We have tested whether action potential-evoked Ca2+ influx is required to initiate clathrin-mediated synaptic vesicle endocytosis in the lamprey reticulospinal synapse. Exo- and endocytosis were temporally separated by a procedure involving tonic action potential stimulation and subsequent removal of extracellular Ca2+ (Ca2+e). A low concentration of Ca2+ ([Ca2+]e of 11 microM) was found to be required for the induction of early stages of endocytosis. However, the entire endocytic process, from the formation of clathrin-coated membrane invaginations to the generation of synaptic vesicles, proceeded in the absence of action potential-mediated Ca2+ entry. Our results indicate that the membrane of synaptic vesicles newly incorporated in the plasma membrane is a sufficient trigger of clathrin-mediated synaptic vesicle endocytosis.

SUMMARY:Salicylate has recently been demonstrated to protect against the auditory and vestibular side effects of aminoglycoside antibiotics. Similarities in the toxic mechanisms suggest salicylate as a treatment strategy to prevent the ototoxic side effects of cisplatin (CDDP). We first tested protection of the inner ear in Wistar rats receiving a single infusion of 16 mg CDDP/kg body weight with or without treatment with 100 mg/kg salicylate (bid) for 5 days beginning one day before the CDDP infusion. Cisplatin induced a threshold shift of more than 30 dB (at 14 kHz; measured by auditory evoked brain stem response) that was significantly reduced by salicylate. We then examined the protective potential of salicylate on the cochlea, peripheral nerves, and kidney in a rat model of breast cancer-Fisher344 rats implanted with highly metastatic MTLn3 breast cancer cells. Animals received 3 ϫ 5 mg CDDP/kg (given every third day), and salicylate was administered at 100 mg/kg (bid) from 2 days before to 3 days after CDDP treatment. Salicylate significantly attenuated the CDDP-induced threshold shift from approximately 20 dB (at 16 and 24 kHz) to approximately 5 dB, and drastically reduced the loss of cochlear outer hair cells. Likewise, salicylate protected kidney function (measured as plasma blood urea nitrogen and creatinine levels) from CDDP toxicity. Protection of nerve conduction velocities of both sensory and motor nerves was minimal. The chemotherapeutic efficacy of CDDP on suppression of tumor mass and cancer cell metastasis remained unaffected by salicylate. The results suggest that administration of salicylate may become the basis of an effective therapeutic intervention against the ototoxic and nephrotoxic side effects associated with CDDP chemotherapy. (Lab Invest 2002, 82:585-596).

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RESULTSThere was an early (first month) abnormality of both erectile and bladder function that persisted through the 8 months of the study. The erectile dysfunction was manifest as reduced intracavernous pressure/blood pressure ratio, and the bladder dysfunction as a persistent increase in detrusor overactivity with no detrusor decompensation. Insulin treatment prevented or modified the abnormality in each organ. Hyperglycaemia caused a progressive decrease in caudal nerve conduction velocity. The mean digital sensory and tibial motor nerve conduction velocity did not deteriorate over time. Correlation measurements of nerve and organ function were not consistent. CONCLUSIONSThe results of this extensive long-term study show early and profound effects of hyperglycaemia on the smooth muscle of the penis and bladder, that were persistent and stable in surviving rats over the 8 months. The physiological changes did not correlate well with neurological measurements of those organs. Significantly, diverse smooth-muscle cellular and subcellular events antedated the measured neurological manifestations of the hyperglycaemia by several months. Although autonomic diabetic neuropathy is a primary life-threatening complication of long-term diabetes in humans, this rat model of STZinduced diabetes showed that the rapid onset of physiological manifestations was based on many molecular changes in the smooth muscle cells in this model of type 1 DM. KEYWORDSdiabetes mellitus, erectile dysfunction, detrusor overactivity, streptozotocin, diabetic autonomic neuropathy OBJECTIVESTo provide sensitive physiological endpoints for the onset and long-term progression of deficits induced by diabetes mellitus (DM) in bladder and erectile function in male rats, and to evaluate parallel changes in urogenital and nerve function induced by hyperglycaemia over a protracted period as a model for chronic deficits in patients with diabetes. MATERIALS AND METHODSThe study comprised in 877 male, 3-monthold, Fischer 344 rats; 666 were injected intraperitoneally with 35 mg/kg streptozotocin (STZ) and divided into insulintreated and untreated diabetic groups. The rats were studied over 8 months and measurements made of both erectile and bladder function, as well as nerve conduction studies over the duration of the study.

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA(B) receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (-ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA(B) agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA(B) antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.