Heterogeneous presynaptic Ca2+ channel types triggering GABA release onto medial preoptic neurons from rat

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Heterogeneous presynaptic Ca²⁺ channel types triggering GABA release onto medial preoptic neurons from rat

David Haage , Urban Karlsson ¹ and Staffan Johansson

* Department of Physiology, Umeå University, S-901 87 Umeå and ¹ Astra Pain Control AB, Novum Unit, S-141 57 Huddinge, Sweden

Received 18 July 1997; accepted after revision 14 October 1997.

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Voltage-dependent Ca²⁺ channels triggering GABA release onto neurons from the medial preoptic nucleus of rat were investigated. Acutely dissociated neurons with adherent functional synaptic terminals were investigated by tight-seal whole-cell recordings from the postsynaptic cells.

Spontaneous current events similar to miniature postsynaptic currents were recorded. They were blocked by bicuculline (100 µM), showed a roughly unimodal amplitude distribution and a reversal potential consistent with a Cl^- current, and were therefore attributed to GABA_A receptors activated by synaptically released GABA.

Application of 140 mM KCl, expected to depolarize presynaptic terminals, evoked currents that were ascribed to a more massive release of GABA. The KCl-induced synaptic currents were abolished in Ca²⁺-free solutions and showed a roughly hyperbolic relation to external Ca²⁺ concentration with half-saturation at 0�15 mM. They further depended on the concentration of applied KCl in a way expected for high-threshold Ca²⁺ channels.

The KCl-evoked synaptic currents were completely blocked by 200 µM Cd²⁺, but only partially blocked by 200 µM Ni²⁺.

The KCl-evoked synaptic currents were insensitive to the L-type Ca²⁺ channel blocker nifedipine (10 µM). However, the synaptic currents were sensitive to either 1 µM $omega$ -conotoxin GVIA, 25 nM $omega$ -agatoxin IVA or 1 µM $omega$ -conotoxin MVIIC.

It was concluded that, in many presynaptic terminals, the Ca²⁺ influx triggering GABA release onto medial preoptic neurons is mainly mediated by one predominant type of high- threshold Ca²⁺ channel that may be either of N-, P- or Q-type.

It was further concluded that terminals with similar predominant channel types often were clustered on the same postsynaptic cell.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Neurotransmitter release is known to be triggered by Ca²⁺ that enters presynaptic terminals through voltage-gated Ca²⁺ channels. Voltage-gated Ca²⁺ channels have been classified as T-, L-, N-, P-, Q- or R-type, depending on their voltage dependence, kinetic and pharmacological properties. A majority of electrophysiological reports suggest that high-threshold channels of the N-type in combination with P-type channels are responsible for transmitter release at central mammalian synapses (Horne & Kemp, 1991; Luebke, Dunlap & Turner, 1993; Takahashi & Momiyama, 1993; Yamamoto, Sawada & Ohno-Shosaku, 1994; Ohno-Shosaku, Hirata, Sawada & Yamamoto, 1994; Regehr & Mintz, 1994; Mintz, Sabatini & Regehr, 1995; Poncer, McKinney, Gähwiler & Thompson, 1997). Involvement of Q-type channels (Wheeler, Randall & Tsien, 1994), and to a smaller extent L-type channels (Reuter, 1995), has also been suggested. In several of the above reports, the cells show a considerable homogeneity in the relative fraction of presynaptic Ca²⁺ influx that each channel type is responsible for. A few recent reports, however, have suggested a heterogeneous distribution of Ca²⁺ channel types in presynaptic terminals of the hippocampus (Reuter, 1995; Poncer et al. 1997; Reid, Clements & Bekkers, 1997).

The medial preoptic nucleus (MPN) is possibly involved in several of the major functions ascribed to the preoptic area, such as control of sexual behaviour, thermoregulation, slow-wave sleep and feeding. The inhibitory neurotransmitter $gamma$ -aminobutyric acid (GABA) has been suggested to be important for several of these functions. GABAergic synaptic contacts are widespread in the MPN, possibly forming local circuits in this region (Hoffman, Kim, Gorski & Dudek, 1994a). The importance of GABA in the control of gonadotropin-releasing hormone secretion from preoptic neurons is clearly documented (see e.g. Leonhardt, Seong, Kim, Thorun, Wuttke & Jarry, 1995). How GABAergic activity is regulated in this area is therefore of great interest. The presynaptic mechanisms controlling GABA release in the MPN have not yet been investigated.

