Synaptic calcium regulation in hair cells of the chicken basilar papilla.

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling.

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay.

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

An electrophysiologic model for functional assessment of effects of neurotrophic factors on facial nerve reinnervation.

OBJECTIVES: To establish a sound objective model for assessing the effects of neurotrophic factors on facial nerve function after injury and to compare the effects of brain-derived neurotrophic factor (BDNF) with its neutralizing antibody on facial nerve function after injury. DESIGN: Prospective electrophysiologic analysis of recovery of function 4 weeks after axotomy involving facial nerve transection and primary end-to-end reanastomosis in adult rats and blind comparison with randomized intramuscular injection of either BDNF, monoclonal antibody to BDNF in neutralizing concentration, or control solution. RESULTS: There were no statistically significant differences between groups in latencies, duration, amplitude, area, or conduction velocity before axotomy, and recorded conduction velocities were consistent with previously reported values, which suggests that the recordings were reliable and reproducible. After transection, there was a mean increase in latency 1 and decreases in latency 2, integrated average area, muscle action potential duration, amplitude, and conduction velocity for all 3 groups. When the groups were compared after transection, the anti-BDNF group showed a significant decrease in conduction velocity and muscle action potential duration (Kruskal-Wallis P = .01 and P = .008, respectively) compared with the other groups. There were no statistically significant differences in latencies, amplitude, or area among the groups. CONCLUSIONS: We have established an electrophysiologic model for objective assessment of facial nerve function in the rat. Future studies should combine functional electrophysiologic assessment and histologic examination to provide a more robust model for studying the effects of neurotrophic factors on facial nerve reinnervation and synkinesis.