SUMMARY1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2" indicator, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells.2. Depolarization with a patch pipette produced radial gradients of Ca21 within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca21 channels were closed.3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2 .4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected.5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99 % of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 /LMCa2+ inside the cell.6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (04 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions ratiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.MS 9349

Hormone release from nerve terminals in the neurohypophysis Is a sensitive function of action potential frequency. We have investigated the cellular mechanisms responsible for this frequency-dependent facilitation by combining patch clamp and fluorimetric Ca2+ measurements in single neurosecretory terminals in thin slices of the rat posterior pituitary. In these terminals both action potential-induced changes in the intracellular Ca2+ concentration ([Ca2+] Little is known about the mechanisms that regulate secretion from nerve terminals, despite the importance of these mechanisms to many physiological processes. For example, a presynaptic site has been proposed as an important locus for change in several forms of use-dependent synaptic plasticity, including facilitation and posttetanic potentiation at neuromuscular synapses (1) and long-term potentiation in the mammalian hippocampus (2, 3). However, in these and most other systems one can do little more than infer that some presynaptic change occurs, because technical difficulties preclude a direct physiological characterization of the nerve terminals. Although a direct physiological characterization is possible in some invertebrate systems (4, 5), until recently there was no comparable vertebrate system for the study of presynaptic physiology.In an effort to bridge this technical gap, some investigators have made electrical and optical recordings from the nerve terminals of the vertebrate neurohypophysis (6-12). These terminals release the neuropeptides vasopressin and oxytocin and have received much attention as a model system for the study of secretion (6-12). Peptide secretion from these terminals also exhibits a striking form of use-dependent plasticity: secretion is a sensitive function of action potential frequency (11,(13)(14)(15)(16)(17). This frequency dependence is essential to the input-output properties of the hypothalamichypophyseal axis (18) and has been proposed to be due to frequency-dependent modulation of action potential duration (10,11,13,14,(19)(20)(21)(22)(23). In the present study, we use patch clamp techniques and fluorimetric calcium indicators to test this hypothesis in individual nerve terminals in pituitary slices. A preliminary account of this work has appeared (24). METHODSNeurohypophysial Slices. The neurointermediate lobe ofthe pituitary was removed from male rats with ages ranging from 2 to 4 months and placed in carbogen (95% 02/5% C02)-saturated rat Ringer's solution (125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2, 1 mM MgCl2, and 20 mM glucose) at 0WC for at least 1 min. Slices 70-80 j&m thick were then cut with an FTB vibratome, with continued bathing in 0WC rat Ringer's solution. Slices were either kept in a 34WC bath or transferred to a recording chamber at room temperature (20-220C) tTo whom reprint requests should be addressed. 380The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with...

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