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. 1999 Oct 15;520(Pt 2):527–537. doi: 10.1111/j.1469-7793.1999.00527.x

Quantal evoked depolarizations underlying the excitatory junction potential of the guinea-pig isolated vas deferens

Rohit Manchanda 1, K Venkateswarlu 1
PMCID: PMC2269600  PMID: 10523420

Abstract

  1. The effects of a putative gap junction uncoupling agent, heptanol, on the intracellularly recorded junction potentials of the guinea-pig isolated vas deferens have been investigated.

  2. After the stimulation-evoked excitatory junction potentials (EJPs) had been suppressed by heptanol (2.0 mm) to undetectable levels, a different pattern of evoked activity ensued. This consisted of transient depolarizations that were similar to EJPs in being stimulus locked and in occurring at a fixed latency, but differed from EJPs in that they occurred intermittently and had considerably briefer time courses.

  3. Analysis of the amplitudes and temporal parameters of the rapid residual depolarizations revealed a close similarity with spontaneous EJPs (SEJPs). There was no statistically significant difference between the rise times, time constants of decay and durations of the rapid residual depolarizations and of SEJPs.

  4. Selected evoked depolarizations were virtually identical to SEJPs occurring in the same cell. Evoked depolarizations of closely similar amplitude and time course also occurred, usually within a few stimuli of each other.

  5. These depolarizations appear to represent the individual quantal depolarizations that normally underlie the EJP and are therefore termed ‘quantal excitatory junction potentials’ (QEJPs) to distinguish them from both EJPs and SEJPs.

  6. We examined the possibility that heptanol revealed QEJPs by disrupting electrical coupling between cells in the smooth muscle syncytium. Heptanol (2.0 mm) had no effect on the amplitude distribution, time courses, or the frequency of occurrence of SEJPs. Intracellular input impedance (Rin) of smooth muscle cells was left unaltered by heptanol.

  7. ‘Cable’ potentials of the vas deferens, recorded using the partition stimulation method, also remained unchanged in the presence of heptanol. Thus, heptanol appeared not to modify syncytial electrical properties of the smooth muscle in any significant way.

  8. Our observations show directly that the quantal depolarizations underlying the EJP in syncytial smooth muscle are SEJP-like events. However, no unequivocal statement can be made about the mechanism by which heptanol unmasks QEJPs from EJPs.

According to the classic ‘quantal hypothesis’ of transmitter action at synapses, the intracellularly recorded spontaneous postjunctional potential represents an irreducible unit of transmitter action, and the evoked potential the summation of the effects of one to several quanta, as observed at neuronal synapses and at the skeletal neuromuscular junction (del Castillo & Katz, 1954; Martin, 1977). At autonomic neuromuscular junctions (ANJs) in smooth muscle organs such as the mammalian vas deferens, however, whether the spontaneous excitatory junction potential (SEJP) is the basic quantal unit of the evoked excitatory junction potential (EJP) has been less clear (Brock & Cunnane, 1992; Manchanda, 1995). This is because of certain problematic features of these junction potentials: (1) the peak amplitudes of SEJPs and EJPs fall in the same range, the EJP is not an integral multiple of the SEJP; (2) the EJP is up to ten times more prolonged than the SEJP (Burnstock & Holman, 1961, 1962). On both grounds, the construction of the intracellular EJP in terms of a multi-quantal SEJP is not obvious. In the past a range of indirect lines of evidence, both experimental and theoretical, have prompted the suggestion that an SEJP-like event occurring during the rising phase of the EJP may in fact constitute the quantal depolarization underlying the EJP, but is obscured by the more prolonged evoked depolarization (Tomita, 1970; Purves, 1976; Blakely & Cunnane, 1979; Bywater & Taylor, 1980; Cunnane & Manchanda, 1989, 1990). However, a stimulus-locked depolarization possessing the time course of the SEJP has not been recorded experimentally, leaving this hypothesis unsubstantiated.

We have recently investigated the effects on EJPs of a presumptive gap junction blocking agent, 1-heptanol (Christ, 1995), with a view to determining the influence of intercellular electrical coupling on smooth muscle junction potentials. Heptanol abolished rapidly and reversibly the EJP of the guinea-pig vas deferens (Manchanda & Venkateswarlu, 1997). However, on close inspection of individual records it was noticed that although the prolonged EJP was suppressed, an unusual pattern of stimulus-locked activity continued to occur in the presence of heptanol. This pattern was characterized by the intermittent occurrence of brief depolarizations that resembled SEJPs in several of their properties, suggesting that they were quantal in nature. On analysis, they seemed to provide the first direct evidence that the individual unitary evoked depolarization underlying the EJP recorded intracellularly is indeed an SEJP-like event. Our findings help clarify certain features of the electrical behaviour of the smooth muscle syncytium during neurotransmission.

