Electrical and optical study of nerve impulse-evoked ATP-induced, P2X-receptor-mediated sympathetic neurotransmission at single smooth muscle cells in mouse isolated vas deferens (original) (raw)
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Neuroscience, 2007
The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other electrically-coupled smooth muscle syncytia has been attributed to electrically-attenuated depolarizations resulting from the spontaneous release of quantized packets of ATP acting on remote smooth muscle cells (SMCs). However, in the present investigation surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176±18 MΩ, range: 141-221 MΩ, n=4) similar to those of dissociated cells. Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001). Using simultaneous electrophysiology and confocal microscopy to investigate these poorly-coupled cells, it is shown that α-latrotoxin-stimulated sEJPs correlate, in timing (median delay ranged from −30 to −57 ms, P<0.05 in all experiments, n=5) and amplitude (Pearson product moment correlation, ρ>0.55 and P<0.001), with purinergic neuroeffector Ca 2+ transients (NCTs) in SMCs. The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC. The amplitude correlation of sEJPs and NCTs argues against the attenuation of electrical signal amplitude along the length of a single SMC. The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.
1993
At the mammalian neuromuscular junction, perhaps the most studied synapse, many aspects of neurotransmitter release and stimulation-induced enhancement of release are still poorly understood. Central hypotheses include: about release, the Ca^ hypothesis (del Castillo and Katz, 1954), and the Ca'' -voltage hypothesis (Parnas and Parnas, 1988), and about enhancement, the residual Ca^ hypothesis (Katz & Miledi, 1968). In the present work, these hypotheses were tested by analysis of the magnitude and timing of release with the technical advantages of computer-assisted analysis of data for large numbers of stimuli and responses and an emphasis on the relative magnitude of phasic and non-phasic release components. In mouse nerve-diaphragm in vitro, phasic neurotransmitter release evoked by action potentials grew with r«0.1 ms and decayed with r«0.3 ms, consistent for Ca, Sr and Ba. Non-phasic release decayed, with a polyphasic time course that varied with the divalent cation. The ...
Doklady Biological Sciences, 2004
Understanding the principles of brain work is impossible without understanding the functioning of its main elements, such as synaptic contacts between neurons. The history of the study of mechanisms responsible for the information processing and transmission in the synapses is already several decades long; therefore, a number of methods and lines of research "classical" for this field have been elaborated. One of these lines is investigation of the properties of synaptic transmitter quantal release, and one of standard methods for the estimation of quantal release content (the average number of quanta released during one transmission act) is the estimation of the inverse squared coefficient of variation of the evoked postsynaptic response amplitude ( CV -2 ). Here, we describe the type of synapses for which a change in CV -2 is not necessarily accompanied by a change in quantal content, set the question as to the cause of this phenomenon, and offer an explanation for this problem, illustrating it with a computer model.
The kinetics of nerve-evoked quantal secretion
Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 1999
Current views on quantal release of neurotransmitters hold that after the vesicle migrates towards release sites (active zones), multiple protein interactions mediate the docking of the vesicle to the presynaptic membrane and the formation of a multimolecular protein complex (the ‘fusion machine’) which ultimately makes the vesicle competent to release a quantum in response to the action potential. Classical biophysical studies of quantal release have modelled the process by a binomial system where n vesicles (sites) competent for exocytosis release a quantum, with probability p , in response to the action potential. This is likely to be an oversimplified model. Furthermore, statistical and kinetic studies have given results which are difficult to reconcile within this framework. Here, data are presented and discussed which suggest a revision of the biophysical model. Transient silencing of release is shown to occur following the pulse of synchronous transmitter release, which is ev...
Further evidence for the dynamic formation of transmitter quanta at the neuromuscular junction
Journal of Neuroscience Research, 1992
Fatt and Katz (Nature 166597498, 1950; J Physiol 117:109-128,1952) attributed miniature endplate potentials (MEPPs) to the action of a standard quantity of transmitter, the quantum (Del Castillo and Katz, J Physiol124:560-573,1954). Quanta1 packets of transmitter were proposed to be preformed (Del Castillo and Katz, In CNRS Paris (Ed): "Microphysiologie comparee des elements excitables" 67:245-258, 1957) and stored in large numbers in the motor nerve terminal. Statistical analyses of intervals between ME-PPs and numbers of quanta composing small endplate potentials indicated that quantal release was a random process and that release sites functioned independently of each other. With the discovery of synaptic vesicles it was proposed that each contained one quantum of transmitter. The quantal-vesicular hypothesis (Del Castillo and Katz, as cited above) fails, however, to explain amplitude distributions of MEPPs that are skewed and/or that show multiple peaks (Kriebel et al., Brain Res Review 15167-178, 1990). The drop formation process (Shaw, "The Dripping Faucet as a Model Chaotic System," Santa Cruz, CA: Aerial Press, Inc., 1984) was shown to generate amplitude classes of drops that were similar to classes of MEPPs which suggested that rapid changes in quantal size and ratios of skew-to bell-MEPPs could be explained with a simple dynamic process which determines quantal size at the moment of release (Kriebel et al., as cited above, 1990). Further similarities between miniature endplate currents (MEPCs) and the formation of drops are reported here. We found that rapid changes in MEPC amplitudes and time courses, which accompany an increase in frequency, mimic changes in drop sizes that accompany increases in flow rate. MEPC intervals have a minimum and their distributions are comparable to those of drop intervals. During an increased rate of transmitter release, MEPP amplitudes and intervals were positively correlated. The results suggest that spontaneously released transmitter "packets" are formed at the moment of release and that transmitter supply to the process that forms packets is continuous. 0 1992 Wiley-Liss, Inc.
