News Physiol Sci 13: 79-84, 1998;
1548-9213/98 $5.00
News in Physiological Sciences, Vol. 13, No. 2, 79-84,
April 1998
© 1998 Int. Union Physiol. Sci./Am. Physiol. Soc.
Transmission at Sympathetic Varicosities
Max R. Bennett
M. R. Bennett is at the Neurobiology Laboratory, Institute for Biomedical Research and the Dept. of Physiology, University of Sydney, NSW 2006, Australia.
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Abstract
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The development of techniques for recording the electrical signs of transmission at single sympathetic varicosities has revealed considerable heterogeneity in the properties of transmission at different varicosities. The origin of these heterogeneities is considered in this short review.
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Introduction
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The concept of chemical transmission at synapses was initiated through studies of the action of sympathetic nerves on smooth muscle. These nerves end in strings of bulbs or varicosities from which the transmitter is released on arrival of the nerve impulse. As these varicosities are ~1 µm in extent and separated by ~4 µm, it is possible to place a 4-µm-diameter microelectrode over an individual visualized varicosity and record the electrical signs of the postjunctional action of the transmitter. In this way, the sympathetic nerve terminal has provided the first recordings to be made of transmission at an intact single synapse or release site rather than from multiple release sites. This has allowed answers to be provided for the following major questions concerning the mechanism of synaptic transmission. Are all the synapses of a terminal equivalent in their capacity to secrete a packet of transmitter on arrival of a nerve impulse? Is the receptor patch beneath a single synapse always saturated by the release of a packet of transmitter? What evidence is there that transmitter packets are always of relatively uniform size? This short review summarizes the observations so far obtained for visualized synaptic varicosities. The results indicate that individual synapses of a terminal are heterogeneous, so that the physiology of transmission must be considered at the level of the single synapse or release site rather than that of the entire terminal's arborization of an axon.
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Transmitter release at varicosities with relatively small numbers of receptors
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On some occasions, both spontaneous and evoked transmitter releases appear to saturate the receptor patch beneath a visualized varicosity during recording with loose-patch electrodes. These glass electrodes, ~4 µm in diameter, are placed over a visualized varicosity with slight negative pressure applied so as to produce a loose seal around the varicosity without severing its connections with the parent nerve trunk. This is done by prior fluorescent staining of the mitochondria in the varicosity and then manipulating the loose-patch electrode into place over the chosen fluorescent varicosity for recording. Histograms of the amplitude of the spontaneous electrical signals observed, namely, the junctional currents due to the spontaneous release of packets of transmitter, are sometimes described by a Gaussian distribution with small variance (Ref. 13; Fig. 1B
). The most plausible explanation for such a distribution is that it arises as a consequence of the paucity of receptors in a patch beneath the varicosity compared with the amount of transmitter in a packet. It is therefore not known whether or not the transmitter is released in packets of fairly uniform size, called quanta, at these varicosities.
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FIGURE 1. Amplitude-frequency histograms of spontaneous junctional currents recorded with loose-patch electrodes placed over a visualized varicosity. These currents are recorded as a potential drop across the electrode rim so that abscissa is labeled in µV. A: histogram from a varicosity that is very positively skewed. B: histogram that follows a Gaussian distribution.
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The analysis of simultaneous recordings of transmission from a large number of varicosities with a large-diameter loose-patch electrode supports the idea that transmitter release saturates the receptors at some varicosities. In this case, occasional recordings are made in which the frequency of evoked release is very low and the junctional currents that are observed are of like amplitude and often occur close together in a long train; these are similar to that recorded with an electrode placed over a single varicosity and are well described by a Gaussian distribution with small variance. This implies that most of the varicosities within the electrode do not respond to stimulation, but one does that possesses a relatively small number of receptors compared with the amount of transmitter released in a packet (11). A recent analysis of the occurrence of junctional currents of like amplitude recorded in this way shows that in many recordings successive junctional currents of similar amplitude may arise at different varicosities, but in a few cases they arise from the same varicosity. In this latter case, it is likely that the receptor patch is saturated (Ref. 12; Fig. 2).
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FIGURE 2. Tests for the proposition that junctional currents of like amplitude are dependent and therefore likely to come from the same varicosity. P value (P1) for the test that successive junctional currents of like amplitude are independent of each other during a long train of impulses (500–1,000) is plotted against the percentage of junctional currents of any amplitude evoked during the train. Results are given for 12 experiments, with large-diameter electrodes that showed any successive pairs of like amplitude; of these, 10 possessed a P1 > 0.15, indicating that the pairs were probably independent, whereas 2 had a P1 < 0.15, indicating that the pairs probably came from the same varicosity. [From Karunanithi et al. (12).]
