Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca 2؉ -dependent activator protein for secretion), a protein required for Ca 2؉ -dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca 2؉ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca 2؉ dependencies. A threshold of Ϸ10 M Ca 2؉ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca 2؉ activity < 10 M. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca 2؉ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.capacitance ͉ pituitary cells ͉ rat melanotrophs

Neuronal network topologies and connectivity patterns were explored in control and glutamate-injured hippocampal neuronal networks, cultured on planar multielectrode arrays. Spontaneous activity was characterized by brief episodes of synchronous firing at many sites in the array (network bursts). During such assembly activity, maximum numbers of neurons are known to interact in the network. After brief glutamate exposure followed by recovery, neuronal networks became hypersynchronous and fired network bursts at higher frequency. Connectivity maps were constructed to understand how neurons communicate during a network burst. These maps were obtained by analysing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and glutamate-injured networks, respectively. Although both the networks showed random features, smallworld properties in these networks were different. These results suggest that functional two-dimensional neuronal networks in vitro are not scale-free. After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. The small-world network property was lost and this was accompanied by a change from an exponential to a Gaussian network.

The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo-and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2÷-containing solutions. The resting Cm of 6.05 --+ 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 lzF/cm ~. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 Izm in diameter). High Ca 2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca~+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca ~+ through the membrane. This is supported by the following findings: addition of Co ~÷ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca 2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.

SUMMARY1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual-wavelength, ratiometric video imaging system and low light level charge-coupled device (CCD) camera visualizing Fura-2 in dispersed male rat anterior pituitary cells.2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca21] localized in the subplasmalemmal region of the cell. 9. From these results, taken together with previous findings, we propose the possibility that hGRF activates tetrodotoxin-insensitive Na' (or non-selective cationic) channels via cyclic AMP, which in turn causes depolarization of the somatotroph leading to activation of Ca21 channels, Ca2" influx and exocytotic secretion of growth hormone.

Secretory responses were measured in single rat pituitary melanotrophs as the relative increase in membrane capacitance (Cm) 8 min after the start of dialysis with solutions containing 0.45 μm Ca2+. In the added presence of cAMP (0.2 mM) in the patch pipette solution, capacitance responses increased 2‐ to 3‐fold in comparison with controls. To study whether cAMP‐dependent mechanisms affect cytosolic calcium activity ([Ca2+]i), dibutyryl cyclic AMP (dbcAMP, 10 mM) was added to intact melanotrophs and [Ca2+]i was measured using fura‐2 AM. Addition of dbcAMP caused a transient reduction in [Ca2+]i to 82 ± 21 nM from a resting value of 100 ± 19 nM (mean ± s.e.m., n= 32, P < 0.002), indicating that the cAMP‐induced increase in secretory activity was not the result of cAMP acting to increase [Ca2+]i, which then increased secretory activity. To investigate whether cAMP affects the secretory apparatus directly, the interaction of a single secretory granule with the plasmalemma was monitored by measuring discrete femtofarad steps in Cm. The signal‐to‐noise ratio of recordings was increased by pre‐incubating the cells with a hydrophobic anion, dipicrylamine. Recordings of unitary exocytic events (discrete ‘on’ steps in Cm) showed that the amplitude of ‘on’ steps ‐ a parameter correlated to the size of exocytosing secretory granules ‐ increased from 4.2 ± 0.2 fF (n= 356) in controls to 7.9 ± 0.2 fF in the presence of cAMP (n= 329, P < 0.001), while the frequency of unitary exocytic events was similar in controls and in the presence of cAMP. The results suggest that a cAMP‐dependent mechanism mediates the fusion of larger granules with the plasmalemma.