In this study, the effects of organic and inorganic Ca²⁺ channel blockers on KCl-induced GABA-mediated synaptic currents, recorded from acutely dissociated medial preoptic neurons with co-isolated synaptic boutons, were investigated. The preparation is especially suitable for studies of synaptic terminals since most other presynaptic structures are eliminated. Thus, influence of relatively remote presynaptic Ca²⁺ channels is largely excluded. We report here that N-, P- and Q-type Ca²⁺ channels are responsible for triggering GABA release onto neurons of the MPN. The results indicate that the Ca²⁺ influx that triggers transmitter release in many synaptic terminals is mainly mediated by one predominant type of channel that may be either of N-, P- or Q-type. Thus, it is suggested that the channel types are heterogeneously distributed, although terminals with similar predominant channel types often are clustered on the same postsynaptic cell.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of slices and dissociation of cells

Young male Sprague-Dawley rats, weighing 50-100 g, were killed by decapitation without the use of anaesthetics. Ethical approval was given by the local ethics committee for animal research. Hypothalamic tissue slices were prepared as previously described (Johansson, Sundgren & Klimenko, 1995). In short, the brain was rapidly removed and placed in pre-oxygenated ice-cold incubation solution (see below). A block of tissue containing the preoptic area and anterior hypothalamus was cut out, and coronal slices, 250-300 µm thick were prepared by using a vibroslicer (752M, Campden Instruments UK). The slices were incubated for at least 1 h in incubation solution at 32�C, before mechanical vibrodissociation of cells. The method by Vorobjev (1991) was used after slight modification (Karlsson, Sundgren, Näsström & Johansson, 1997). No enzymes were used. Mechanical vibration was applied via a glass rod (about 0�5 mm in diameter), mounted on a piezo-electric bimorph crystal, at the site of the medial preoptic nucleus. The dissociated cells were allowed to settle at the bottom of a Petri dish for 20 min. The cell bodies were 10-15 µm at their longest axis, rounded or elongated in shape. Although some dissociated cells had remaining neurites of up to 100 µm length, the majority of cells used in the present study had neurites that were less than 15 µm long.

Electrophysiology

The amphotericin-B perforated-patch technique (Rae, Cooper, Gates & Watsky, 1991) was used to record whole-cell currents under voltage-clamp conditions. Borosilicate glass pipettes (GC150, Clark Electromedical Instruments), with a resistance of 3-7 M $Omega$ when filled with intracellular solution and immersed in extracellular solution (see below), were used. The liquid-junction potential, measured as described by Neher (1992), has been subtracted in all potential values given. The signals were recorded using an Axopatch 200A amplifier, a Digidata 1200 interface and the pCLAMP software (version 6.03; all from Axon Instruments) controlled via a 486-processor based personal computer. Recorded signals were low-pass filtered at 2-10 kHz (-3 dB). Series-resistance compensation was not used, due to its introduction of extra noise and due to the fact that absolute voltage control was not critical to the present experiments. However, to avoid experiments with large changes in series resistance, the time course of capacitative current elicited by a -5 mV voltage step was repeatedly monitored during the experiments. The KCl and extracellular solutions were applied by a gravity-fed fast perfusion system, controlled by solenoid valves operated from the computer. The solution exchange time, as indicated by the current change measured from a patch pipette in alternating extracellular solution and 140 mM KCl, was in good cases less than 10 ms. However, especially at short solution exchange intervals, it was sensitive to the exact location of the perfusion pipette with respect to the cell and was not measured for each cell. Therefore, the duration of KCl applications indicated should be regarded as approximate. (The peak of the KCl-evoked currents described below, however, was not sensitive to the exact location of the perfusion pipette.) All experiments were performed at room temperature (21-23�C).