The Journal of Physiology, 1997
1. Action potential (AP)-induced fluorescence transients were measured, using Ca2+ indicators and a spot-detection method, at single nerve terminals of a cultured Xenopus neuromuscular junction preparation with simultaneous measurement of neurotransmitter release. 2. Transients obtained using the low affinity Ca2+ indicator Oregon Green.T 488 BAPTA-5N (OGB-5N) exhibited rapid rising (1½ (time at which one-half of the peak fluorescence was attained) = 054 ms) and decaying (Tfast = 1 9 ms) phases. The higher affinity indicator Oregon GreenTm 488 BAPTA-2 (OGB-2) produced transients with significantly slower kinetics (t1 = 2 ms; r810w = 73 ms). 3. Tetanic stimulation elicited distinct increases in fluorescence in response to each AP. Each OGB-5N fluorescence increase was more rapid than those observed using OGB-2. Furthermore, a smaller proportion of residual fluorescence at the end of the train was observed using OGB-5N. 4. When OGB-5N was used, a significant [Ca2+] increase was observed prior to the release of neurotransmitter. This was not observed when OGB-2 was used.
Quelling of spontaneous transmitter release by nerve impulses in low extracellular calcium solutions
The Journal of …, 1978
1. The effect of nerve stimulation on spontaneous transmitter release was studied at the frog neuromuscular synapse which was bathed in a solution containing very low extracellular calcium concentration. Conventional methods for intracellular and extracellular recording were used and the pattern of quantal liberation following the nerve stimulus was determined. 2. Stimulation of the motor nerve (at rates between 009 and 2 Hz) caused a reduction in the frequency of the miniature e.p.p.s in comparison to the prestimulation values. 3. The mean distribution of the time of occurrence of the miniature e.p.p.s during the interstimulus period showed periodic oscillations. 4. The quelling effect of nerve stimulation on transmitter release is explained by the hypothesis that at low [Ca]0 a reversed electrochemical gradient for calcium occurs and nerve stimulation causes an increased calcium conductance leading to calcium efflux which in turn temporarily reduces [Ca]i and transmitter release.
Electrical Activity of Individual Neurons in Situ: Extra- and Intracellular Recording
Modern Techniques in Neuroscience Research, 1999
Microelectrode recording of electrical activity provides a means to measure the discharge patterns of nerve cells with high spatial and temporal resolution and with minimal damage to nervous tissue. For these reasons it has long been the principal method for analyzing the behavior and function of neurons and neural networks. An additional and extremely useful application of microelectrode technology is the ability to inject tracers directly into neurons through an intracellular microelectrode in order to label the cells and identify their location, morphology, and synaptic contacts with other neurons and effectors. The first investigations of single neuron activity were carried out with microelectrodes for extracellular recording, which led to the identification of previously uncharacterized cell types and synaptic circuits (e.g., Lorente de Nó, 1938; Renshaw, 1946; see Eccles (1964) and McLennan (1970) for additional examples). Shortly thereafter, micropipettes for intracellular recording were developed (Ling and Gerard 1949). These were used to measure membrane potentials and to uncover voltage-and time-dependent properties that determine neuron excitability. Intracellular recording also revealed the nature and functional significance of excitatory and inhibitory postsynaptic potentials and helped to identify the underlying membrane conductance mechanisms (Combs, Eccles and Fatt 1955a-c). Subsequent adaptation of voltage-clamp technology for use with microelectrodes (Brennecke and Lindemann 1974a,b; Wilson and Goldner 1975; Adams and Gage 1979; Finkel and Redman 1984) permitted measurement of membrane currents and estimates of conductances in vitro (Adams et al. 1982a,b; Johnston et al. 1980) and in vivo (Dunn and Wilson 1977; Finkel and Redman 1983a; Richter et al. 1996). Further advances in neurophysiological investigation came with the development of techniques which enabled investigators to record the electrical activity of neurons and then label them by intracellular injections of fluorescent dyes (Thomas and Wilson 1966; Stretton and Kravitz 1968). Microelectrode recording of electrophysiological properties and labeling of neurons continue to be important tools for analyzing the behavior and function of single nerve