Here we present an account of these observations along with others that concern the mechanism of action of heptanol. As first steps towards elucidating this mechanism, we have analysed the properties of SEJPs, and examined intracellular input impedance and cable potentials in the presence of heptanol. Our results suggest that mechanisms other than cell-to-cell electrical uncoupling may need to be invoked in order to explain consistently the range of observations in the presence of heptanol, and we discuss the implications of these findings.

METHODS

Intracellular and extracellular recording

The preparation, apparatus and procedures for electrophysiological recordings were essentially as described previously (Cunnane & Manchanda, 1990; Manchanda & Venkateswarlu, 1997) and need not be elaborated here except to indicate points of departure in methodology. Briefly, male guinea-pigs weighing 350-600 g were killed by stunning and exsanguination in accordance with national guidelines, and both vasa dissected out through a mid-line incision in the abdomen. EJPs were recorded using intracellular glass capillary microelectrodes with tip impedances of 40-60 MΩ interfaced with a high input impedance electrometer (IE 201, Warner Instruments, USA). Focal extracellular recordings of nerve action potentials were made conventionally (Cunnane & Stjärne, 1984a), using glass microelectrodes with tips broken back to diameters of 20-80 μm and filled with normal Krebs solution, the signals being led to an AC amplifier. A small branch of the vas deferens nerve was dissected free of attachments and drawn into the electrode with gentle suction. The impulse of a single unit was isolated from clusters of a few by fine adjustment of stimulation strength and verification of the all-or-none nature of the impulse. The output of the amplifiers was observed on an oscilloscope (TDS 310, Tektronix, USA) and stored simultaneously on a digital tape recorder (DTR-1204, Biologic, Claix, France).

Experiments were carried out at 36-37°C. Stimuli supramaximal for EJPs were delivered to the hypogastric nerve at 0.7 Hz using rectangular voltage pulses (amplitude 6-10 V, pulse width 0.05-0.5 ms) via bipolar Ag-AgCl ring electrodes placed 1-3 cm proximal to the point of nerve entry. Two millimolar heptanol solution was used in all experiments, as heptanol appears to act specifically and optimally on EJPs at this concentration (Manchanda & Venkateswarlu, 1997; Venkateswarlu et al. 1999; see also Christ, 1995).

Input impedance (Rin) was measured using the method of simultaneous current injection and voltage recording using a single microelectrode. Estimation of the absolute value of Rin of smooth muscle cells by this method is problematic; however, our emphasis in these studies was to detect changes, if any, of Rin in individual cells during the action of heptanol. Accordingly, measurements were accepted for analysis only if (a) microelectrode insertion was maintained before and during the application of heptanol solution, while measurements of Rin were simultaneously made and (b) microelectrode impedance varied by less than 10 % before insertion and after withdrawal from the cell, so as to prevent contributions from changes in tip impedance to the intracellularly recorded impedance (Purves, 1981). Inhibition of EJPs was used as an index of heptanol action.

Cable potentials

Cable potentials of the smooth muscle of the vas deferens were recorded using the plate-partition stimulation method of Abe & Tomita (Tomita, 1967, 1970; Bywater & Taylor, 1980), which appears to impart one-dimensional cable properties along the axial direction of smooth muscle. The Ag-AgCl stimulation plate (thickness 250 μm) was insulated with Araldite (Ciba-Geigy) on the side facing the recording compartment. The quality of electrical insulation between stimulation and recording compartments was tested by recording electrotonic potentials in the extracellular fluid after the vas had been drawn gently through the aperture in the stimulation plate. In the recordings accepted for analysis, the drops in extracellular potential were negligible in amplitude (0.1-0.2 mV) compared with the amplitudes of intracellularly recorded cable potentials (2-10 mV), indicating insignificant leakage of current into the extracellular compartment. In addition, intracellular cable potentials were accepted for analysis only if they were uncontaminated by resistive potential drops across the series resistance of the extracellular fluid. The electrical constants of the tissue (e.g. time constant and length constant) were not evaluated, since our emphasis was on seeing whether cable potentials recorded at any particular site, and preferably in the same cell, were altered by heptanol.

Care was taken to place at least five length constants (∼4-5 mm, see Bywater & Taylor, 1980) of the tissue in the stimulation compartment so as to avoid effects due to ‘reflection’ of injected current (Bywater & Taylor, 1980). Distances were measured using a calibrated reticle affixed to the eyepiece of a binocular microscope. To minimize electrode polarization, firstly, the stimulation plate was freshly chloridized at the start of each experiment. Secondly, alternating sets of depolarizing and hyperpolarizing currents were delivered, each set comprising four to eight rectangular pulses (duration 750 ms to 1.2 s). Some degree of polarization was evident in a few recordings in which the steady-state value of the cable potential failed to be maintained; these records were not included for analysis.

