Glutamate application at synapses between hippocampal neurons in culture produces long-term potentiation of the frequency of spontaneous miniature synaptic currents, together with long-term potentiation of evoked synaptic currents. The mini frequency potentiation is initiated postsynaptically and requires activity of NMDA receptors. Although the frequency of unitary quantal responses increases strongly, their amplitude remains little changed with potentiation. Tests of postsynaptic responsiveness rule out recruitment of latent glutamate receptor clusters. Thus, postsynaptic induction can lead to enhancement of presynaptic transmitter release. The sustained potentiation of mini frequency is expressed even in the absence of Ca2+ entry into presynaptic terminals.

Abstract. The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca 2+ ([Ca2+]~) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37~ with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15~ temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37~ for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15~ demonstrated in spotty cells a marked apparent blunting of Ca 2 § transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]~ was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca 2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]~ measurements. (34) and, more recently, fura-2 (14, 33). The remarkable advantages of the latter dye with respect to its older congener, discussed in detail by Tsien and his associates (14,33,(35)(36)(37), include higher fluorescence and quantum yield, lower affinity for Ca 2 § and favorable spectral properties, including shift of the exci-1. Abbreviations used in this paper: AM, acetoxy-methyl ester; AO, acridine orange; [Ca2+]i, free ionized Ca2+; HSE human skin fibroblasts; KRH, Krebs-Ringer medium buffered with Hepes. tation spectrum induced by Ca 2 § binding, with reciprocal changes of fluorescence across the isos...

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca21],.Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[y-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[fithio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]1 at rest but caused a partial blunting of the initial [Ca2+], peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.In various cellular systems the function of voltage-gated Ca2l channels is known to be modulated by intracellular events [phosphorylations by cAMP-and cGMP-dependent protein kinases (1-3), and protein kinase C (4, 5)] triggered by the activation of the receptors for various neurotransmitters [e.g., norepinephrine, y-aminobutyric acid, serotonin, adenosine, and acetylcholine (AcCho) (6-13)]. Studies carried out during the last few years have shown that Ca2+ channels are heterogeneous. In sensory neurons of the chicken dorsal root ganglion (DRG), three types of channels (L, N, and T) have been identified that differ in their unitary conductance, voltage dependence ofactivation, and kinetics of inactivation (14). Because of their different properties, these channels may play different physiological roles, and it is therefore imp...

Synapses in the central nervous system are typically studied by recording electrical responses from the cell body of the postsynaptic cell. Because neurons are normally connected by multiple synaptic contacts, these postsynaptic responses reflect the combined activity of many thousands synapses, and it remains unclear to what extent the properties of individual synapses can be deduced from the population response. We have therefore developed a method for recording the activity of individual hippocampal synapses. By capturing an isolated presynaptic bouton inside a loose-patch pipette and recording from the associated patch of postsynaptic membrane, we were able to detect miniature excitatory postsynaptic currents ('minis') arising from spontaneous vesicle exocytosis at a single synaptic site, and to compare these with minis recorded simultaneously from the cell body. The average peak conductance at a single synapse was about 900 pS, corresponding roughly to the opening of 90 AMPA-type glutamate-receptor channels. The variability in this conductance was about 30%, matching the value reported for the neuromuscular junction. Given that our synapses displayed single postsynaptic densities (PSDs), this variability is larger than would be predicted from the random opening of receptor channels, suggesting that they are not saturated by the content of a single vesicle. Therefore the response to a quantum of neurotransmitter at these synapses is not limited by the number of available postsynaptic receptors.