Kiyomitsu Shoudai

Thermally Active TRPV1 Tonically Drives Central Spontaneous Glutamate Release

Central synapses spontaneously release neurotransmitter at low rates. In the brainstem, cranial visceral afferent terminals in caudal solitary tract nucleus (NTS) display pronounced, activity-dependent, asynchronous release of glutamate and this extra release depends on TRPV1 receptors (TRPV1+). Asynchronous release is absent for afferents lacking TRPV1 (TRPV1−) and resting EPSC frequency was greater in TRPV1+. Here, we studied this basal activity difference by assessing thermal sensitivity of spontaneous and miniature synaptic events in TRPV1+ and TRPV1− second-order NTS neurons. The spontaneous EPSC rate decreased when temperature was decreased, increased steeply between 30 and 42°C only in TRPV1+ neurons, and was calcium-dependent. TRPV1-specific antagonist SB366791, but not TTX, strongly attenuated thermal responses. Temperature changes failed to alter EPSC frequency in TRPV1− neurons. EPSC amplitudes and decay kinetics changed little with temperature. IPSCs in these second-order NTS neurons were unaltered by temperature. Such results suggest that activated, presynaptic TRPV1+ receptors trigger continuous resting release of glutamate vesicles at physiological temperatures only in capsaicin-responsive terminals. In mechanically isolated individual neurons harvested from medial NTS, increases in temperature increased the rate of glutamate release only in TRPV1+ neurons, whereas IPSC rates were unaffected. Cadmium failed to block thermal increases in glutamate release, suggesting that calcium entry through TRPV1 channels may trigger glutamate release independently of voltage-activated calcium channels. Together, our findings indicate a new form of afferent signaling in which TRPV1 channels within central terminals of peripheral afferents tonically generate glutamate release in NTS at 37°C in the absence of afferent action potentials.

P- and R-type Ca2+ channels regulating spinal glycinergic nerve terminals

The contributions of P- and R-type Ca2+ channels on glycinergic nerve endings (boutons) projecting to the rat spinal sacral commissural nucleus (SDCN) neurons are not understood. Thus, we investigated the functional role of P- and R-type Ca2+ channels by measuring the inhibitory postsynaptic currents (eIPSCs) evoked from individual nerve endings (boutons) by focal electrical stimulation. The current amplitude and failure rate (Rf) of glycinergic eIPSCs varied directly with changes in [Ca2+](o). Low concentration of omega-Aga IVA (P-type selective antagonist) suppressed eIPSCs as much as high concentration (both P- and Q-type selective) indicating little contribution of Q-type Ca2+ channels. Antagonism of R-type Ca2+ channels with SNX-482 and Ni2+ greatly decreased the current amplitude and increased failure rate (Rf) of glycinergic eIPSCs. Overall, our results suggest that the dominant control of glycine release depends on Ca2+ entry through P- and R-type Ca2+ channels that ubiquitously populate spinal glycine release sites.

Excitatory and Inhibitory Neural Control of Airway Smooth Muscles and a Braking System for Airway Constriction

The importance of parasympathetic excitatory and inhibitory neural control of airway smooth muscles has been demonstrated. It is concluded that excitatory (acetylcholine, ACh) and inhibitory (NO- and VIP-related) neurotransmitters co-exist and are co-released from vagus nerve terminals. The latter transmitters can directly relax airway smooth muscles and, at the same time, inhibit ACh release, playing the role of the braking system against bronchoconstriction.

A Functional Study of Single Mammalian CNS “Synaptic Bouton”

Single CNS neurons could be dissociated with adherent functional synaptic boutons without using any enzyme, namely when preparing a “synaptic bouton.” This allows experimenters to investigate the effects of presynaptic modulators of synaptic transmission with unprecedented case and accuracy. Moreover, a single bouton can be visualized using fluorescent markers and can also be focally stimulated with electrical pulses. In this communication, high voltage-dependent Ca2+ channels of nerve endings, as one of experimental examples using the “synaptic bouton” preparation, are described. Ca2+ channels belonging to different subtypes, which trigger GABA release from nerve terminals (boutons) projecting to rat hippocampal CA1 pyramidal neurons, were studied. GABA-ergic evoked inhibitory postsynaptic currents (eIPSCs) were recorded; these currents were evoked by focal stimulation of single boutons in mechanically dissociated neurons and by stimulation of a nerve bundle in slice preparations. Nilvadipine, an L-type Ca2+ channel blocker, completely inhibited eIPSCs evoked by stimulation of single boutons but exerted no effect on eIPSCs evoked by low-frequency stimulation of the nerve bundle. Nilvadipine did, however, prevent potentiation of the eIPSC amplitude following high-frequency stimulation of the nerve bundles in slice preparations. ω-Conotoxin-GVIA, an N-type Ca2+ channel blocker, and ω-Agatoxin-IVA, a P/Q-type Ca2+ channel blocker, completely inhibited the eIPSCs in 33.3 and 83.3% of the recordings from single boutons, respectively. In response to low-frequency nerve bundle stimulation in the slice preparation, both ω-Conotoxin-GVIA and ω-Agatoxin-IVA partially reduced the amplitude of eIPSC, and the residual component could be abolished by Cd2+. From these results, the following hypotheses could be drawn. (i) The distribution of P/Q- and N-type Ca2+ channels at a single bouton is nonuniform; (ii) when a focal stimulation is applied to a single bouton, L-type Ca2+ channels play a significant role in generation of action potentials, which subsequently activate P/Q- and N-type Ca2+ channels at GABA release sites; and (iii) action potentials conducted through axons in the slice preparation are sufficient to depolarize the bouton membrane, even when L-type Ca2+ channels are suppressed.