Data collection and analysis

Electrophysiological signals were filtered (low pass, -3 dB cutoff 1 kHz), amplified as required, and collected as described previously (Manchanda & Venkateswarlu, 1997). Data collection and automated analysis on computer was done with the help of standard routines available in the customized software used (SCAN; Dempster, 1993). In the present work, the most critical element of analysis is the comparison between spontaneously occurring and stimulation-evoked events (SEJPs and QEJPs, respectively) of an apparently similar nature. We therefore rejected events that were contaminated by distortions and artifacts that might confound the analysis. The time courses of QEJPs that occurred while the EJP was not fully abolished were distorted by the residual background depolarization. In such cases the background depolarization was algebraically subtracted from the net depolarization, and the resulting record used for analysis (see Fig. 4). Similarly, only those SEJPs were accepted for analysis which were free of modulating drift. All parameters returned by automated analysis were cross checked by careful visual examination of the signals. Data are expressed as means ±s.e.m. (no. of observations). Student's unpaired t test was used to assess the statistical significance of differences between means, with P < 0.05 being taken to indicate a statistically significant difference.

Figure 4. Extraction and properties of QEJPs.

Figure 4

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Aa, superimposed traces of background EJP alone after inhibition by heptanol (R) and background EJP with a QEJP (R + Q). Ab, QEJP alone (Q), obtained after subtraction of R from R + Q in a. B, two pairs of apparently identical QEJPs superimposed, each pair drawn from a different cell (a and b). Ca and b, two examples of a QEJP (with stimulus artifact) and an SEJP that matched closely, superimposed. Each pair of signals recorded from the same cell.

RESULTS

Emergence of QEJPs in the presence of heptanol

In all cells investigated (n= 45) the application of 2.0 mm heptanol resulted in rapid, reversible reduction of the amplitude of the EJP without change of resting membrane potential, which ranged between -60 and -70 mV (Manchanda & Venkateswarlu, 1997). In about 25 % of the cells, when the EJP had been suppressed to less than 70 % of its peak control amplitude, the pattern of evoked activity took on a strikingly different appearance. Figure 1A shows an example of the gradual replacement of the EJP with this pattern. After ∼70 % inhibition, the EJP consisted of a low-amplitude background depolarization superimposed on which there occurred, intermittently during the initial part of the EJP, stimulus-locked depolarizations that developed and decayed relatively rapidly (Fig. 1Ac). These rapid initial depolarizations, whose falling phases were interrupted and modified by the more slowly decaying background depolarization, seemed similar in shape and time course to SEJPs, and varied in configuration from one event to the next (Fig. 1Ac).

Figure 1. Emergence of rapid evoked depolarizations (QEJPs) following suppression of EJPs by heptanol.

Figure 1

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Three records superimposed in each panel. A, control EJPs (a) and EJPs inhibited to ≈50 (b), 25 (c) and 10 % (d) of control peak amplitude. B, records from another cell showing control EJPs (a) and QEJPs after EJP suppression (b). *, QEJPs; •, SEJPs; S, stimulation artifact.

Once inhibition of the EJP by heptanol was almost complete (Fig. 1Ad and B b) virtually no background depolarization remained (< 1 mV). At this point the resolution of the transient depolarizations improved (Fig. 1, asterisks), and a conspicuous resemblance to SEJPs (Fig. 1, filled circles) emerged. For this reason, and others to be elaborated below, these events will henceforth be referred to as ‘quantal EJPs’ (QEJPs).

Following suppression of the EJP, the peak amplitudes of QEJPs displayed prominent event-to-event variation, more markedly so than did control EJPs. After the EJP had declined to negligible levels, evoked activity (in the form of QEJPs) appeared as a series of success-or-failure events (Fig. 2, record no. 37 onwards). They were thus reminiscent of skeletal muscle end-plate potentials at low probabilities of transmitter release (Liley, 1956), but differed from the latter in that the distribution of QEJP amplitudes was not Gaussian but positively skewed, as for SEJPs in smooth muscle (not shown).

Figure 2. Effect of heptanol on EJP amplitudes.

Figure 2

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Peak EJP amplitudes in control solution (▵) and after 2.0 mm heptanol application (3.5 min, □), i.e. during emergence of QEJPs. Note the marked variation in amplitudes of QEJPs compared with EJPs, and the intermittent failures (record 37 onwards).