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Other evidence supporting the idea that a proportion of varicosities has relatively few receptors comes from observations on both spontaneous and evoked junctional currents recorded simultaneously from a large number of varicosities (10). In this case, it is sometimes possible to match the amplitude and time course of a particular set of spontaneous and evoked junctional currents, implying that they come from the same varicosity with a particular number of receptors that are saturated. Other sets in the same recording may then arise from other varicosities with different numbers of saturated receptors. The identification of junctional currents with a particular varicosity is greatly assisted by this method of recording with large-diameter electrodes. In this case, junctional currents arising from varicosites near the rim of the electrode give rise to much larger recorded potential responses than the same size junctional currents recorded near the center of the electrode (5).
Recent Monte Carlo simulations of the action of evoked transmitter on receptors after it has been released from a varicosity do not support the idea that different-sized receptor patches are likely to give rise to markedly differently shaped junctional currents (3). These simulations do support the possibility that the release of a packet of transmitter from varicosities at different distances from the receptor patch will give rise to characteristically different amplitude and time course junctional currents. The distance between the closest region of apposition of the prejunctional membrane of most varicosities and the postjunctional smooth muscle membrane varies from 20 nm up to ~100 nm. If the release of a packet of transmitter occurs at these regions of closest apposition, there will be considerable variation in the amplitude and time course of the junctional currents generated at different varicosities (3). The ability to match evoked and spontaneous junctional currents might then arise from the release of transmitter from varicosities at a particular distance from a receptor patch that is then saturated by the transmitter.
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Transmitter release at varicosities with relatively large numbers of receptors
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At many varicosities neither spontaneous nor evoked release of packets of transmitter gives rise to amplitude-frequency histograms of junctional currents that can be described by a Gaussian distribution. Rather, very positively skewed histograms, with some very large junctional currents, are observed (Ref. 1; Fig. 1A
). In this case, the receptors beneath the varicosity are not saturated by most of the packets of transmitter released. One possible explanation for these skewed distributions is that transmitter is released in uniform packets or quanta and that there is multiquantal release of transmitter from the varicosities. If synaptic vesicles are randomly distributed in the presynaptic membrane of these varicosities, together with the channels through which calcium must enter to trigger the release of a packet of transmitter in vesicles, spontaneous multiquantal release would be expected to occur (6). Another possibility is that transmitter is not released in quanta at all but that the large variation in junctional currents arises from large differences in the amount of transmitter that is packaged for release.
Tests are now available for determining whether the positively skewed histograms of junctional currents are most likely derived from several transmitter packets of uniform size (multiquantal release) or from single packets with variable amounts of transmitter. This involves determining whether a single unimodal distribution with a long tail best fits the histograms of spontaneous junctional currents recorded at a single varicosity (the nonuniform size packets hypothesis) or whether they are best fitted by a mixture distribution (the multiquantal hypothesis). The simplest mixture distribution is a Poisson mixture of Gaussians like that first introduced to analyze the histograms of evoked currents recorded at the somatic neuromuscular junction. If this distribution best describes the histogram of spontaneous currents at varicosities, then the inference is that release is multiquantal. However, if distributions with a long tail, such as a gamma distribution, provide the best fit, then it is likely that the released packages contain highly nonuniform amounts of transmitter. Analysis of the histograms of spontaneous junctional currents recorded at single varicosities gives equivocal results at this time: it is uncertain whether the mixture distributions are superior to the unimodal distributions (7). For the present then, it is not clear if the long-tailed histograms of junctional currents at single varicosities arise from multiple release of uniform transmitter packets or from nonuniform transmitter packets.
Other evidence exists for the idea of multiquantal releases from single varicosities. Intracellular recordings of junction potentials provide evidence for both spontaneous and evoked multiquantal release from varicosities. Recordings of these potentials have been made in high-input-resistance smooth muscle cells. Such cells are likely to have very few couplings with other cells in the smooth muscle syncytium; they may therefore be considered as innervated muscle cells in relative electrical isolation from the other cells in the syncytium. In this case the amplitude-frequency histogram of spontaneous junction potentials is multimodal, as is the histogram of evoked junction potentials; furthermore, the modes in both histograms occur at the same amplitudes (9). This provides direct evidence for the existence of multiquantal spontaneous release, if it is accepted that two or more varicosities do not each synchronously release single quanta spontaneously.
Intracellular recordings from muscle cells that do not have a high-input impedance reveal that the rate of rise of the junction potential fluctuates from relatively low to very high. Differentiation of these potentials reveals that they are composed of a relatively large and fast component, which occurs stochastically, superimposed on a constant-size slow component. Analysis of these indicates that the fast component arises from a large conductance change in the impaled cell, whereas the slow component is due to the additive effects of many small conductance changes in the cell that occur with considerable temporal dispersion (Ref. 4; Fig. 3). One interpretation of this is that the large conductance change is due to the release of transmitter from a site on a varicosity that is in particularly close apposition to the smooth muscle cell, whereas the many small conductance changes are due to transmitter release from sites on varicosities that are at much greater distances. Multimodal amplitude-frequency histograms of the fast differential are often observed for both evoked transmission and spontaneous transmission (9). These modes appear to occur at multiples of the smallest mode and have therefore been taken as evidence for multiquantal release from varicosities.
