Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors.

GABA(A) receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α(5) has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α(5) subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α(5) subunit-containing GABA(A) receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α(4/6) subunit-containing GABA(A) receptors, but is sensitive to L-655708, an antagonist of the GABA(A) receptors containing α(5) subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α(5) subunit in the turtle spinal cord. Together, these results suggest that GABA(A) receptors containing the α(5) subunit mediate the tonic inhibitory currents observed in VHIs.

Involvement of R-type Ca2+ channels in neurotransmitter release from spinal dorsolateral funiculus terminals synapsing motoneurons.

Molecular studies have revealed the presence of R-type voltage-gated Ca(2+) channels at pre- and postsynaptic regions; however, no evidence for the participation of these channels in transmitter release has been presented for the spinal cord. Here we characterize the effects of SNX-482, a selective R channel blocker, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of dorsolateral funiculus (DLF) terminals in a slice preparation from the adult turtle spinal cord. SNX-482 inhibited neurotransmission in a dose-dependent manner, with an IC(50) of approximately 9 +/- 1 nM. The EPSP time course and membrane time constant of the motoneurons were not altered, suggesting a presynaptic mechanism. The toxin inhibited the residual component of the EPSPs recorded in the presence of N- and P/Q-type Ca(2+) channel blockers, strongly suggesting a role for the R channels in neurotransmission at the spinal cord DLF terminals. Consistently with this, RT-PCR analysis of turtle spinal cord segments revealed the expression of the Ca(V)2.3 pore-forming (alpha(1E)) subunit of R channels, whereas the use of anti-alpha(1E)-specific antibodies resulted in its localization in the DLF fibers as demonstrated by immunohistochemistry coupled with laser confocal microscopy.

G-protein-coupled GABAB receptors inhibit Ca2+ channels and modulate transmitter release in descending turtle spinal cord terminal synapsing motoneurons.

Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.

N- and P/Q-type Ca2+ channels regulate synaptic efficacy between spinal dorsolateral funiculus terminals and motoneurons.

Ca2+ influx through voltage-gated Ca2+ channels mediates synaptic transmission at numerous central synapses. However, electrophysiological and pharmacological evidence linking Ca+ channel activity with neurotransmitter release in the vertebrate mature spinal cord is scarce. In the current report, we investigated in a slice preparation from the adult turtle spinal cord, the effects of various Ca+ channel antagonists on neurotransmission at terminals from the dorsolateral funiculus synapsing motoneurons. Bath application of tetrodotoxin or NiCl2 prevented the monosynaptic excitatory postsynaptic potentials (EPSPs), and this effect was mimicked by exposure to a zero-Ca2+ solution. Application of polypeptide toxins that block N- and P/Q-type channels (omega-CTx-GVIA and omega-Aga-IVA) reduced the EPSP amplitude in a dose-dependent manner. By analyzing the input resistance and the EPSP time course, and using a paired pulse protocol we determined that both toxins act at presynaptic level to modulate neurotransmitter release. RT-PCR studies showed the expression of N- and P/Q-type channel mRNAs in the turtle spinal cord. Together, these results indicate that N- and P/Q-type Ca2+ channels may play a central role in the regulation of neurotransmitter release in the adult turtle spinal cord.