In all cells in which QEJPs were observed (n= 11), they were characterized in addition by the following properties. (i) In each cell, they were confined to a relatively narrow latency band that fell within the wider band for EJPs. In the cell represented in Fig. 1A, control EJPs arose at latencies of 30-45 ms (34.1 ± 0.55 ms, n= 30), whereas QEJPs had latencies of 39-43 ms (41.7 ± 0.4 ms, n= 14). These latencies are displayed on an expanded time scale in Fig. 3A. The relative latencies of QEJPs and EJPs indicate that QEJPs probably reflect pick-up of the responses to a subset of the prejunctional axons that generate the EJP. To verify active invasion of prejunctional axons the single-unit action potential was recorded extracellularly directly from branches of the vas deferens nerve. Recordings were made from regions of the tissue similar to those used for intracellular recordings. The impulse persisted unchanged in the presence of heptanol, in each of the six trials conducted in four preparations (Fig. 3B). A small increase in latency of the impulse (∼1 ms) was occasionally noticeable; however, this can be attributed entirely to the ‘walking’ of the axonal action potential during continuous stimulation over comparable periods (Cunnane & Stjärne, 1984a), and is therefore probably unrelated to the actions of heptanol.

Figure 3. Effect of heptanol on latencies of EJPs.

Figure 3

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A, latencies of EJPs (a) and QEJPs (b and c). QEJPs (*) shown with associated intervening ‘failures’ (arrows). B, the nerve terminal impulse from a single postganglionic nerve fibre before and after heptanol application. Each trace is an average of 25 records.

To rule out the possibility that QEJPs were just SEJPs occurring coincidentally at a particular latency, the hypogastric nerve was stimulated at an intensity subthreshold for the generation of EJPs. Responses were monitored in cells in which SEJPs occurred at normal frequencies. When ‘blank’ stimulation of this kind was delivered, in three trials with 300 stimuli each (at 0.7 Hz), not a single stimulus was followed by an SEJP-like depolarization within the latency band of the EJP, although 33, 85 and 110 SEJPs occurred in the respective trials during this period. It was therefore reasonable to identify QEJPs on the basis of their fixed latencies.

(ii) QEJPs occurred intermittently (Figs 1 and 3Ab and Ac). After the EJP was suppressed by more than 90 %, in two cells which lent themselves to detailed analysis (henceforth referred to as ‘Cell 1′ and ‘Cell 2′), QEJPs were elicited by only 14 of 100 stimuli in Cell 1, and by 16 of 110 stimuli in Cell 2. Considerably lower probabilities of occurrence (down to ∼0.01) were also observed in other cells. These levels of intermittence fall within the range of those of ‘discrete events’ (DEs; Cunnane & Stjärne, 1984b) or of extracellularly recorded excitatory junction currents in the vas deferens (EJCs; Brock & Cunnane, 1988; Lavidis & Bennett, 1992), both of which represent secretory activity from a local population of release sites.

(iii) The decay phases of QEJPs that rode atop residual background EJPs were modified noticeably by the slower background depolarization (Fig. 4Aa). Algebraic subtraction of the latter from the former yielded the true time course of the QEJP (Fig. 4Ab), which was used for detailed analysis (see Methods).

(iv) Amplitudes and time courses of QEJPs varied from one event to another. Occasionally, however, one QEJP was followed within a few stimuli by an apparently identical one, within the limits of resolution imposed by recording noise (Fig. 4B).

(v) The rise times, time constants of decay and total durations of QEJPs were not significantly different when compared statistically to those of SEJPs recorded in the same cell (Table 1).

Table 1. Comparison of SEJPs and QEJPs in two cells.

Cell 1 Cell 2
Rise time τdecay Duration Rise time τdecay Duration
SEJPs 11.7 ± 0.9 (18) 30.2 ± 1.2 (11) 89.4 ± 4.2 (11) 15.0 ± 0.6 (30) 40.3 ± 2.2 (18) 126.4 ± 4.1 (18)
QEJPs 13.2 ± 0.8 (14) 28.1 ± 2.9 (10) 91.5 ± 5.1 (10) 15.7 ± 1.2 (16) 34.1 ± 2.3 (9) 137.1 ± 4.2 (9)
P 0.29 0.45 0.75 0.59 0.09 0.12
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All values (except P) expressed in milliseconds and given as means ±s.e.m. with number of observations in parentheses. The number of observations for τdecay and duration are fewer than for rise time because the decays of some events could not be analysed satisfactorily due to contamination by noise.

(vi) Certain QEJPs could be matched very closely with SEJPs occurring in the same cell. Figure 4C shows two such examples, taken from different cells; in each the superimposed QEJP and SEJP are so alike as to be scarcely differentiable.

Three of the features of QEJPs enumerated above, i.e. intermittency, closely spaced occurrence of the same event, and the accurate match between an evoked and a spontaneous event, are characteristic also of DEs and EJCs. These latter signals are believed to represent quantal transmitter release events from the autonomic innervation (Cunnane & Stjärne, 1984b; Brock & Cunnane 1988, 1992), hence our choice of the term ‘quantal EJPs’ to describe the rapid evoked depolarizations observed in the presence of heptanol.

