The depolarization of feline ventral horn group Ia spinal afferent terminations by GABA

Summary The unmyelinated terminal regions of extensor muscle Ia afferent fibres were stimulated electrically near lumbar motoneurones in anaesthetised cats using 300 s pulses of less than 1 A passed through the central NaCl barrel of seven barrel micropipettes. Such terminations were identified by anodal blocking factors of less than four and the latency of the antidromic impulse recorded in the appropriate peripheral muscle nerve.Although the effects of microelectrophoretically administered GABA were occasionally complex, the most consistent finding was a reduction in termination threshold followed by an increase. Both this reduction in threshold by GABA, and that produced by tetanic stimulation of low threshold flexor afferents (PAD) were diminished by microelectrophoretic bicuculline methochloride. This GABA antagonist alone elevated the threshold of some terminations but did not reduce the depolarizing action of either potassium or L-glutamate. Furthermore, since reductions in threshold by GABA, but not by either potassium or L-glutamate, were associated with a decrease in PAD, GABA appears to increase terminal membrane conductance.Since neither GABA nor bicuculline methochloride influenced the threshold or afferent depolarization of non-terminal regions of Ia fibres, these results are consistent with the function of GABA as a depolarizing transmitter at gabergic axoaxonic synapses upon the terminals of Ia afferent fibres synapsing with motoneurones.     

Enhancement by GABA of glutamate depolarization-induced release from cerebellar nerve endings

Cerebellar synaptosomes were superfused in the presence of D-[3H]aspartate (to label the glutamate 'reuptake pool') and [14C]glutamine (to label the 'new synthesis pool'). The depolarization-induced release of D-[3H]aspartate and of newly synthesized [14C]glutamate were potentiated by low concentrations of GABA (2--20 microM) or muscimol. The effect was probably mediated by the interaction of GABA with presynaptic GABA receptors localized in 'glutamergic' nerve endings, since it was antagonized by the GABA antagonists picrotoxin and bicuculline.     

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

Presynaptic excitability changes produced in brain stem endings of tooth pulp afferents by raphe and other central and peripheral influences

1. (1) In view of the association of tooth pulp stimulation with pain, and the possibility that nociceptive transmission may be presynaptically modulated by endogenous opiate-related mechanisms, we examined the presynaptic modulatory effects of peripheral and central conditioning stimuli on the excitability of brain stem endings of tooth pulp primary afferents in anesthetized or decerebrate cats. A presynaptic excitability effect was measured as a change in the amplitude of the antidromic compound action potential recorded from the pulp or as a change in the probability of antidromic activation of single pulp primary afferents recorded either in the trigeminal (V) ganglion or directly from the pulp.

2. (2) A total of 195 single tooth pulp afferents supplying the ipsilateral maxillary or mandibular canine teeth was recorded. The majority conducted in the A-delta range, although about 25 % had conduction velocities indicative of faster conducting axons. The excitability of the brain stem endings of the pulp afferents could be increased by conditioning stimuli delivered to the periaqueductal gray (PAG) and nucleus raphe magnus (NRM), as well as by noxious and non-noxious stimuli applied to oral-facial and more remote sites, and by stimulation of the cerebral cortex.

3. (3) The presynaptic effects induced in pulp endings in V subnucleus oralis by raphe stimulation do not rely on the integrity of V subnucleus caudalis which is considered the important brain stem relay site for oral-facial pain. This was shown by the inability of reversible cold block of synaptic transmission in caudalis to alter presynaptic excitability increases induced in oralis. Nonetheless, the cold block procedure did reveal that caudalis exerts a tonic presynaptic ascending facilitatory influence on the pulp afferent input to oralis neurons.

4. (4) Since tooth pulp afferents are predominantly, if not exclusively, related to pain, it is noteworthy that our studies with the opiate antagonist naloxone failed to provide any substantive evidence that the presynaptic modulatory effects on pulp afferent endings are opiate-related. Only in one of 25 pulp afferents tested did naloxone reduce the PAD effect induced by PAG, NRM or afferent conditioning. These findings do not support the view that the descending raphe influences operate through a presynaptic opiate-related mechanism on nociceptive transmission, at least as far as the tooth pulp afferent input to the brain stem is concerned.

