The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Under depolarizing voltage clamp of Paramecium an inward calcium current developed and subsequently relaxed within 10 milliseconds. The relaxation was substantially slowed when most of the extracellular calcium was replaced by either strontium or barium. Evidence is presented that the relaxation is not accounted for by a drop in electromotive force acting on calcium, or by activation of a delayed potassium current. Relaxation of the current must, therefore, result from an inactivation of the calcium channel. This inactivation persisted after a pulse, as manifested by a reduced calcium current during subsequent depolarization. Inactivation was retarded by procedures that reduce net entry of calcium, and was independent of membrane potential. The calcium channel undergoes inactivation as a consequence of calcium entry during depolarization. In this respect, inactivation of the calcium channel departs qualitatively from the behavior described in the Hodgkin-Huxley model of the sodium channel.

No abstract

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.active zone | exocytosis | synapse | acetylcholine receptor A hallmark of synaptic transmission is the synchrony between the neuronal action potential and the evoked release of transmitter. However, an asynchronous release mode, first described at the nerve muscle junction (1, 2), also participates in neurotransmission at certain synapses (3-5). Although asynchronous release usually contributes less than 10% of the overall synaptic charge at low stimulus frequencies, it often plays a prominent role at higher frequencies (3, 6), prolonging both inhibitory (7) and excitatory (8, 9) postsynaptic responses through sustained release. Indeed, inhibitory deep cerebellar neurons, which are tuned for high-frequency signaling, rely exclusively on asynchronous synaptic transmission at contacts with inferior olive neurons (10). The mechanisms underlying synchronous and asynchronous release appear to be distinct but share a requirement for calcium (6,11). It is generally thought that local transient calcium signals govern synchronous release, whereas elevated residual calcium levels associated with highfrequency stimulation lead to asynchronous release (12, 13).A vesicular calcium sensor, synaptotagmin, couples synchronous release to presynaptic calcium entry in both mammals (14) and flies (15, 16). In hippocampus, the responsible isoform is syt1 (14, 17), whereas at neuromuscular synapses (18) and calyx of Held (11,19), it is syt2. The mechanisms underlying asynchronous release are not known. Our findings from zebrafish neuromuscular junction provide identification of a synaptotagmin isoform as a signaling component in asynchronous release. Results Paired Recordings Reveal a Small Quantal Content with ReleaseProbability Near "1." Paired whole-cell recordings between the caudal primary motor neuron (CaP) and ventral target skeletal muscle were performed on 72-to 96-h-postfertilization (hpf) zebrafish as previously described (20). The motor neuron was current clamped to −80 mV and stepped positive for 2 ms to elicit an action potential. The fast-type skeletal muscle was voltage clamped to −50 mV to inactivate sodium channels. The amplitude for spontaneous unitary events (...

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

The amphibian tetradecapeptide bombesin and its mammalian homolog gastrin-releasing peptide are neurotransmitters and paracrine hormones, and are mitogenic for fibroblast and small cell lung carcinoma cell lines. cDNAs encoding the bombesin/gastrin-releasing peptide receptor (BR) expressed by murine Swiss 3T3 fibroblasts were isolated using electrophysiological and luminometric Xenopus oocyte expression assays. Oocytes microinjected with BR transcripts responded to concentrations of bombesin from 1 x 10(-10) to 1 x 10(-6) M. These responses showed homologous desensitization and could be specifically blocked by bombesin antagonists. Sequence analysis showed that the BR has seven membrane-spanning domains and five potential N-linked glycosylation sites. Data base analysis showed that the BR is most homologous to the tachykinin receptors. Although tyrosine kinase activity has been associated with BR function, no tyrosine kinase homologies occur within the BR sequence.

The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes. A 1 min exposure of PC12 cells to interferon-gamma also causes PN1 gene induction, suggesting that the "triggered" NGF and interferon-gamma signaling pathways share common molecular intermediates.

SUMMARY1. The Ca current seen in response to depolarization was investigated in Paramecium caudatum under voltage clamp. Inactivation of the current was measured with the double pulse method; a fixed test pulse of an amplitude sufficient to evoke maximal inward current was preceded by a conditioning pulse of variable amplitude (0-120 mV).2. The amplitude of the current recorded during the test pulse was related to the potential of the conditioning pulse. Reduction of test pulse current was taken as an index of Ca current inactivation. The current recorded during a test pulse showed a progressive decrease to a minimum as the potential of the conditioning pulse approached + 10 to + 30 mV. Further increase in conditioning pulse amplitude was accompanied by a progressive restoration of the test pulse current. Conditioning pulses near the calcium equilibrium potential had only a slight inactivating effect on the test pulse current.3. Injection of a mixture of Cs and TEA which blocked late outward current had essentially no effect on the inward current or its inactivation.4. Elevation of external Ca from 0 5 to 5 mM was accompanied by increased inactivation of the test pulse current. The enhanced inactivation of the test pulse current was approximately proportional to the increase in current recorded during the conditioning pulse.5. Following injection of the Ca chelating agent, EGTA, the inactivation of the test pulse current was diminished; in addition, the transient inward current relaxed slightly more slowly, and the transient was followed by a steady net inward current.6. The time course of recovery from inactivation in the double pulse experiment approximated a single exponential having a time constant of 80-110 msec. Injection of EGTA shortened the time constant by as much as 50 %.7. It is concluded that interference with the entry of Ca or enhanced removal of intracellular free Ca2+ interferes with the process of Ca current inactivation, while enhanced entry of Ca promotes the process of inactivation. While the mechanism of inactivation is unknown, arguments are presented that the accumulation of intracellular Ca influences the Ca channel conductance.