To see if the emergence of QEJPs in the presence of heptanol could be attributed to its well-known intercellular uncoupling actions (Christ, 1995; Bastide et al. 1995), we studied the effects of heptanol on SEJPs, Rin and cable potentials, since the properties of these signals and parameters are dictated by the degree of syncytial coupling (Purves, 1976; Manchanda, 1995).

SEJPs and Rin in the presence of heptanol

Heptanol (2.0 mm) did not affect any of the properties of SEJPs in any experiment (n= 45). The amplitude distribution of SEJPs in the presence of heptanol remained typically skewed towards the low amplitude events, as in control Krebs solution (Fig. 5A). These data have been pooled from five cells, including Cell 1 and Cell 2. The insets in Fig. 5A, in which traces of SEJPs recorded in control and heptanol solutions are displayed, show that the configurations of SEJPs also remained unchanged in the presence of heptanol. Statistical analysis of the temporal parameters of SEJPs confirmed that they were not changed significantly by heptanol, as shown by the data in Table 2 for Cell 1 and Cell 2.

Figure 5. Properties of SEJPs in the presence of heptanol.

Figure 5

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A, amplitude histograms of SEJPs in control (left) and 2.0 mm heptanol solution (right) (data pooled from 5 cells). Insets, examples of SEJPs recorded in these solutions (4-5 traces superimposed in each). B, frequency of occurrence of SEJPs before (left) and during (right) the action of heptanol, in two trials in separate cells (a and b). Each bin is a 50 s observation period in a, and a 30 s period in b.

Table 2. Temporal parameters of SEJPs before and during application of heptanol.

Rise time τdecay Duration
Control 14.2 ± 0.7 (27) 35.3 ± 1.7 (16) 106.6 ± 4.7 (16)
Heptanol 13.3 ± 1.0 (21) 37.8 ± 3.0 (14) 117.4 ± 7.8 (14)
P 0.43 0.48 0.23
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All values (except P) expressed in milliseconds and given as means ±s.e.m. with number of observations in parentheses. Data obtained from a single cell.

Figure 5B shows two pairs of time series histograms for the frequency of occurrence of SEJPs in normal Krebs solution and in the presence of 2.0 mm heptanol, each pair obtained from a different cell. There was no evidence of a decrease in the rate of occurrence of SEJPs due to heptanol; if anything, a slight increase appeared to have taken place in the cell represented in Fig. 5B b.

Rin was measured while the intracellular insertion was maintained during heptanol action (verified by observing the concomitant abolition of the EJP). Satisfactory data (see Methods) were obtained in eight cells from five preparations. Rin fell in the range 5-24 MΩ, similar to that reported earlier for the guinea-pig vas deferens (Bennett, 1967; Holman et al. 1977). Of the eight cells, five showed no consistent effect of heptanol; Rin either remained steady or fluctuated about a mean level during heptanol action. In a further two cells, Rin increased in the presence of heptanol but the increase was not reversible on washout, indicating a non-specific change. Furthermore, there was no difference in the amplitudes of EJPs in these cells after washout compared with those before application of heptanol. An increase of Rin in the presence of heptanol, which was also reversible on washout, was observed in only one cell. Taken together, these data fail to show any consistent effect of heptanol on Rin.

Cable potentials

Decisive observations on cable potentials were obtained in eight trials conducted in a total of six cells (four preparations). In these trials, it was possible to retain the intracellular insertion during the application of heptanol (and in five of these also during its washout) while cable potentials were recorded. Intracellular recordings were carried out at distances of between 200 and 1300 μm from the edge of the stimulation plate in the recording compartment.

In all trials but two, cable potentials failed to be suppressed or otherwise altered by heptanol at a concentration (2.0 mm) and duration of exposure (3-5 min) that were invariably sufficient to abolish EJPs fully, and reveal QEJPs, under the same conditions. Relatively prolonged exposure to heptanol (up to 10 min) in two trials also failed to affect cable potentials. This was the case regardless of the distance from the stimulation plate at which cable potentials were recorded. Figure 6 shows representative examples of cable potentials recorded in one such trial, carried out during a single insertion, before and during the application of 2.0 mm heptanol. These potentials were recorded 1300 μm from the site of current injection, i.e. at a distance greater than about three average cell lengths (Merrillees et al. 1963; Merrillees, 1968) from the stimulation plate. It is evident that the cable potentials in the absence and presence of heptanol are very similar, both in steady-state amplitude and in time course of polarization and repolarization, indicating a lack of effect of heptanol on current flow between smooth muscle cells. In one of the eight trials, the steady-state amplitude of cable potentials was significantly enhanced, by about 15 %, by heptanol, while in the remaining trial the amplitude was suppressed, by about 20 %. In sum, therefore, we failed to find any evidence that heptanol affected the cell-to-cell spread of current in guinea-pig vas deferens.

