4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) modifies synaptic transmission in hippocampal CA3 neurons of rats.

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an alternative to organotin antifoulants, such as tributyltin and triphenyltin. Since DCOIT is found in harbors, bays, and coastal areas worldwide, this chemical compound may have some impacts on ecosystems. To determine whether DCOIT possesses neurotoxic activity by modifying synaptic transmission, we examined the effects of DCOIT on synaptic transmission in a 'synaptic bouton' preparation of rat brain. DCOIT at concentrations of 0.03-1 µM increased the amplitudes of evoked synaptic currents mediated by GABA and glutamate, while it reduced the amplitudes of these currents at 3-10 µM. However, the currents elicited by exogenous applications of GABA and glutamate were not affected by DCOIT. DCOIT at 1-10 µM increased the frequency of spontaneous synaptic currents mediated by GABA. It also increased the frequency of glutamate-mediated spontaneous currents at0.3-10 µM. The frequencies of miniature synaptic currents mediated by GABA and glutamate, observed in the presence of tetrodotoxin under external Ca2+-free conditions, were increased by 10 µM DCOIT. With the repetitive applications of DCOIT, the frequency of miniature synaptic currents mediated by glutamate was not increased by the second and third applications of DCOIT. Voltage-dependent Ca2+ channels were not affected by DCOIT, but DCOIT slowed the inactivation of voltage-dependent Na+ channels. These results suggest that DCOIT increases Ca2+ release from intracellular Ca2+ stores, resulting in the facilitation of both action potential-dependent and spontaneous neurotransmission, possibly leading to neurotoxicity.

Modulation of inhibitory and excitatory fast neurotransmission in the rat CNS by heavy water (D2O).

The effects of heavy water (deuterium oxide, D2O) on GABAergic and glutamatergic spontaneous and evoked synaptic transmission were investigated in acute brain slice and isolated "synaptic bouton" preparations of rat hippocampal CA3 neurons. The substitution of D2O for H2O reduced the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner but had no effect on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, for evoked synaptic responses in isolated neurons, the amplitude of both inhibitory and excitatory postsynaptic currents (eIPSCs and eEPSCs) was decreased in a concentration-dependent manner. This was associated with increases of synaptic failure rate (Rf) and paired-pulse ratio (PPR). The effect was larger for eIPSCs compared with eEPSCs. These results clearly indicate that D2O acts differently on inhibitory and excitatory neurotransmitter release machinery. Furthermore, D2O significantly suppressed GABAA receptor-mediated whole cell current (IGABA) but did not affect glutamate receptor-mediated whole cell current (IGlu). The combined effects of D2O at both the pre- and postsynaptic sites may explain the greater inhibition of eIPSCs compared with eEPSCs. Finally, D2O did not enhance or otherwise affect the actions of the general anesthetics nitrous oxide and propofol on spontaneous or evoked GABAergic and glutamatergic neurotransmissions, or on IGABA and IGlu. Our results suggest that previously reported effects of D2O to mimic and/or modulate anesthesia potency result from mechanisms other than modulation of GABAergic and glutamatergic neurotransmission.

Potent and direct presynaptic modulation of glycinergic transmission in rat spinal neurons by atrial natriuretic peptide.

Atrial and brain natriuretic peptides (ANP and BNP) exist in the central nervous system and modulate neuronal function, although the locus of actions and physiological mechanisms are still unclear. In the present study we used rat spinal sacral dorsal commissural nucleus (SDCN) and hippocampal 'synaptic bouton' preparations, to record both spontaneous and evoked glycinergic inhibitory postsynaptic currents (sIPSCs and eIPSCs) in SDCN neurons, and the evoked excitatory postsynaptic currents (eEPSCs) in hippocampal CA3 neurons. ANP potently and significantly reduced the sIPSC frequency without affecting the amplitude. ANP also potently reduced the eIPSCs amplitude concurrently increasing the failure rate and the paired pulse ratio response. These ANP actions were blocked by anantin, a specific type A natriuretic peptide receptor (NPR-A) antagonist. The results clearly indicate that ANP acts directly on glycinergic presynaptic nerve terminals to inhibit glycine release via presynaptic NPR-A. The ANP effects were not blocked by the membrane permeable cGMP analog (8Br-cGMP) suggesting a transduction mechanisms not simply related to increasing cGMP levels in nerve terminals. BNP did not affect on glycinergic sIPSCs and eIPSCs. Moreover, both ANP and BNP had no effect on glutamatergic EPSCs in hippocampal CA3 neurons. The results indicate a potent and selective presynaptic inhibitory action of ANP on glycinergic transmission in spinal cord sensory circuits.

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.

Differential effects of divalent cations on spontaneous and evoked glycine release from spinal interneurons.

The effects of Ca2+, Sr2+, and Ba2+ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the "synaptic bouton" preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40 approximately 50% of control followed by a gradual further decline in mIPSC frequency to approximately 30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+>>Ca2+>Sr2+. Moreover, the application of excess external [K+]o solution (30 mM) containing Sr2+ or Ba2+ after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+>or==Ba2+>Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+ was replaced by Sr2+ or Ba2+ (2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+ or Ba2+ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.

Effects of various K+ channel blockers on spontaneous glycine release at rat spinal neurons.

Molecular biology approaches have identified more than 70 different K+ channel genes that assemble to form diverse functional classes of K+ channels. Although functional K+ channels are present within presynaptic nerve endings, direct studies of their precise identity and function have been generally limited to large, specialized presynaptic terminals such as basket cell terminals and Calyx of Held. In the present study, therefore, we investigated the functional K+ channel subtypes on the small glycinergic nerve endings (< 1 microm diameter) projecting to spinal sacral dorsal commissural nucleus (SDCN) neurons. In the presence of TTX, whole-cell patch recording of mIPSCs was made from mechanically dispersed SDCN neurons in which functional nerve endings remain attached. Glycinergic responses were isolated by blocking glutamatergic and GABAergic inputs with CNQX, AP5 and bicuculline. The K+ channel blockers, 4-AP, TEA, delta-dendrotoxin, margatoxin, iberiotoxin, charybdotoxin and apamin, significantly increased 'spontaneous' mIPSC frequency without affecting mIPSC amplitude. The results suggest the existence of the following K+ channel subtypes on glycinergic nerve endings that are involved in regulating 'spontaneous' glycine release (mIPSCs): the Shaker-related K+ channels Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv1.7 and the intracellular Ca2+ -sensitive K+ channels BKCa, IKCa and SKCa. Ca2+ channel blockers by themselves, including L-type (nifedipine), P/Q-type (omega-agatoxin IVA, AgTX) and N-type (omega-conotoxin GVIA, CgTX), did not alter the 'spontaneous' mIPSC frequency or amplitude, but inhibited the increase of the mIPSC frequency evoked by 4-AP, indicating the participation of L-, P/Q- and N-type Ca2+ channels regulating 'spontaneous' glycine release from the nerve terminals.