Single-channel currents underlying glycinergic inhibitory postsynaptic responses in spinal neurons.

Single-channel properties of glycine receptors have been characterized so far only in cultured neurons. To characterize the glycine receptor channels in situ, we applied the patch-clamp technique to spinal neurons in slice preparations. Glycine-gated, single-channel currents were recorded in outside-out patches excised from spinal neurons. In the falling phase of glycinergic inhibitory synaptic currents, single-channel currents were resolved as discrete steps. In both cases, the glycine-gated channels showed similar multiple conductance levels. These results suggest that the receptor channel properties are indistinguishable in the synaptic and extrasynaptic sites. We conclude that multiple conductance states of a receptor channel are the native feature of the glycine receptor in situ.

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels.

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

Developmental changes in calcium channel types mediating central synaptic transmission.

Multiple types of high-voltage-activated Ca(2+) channels trigger neurotransmitter release at the mammalian central synapse. Among them, the omega-conotoxin GVIA-sensitive N-type channels and the omega-Aga-IVA-sensitive P/Q-type channels mediate fast synaptic transmission. However, at most central synapses, it is not known whether the contributions of different Ca(2+) channel types to synaptic transmission remain stable throughout postnatal development. We have addressed this question by testing type-specific Ca(2+) channel blockers at developing central synapses. Our results indicate that N-type channels contribute to thalamic and cerebellar IPSCs only transiently during early postnatal period and P/Q-type channels predominantly mediate mature synaptic transmission, as we reported previously at the brainstem auditory synapse formed by the calyx of Held. In fact, Ca(2+) currents directly recorded from the auditory calyceal presynaptic terminal were identified as N-, P/Q-, and R-types at postnatal day 7 (P7) to P10 but became predominantly P/Q-type at P13. In contrast to thalamic and cerebellar IPSCs and brainstem auditory EPSCs, N-type Ca(2+) channels persistently contribute to cerebral cortical EPSCs and spinal IPSCs throughout postnatal months. Thus, in adult animals, synaptic transmission is predominantly mediated by P/Q-type channels at a subset of synapses and mediated synergistically by multiple types of Ca(2+) channels at other synapses.

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors.

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.

Presynaptic inhibitory action of a metabotropic glutamate receptor agonist on excitatory transmission in visual cortical neurons.

A family of metabotropic glutamate receptors (mGluRs) has been elucidated by molecular cloning. To study the possible modulatory role of mGluRs in synaptic transmission, we tested the effect of a mGluR agonist, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), on the excitatory post-synaptic currents (EPSCS) recorded from neurons in thin slices of rat visual cortex, by using the whole-cell patch-clamp method. We found that trans-ACPD markedly suppressed the evoked EPSCS without affecting the mean amplitude of spontaneous miniature EPSCS. This effect on the evoked EPSCS was blocked by a potassium channel blocker, 4-aminopyridine (4-AP) in a dose-dependent manner. We suggest that trans-ACPD presynaptically inhibits EPSCS by a mechanism involving the 4-AP-sensitive channels.

Glycine-gated Cl- channels underlying synaptic currents.

Glycine receptor Cl- channels underlying inhibitory synaptic currents (IPSCs) were characterized with patch clamp methods applied to thin slices of rat spinal cord. The channels had multiple conductance states with their mean open time being similar to the mean decay time constant of IPSCs. During postnatal development, channel open time became gradually shorter, concomitantly with the decay time of IPSCs. Single channel currents of alpha-homomeric glycine receptors expressed in Xenopus oocytes from cRNAs showed that the adult type receptor had much faster kinetics than the fetal one. It is suggested that the molecular switching of glycine receptor alpha subunits accelerate the time course of inhibitory synaptic responses.

Distinct synaptic and extrasynaptic NMDA receptors identified in dorsal horn neurones of the adult rat spinal cord.

1. Using patch-clamp recordings, properties of single-channel and synaptic currents mediated by N-methyl-D-aspartate receptors (NMDARs) were examin ed in substantia gelatinosa (SG) neurones of adult rat spinal cord slices. 2. In somatic outside-out patches, high- and low-conductance NMDAR channels were present. The low-conductance channels exhibited asymmetrical transitions between the main (44 pS) and subconductance (19 pS) levels, suggesting that they arise from NR2D subunit-containing receptors. The high-conductance channels (main conductance, 57 pS) were blocked by ifenprodil, an NR2B subunit selective blocker. 3. Ifenprodil had no effect on NMDA-EPSCs. The double-exponential decay time course and the apparent Kd for Mg2+ of NMDA-EPSCs suggested the expression of NR2A subunit-containing receptors at the synapse. 4. These results indicate that different NMDAR subtypes are expressed in subsynaptic and extrasynaptic regions of adult SG neurones, which may have differential roles in nociception.

Calcium channels responsible for potassium-induced transmitter release at rat cerebellar synapses.

