Robert S. Zucker

Calcium- and activity-dependent synaptic plasticity

Calcium ions play crucial signaling roles in many forms of activity-dependent synaptic plasticity. Recent presynaptic [Ca2+]i measurements and manipulation of presynaptic exogenous buffers reveal roles for residual [Ca2+]i following conditioning stimulation in all phases of short-term synaptic enhancement. Pharmacological manipulations implicate mitochondria in post-tetanic potentiation. New evidence supports an influence of Ca2+ in replacing depleted vesicles after synaptic depression. In addition, high-resolution measurements of [Ca2+]i in dendritic spines show how Ca2+ can encode the precise relative timing of presynaptic input and postsynaptic activity and generate long-term synaptic modifications of opposite polarity.

Multiple calcium-dependent processes related to secretion in bovine chromaffin cells

We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient--slow secretion for up to 1 min due to displacement of Ca2+ by cytoplasmic Mg2+. Flash photolysis elicited 100 microM [Ca2+]i steps that evoked intense secretion, lasting a few seconds. In cells experiencing a loading transient, [Ca2+]i steps evoked an especially fast secretion. A persistent, slow secretion often followed these fast phases. Distinct kinetic components may reflect secretion from pools that are differentially capable of release. Both secretion and movement of vesicles between pools appear to be [Ca2+]i sensitive. Later [Ca2+]i steps sometimes evoked a rapid capacitance decrease, indicating a fast, [Ca2+]i-dependent phase of endocytosis.

Intracellular calcium release at fertilization in the sea urchin egg

Abstract Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μ M free calcium 45–60 sec after activation and to last 2 3 min, assuming equal Ca 2+ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zerocalcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μ M Ca 2+ . Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.

Mitochondrial Involvement in Post-Tetanic Potentiation of Synaptic Transmission

Posttetanic potentiation (PTP) is an essential aspect of synaptic transmission that arises from a persistent presynaptic [Ca2+]i following tetanic stimulation. At crayfish neuromuscular junctions, several inhibitors of mitochondrial Ca2+ uptake and release (tetraphenylphosphonium or TPP+, carbonyl cyanide m-chlorophenylhydrazone or CCCP, and ruthenium red) blocked PTP and the persistence of presynaptic residual [Ca2+]i, while endoplasmic reticulum (ER) Ca2+ pump inhibitors and release channel activators (thapsigargin, 2,5-di-(tert-butyl)-1,4-benzohydroquinone or BHQ, and caffeine) had no effects. PTP apparently results from the slow efflux of tetanically accumulated mitochondrial Ca2+.

Exocytosis: A Molecular and Physiological Perspective

At synapses between nerve cells, high concentrations From 1952 to about 1985, exocytosis in neurons was of [Ca]i occur only during action potentials and are studied mainly at neuromuscular junctions and selected localized at active zones where vesicles are docked in giant synapses by electrophysiology and electron mithe neighborhood of clusters of Ca channels.Evidence croscopy. Early advances were followed by a period for the existence of such “Ca microdomains” has of relative stagnation as these techniques appeared to come from three types of experiments: 1) Simulations reach their full potential. Then, in the last few years, of Ca21 diffusion from Ca21 channel clusters in the preseverything changed. New methods for measuring and ence of a slowly diffusible buffer predict a complex patcontrolling presynaptic calcium concentration ([Ca]i) tern of peaks and troughs of [Ca]i transients in active using fluorescent dyes and photosensitive Ca-binding zones during action potentials (Figure 1). In these simucompounds emerged, as did techniques for simulating lations, [Ca]i reaches about 150 mM in the spaces the diffusion of Ca in the presynaptic neuron. New between rows of Ca channels where vesicles are methods of monitoring secretion were developed, indocked. These predictions have been confirmed in accluding measurement of presynaptic membrane area by tual experiments on frog saccular hair cells by measurmeans of membrane capacitance, and of uptake and ing the activity of Ca-activated potassium channels release by means of fluorescent dyes trapped in endocythat are co-localized with presynaptic Ca channels tosed vesicles. Modern methods of membrane biology (Roberts, 1994). 2) Local Ca domains also have been and genetic engineering were applied to proteins observed in turtle cochlear hair cells by confocal imthought to be involved in neurosecretion. Meanwhile the aging of fast-binding low affinity fluorescent Ca buffpatch clamp permitted electrophysiological analysis of ers (Tucker and Fettiplace, 1995) and at squid giant previously inaccessible tissues. As a result, exocytosis synapses by imaging phosphorescence from low affinity emerged from a backwater in relative obscurity to bephotoproteins (Llinás et al., 1995). 3) Secretion is often come a new frontier in biological research. This review triggered by Ca1 entering through mixtures of Nand summarizes some of the recent advances and indicates P/Q-type high-voltage-activated Ca channels, ascurrent directions of research in this active field. sessed by actions of specific channel blockers on trans-

Changes in the statistics of transmitter release during facilitation

1. The statistical nature of transmitter release during facilitation was studied at single synaptic sites by recording extracellular excitatory junctional potentials from the claw opener muscle in crayfish.

