Fumihito Saitow

Synaptic activation of AMPA receptors inhibits GABA release from cerebellar interneurons

A single neurotransmitter elicits diverse physiological responses through activation of multiple receptor subtypes and/or heterosynaptic interactions involving distinct synaptic targets. We found that a typical excitatory transmitter released from the climbing fiber (CF) in the cerebellar cortex not only excited Purkinje cells directly but also presynaptically inhibited GABAergic transmission from interneurons converging on the same Purkinje cells. Both homosynaptic and heterosynaptic actions of the CF transmitter (possibly glutamate) were mediated by activation of AMPA receptors. Dual AMPA receptor-mediated functions of excitation and disinhibition may ensure transmission of cerebellar CF signals controlling sensorimotor coordination.

GABAB receptor-mediated presynaptic inhibition of glutamatergic and GABAergic transmission in the basolateral amygdala

The information processing at central synapses is mediated not only by homosynaptic transmission with direct synaptic connections but also by heterosynaptic interactions between distinct synaptic inputs. Using rat brain slices and whole-cell recordings this study aimed to examine the roles of GABA(B) receptors in synaptic interactions in the basolateral amygdala (BLA), a critical brain structure related to fear and anxiety. Stimulation in the BLA produced non-NMDA type glutamate receptor antagonist-sensitive excitatory postsynaptic currents (EPSCs) and bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in the BLA neurons. The GABA(B) receptor agonist baclofen markedly inhibited both EPSCs and IPSCs in a concentration-dependent manner, and the baclofen-induced inhibition was selectively abolished by the GABA(B) receptor antagonist CGP55845A. The paired-pulse ratio of EPSC and IPSC amplitude was increased by baclofen. The effect of baclofen was mimicked by lowering the external Ca2+ concentration but not by glutamate- and GABA(A)-receptor antagonists. The frequency but not the mean amplitude of miniature EPSCs and IPSCs was decreased by baclofen. The findings suggest that activation of GABA(B) receptors by baclofen reduces the strength of excitatory and inhibitory transmission in the BLA by a presynaptic mechanism. Repetitive conditioning stimulation applied to GABAergic synaptic inputs exerted an inhibitory action on glutamatergic excitatory transmission, and the stimulation-induced inhibition was abolished by CGP55845A. Furthermore, the paired-pulse ratio of EPSCs was increased during the stimulation-induced inhibition. The results in this study provide evidence that synaptic activation of GABA(B) heteroreceptors elicits presynaptic inhibition of glutamatergic excitatory transmission in the BLA.

miR-7a alleviates the maintenance of neuropathic pain through regulation of neuronal excitability.

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 β2 subunit of the voltage-gated sodium channel, and decreased miR-7a associated with neuropathic pain caused increased β2 subunit protein expression, independent of messenger RNA levels. Consistently, miR-7a overexpression in primary sensory neurons of injured dorsal root ganglion suppressed increased β2 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.

AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanisms

A major subtype of glutamate receptors, AMPA receptors (AMPARs), are generally thought to mediate excitation at mammalian central synapses via the ionotropic action of ligand‐gated channel opening. It has recently emerged, however, that synaptic activation of AMPARs by glutamate released from the climbing fibre input elicits not only postsynaptic excitation but also presynaptic inhibition of GABAergic transmission onto Purkinje cells in the cerebellar cortex. Although presynaptic inhibition is critical for information processing at central synapses, the molecular mechanisms by which AMPARs take part in such actions are not known. This study therefore aimed at further examining the properties of AMPAR‐mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum. Our data provide evidence that the climbing fibre‐induced inhibition of GABA release from interneurons depends on AMPAR‐mediated activation of GTP‐binding proteins coupled with down‐regulation of presynaptic voltage‐dependent Ca2+ channels. A Gi/o‐protein inhibitor, N‐ethylmaleimide, selectively abolished the AMPAR‐mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR‐mediated excitatory actions on Purkinje cells. Furthermore, both Gi/o‐coupled receptor agonists, baclofen and DCG‐IV, and the P/Q‐type calcium channel blocker ω‐agatoxin IVA markedly occluded the AMPAR‐mediated inhibition of GABAergic transmission. Conversely, AMPAR activation inhibited action potential‐triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons. By suppressing the inhibitory inputs to Purkinje cells, the AMPAR‐mediated presynaptic inhibition could thus provide a feed‐forward mechanism for the information flow from the cerebellar cortex.

Modulation of Presynaptic Ca2+ Entry by AMPA Receptors at Individual GABAergic Synapses in the Cerebellum

Cerebellar Purkinje cells (PCs) receive GABAergic input that undergoes powerful retrograde modulation by presynaptic cannabinoid and glutamate receptors. Here we examine a distinct modulatory mechanism at these synapses, which does not require postsynaptic depolarization and acts via presynaptic AMPA receptors. We find that this mechanism operates mainly in the somatic vicinity of PCs in which large boutons of basket cell axons form synapses on the PC soma. We use fast confocal microscopy and detailed kinetic modeling to estimate that, in these boutons, an action potential opens 100-200 Ca2+ channels, eliciting a brief 3-5 μm transient, followed by a longer-term, 15-30 nm rise of free Ca2+ (above the resting level of ∼100 nm). Brief activation of local AMPA receptors suppresses Ca2+ entry (probably by silencing 20-40 P/Q-type channels) in a subgroup of terminals that tend to show a higher dynamic range of Ca2+ signaling. The results provide the first quantitative description of presynaptic Ca2+ kinetics and its modulation by AMPA receptor activation (most likely via a glutamate spillover-mediated mechanism) at identified GABAergic synapses.

