Cysteine-string proteins (CSPs) are associated with secretory vesicles and critical for regulated neurotransmitter release and peptide exocytosis. At nerve terminals, CSPs have been implicated in the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. Here we show that the individual expression of various CSP isoforms in Drosophila similarly rescues the loss of evoked neurotransmitter release at csp null mutant motor nerve terminals, suggesting widely overlapping functions for each isoform. Thus, the structural difference of CSP variants may not explain the opposing putative functions of CSP in neurotransmitter and peptide exocytosis. Consistently, the individual overexpression of each CSP isoform in wild-type Drosophila shows similar effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP are suppressed by the simultaneous overexpression of syntaxin-1A but not by the coexpression of SNAP-25. Although overexpression of CSP itself has no apparent effect on the synaptic physiology of larval motor nerve terminals, it fully suppresses the decrease of evoked release induced by the overexpression of syntaxin-1A. A direct protein-protein interaction of CSP with syntaxin is further supported by coimmunoprecipitations of syntaxin with CSP and by protein binding assays using recombinant fusion proteins. Together, the genetic and biochemical interactions of CSP and syntaxin-1A suggest that CSP may chaperone or modulate protein-protein interactions of syntaxin-1A with either calcium channels or other components of the regulatory machinery mediating depolarization-dependent neurotransmitter exocytosis.

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Nitric oxide (NO) is an endogenous neuromodulator that may mediate neurotoxic effects of glutamate. NO-synthesizing neurons are, however, resistant to NO- and glutamate-induced neurotoxicity. We now show that NO synthase neurons are selectively spared in patients with Alzheimer's disease, even in a severely affected region of the brain such as the hippocampal formation.

We report an anatomically defined opioid-responsive site in the rostral agranular insular cortex (RAIC) of the rat and characterize the antinociception produced by morphine acting within this region. Immunohistochemistry for the mu-opioid receptor identified a discretely localized cluster of densely labeled dendrite-like processes in the agranular insular cortex. The antinociceptive effect of morphine microinjected unilaterally into this area was evaluated using the formalin test. Antinociception was observed in both ipsilateral and contralateral hindpaws. Local pretreatment with naltrexone in the RAIC blocked the antinociception of local morphine injection, confirming that morphine was acting at an opioid receptor. Unilateral injection of naloxone methiodide into the RAIC reversed the behavioral antinociception of systemic morphine bilaterally in the formalin test. Evidence for a descending inhibitory mechanism acting on spinal nociceptive neurons was obtained by monitoring noxious stimulus-induced c-fos expression in rats having undergone formalin testing and by electrophysiological recording of single units in the lumbar dorsal horn after localized application of morphine into the RAIC. A significant reduction in the number of Fos-like immunoreactive neurons was found ipsilateral to the formalin stimulus in nociresponsive areas of the dorsal horn after on-site injections of morphine into the RAIC. Electrophysiological recording of nociresponsive dorsal horn neurons demonstrated a naloxone-reversible reduction in noxious thermal stimulus-evoked firing after morphine injection into this same area. These results suggest that the RAIC contributes to opioid-receptor-mediated antinociception after either local or systemic morphine administration and that these effects may be associated with an increased descending inhibition of dorsal horn neurons.