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GABA A receptors mediating tonic inhibitory currents are present in neurons from hippocampus, cerebellum, sensory cortex and thalamus. These receptors located at peri- and extra-synaptic sites are constituted mainly by α 4/6 and α 5 subunits which confer them high affinity for GABA and low desensitization. Immunohistochemical and in vitro hybridization studies have shown the expression of these subunits, while functional studies have reported the presence of GABAergic tonic currents in spinal dorsal horn neurons. However, the presence of this inhibitory current has not been documented in motoneurons. In addition, we previously reported that the monosynaptic reflex is facilitated by furosemide, an antagonist of the α 4/6 GABA A receptors, without affecting the dorsal root potential, which suggests the presence of a GABAergic tonic inhibitory current in motoneurons. The aim of this work was to investigate the presence of high affinity GABA A receptors in motoneurons. By intracellular recordings made with sharp electrodes and the whole-cell patch clamp recording technique we show here that the membrane input resistance and the monosynaptic excitatory post-synaptic potential (EPSPs) are significantly increased by bicuculline. Likewise, the depression of the EPSPs and the input membrane resistance normally induced by muscimol was partially reverted by 20 μM bicuculline and abolished when the concentration of the antagonist was raised to 100 μM. Last, bicuculline at low concentration did not affect the holding current as occur with the high concentration that block the tonic inhibitory GABAergic current. Together these results suggest that the excitability in motoneurons may be tonically inhibited by high affinity 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.

There is growing evidence that activation of high affinity extrasynaptic GABAA receptors in the brain, cerebellum and spinal cord substantia gelatinosa results in a tonic inhibition controlling postsynaptic excitability. The aim of the present study was to determine if GABAA receptors mediating tonic inhibition participate in the modulation of monosynaptic reflex (MSR) in the vertebrate spinal cord. Using an in vitro turtle lumbar spinal cord preparation, we show that conditioning stimulation of a dorsal root depressed the test monosynaptic reflex (MSR) at long condition–test intervals. This long duration inhibition is similar to the one seen in mammalian spinal cord and it is dependent on GABAA as it was completely blocked by 20 μm picrotoxin (PTX) or bicuculline (BIC) or 1 μm gabazine, simultaneously depressing the dorsal root potential (DRP) without MSR facilitation. Interestingly 100 μm picrotoxin or BIC potentiated the MSR, depressed the DRP, and produced a long lasting motoneurone after-discharge. Furosemide, a selective antagonist of extrasynaptic GABAA receptors, affects receptor subtypes with α4/6 subunits, and in a similar way to higher concentrations of PTX or BIC, also potentiated the MSR but did not affect the DRP, suggesting the presence of α4/6 GABAA receptors at motoneurones. Our results suggest that (1) the turtle spinal cord has a GABAA mediated long duration inhibition similar to presynaptic inhibition observed in mammals, (2) GABAA receptors located at the motoneurones and primary afferents might produce tonic inhibition of monosynaptic reflex, and (3) GABAA receptors modulate motoneurone excitability reducing the probability of spurious and inappropriate activation.

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.

Motoneurons are furnished with a vast repertoire of ionotropic and metabotropic receptors as well as ion channels responsible for maintaining the resting membrane potential and involved in the regulation of the mechanisms underlying its membrane excitability and firing properties. Among them, the GABAA receptors, which respond to GABA binding by allowing the flow of Cl− ions across the membrane, mediate two distinct forms of inhibition in the mature nervous system, phasic and tonic, upon activation of synaptic or extrasynaptic receptors, respectively. In a previous work we showed that furosemide facilitates the monosynaptic reflex without affecting the dorsal root potential. Our data also revealed a tonic inhibition mediated by GABAA receptors activated in motoneurons by ambient GABA. These data suggested that the high affinity GABAA extrasynaptic receptors may have an important role in motor control, though the molecular nature of these receptors was not determined. By combining electrophysiological, immunofluorescence and molecular biology techniques with pharmacological tools here we show that GABAA receptors containing the α6 subunit are expressed in adult turtle spinal motoneurons and can function as extrasynaptic receptors responsible for tonic inhibition. These results expand our understanding of the role of GABAA receptors in motoneuron tonic inhibition.

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.

Ca 2+ influx through voltage-gated Ca 2+ channels mediates synaptic transmission at numerous central synapses. However, electrophysiological and pharmacological evidence linking Ca 2+ 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 2+ channel antagonists on neurotransmission at terminals from the dorsolateral funiculus synapsing motoneurons. Bath application of tetrodotoxin or NiCl 2 prevented the monosynaptic excitatory postsynaptic potentials (EPSPs), and this effect was mimicked by exposure to a zero-Ca 2+ solution. Application of polypeptide toxins that block N- and P/Q-type channels (ω-CTx-GVIA and ω-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 Ca 2+ channels may play a central role in the regulation of neurotransmitter release in the adult turtle spinal cord.