Solutions and chemicals

The incubation solution used during the preparation contained (mM): 150 NaCl, 5�0 KCl, 2�0 CaCl₂, 10 Hepes, 10 glucose and 4�93 Tris-base. This solution was used supplemented with a gas mixture containing 95 % O₂-5 % CO₂. The standard extracellular solution used during electrophysiological recording contained (mM): 137 NaCl, 5�0 KCl, 1�0 CaCl₂, 10 Hepes and 10 glucose. Glycine (3 µM) and tetrodotoxin (2 µM; from Sigma) were routinely added, and pH adjusted to 7�4 with NaOH. In the majority of experiments, including all with organic Ca²⁺ channel blockers, the extracellular solution was supplemented with 0�01 % (w/v) bovine serum albumin. A few experiments were performed without albumin, with no significant difference in recorded parameters. The intracellular solution, used for filling the pipette, contained (mM): 140 potassium gluconate, 3�0 NaCl, 1�2 MgCl₂, 1�0 EGTA, 10 Hepes; pH was adjusted to 7�2 with KOH. Amphotericin B (Sigma), prepared from a stock solution (6 mg amphotericin B dissolved in 100 µl dimethyl sulphoxide), was added to a final concentration of 120 µg amphotericin B per millilitre intracellular solution. Nifedipine was purchased from Sigma and peptide toxins ( $omega$ -conotoxin GVIA, $omega$ -conotoxin MVIIC and $omega$ -agatoxin IVA) from Alomone Labs (Jerusalem, Israel).

KCl-evoked depolarization

KCl was in many experiments applied externally (as described above) to induce depolarization of presynaptic terminals (see Results). To give a relatively short Ca²⁺ transient in response to the depolarization and in order not to impose an unnecessary load on the synaptic machinery, the KCl solution was not supplemented with Ca²⁺. For KCl concentrations lower than 140 mM, the reduction (from 140 mM) in K⁺ concentration was compensated by an equimolar increase in Na⁺ concentration. The expected change in presynaptic membrane potential was estimated from the Goldman-Hodgkin-Katz equation (Goldman, 1943; Hodgkin & Katz, 1949) under the assumption that presynaptic membrane potential is determined by permeabilities for K⁺ and Na⁺ with relative magnitudes of 20 : 1. It was further assumed that the intraterminal K⁺ and Na⁺ concentrations were about 140 mM and 10 mM, respectively. (Intraterminal Na⁺ concentrations in the range 1-50 mM resulted in essentially similar potential estimates.) Application of 208 and 308 mM KCl implied an increased osmolarity. To exclude the effects of osmolarity of these solutions, control experiments with 140 mM KCl plus a corresponding concentration of sucrose were performed (n = 5). The results did not differ significantly from those with 140 mM KCl alone.

Analysis

All analysis, including curve fitting, was performed by use of the pCLAMP software (see above) and the Origin software (version 4.00, Microcal Software, Northampton, MA, USA). Spontaneous synaptic currents were detected by visual inspection and the amplitude of each event was measured semi-manually by using cursors. The KCl-evoked synaptic currents were quantified by measuring (also by using cursors) the amplitude of the initial peak as described in the Results section. The steady leak current in control solution has been subtracted in the figures.

RESULTS

Top
Abstract
Introduction
Methods
Results
Discussion
References

Spontaneous synaptic currents

Spontaneous outward currents were recorded at a holding potential of -34 mV. These currents had a fast rising phase and slower, roughly exponential decay phase (Fig. 1A; time constant, 21 � 1 ms (mean � S.E.M.); 155 events from 13 cells). The peak amplitudes reached values of up to 100 pA with an average of 21 � 1 pA (mean � S.E.M.; 546 events from 30 cells; lower limit for detection was about 5 pA). The amplitude distribution was roughly unimodal and somewhat skewed, with no obvious signs of quantization or distinct subpopulation of events (Fig. 2). The spontaneous events occurred with an average frequency of six per minute, but bursts of higher frequency as well as silent periods of more than 5 min were also observed. The currents reversed from outward to inward near -80 mV (Fig. 3). Although the recordings were obtained from isolated cells, the spontaneous currents had a time course similar to miniature postsynaptic currents. The spontaneous currents were thus interpreted as originating from synaptic boutons that survived the dissociation procedure, adhering to the isolated cells and spontaneously releasing transmitter, as has been reported earlier for dissociated neurons (Drewe, Childs & Kunze, 1988; Akaike, Harata, Ueno & Tateishi, 1992). This interpretation was supported by the effect of blockers of synaptic transmission described below. It was further supported by the effect of the GABA_A receptor antagonist bicuculline methiodide (for simplicity called bicuculline hereafter). When added to the external solution, 100 µM bicuculline reversibly blocked the spontaneous current events (Fig. 1; n = 6). This indicates that the current events are spontaneous (miniature) synaptic currents mediated by GABA_A receptors.

