Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (

Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.The coupling between presynaptic membrane depolarization and transmitter release in chemical synaptic transmission has been the subject of a large number of studies over several decades (cf. ref. 1). It is clear that this depolarization causes the opening of voltage-sensitive calcium channels, entry of calcium into the nerve terminal, and calcium-dependent release of neurotransmitter (cf. refs. 1 and 2). The present study represents a step towards elucidating the molecular mechanism(s) by which the increase in intracellular calcium regulates transmitter availability or release.Synapsin I is a neuron-specific phosphoprotein localized to presynaptic terminals, where it is associated with synaptic vesicles (3)(4)(5)(6) (3-6, 16, 17). In the present study the possible roles of synapsin I and of calmodulin kinase II in synaptic transmission were examined by injecting these substances into the presynaptic terminal of the squid giant synapse and measuring the effect on calcium entry and neurotransmitter release. For this purpose, well-established electrophysiological methods for studying the squid giant synapse were used (18-21). METHODS Intracellular Injection and Electrophysiological Recording.The experiments were performed on Loligo pealii at the Marine Biological Laboratory (Woods Hole, MA), and the techniques for the electrophysiological portion of the work were similar to those described previously (19). The stellate ganglion was held in a three-compartment chamber that was superfused with Tris-buffered a...

Four mammalian Kv3 genes have been identified, each of which generates, by alternative splicing, multiple protein products differing in their C-terminal sequence. Products of the Kv3.1 and Kv3.2 genes express similar delayed-rectifier type currents in heterologous expression systems, while Kv3.3 and Kv3.4 proteins express A-type currents. All Kv3 currents activate relatively fast at voltages more positive than -10 mV, and deactivate very fast. The distribution of Kv3 mRNAs in the rodent CNS was studied by in situ hybridization, and the localization of Kv3.1 and Kv3.2 proteins has been studied by immunohistochemistry. Most Kv3.2 mRNAs (approximately 90%) are present in thalamic-relay neurons throughout the dorsal thalamus. The protein is expressed mainly in the axons and terminals of these neurons. Kv3.2 channels are thought to be important for thalamocortical signal transmission. Kv3.1 and Kv3.2 proteins are coexpressed in some neuronal populations such as in fast-spiking interneurons of the cortex and hippocampus, and neurons in the globus pallidus. Coprecipitation studies suggest that in these cells the two types of protein form heteromeric channels. Kv3 proteins appear to mediate, in native neurons, similar currents to those seen in heterologous expression systems. The activation voltage and fast deactivation rates are believed to allow these channels to help repolarize action potentials fast without affecting the threshold for action potential generation. The fast deactivating current generates a quickly recovering after hyperpolarization, thus maximizing the rate of recovery of Na+ channel inactivation without contributing to an increase in the duration of the refractory period. These properties are believed to contribute to the ability of neurons to fire at high frequencies and to help regulate the fidelity of synaptic transmission. Experimental evidence has now become available showing that Kv3.1-Kv3.2 channels play critical roles in the generation of fast-spiking properties in cortical GABAergic interneurons.

Compelling evidence links the recently discovered hypothalamic peptides Hypocretin/Orexin (Hcrt/Orx) to rapid eye movement sleep (REM) control and the sleep disorder narcolepsy, yet how they influence sleep-related systems is not well understood. We investigated the action of Hcrt/Orx on mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT), a target group whose function is altered in canine narcolepsy and appears pivotal for normal REM and wakefulness. Extracellular recordings from mouse brainstem slices revealed that Hcrt/Orx evoked prolonged firing of LDT neurons. Whole-cell recordings revealed that Hcrt/Orx had actions on both presynaptic neurons and at postsynaptic sites. Hcrt/Orx produced an increase in frequency and amplitude of spontaneous EPSCs without equivalent effect on IPSCs, by triggering action potentials and enhancing spike-evoked synaptic transmission in glutamatergic afferents. Postsynaptically, Hcrt/Orx produced an inward current and an increase in membrane current noise, which were accompanied by a conductance increase. These persisted in TTX, ionotropic glutamate receptor antagonists, and low extracellular calcium. Both presynaptic and postsynaptic actions were specific because they were not mimicked by an Hcrt/Orx fragment, and both actions were observed for cholinergic and noncholinergic LDT neurons. Finally, extracellular recordings during postsynaptic potential blockade demonstrated that postsynaptic actions of Hcrt/Orx alone could evoke prolonged firing. In the context of other recent work, our findings suggest that Hcrt/Orx neurons may coordinate the activity of the entire reticular activating system during waking. Moreover, these findings address specific hypotheses regarding the cellular mechanisms underlying REM disregulation in narcolepsy.