Metabotropic P2Y Purinoceptor-Mediated Presynaptic and Postsynaptic Enhancement of Cerebellar GABAergic Transmission

Cerebellar GABAergic inhibitory transmission is under heterosynaptic control mediated by diverse chemical messengers. Here, we investigated roles of metabotropic P2Y purinoceptors (P2YRs) on GABAergic synapses between cerebellar interneurons and Purkinje cells (PCs). Activation of P2Y purinoceptors by two selective agonists, ADP and 2-methylthio-ADP (2MeSADP), elicited two distinct forms of synaptic plasticity of GABAergic transmission in the cerebellar cortex. First, the two agonists induced long-lasting enhancement of stimulation-evoked GABAergic IPSCs as well as GABAA receptor currents in PCs. This effect was completely abolished by intracellular infusion of the Ca2+-chelating agent BAPTA. Measurements of intracellular Ca2+ ([Ca2+]i) dynamics showed that puff application of 2MeSADP produced an increase in [Ca2+]i of PCs and that this increase persisted in an external Ca2+-deficient medium. These results suggest that P2Y activation postsynaptically elicits long-term enhancement of GABAA receptor sensitivity of PCs through a Gq-mediated increase in [Ca2+]i. The other action of P2YR agonists on cerebellar GABAergic synapses was that they produced a short-term increase in the frequency and the amplitude of spontaneous GABAA receptor-mediated IPSCs in PCs in a manner sensitive to a P2Y1R antagonist, N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate. This action appeared to be attributable to an excitability increase in presynaptic GABAergic interneurons, because ADP excited all Lugaro cells examined and some of interneurons in the molecular layer. These results suggest that activation of cerebellar P2Y purinoceptors leads to modulation of GABAergic transmission in different spatial and temporal domains, namely short-term and long-term plasticity through presynaptic and postsynaptic mechanisms at interneuron→PC inhibitory synapses in the rat cerebellar cortex.

β-Adrenoceptor-mediated long-term up-regulation of the release machinery at rat cerebellar GABAergic synapses

Properly regulated interactions among excitatory and inhibitory synapses are critical for brain function. Compared to excitatory synapses, much less is known about the gain control mechanisms at inhibitory synapses. Herein we report a mechanism of noradrenergic long‐term potentiation (LTP) at inhibitory synapses following presynaptic β‐adrenoceptor activation. Stimulation of β‐adrenoceptors elicited LTP of GABA release from terminals of cerebellar interneurones. This action was dependent on the cAMP/protein kinase A signalling cascade and independent of the β‐adrenoceptor‐mediated acceleration of hyperpolarization‐activated cyclic nucleotide‐gated cation (HCN) channel. Furthermore, the β‐adrenoceptor‐ and protein kinase A‐mediated LTP was triggered by enhancement of the Ca2+ sensitivity of the release machinery and increase in the readily releasable pool. β‐Adrenoceptor activation also accelerated the recruitment of GABA into the releasable pool and enhanced synchronous and asynchronous release of GABA from the presynaptic terminal. Thus, the up‐regulation of GABA release machinery mediated by noradrenaline and β‐adrenoceptor activation provides a likely mechanism of feedforward inhibition of the cerebellar output neurone Purkinje cell, leading to a profound effect on motor control and learning associated with the cerebellum.

Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus

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-HT1A or 5-HT2A/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.

Modulatory effects of serotonin on GABAergic synaptic transmission and membrane properties in the deep cerebellar nuclei.

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.

Distribution and pharmacological characterization of primate NK‐1 and NK‐3 tachykinin receptors in the central nervous system of the rhesus monkey

Much attention has focused on tachykinin receptors as therapeutic targets for neuropsychiatric disorders, although their expressional distributions in the primate central nervous system (CNS) remain unclear. We cloned the genes encoding the NK‐1 and NK‐3 tachykinin receptors (referred to as rmNK‐1 and rmNK‐3) from the rhesus monkey (Macaca mulatta) brain and examined their pharmacological profiles and regional distributions in the CNS. The deduced rmNK‐1 amino‐acid sequence differed by only two amino acids from the human NK‐1 (hNK‐1). The deduced rmNK‐3 amino‐acid sequence was two amino acids shorter than human NK‐3 (hNK‐3), with a seven‐amino‐acid difference in sequence. Ligand binding studies revealed that the affinity of rmNK‐1 to substance P (SP) was comparable to that of hNK‐1 in cell lines that expressed individual receptors stably. Nonpeptide antagonists had similar effects on the binding of rmNK‐1 and hNK‐1. Affinity of rmNK‐3 for NKB was stronger than for SP and the IC50 value was comparable with that of hNK‐3. Ca2+ imaging showed that activations of both rmNK‐1 and rmNK‐3 by specific ligands, SP and senktide, induced increased intracellular Ca2+ in cell lines that stably expressed individual primate tachykinin receptors. The amounts of rmNK‐1 and rmNK‐3 mRNAs were quantitatively determined in the monkey CNS. The expression of rmNK‐1 was observed in all of the cortical and subcortical regions, including the hippocampus and the amygdala. The putamen contained the most NK‐1 mRNA in the brain, with less rmNK‐3 mRNA found in the cortex compared to rmNK‐1 mRNA. In the monkey hippocampus and amygdala, rmNK‐1 mRNA was present at markedly higher concentrations than rmNK‐3 mRNA. The present results provide an insight into the distinct physiological nature and significance of the NK‐1 and NK‐3 tachykinin systems in the primate CNS. These findings are indispensable for establishing model systems in the search for a subtype‐specific tachykinin receptor agonist and antagonist for the treatment of neuropsychiatric disorders.