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Figure 1. Spontaneous current events blocked by bicuculline

Currents recorded at a holding potential of -34 mV. A, currents recorded in control solution. B, currents from the same cell as in A, but with 100 µM bicuculline added to the external solution. C, currents as in A and B, but in control solution after washout of bicuculline. The records were taken from a cell with an atypically high frequency of spontaneous current events.

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Figure 2. Amplitude distribution of spontaneous current events

Distribution of amplitudes of 546 spontaneous currents, pooled from 30 different neurons. All currents recorded at a steady holding potential of -34 mV. Lower limit for detection was about 5 pA.

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Figure 3. Spontaneous current events show a reversal potential near -80 mV

Three superimposed current traces at each potential indicated (-104, -84, -64 and -44 mV).

KCl-evoked currents

In order to evoke transmitter release mediated by presynaptic Ca²⁺ channels, the cells were perfused with 140 mM KCl for 20-800 ms. This is expected to depolarize the synaptic boutons but not the postsynaptic cell, which was held in the voltage-clamp mode at a membrane potential in the range -34 to +26 mV. The KCl application resulted in several current components (Fig. 4A, upper trace): a transient outward current was followed by a more steady current. At the end of the KCl application, a second transient outward current was often recorded.

The first transient outward current component showed a fast rising phase (10-90 % rise time usually 5-40 ms) followed by a slower, roughly exponential decay phase (time constant, 40-130 ms). In this response, individual current deflections could been seen that had the same shape as the spontaneous synaptic currents described above. Furthermore, the outward current component was reversibly blocked by 100 µM bicuculline (Fig. 4A; n = 8), like the spontaneous synaptic currents. The outward current component evoked by KCl application was thus interpreted as having been caused by synaptically released GABA acting on postsynaptic GABA_A receptors. The current had a peak amplitude (measured as described below) of 70-585 pA at -34 mV (204 � 32 pA (mean � S.E.M.); n = 18), about 4-28 times the average amplitude of the spontaneous currents. This suggests that four to twenty-eight quanta are simultaneously released onto the postsynaptic cell, when KCl is applied.

The second transient outward current component was often, but not always, seen after the end of a pulse of applied KCl (see e.g. Figs 10 and 11). Except for a slower time course and a more variable amplitude, this 'tail' current component showed properties similar to the first outward current peak elicited by KCl (e.g. block by 100 µM bicuculline and block by Ca²⁺ channel blockers; see below). It seems likely that it corresponds to a second peak of GABA release triggered by the 'tail' Ca²⁺ current that is expected after the end of the KCl-induced depolarization of the presynaptic terminals.

Between the two outward current transients evoked by KCl application, a steadier current component was usually recorded. When bicuculline or non-specific blockers of synaptic transmission were used (see below), a relatively steady inward current was revealed (Fig. 4A, lower trace). This current component did not change much with time during the KCl application, but followed a time course similar to that expected for the solution exchange. Furthermore, the current increased in amplitude (as measured from the baseline in standard extracellular solution) when the membrane potential of the postsynaptic cell was clamped at successively more negative levels, but decreased at more positive potentials (Fig. 4B). This behaviour is qualitatively consistent with the change in 'leak' K⁺ current expected from the KCl application (as can be calculated from the Goldman-Hodgkin-Katz current equation under the assumption of constant K⁺ permeability).

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Figure 4. KCl-evoked currents

A, currents recorded at -14 mV during application of 140 mM KCl (as indicated by bar); control solution (upper trace) and in the presence of 100 µM bicuculline (lower trace). B, currents evoked by 140 mM KCl (as indicated by bar) in the presence of 100 µM bicuculline at -14, +6 and +26 mV (as indicated). C, current (average of four traces) at -34 mV during application of 140 mM KCl (bar). The current evoked by 140 mM KCl in the presence of 100 µM bicuculline has been subtracted. A single exponential with a time constant of 60 ms is superimposed. Note the steady current component during KCl application. (A cell with particularly large currents and consequently low relative noise level was chosen.) D, peak currents evoked by repeated (30 s intervals) application of 140 mM KCl over a > 40 min recording period. Holding potential, -34 mV. Different cells were used in A-D.