     

Evidence of the modulatory role of serotonin in acetylcholine release from striatal interneurons

The release of acetylcholine was studied in isolated striatal slices of the rat. The spontaneous and ouabain-stimulated release of acetylcholine was higher in those slices where serotonergic input was somehow impaired: raphe nuclei lesion orp-chlorophenylalanine pretreatment or 5,7-dihydroxytryptamine pretreatment resulted in a higher release.l-(m-chlorophenyl)-piperazine, a pure serotonin receptor stimulant andd-fenfluramine, a serotonin releaser significantly reduced the release of acetylcholine evoked by ouabain. Serotonin antagonists (cyproheptadine, mianserine and methysergide) prevented the effect of serotonin agonists. When the serotonergic neurons were destroyed either byp-chlorophenylalanine or by 5,7-dihydroxytryptamine pretreatmentd-fenfluramine had no inhibitory action; however, the effect ofl(m-chlorophenyl)-piperazine was not affected.It is suggested that there is a link between serotonergic and cholinergic neurons in the striatum: serotonin released from raphe-striatal neurons is able to inhibit the release of acetylcholine from striatal interneurons.     

Imaging LTP of presynaptic release of FM1-43 from the rapidly recycling vesicle pool of Schaffer collateral-CA1 synapses in rat hippocampal slices

Recent studies using the styryl dye FM1-43 and two-photon microscopy to directly visualize transmitter release at CA3-CA1 excitatory synapses in the hippocampus have demonstrated that activity-dependent long-term potentiation (LTP) and long-term depression are associated with alterations in vesicular release. It is not known whether particular vesicle pools preferentially express these alterations or what second messenger cascades are involved. To address these questions, we selectively loaded FM1-43 into the rapidly recycling pool (RRP) of vesicles by use of a brief hypertonic shock to release and load the RRP. We demonstrate here that the induction of LTP can lead to a selective long-lasting enhancement in presynaptic release from the RRP, while reserve pool kinetics remain unchanged. LTP of RRP release was N-methyl-d-aspartate receptor-dependent and also required production of the intercellular messenger NO and activation of receptor tyrosine kinase. Measurement of FM1-43 stimulus-evoked uptake rates following induction of LTP confirmed that LTP produces more rapid recycling of vesicles released by electrical stimulation, consistent with an enhanced release probability from the RRP.     

Electrical synapse formation disrupts calcium-dependent exocytosis, but not vesicle mobilization

Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission.     

Presynaptic muscarinic acetylcholine receptors suppress GABAergic synaptic transmission in the intermediate grey layer of mouse superior colliculus

The intermediate grey layer (the stratum griseum intermediale; SGI) of the superior colliculus (SC) receives cholinergic inputs from the parabrachial region of the brainstem. It has been shown that cholinergic inputs activate nicotinic acetylcholine (nACh) receptors on projection neurons in the SGI. Therefore, it has been suggested that they facilitate the initiation of orienting behaviours. In this study, we investigated the effect of muscarinic acetylcholine (mACh) receptor activation on GABAergic synaptic transmission to SGI neurons using the whole-cell patch-clamp recording technique in slice preparations from mice. The GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked in SGI neurons by focal electrical stimulation were suppressed by bath application of 10 microm muscarine chloride. During muscarine application, both the paired-pulse facilitation index and the coefficient of variation of IPSCs increased; however, the current responses induced by a transient pressure application of 1 mm GABA were not affected by muscarine. Muscarine reduced frequencies of miniature IPSCs (mIPSCs) while the amplitudes of mIPSCs remained unchanged. These results suggested that mAChR-mediated inhibition of IPSCs was of presynaptic origin. The suppressant effect of muscarine was antagonized by an M1 receptor antagonist, pirenzepine dihydrochloride (1 microM), and a relatively specific M3 receptor antagonist, 4-DAMP methiodide (50 nM). By contrast, an M2 receptor antagonist, methoctramine tetrahydrochloride (10 microM), was ineffective. These results suggest that the cholinergic inputs suppress GABAergic synaptic transmission to the SGI neurons at the presynaptic site via activation of M1 and, possibly, M3 receptors. This may be an additional mechanism by which cholinergic inputs can facilitate tectofugal command generation.