Figure 6. Absence of effect of heptanol on cable potentials of the guinea-pig vas deferens.

Figure 6

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A, depolarizing potentials. B, hyperpolarizing potentials. Each trace is an average of three to four records. The potentials were obtained at a distance of ≈1300 μm from the plane of stimulation.

DISCUSSION

Two main points arise on considering the results presented here. The first is the significance of the observation of QEJPs in relation to the nature of the quantal depolarization underlying the EJP in smooth muscle. The second is the explanation of the range of observations in the presence of heptanol, in relation to its proposed intercellular uncoupling actions (Christ, 1995).

The nature of quantal EJPs

In the smooth muscle of the vas deferens and other organs, the SEJP is believed to be the basic quantal electrical event recorded intracellularly. SEJPs are presumed to be generated by individual packets of neurotransmitter randomly secreted from release sites that are electrically close to the site of recording, probably close-contact varicosities (CCVs) impinging either on the penetrated cell or on close neighbours. The EJP in contrast follows nerve stimulation and is generated by the action of transmitter released within a brief latency band from numerous release sites spatially distributed throughout the ground plexus (Cunnane & Stjärne, 1984b; Cunnane & Manchanda, 1990; Bennett & Gibson, 1995).

Because of the difference in time courses of the EJP and SEJP and the similarity of their ranges of amplitudes, the precise quantal relation between these events has resisted elucidation. However, it has been inferred, on the following lines of evidence, that the unitary intracellular depolarization underlying the EJP may be an SEJP-like event. (1) Stimulation-evoked discrete events (DEs), which serve as electrical fingerprints of evoked quantal release (Cunnane & Stjärne, 1984b) can be matched closely with selected spontaneous DEs in the same cell. (2) Quantal evoked excitatory junction currents (EJCs) recorded extracellularly, which underlie the EJP, have time courses similar to SEJPs (Brock & Cunnane, 1988; Cunnane & Manchanda, 1989, 1990). (3) Theoretical work suggests that a junctional current comparable in time course to an SEJP can generate, under conditions appropriate for multi-point transmitter action, a membrane potential change that resembles an EJP (Purves, 1976; Cunnane & Manchanda, 1990; Bennett & Gibson, 1995).

Though these arguments are compelling, they have not yet been confirmed with intracellular recording. By using a putative intercellular uncoupling agent, heptanol, we have found that it is possible directly to observe the electrical events that are apparently the quantal depolarizations underpinning evoked activity. The depolarizations thus detected were characterized by a novel amalgamation of the properties of various types of electrical activity previously recorded, i.e. EJPs (stimulus locking), DEs and EJCs (stimulus locking, intermittence and closely spaced repetition), and SEJPs (time courses and amplitudes). Their close correspondence to the signs of quantal release, the evoked DEs and EJCs (Cunnane & Stjärne, 1984b,Brock & Cunnane, 1988), leads us to believe that QEJPs reflect evoked quantal release events occurring in the electrical vicinity of the recording microelectrode.

In time course, QEJPs were indistinguishable from SEJPs. Our observations therefore support the hypothesis that a depolarization of the time course of the SEJP is the basic unit of evoked quantal transmitter action at the autonomic neuroeffector junction of the guinea-pig vas deferens. As such the QEJP, like the EJC, would occupy mainly the rising phase of the EJP. The remainder of the EJP should then be generated primarily by passive decay of injected charge through the membrane resistance, as postulated earlier (Cunnane & Manchanda, 1990; Manchanda, 1995). It may be added here that QEJPs, like the other signs of postjunctional electrical activity recorded intracellularly or extracellularly in the vas deferens, are most probably generated by the action of adenosine 5′-triphosphate (ATP) released as a co-transmitter with noradrenaline from the sympathetic innervation (Burnstock, 1985; Kennedy et al. 1996).

Mechanism of action of heptanol

The mechanism by which QEJPs may be unmasked by heptanol following the suppression of EJPs merits consideration. There is persuasive evidence that heptanol uncouples cells from one another, including smooth muscle cells (Christ, 1995; Christ et al. 1996), by specifically disrupting intercellular electrical communication mediated by gap junctional channels (Bukauskas et al. 1992; Bastide et al. 1995). A straightforward explanation of the suppression of the EJP would be that since the EJP recorded in any one cell is a syncytial depolarization, reflecting largely the passively transmitted depolarization generated in neighbouring cells, uncoupling the penetrated cell from its neighbours might prevent its pick-up (Manchanda & Venkateswarlu, 1997). Thus the background depolarization of the EJP, possibly generated by loose contact varicosities (LCVs; Bennett & Gibson, 1995), may be removed, leaving behind only locally generated quantal depolarizations caused by activation of nearby CCVs. This would accommodate the observation that QEJPs were observed in just 25 % of cells, since only a comparable fraction of cells in the guinea-pig vas deferens appear to receive innervation by CCVs (Merrillees et al. 1963; Merrillees, 1968).