Role of presynaptic calcium ions and channels in synaptic facilitation and depression at the squid giant synapse.

1. The roles of presynaptic calcium influx and calcium accumulation in synaptic facilitation and depression were explored at the giant synapse in the stellate ganglion of the squid. 2. Calcium currents were recorded in the presynaptic terminal, using a three‐electrode voltage clamp and blocking sodium and potassium currents pharmacologically. The calcium influx was constant during pairs or trains of brief depolarizing pulses that elicited facilitating or depressing excitatory post‐synaptic potentials (e.p.s.p.s). 3. The relationship between calcium influx and transmitter release during brief depolarizing pulses of varying amplitude resembled a power function with exponent of about 2. 4. Presynaptic calcium concentration transients were measured by injecting the dye arsenazo III and detecting absorbance changes microspectrophotometrically. Increments in intracellular free calcium accompanying single action potentials appeared constant for repeated action potentials that elicited facilitating e.p.s.p.s. 5. The presynaptic calcium concentration remains elevated for several seconds following action potentials. 6. Presynaptic injection of calcium ions by interbarrel ionophoresis evokes a postsynaptic depolarization, apparently reflecting a large increase in miniature e.p.s.p. frequency. Presynaptic action potentials remain unaffected by this treatment, but e.p.s.p.s triggered by them are facilitated for several seconds, and then depressed. 7. The results are consistent with the hypothesis that synaptic facilitation is due to the action of residual calcium or a calcium complex remaining in the presynaptic terminal after electrical activity. The late depression of release during calcium injection may be a result of the continual release of transmitter and consequent depletion of a presynaptic store.

Enhancement of synaptic transmission by cyclic AMP modulation of presynaptic Ih channels.

Presynaptic activation of adenylyl cyclase and subsequent generation of cAMP represent an important mechanism in the modulation of synaptic transmission. In many cases, short- to medium-term modulation of synaptic strength by cAMP is due to activation of protein kinase A and subsequent covalent modification of presynaptic ion channels or synaptic proteins. Here we show that presynaptic cAMP generation via serotonin receptor activation directly modulated hyperpolarization-activated cation channels (Ih channels) in axons. This modulation of Ih produced an increase in synaptic strength that could not be explained solely by depolarization of the presynaptic membrane. These studies identify a mechanism by which cAMP and Ih regulate synaptic plasticity.

Time course of transmitter release calculated from simulations of a calcium diffusion model.

A three-dimensional presynaptic calcium diffusion model developed to account for characteristics of transmitter release was modified to provide for binding of calcium to a receptor and subsequent triggering of exocytosis. When low affinity (20 microM) and rapid kinetics were assumed for the calcium receptor triggering exocytosis, and stimulus parameters were selected to match those of experiments, the simulations predicted a virtual invariance of the time course of transmitter release to paired stimulation, stimulation with pulses of different amplitude, and stimulation in different calcium solutions. The large temperature sensitivity of experimental release time course was explained by a temperature sensitivity of the models final rate limiting exocytotic process. Inclusion of calcium tail currents and a saturable buffer with finite binding kinetics resulted in high peak calcium transients near release sites, exceeding 100 microM. Models with a single class of calcium binding site to the secretory trigger molecule failed to produce sufficient synaptic facilitation under this condition. When at least one calcium ion binds to a different site having higher affinity and slow kinetics, facilitation again reaches levels similar to those seen experimentally. It is possible that the neurosecretory trigger molecule reacts with calcium at more than one class of binding site.

Temporal limits on the Rise in Postsynaptic Calcium Required for the Induction of Long-Term Potentiation

The induction of long-term potentiation (LTP) in hippocampal CA1 pyramidal cells requires a rise in postsynaptic intracellular Ca2+ concentration ([Ca2+]i). To determine the time for which Ca2+ must remain elevated to induce LTP, the photolabile Ca2+ buffer diazo-4 was used to limit the duration of the rise in postsynaptic [Ca2+]i following a tetanus. The affinity of diazo-4 for Ca2+ increases approximately 1600-fold upon flash photolysis, permitting almost instantaneous buffering of [Ca2+]i without disturbing resting [Ca2+]i prior to the flash. Photolysis of diazo-4 1 s following the start of the tetanus blocked LTP, while delaying photolysis for more than 2 s had no discernible effect on LTP. Photolyzing diazo-4 at intermediate delays (1.5-2 s) or reducing photolysis of diazo-4 often resulted in short-term potentiation (STP). These results indicate that a tetanus-induced rise in postsynaptic [Ca2+]i lasting at most 2-2.5 s is sufficient to generate LTP. Smaller increases or shorter duration rises in [Ca2+]i may result in STP.