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FIGURE 3. Time derivatives of experimental excitatory junction potential (EJP; A) compared with that generated in a model of the smooth muscle syncytium for which there was a particular distribution of varicosities (B). A: intracellular records of rising phase of EJP in a smooth muscle cell in guinea pig vas deferens (noiseless trace, i) as well as time derivative of the EJP (noisy trace, ii). EJPs were evoked by trains of submaximal stimuli (0.91 Hz) to the postganglionic hypogastric nerves; successive records of EJP are shown after facilitation was complete. [Records are from Fig. 1 (II), Blakely and Cunnane (8).] B: model results for effect of probabilistic secretion of transmitter from both close-contact and loose-contact varicosities on the EJP and its time derivative (DEJP). Every cell in the model syncytium received an innervation from a close-contact varicosity with a probability for secretion drawn from a ß-distribution with parameters (1,50), giving rise to a conductance value drawn from a  -distribution with a mean 30 ns after a delay drawn from an exponential distribution with mean of 30 ms. Each cell in the syncytium also received an innervation from 20 loose-contact varicosities, each of which secreted with a probability drawn from a ß-distribution with parameters (2,3) after a delay drawn from an exponential distribution with a mean of 30 ms; 10 loose-contact varicosities produced a conductance change drawn from a -distribution with mean 0.1 ns and the other 10 from a -distribution with parameters (120,3). B: results for 4 different impulses to cell (6, 3, –3) in the syncytium [from Bennett and Gibson (4).]
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The question arises as to what may determine differences in the number of receptors beneath varicosities. A possible answer to this is provided by recent observations on the receptor density beneath different boutons in cultures of hippocampal neurons (14). In this case, the receptors appear to be in relatively small numbers beneath boutons on densely innervated neurons, but in much higher density beneath boutons on neurons that receive a sparse innervation. The same condition might apply for smooth muscle cells that receive very different densities of innervation from varicosities.
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Nonuniform probability for transmitter release at different varicosities
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Another form of heterogeneity between varicosities is that they have very different probabilities for the evoked release of a packet of transmitter, even along the length of the same nerve terminal branch. Placing a loose-patch electrode sequentially over neighboring varicosities along a single terminal branch and recording the electrical signs of evoked transmitter release from each of these in turn during a long train of impulses shows that some varicosities give very little, if any, evoked release, whereas others release frequently (1). The vesicle-associated protein synaptotagmin appears to be the calcium sensor in the nerve terminal that triggers transmitter release on an influx of calcium ions. Studies on the development of the synaptotagmin molecule in both vertebrate and invertebrate synapses show that the mature molecule is missing at early stages of synapse formation and that at this time there is about a twofold dependence of transmitter release on calcium; with the appearance of the mature molecule, a fourfold dependence of release on calcium is established. One possibility then is that some varicosities of sympathetic nerve terminals do not possess the mature form of synaptotagmin, so that there is a relatively low dependence of secretion on calcium, whereas others do possess the mature molecule and the ususal fourfold dependence on calcium is present. However, determination of the calcium sensitivity of release at individual varicosities with a loose-patch electrode shows that there is a fourfold dependence of release on the external calcium concentration at varicosities with both low and high probabilities for the evoked release of transmitter (15). Although this indicates that differences in the probability for evoked release do not seem to be associated with differences in the power relationship between calcium and synaptotagmin, the variation in the affinity of this protein for calcium that has been observed during development may occur between different sympathetic varicosities.
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Conclusion
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A wide range of experimental observations on transmission at sympathetic nerve varicosities indicates that different size nerve packets of transmitter may be released from varicosities with different probabilities on arrival of the nerve impulse and that these varicosities possess different numbers of postjunctional receptors (Fig. 4). A survey of transmission at other synapses in the peripheral and central nervous system suggests that such variability might be the norm rather than the exception (2), a conjecture that is supported by recent recordings from single boutons on hippocampal pyramidal neurons (14).