The effects of calcium channel blockers on potassium-induced transmitter release were studied in thin slices of cerebellum from neonatal rats using whole-cell patch clamp methods. Miniature inhibitory postsynaptic currents (mIPSCs) mediated by gamma-aminobutyric acid (GABA) were recorded from deep cerebellar nuclear neurones in the presence of tetrodotoxin. The frequency of mIPSCs was reproducibly increased by a brief application of high-potassium solution. In the presence of the L-type Ca2+ channel blocker nicardipine (10 microM), the potassium-induced increase in mIPSC frequency was suppressed by 49%. Neither the mean amplitude nor the time course of mIPSCs was affected by the blocker. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTX, 3 microM) had no effect on the frequency of potassium-induced mIPSCs. The P-type Ca2+ channel blocker omega-Aga-IVA (200 nM) suppressed the potassium-induced increase in mIPSC frequency by 83% without affecting the mean amplitude or time course of mIPSCs. Comparing these data with previous studies of neurally evoked transmission, it is concluded that the Ca2+ channel subtypes responsible for potassium-induced transmitter release may be different from those mediating fast synaptic transmission.

Different types of calcium channels mediate central synaptic transmission.

Synaptic transmission is mediated by calcium entry through voltage-dependent calcium channels in presynaptic nerve terminals. Various types of calcium channel have been characterized in neuronal somata, but it is not clear which subtypes induce transmitter release at central synapses. The N-type Ca2+ channel blocker omega-conotoxin GVIA (omega-CgTx) suppresses the excitatory postsynaptic responses only partially, whereas potassium-induced release of glutamate from brain synaptosomes can be blocked by omega-Aga-VIA (ref. 9), a blocker of P-type calcium channels and possibly of other types of calcium channels. Here we test type-specific calcium-channel blockers on postsynaptic currents recorded from neurons in thin slices of rat central nervous system. Inhibitory postsynaptic currents in cerebellar and spinal neurons and excitatory postsynaptic currents in hippocampal neurons are markedly suppressed by omega-Aga-IVA and reduced to a lesser extent by omega-CgTx. The L-type calcium channel blocker nicardipine had no effect. Our results indicate that at least two types of calcium channel mediate synaptic transmission in the mammalian central nervous system.

Re-evaluation of calcium currents in pre- and postsynaptic neurones of the chick ciliary ganglion.

1. Presynaptic nerve terminals of ciliary ganglia of the chick embryo were identified by the accumulation of dextran-tetramethylrhodamine applied to the cut end of the oculomotor nerve. Ca2+ currents were then recorded from the identified nerve terminals. 2. Whole-cell recordings were carried out simultaneously from a presynaptic terminal and its postsynaptic cell. The generation of presynaptic Ca2+ currents induced a postsynaptic response with a short delay. Electrical coupling was present in eight of fifteen pairs. The coupling ratio did not exceed 5%. 3. High-threshold Ba2+ currents were observed in presynaptic terminals without any evidence for the presence of low-threshold Ca2+ channels. The Ba2+ current was completely blocked by 50 microM Cd2+. 4. The presynaptic Ca2+ current induced by a long depolarizing pulse showed inactivation, but this inactivation was diminished when Ca2+ was replaced with Ba2+. 5. The presynaptic Ba2+ current was insensitive to dihydropyridines (DHPs). omega-Conotoxin GVIA (omega CgTX) suppressed a large fraction of the Ba2+ current irreversibly. About 10% of the Ba2+ current was resistant to both DHPs and omega CgTX. 6. The omega CgTX-sensitive component was not sensitive to changes in the holding potential between -120 and -50 mV. The omega CgTX-resistant component tended to be inactivated at depolarized holding potentials. 7. In some perisynaptic Schwann cells, small Ca2+ currents were observed. These Ca2+ currents increased monotonically with depolarization. 8. Only high-threshold Ca2+ channel currents were observed in postsynaptic ciliary cells. Exposure to 50 microM Cd2+ completely abolished the Ca2+ current. 9. About 25% of the Ba2+ currents were blocked by nifedipine (10 microM) in ciliary cells. The nifedipine-resistant component was partly blocked by omega CdTX (10 microM) leaving a small component (about 20%) which was resistant to both nifedipine and omega CgTX. 10. In ciliary cells, the fraction of Ba2+ currents blocked by omega CgTX was not affected by the presence or absence of nifedipine. Similarly, nifedipine blocked the Ba2+ currents to the same extent whether omega CgTX was present or not. The Ba2+ currents potentiated by Bay K 8644 were eliminated by nifedipine. 11. It is concluded that the presynaptic terminal of chick ciliary ganglion did not possess DHP-sensitive Ca2+ channels in contrast with the postsynaptic cell. Two subpopulations of presynaptic Ca2+ channels were distinguishable by their sensitivity to omega CgTX and membrane potential.

Identification of a native low-conductance NMDA channel with reduced sensitivity to Mg2+ in rat central neurones.

