No abstract

Disruptions in brain-derived neurotrophic factor (BDNF) expression are proposed to contribute to the molecular pathogenesis of Rett syndrome (RTT), a severe neurological disorder caused by loss-of-function mutations in methyl-CpG-binding protein-2 (MeCP2). Although MeCP2 is a transcriptional regulator of BDNF, it is unknown how MeCP2 mutations affect transynaptic BDNF signaling. Our findings demonstrate an early, abnormal neurosecretory phenotype in MeCP2-deficient neurons characterized by significant increases in the percentage of cellular BDNF content available for release. However, loss of MeCP2 also results in deficits in total cell BDNF content that are developmentally regulated in a cell-type-specific manner. Thus, the net effect of MeCP2 loss on absolute BDNF secretion changes with age and is determined by both the amount of BDNF available for release and progressive declines in total cellular BDNF. We propose, therefore, that loss of MeCP2 function disrupts transynaptic BDNF signaling by perturbing the normal balance between BDNF protein levels and secretion. However, mutant neurons are capable of secreting wild-type levels of BDNF in response to high-frequency electrical stimulation. In addition, we found elevated exocytic function in Mecp2 Ϫ/y adrenal chromaffin cells, indicating that the Mecp2 null mutation is associated with alterations of neurosecretion that are not restricted to BDNF. These findings are the first examples of abnormal neuropeptide and catecholamine secretion in a mouse model of RTT.

1. Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. 2. The sAPs evoked inward Na(+) and Ca(2+) currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (tau = 560 ms). 3. Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. 4. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. 5. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506.

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic‐chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential‐dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP‐evoked stimulation involves a phospholipase C and protein kinase C‐dependant pathway to facilitate calcium influx through a Ni2+ and mibefradil‐sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho‐adrenal synapse under the stress response.

1 The nature of the binding site mediating the insulin secretagogue activity of certain imidazoline compounds remains unclear and the pharmacology of the I,-and I2-imidazoline sites