Calcium influx through voltage-gated Ca2+ channels plays an important role in neuronal function. In a thin-slice preparation of neonatal rat hypoglossal motoneurons (HMs) we recorded Ba2+ currents through voltage-gated Ca2+ channels using the whole-cell configuration of the patch-clamp technique. We found that HMs have low-voltage-activated (LVA) and at least three types of high-voltage-activated (HVA) Ca2+ channels (omega-Aga-IVA sensitive, omega CgTx sensitive, and dihydropyridine sensitive), based on pharmacological and voltage-dependent properties. Of the Ca2+ current activated at 0 mV from a holding potential of -70 mV, approximately one-half was omega-Aga-IVA (200 nM) sensitive, one-third was omega-CgTx (3 microM) sensitive, whereas only 6% was DHP (nimodipine; 10 microM) sensitive. The residual current, after applying these three antagonists, had characteristics of LVA Ca2+ currents. Based on this pharmacology we found that Ca2+ entry during a single action potential (AP) through LVA Ca2+ channels has a different role from CA2+ entry through HVA Ca2+ channels. Ca2+ influx through omega-Aga-IVA-sensitive and omega-CgTx-sensitive HVA Ca2+ channels activates Ca(2+)-activated K+ channels responsible for the AP afterhyperpolarization. On the other hand, Ca2+ entry through LVA Ca2+ channels is responsible for spike afterdepolarization and provides Ca2+ for the Ca(2+)-activated K+ channels that contribute to AP repolarization.

1. We investigated the effects of serotonin (5-hydroxytryptamine, 5-HT) on whole-cell barium currents through calcium channels in visualized neonatal rat hypoglossal motoneurones (HMs) in a thin brainstem slice preparation. 2. High voltage-activated (HVA) currents were elicited by depolarizing voltage steps from -70 to 0 mV; low voltage-activated (LVA) currents were evoked using steps to between -30 and -40 mV from hyperpolarized potentials (< -80 mV). 5-HT (1P0G M) inhibited HVA currents by at least 10% in 70% of HMs tested (n = 99); in those responsive neurones, 5-HT decreased HVA current by 22 + 1P3 % (mean + S.E.M.). In contrast, 5-HT had no effect on LVA current amplitude in HMs (n = 7). IVA), to block N-and P-type components of calcium current, the 5-HT-sensitive current was reduced; 5-HT had no effect on the current remaining after application of both toxins. Thus, 5-HT inhibits both N-and P-type calcium currents in neonatal HMs. 5. Inhibition of HVA current by 5-HT was irreversible, and subsequent applications of 5-HT were occluded, when GTPyS was substituted for GTP in the pipette. In addition, inhibition of HVA current by 5-HT was relieved following depolarizing prepulses. These data indicate that inhibition of calcium channels by 5-HT is mediated by G proteins. 6. Under current clamp, both 5-HT and 8-OH-DPAT decreased the amplitude of the afterhyperpolarization (AHP) that followed action potentials, indicating involvement of a 5-HTlA receptor. The AHP was decreased by > 10 % in 69 % of cells tested with 5-HT and/or 8-OH-DPAT (n = 16), and in responsive cells the inhibition averaged 26-1 + 6-3 and 32-3 + 9.3 % of control, respectively. 7. We conclude that inhibition of calcium current by a 5-HTlA receptor contributes, at least in part, to the 5-HT-induced decrease in the calcium-dependent AHP in neonatal HMs. The decrease in calcium current and the AHP caused by 5-HT may enhance the overall excitability of HMs by increasing the input-output gain of motoneurones.

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A combination of Ca2+ imaging and current clamp recording in cultured cortical neurons was used to evaluate the reliability of coupling between the action potential and rises in Ca2+ at distal release sites as a possible source of variability in CNS synaptic transmission. Local domains of enhanced Ca2+ influx were observed at varicosities on axon collaterals. Functional assay of vesicle turnover using FM1-43 and parallel electron microscopy confirmed that these varicosities were release sites. Single action potentials reliably ( > 95% of the time) resulted in a presynaptic Ca2+ transient at all presumed release sites including those on distal collaterals. Variability in the amplitude of presynaptic Ca2+ transients at individual boutons was estimated to be on average less than 20%. We conclude that the coupling of somatic action potentials to distal release sites is generally a reliable process, although nonlinearity in the relationship between Ca2+ influx and neurotransmitter release may amplify the effects of relatively small fluctuations in Ca2+ influx.

Gamma-aminobutyric acid (GABA)-containing medium spiny neurons constitute approximately 90% of the neuronal population in the neostriatum (caudate and putamen) and play an important role in motor programming. Cortical glutamatergic afferents provide the main excitatory drive for these neurons, whereas nigral dopaminergic neurons play a crucial role in regulating their activity. To further investigate the mechanisms underlying the dopaminergic modulation of medium spiny neuronal activity, we tested the effect of dopamine receptor agonists on excitatory synaptic transmission recorded from these neurons. Excitatory postsynaptic currents (EPSCs) were evoked by local stimulation and recorded from medium spiny neurons in postnatal rat striatal thin brain slices. Recordings were made using the whole cell patch-clamp technique under voltage clamp and conditions that selected for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type glutamate receptor-mediated components of the EPSC. Incubation of slices in 10 microM dopamine resulted in a 33 +/- 11% (mean +/- SE) decrease in the amplitude of evoked EPSCs, an effect that developed during seconds. The relative variability in amplitude of dopamine's effects on medium spiny neuron EPSCs may reflect activation of different receptor subtypes with opposing effects. In contrast to the results with dopamine, incubation of slices in SKF 38393, a D1-type dopamine receptor selective agonist, resulted in dose-dependent potentiation of the medium spiny neuron EPSC that developed during several minutes. At a concentration of 5 microM, SKF 38393 resulted in a 29 +/- 4.5% increase in EPSC amplitude, an effect that was blocked by preincubation with the D1-selective antagonist, SCH 23390 (10 microM). On the other hand, 5 microM SKF 38393 had no apparent effect on medium spiny neuron currents activated by exogenous application of glutamate or kainate. However, because of the inherent limitations of rapid agonist perfusion in the brain slice preparation (caused by slow agonist diffusion and rapid glutamate receptor desensitization) and because of anatomic evidence that colocalizes D1 and glutamate receptors to medium spiny neuron dendrites, our results leave open the possibility that the effect of D1 receptor activation on the EPSC is mediated via modulation of postsynaptic glutamate receptor responsiveness. The significant potentiation by D1 receptor agonists of EPSC amplitude at the cortico-striatal medium spiny synapse that we observed, in part, may underlie the role of D1 receptors in facilitating medium spiny neuronal firing, with implications for understanding regulation of movement.