by nerve inputs originating from other brain regions within and outside the cerebellum. In this study, we examined the modulatory effects of 5-HT on GABAergic synapses in the DCN. 5-HT decreased the amplitude of stimulation-evoked inhibitory postsynaptic currents (eIPSCs) in DCN neurons, and this effect was abolished by a 5-HT(1B) antagonist, SB 224289. The decrease in IPSC amplitude was associated with an increased paired-pulse ratio of the IPSC. 5-HT also decreased the frequency of miniature IPSCs without altering the amplitude. These data suggest that 5-HT presynaptically inhibited GABA release. Furthermore, 5-HT elicited a slow inward current in DCN neurons. Pharmacological studies showed that 5-HT activated the 5-HT(5) receptor, which is positively coupled to G protein and elicited the slow inward current through enhancement of hyperpolarization-activated cation channel activation. Finally, we examined the effects of 5-HT on the spike generation that accompanies repetitive stimulation of inhibitory synapses. 5-HT increased the spontaneous firing rate in DCN neurons caused by depolarization. Increase in the 5-HT-induced tonic firing relatively decreased the contrast difference from the rebound depolarization-induced firing. However, the inhibitory transmission-induced silencing of DCN firing remained during the conditioning stimulus. These results suggest that 5-HT plays a regulatory role in spike generation and contributes to the gain control of inhibitory GABAergic synapses in DCN neurons.
Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.
The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67-green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT(1A) or 5-HT(2A/2C) receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.
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