Any such explanation is complicated, however, by the failure of heptanol to affect any of the indices of syncytial function subjected to scrutiny here. Syncytial spread of current may be assessed in a number of ways (Tomita, 1967, 1970). A useful indirect index is afforded by the temporal and amplitude properties of SEJPs, for two principal reasons. Firstly, the brief time course of the SEJP relative to the EJP is thought to result from the rapid spatial and temporal spread of current injected focally in a three-dimensional syncytium in which the intercellular shunt time constant (of the order of a few milliseconds) is substantially lower than the membrane time constant (of the order of hundreds of milliseconds, probably 200-300 ms in the guinea-pig vas deferens: see Tomita, 1970; Purves, 1976; Cunnane & Manchanda, 1990). If, however, a cell is electrically uncoupled from its neighbours and charge is injected into it, the charge should be constrained to dissipate through the cell's own membrane resistance. Consequently, the time course of repolarization should be dictated by membrane time constant, as happens in electrically isolated cells (Manchanda, 1995). SEJPs would thus be predicted to be prolonged in the presence of heptanol. This is borne out interestingly by the observation that in electrically short segments of arterioles in which the muscular coat is just one cell layer deep, an arrangement that presumably obliges injected current to discharge through the surface membrane, SEJPs follow much the same time course as EJPs (Hirst & Neild, 1980).

Secondly, the positively skewed amplitude distribution of SEJPs is also believed to result largely from syncytial coupling (Purves, 1976; Tomita, 1970). Consequently, one may predict that in an uncoupled cell relatively fewer low-amplitude events should be recorded, and further that their amplitude distribution might be closer to the Gaussian than to the skewed. It also follows that if intercellular coupling is blocked the frequency of ‘occurrence’ (i.e. observation) of SEJPs in any one cell should be dramatically diminished. It is noteworthy that none of these predicted effects of intercellular uncoupling on SEJPs were reflected in the present experimental findings.

A more direct measure of syncytial coupling is the membrane potential response to current injected focally through an intracellular microelectrode, enabling estimation of input impedance, Rin. Following the same line of argument as for SEJPs, Rin for intracellular current injection should increase when cells are uncoupled from one another (Purves, 1976). Indeed, it has been found that while the input impedance measured in syncytial smooth muscle lies in the range 5-30 MΩ (Bennett, 1967; Holman et al. 1977; present observations), Rin for isolated smooth muscle cells isolated from vas deferens is much higher, close to 1 GΩ (Nakazawa et al. 1987). Heptanol, however, produced no significant increase of Rin in our experiments.

Perhaps the most unambiguous index of current flow between cells is afforded by the intracellular cable potentials recorded using the ‘partition stimulation’ method following injection of current through a plate electrode surrounding the smooth muscle circumferentially. The fact that cable potentials can be recorded at axial distances considerably greater than the average cell length in a particular smooth muscle organ constitutes firm evidence for current flow between the muscle cells (Tomita, 1967, 1970). In the present experiments we recorded cable potentials at distances that were both greater and smaller than the average cell length in the guinea-pig vas deferens (∼400 mm; Merrillees, 1968; Burnstock, 1970). In the former case, electrical uncoupling between cells should result in either abolition or significant attenuation of the potentials; in the latter, an increase in amplitude might be expected, due to increased retention of charge in the impaled cells. We found that neither the cable potentials at distances considerably less than the average cell length (200 mm) nor those at distances considerably larger (up to 1300 mm) were affected by heptanol. This argues persuasively that heptanol failed to affect the spread of injected current between smooth muscle cells.

In the presence of heptanol, thus, none of the predicted effects of intercellular uncoupling on the indices of syncytial function employed here was observed. Therefore, although experiments at the cellular level in other cell types show convincingly that heptanol (at concentrations between 1 and 5.6 mm) specifically blocks gap junctional communication (Peracchia, 1991; Bukauskas et al. 1992; Bastide et al. 1995; Christ, 1995), our results do not support uncoupling at the level of the individual cell in smooth muscle, at the concentration of heptanol (2.0 mm) used here.