1. We have identified a new type of NMDA channel in rat central neurones that express mRNA for the NR2D subunit. We have examined single NMDA channels in cerebellar Purkinje cells (which possess NR1 and 2D), deep cerebellar nuclei (NR1, 2A, 2B and 2D) and spinal cord dorsal horn neurones (NR1, 2B and 2D). 2. In Purkinje cells, NMDA opened channels with a main conductance of 37.9 +/- 1.1 pS and a subconductance of 17.8 +/- 0.7 pS, with frequent transitions between the two levels. 3. NMDA activated low-conductance ('38/18 pS') events (along with high-conductance--'50/40 pS'--openings) in some patches from deep cerebellar nuclei and dorsal horn neurones. Our evidence suggests that 38/18 pS and 50/40 pS events arose from distinct types of NMDA receptors. 4. The transitions for 38/18 pS events were asymmetrical: steps from 38 to 18 pS were more frequent (72.2%) than steps from 18 to 38 pS. This feature appeared common to the 38/18 pS events in all three cell types, suggesting similarity in the low-conductance channels. 5. The 38/18 pS channels in Purkinje cells exhibited characteristic NMDA receptor properties, including requirement for glycine, antagonism by D-2-amino-5-phosphonopentanoic acid (D-AP5) and 7-chlorokynurenic acid, and voltage-dependent block by extracellular Mg2+. 6. The mean open time for the 38 pS state (0.74 +/- 0.07 ms) was significantly briefer than that for the 18 pS state (1.27 +/- 0.18 ms). 7. Mg2+ block of low-conductance NMDA channels in Purkinje cells was less marked than block of 50/40 pS channels in cerebellar granule cells. 8. The time course of appearance of 38/18 pS NMDA channels matched the expression of mRNA for the NR2D subunit. Thus 38/18 pS events were present in > 70% of Purkinje cell patches in 0- to 8-day-old animals, and absent by postnatal day 12. 9. We propose that the 38/18 pS NMDA channels identified here (associated with the NR2D subunit), and the other low-conductance NMDA channel associated with the NR2C subunit, may together constitute a functionally distinct subclass of native NMDA receptors.

Locus of frequency-dependent depression identified with multiple-probability fluctuation analysis at rat climbing fibre-Purkinje cell synapses.

1. EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. 2. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This 'multiple-probability fluctuation' (MPF) analysis gave an estimate of 510 +/- 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 +/- 0.03 nS (n = 6). 3. Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 +/- 0.01 nS and a CV of 0.37 +/- 0.01 (n = 4). Similar estimates of q were obtained in 2 mM Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. 4. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (pr) was unusually high (0.90 +/- 0.03 at 0.033 Hz, n = 5), but as the frequency of stimulation was increased, pr fell dramatically (0.02 +/- 0.01 at 10 Hz, n = 4) with no apparent change in either q or N. This indicates that the observed 50-fold depression in EPSC amplitude is presynaptic in origin. 5. Presynaptic frequency-dependent depression was investigated with double-pulse and multiple-pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 +/- 0.09 and 3.2 +/- 0.4 s, n = 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter. 6. The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.

Synthesis of omega-agatoxin IVA and its related peptides.

A potent and selective P type calcium channel blocker isolated from the venom of the funnel web spider Agelenopsis aperta, omega-agatoxin IVA, and its related peptides were synthesized by the solution procedure. Synthetic omega-agatoxin IVA was found to block high-threshold P-type calcium current in rat Purkinje neuron with the same potency as that reported for the natural product. Its disulfide structure was determined by amino acid analysis, gas-phase sequencing and mass spectrometry of the proteolytic fragments. The N-terminus biotinylated and truncated peptides showed the same disulfide-bond-forming profile and the same activities as those of omega-agatoxin IVA, indicating that the N-terminal basic tripeptide, Lys-Lys-Lys, is not important for both the folding and the expression of the biological activity. However, the Trp residue in the molecule might be essential for the toxin to bind tightly with the channel pores.

Role of a metabotropic glutamate receptor in synaptic modulation in the accessory olfactory bulb.

Various functions of glutamate transmission are mediated by both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) consists of at least six different subtypes that are classified into three subgroups, mGluR1/mGluR5, mGluR2/mGluR3, and mGluR4/mGluR6 (refs 1-5), but their physiological roles are largely unknown. Here we report the identification of a very potent agonist for mGluR2/mGluR3, DCG-IV, and the specific localization of mGluR2 in granule cell dendrites that form dendrodendritic synapses with mitral cells in the accessory olfactory bulb. Using the DCG-IV agonist for mGluR2 in combination with slice patch-recording, we demonstrate that the granule cell mGluR2 presynaptically suppresses inhibitory GABA (gamma-aminobutyrate) transmission to the mitral cell. Our results indicate that mGluR2 in granule cells plays an important role in the persistent excitation of olfactory sensory transmission in the accessory olfactory bulb by relieving mitral cells from the GABA inhibition.