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FIGURE 4. Model of the conventional hypothesis (A) and nonuniform hypothesis (B) for transmission at single varicosities. A, top: recordings made with loose-patch electrodes over each of 2 varicosites (1 and 2). Bottom: junctional currents generated spontaneously or evoked by impulses; dots surrounding varicosities indicate extent of spread of receptor patch associated with each varicosity. Note that all varicosities have large receptor patches that are not saturated by the packets of transmitter released from overlying varicosities; electrical signs of both spontaneous and evoked release indicate that uniform size packets of transmitter are released and that, at most, one packet is released by an impulse. Furthermore frequency of successful evoked release of these quanta at varicosities 1 and 2 is about the same, showing that probability of release is the same for each varicosity. B, top: recordings made with loose-patch electrodes over each of 2 varicosites (1 and 2). Bottom: junctional currents generated spontaneously or evoked by impulses; dots surrounding varicosities indicate extent of spread of receptor patch associated with each varicosity. Some varicosities (e.g., 1) have large receptor patches that are not then saturated by packets of transmitter released from the overlying varicosity, whereas other varicosities (e.g., 2) have very small receptor patches that do not extend from beneath the varicosity. Electrical signs of both spontaneous and evoked release indicate that non-uniform-size packets of transmitter are released and can be measured at varicosities with large receptor patches (1), while at other varicosities the receptor patch is so small it is saturated by the released transmitter (varicosity 2), and so both spontaneous and evoked junctional currents are of the same size. Note that the frequency of successful evoked release at varicosities 1 and 2 is different, showing that there is nonuniform probability of release at different varicosities. [Reprinted from Prog. Neurol. 50, M. Bennett, Autonomic neuromuscular transmission at a varicosity, p. 505–532, © 1996, with permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington OX5 1GB, UK.]
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References
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Bennett, M. R. Quantal secretion from single visualized synaptic varicosities of sympathetic nerve terminals. In: Molecular and Cellular Mechanisms of Neurotransmitter Release, edited by L. Stjarne, P. Greengard, S. Grillner, T. Hokfelt, and D. Ottoson. New York: Raven, 1994, p. 399–423.
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Bennett, M. R. The origin of Gaussian distributions of synaptic potentials. Prog. Neurobiol. 46: 331–350, 1995.[Medline]
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Bennett, M. R., L. Farnell, W. G. Gibson, and S. Karunanithi. Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors. Biophys. J. 68: 925–935, 1995.[Abstract/Free Full Text]
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Bennett, M. R., and W. G. Gibson. On the contribution of quantal secretion from close-contact and loose-contact varicosities to the synaptic potentials in the vas deferens. Philos. Trans. R. Soc. Lond. B Biol. Sci. 347: 187–204, 1995.[Medline]
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Bennett, M. R., W. G. Gibson, and R. R. Poznanski. Extracellular current flow and potential during quantal transmission from varicosities in a smooth muscle syncytium. Philos. Trans. R. Soc. Lond. B Biol. Sci. 342: 89–99, 1993.[Medline]
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Bennett, M. R., W. G. Gibson, and J. Robinson. Probabilistic secretion of quanta: spontaneous release at active zones of varicosities, boutons and endplates. Biophys. J. 69: 42–56, 1995.[Abstract/Free Full Text]
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Bennett, M. R., J. Robinson, M. C. Phipps, S. Karunanithi, Y. Q. Lin, and L. Cottee. Quantal components of spontaneous excitatory junction potentials at visualized varicosities. J. Auton. Nerv. Syst. 56: 161–174, 1995.
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Blakeley, A. G. H., and T. C. Cunnane. The packeted release of transmitter from the sympathetic nerves of the guinea-pig vas deferens: an electrophysiological study. J. Physiol. (Lond.) 296: 85–96, 1979.
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Blakeley, A. G. H., P. M. Dunn, and S. A. Petersen. Properties of excitatory junction potentials and currents in smooth muscle cells of the mouse vas deferens. J. Auton. Nerv. Syst. 27: 47–56, 1989.[Medline]
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Brock, J. A., and T. C. Cunnane. Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens. J. Physiol. (Lond.) 399: 607–632, 1988.[Abstract/Free Full Text]
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Cunnane, T. C., and L. Stjarne. Transmitter secretion from individual varicosities of guinea-pig and mouse vas deferens: highly intermittent and monoquantal. Neuroscience 13: 1–20, 1984.[Medline]
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Karunanithi, S., M. C. Phipps, J. Robinson, and M. R. Bennett. Statistics of quantal secretion during long trains of sympathetic nerve impulses in mouse vas deferens. J. Physiol. (Lond.) 489: 171–181, 1995.[Medline]
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Lavidis, N. A., and M. R. Bennett. Probabilistic secretion of quanta from visualized sympathetic nerve varicosities in mouse vas deferens. J. Physiol. (Lond.) 454: 9–26, 1992.[Abstract/Free Full Text]
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Liu, G., and R. W. Tsien. Properties of synaptic transmission at single hippocampal synaptic boutons. Nature 375: 404–408, 1995.[Medline]
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Macleod, G. T., N. A. Lavidis, and M. R. Bennett. Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens. J. Physiol. (Lond.) 490: 61–70, 1994.
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Introduction
Transmitter release at...