Smooth muscle cells are known to be organized into morphologically distinguishable multicellular bundles, in the vas deferens and in other organs (Burnstock, 1970). Electrical interconnections in smooth muscle may exist not just at the level of individual cells, but also between bundles of cells (Burnstock, 1970; Bramich & Brading, 1996). A plausible explanation of our results can be that heptanol selectively disrupts electrical connections between cellular bundles. On this assumption, should coupling between bundles be essential for the EJP to be recorded, it follows that heptanol would suppress the EJP. Furthermore if syncytial properties within a bundle were left unaffected by heptanol then, given a certain critical volume of cells in the bundle (such as to permit local three-dimensional behaviour), SEJPs and Rin would remain unaltered. From exploratory theoretical studies it is estimated that the minimum size of a cubical smooth muscle bundle that demonstrates these features may be of the order of 15 × 15 × 15 cells (R. Manchanda, K. Moudgalya & S. Sen, unpublished observations). Such a proposition must be considered speculative, however, because no known morphological substrate exists for electrical coupling between bundles of cells, although gap junctions are believed to mediate coupling between individual cells (Daniel, 1986).

Inhibition of evoked transmitter release by heptanol?

It follows from the above that in accounting for our observations we cannot at present dismiss actions of heptanol unrelated to electrical uncoupling. Specifically, the results are compatible with the possibility that heptanol reduces the number of active prejunctional release sites recruited following a stimulus (e.g. by lowering the probability of stimulation-evoked, but not spontaneous, release of transmitter). In this event, a lower net syncytial depolarization would result owing to the reduction in quantity of transmitter acting postjunctionally, explaining the suppression of the EJP. Since postjunctional syncytial behaviour still prevails, however, SEJPs and Rin would be left unaffected, as would cable potentials. Interestingly, at low prejunctional densities of release, evoked depolarizations are expected to be quickened in time course (Bennett & Gibson, 1995), and this would explain the rapidity of QEJPs.

The observation that the amplitudes of QEJPs are not distributed closely about a mean ‘quantal’ level but vary continuously from the very small to the large (up to 10-12 mV) would also be accounted for by the persistence of syncytial properties. We propose that QEJPs may be generated essentially by the same population of transmitter quanta released by nerve stimulation as would otherwise have been secreted spontaneously at rest, i.e. they are simply the evoked analogue of SEJPs. Thus, QEJPs would possess the same time courses as SEJPs and similar amplitude distributions. Like SEJPs, they would not strictly be unitary subunits of the EJP in terms of amplitude, but would possess all the other properties expected of the quantal depolarization, as observed here. Finally, the persistence of extracellularly recorded EJCs in the presence of heptanol reported earlier (Manchanda & Venkateswarlu, 1997) is also explicable if one assumes that heptanol is prevented from gaining access to the interior of the recording electrode owing to the electrode-tissue seal, a point yet to be resolved. Inhibition of release may also result from loss of conduction in prejunctional axons; however, heptanol was observed never to exert such an effect (see Fig. 3B), suggesting that it may have affected one or more step(s) in the process of depolarization-secretion coupling.

A mode of action of heptanol involving prejunctional inhibition of evoked release, if corroborated, would represent a novel, hitherto unsuspected biophysical mechanism of interference, and would have significant implications. First, the possibility of prejunctional effects would need to be considered in accounting for any effects of heptanol observed empirically in nerve-muscle preparations where the presence of the innervation could affect interpretation (Venkateswarlu et al. 1999). Second, it will be interesting to identify the precise target of heptanol action in the process of evoked transmitter release, or ‘stimulus-secretion coupling’ in the autonomic varicosity, in which several steps are interposed, starting with Ca2+ influx linked to action potential invasion, triggering fusion and poration of vesicle and surface membranes, and culminating in exocytosis of transmitter.

In conclusion, the detection of QEJPs in the presence of heptanol shows unequivocally that the quantal depolarization underlying the EJP of smooth muscle is identical or very similar to the spontaneous depolarization (the SEJP). Therefore this feature of neurotransmission at the ANJ is essentially similar to that at other synapses, the apparent differences arising from the unique electrical properties imparted to smooth muscle by its syncytial nature. The precise mechanism by which the QEJP is revealed by heptanol requires close scrutiny. In the absence of more decisive evidence we cannot go further than to suggest that if heptanol interferes with electrical coupling in smooth muscle organs, it may do so at the level of syncytial groups of cells rather than individual cells; or that it acts by a mechanism unrelated to electrical coupling, most likely through inhibition of stimulation-evoked transmitter release from the autonomic innervation.

Acknowledgments

We gratefully acknowledge financial support from the Department of Science and Technology, India, under Project SP/SO/NO6/93, and from the All-India Council of Technical Education, India, under the Career Award for Young Teachers scheme. We would like to thank Dr S. Tripathi at Tata Institute of Fundamental Research, Mumbai, for use of his workshop facilities, and Dr J. Dempster, Strathclyde University, Glasgow, for electrophysiological data collection and analysis software.

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