The current component due to GABA_A receptor activation was studied in isolation by subtraction of the KCl-evoked 'leak' current obtained in the presence of 100 µM bicuculline from the control current evoked by KCl without bicuculline. The (averaged) current obtained showed a decay, during KCl application, that was reasonably well fitted by a single exponential with time constant of 63 � 10 ms (mean � S.E.M.; n = 5; Fig. 4C). The leak subtraction procedure also revealed a sustained current component, most probably due to a steadier component of GABA release during KCl application (Fig. 4C). This sustained current component was in many cases obvious without leak subtraction, and was sensitive to the Ca²⁺ channel blockers described below (see e.g. Fig. 10A). However, it was of variable amplitude and was not further investigated in the present study.

For the present series of experiments, which often required relatively long-lasting ( > 30 min) recordings, it was not practical to perform routinely the 'leak subtraction' procedure described above. Instead, the upstroke of the initial KCl-evoked synaptic current was quantified by measuring the amplitude difference between the peak current and the preceding sharp take-off from the initial part of the leak current (or from baseline). The GABA-mediated current evoked by KCl application was studied at postsynaptic membrane potentials in a range between -34 and +26 mV. As expected from the stochastic nature of transmitter release, there was some variability in the KCl-evoked peak current from trial to trial. However, when KCl was applied repeatedly at 30 s intervals, reasonably stable responses were obtained. Figure 4D illustrates the typical variability in individual responses over a > 40 min recording period.

Ca²⁺ dependence of KCl-evoked synaptic currents

The KCl-evoked synaptic current was reversibly abolished by substitution of 1 mM Co²⁺ for 1 mM Ca²⁺ in the extracellular solution (Fig. 5; n = 4). Similar results were obtained in Ca²⁺-free solution without Co²⁺ (n = 2). These observations suggest that the KCl-evoked release of GABA from the synaptic terminals is dependent on Ca²⁺ influx. To further quantify the Ca²⁺ dependence of the KCl-evoked synaptic currents, a range of different Ca²⁺ concentrations ([Ca²⁺]_o) was used for the extracellular solution. In these experiments, no substitution of other ions for Ca²⁺ was made. (This means that interference of other divalent cations with Ca²⁺ channel function was avoided, but possible effects of altered surface potential were neglected.) The peak amplitude of KCl-evoked synaptic current rapidly (within 1 min) reached a new steady-state level after a change in [Ca²⁺]_o. The amplitude increased sub-linearly with increasing [Ca²⁺]_o in the range 0-2 mM (Fig. 6). The relation was roughly hyperbolic and could be described by the equation:

I = I_max([Ca²⁺]_o/([Ca²⁺]_o + K_Ca)),

where the half-saturating concentration (K_Ca) is 0�15 mM and the maximal current (I_max) is 117 % of the control current obtained with 1 mM Ca²⁺. Saturation was thus approached at [Ca²⁺]_o above 0�5 mM, where about 90 % of the amplitude in control solution (with 1 mM Ca²⁺) was obtained.

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Figure 5. Block of KCl-evoked synaptic current in Ca²⁺-free Co²⁺-containing solution

Currents elicited by application of 140 mM KCl (at 900-1500 ms) after perfusion with control solution (upper trace) and an extracellular solution with Co²⁺ substituted for Ca²⁺ (lower trace). Holding potential, -14 mV.

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Figure 6. Ca²⁺ dependence of KCl-evoked synaptic current

The peak of the KCl-evoked synaptic currents elicited with different [Ca²⁺]_o in the extracellular solution were normalized to the responses obtained in the same cell with 1 mM [Ca²⁺]_o and plotted versus [Ca²⁺]_o. cir , currents obtained in Mg²⁺-free solutions (mean values for 2-6 cells, as indicated by numbers next to the symbols; bars denote S.E.M.). , currents obtained with 1 mM Mg²⁺ added to the extracellular solution (average for three cells at 1 mM [Ca²⁺]_o, and one cell each at 0�5 and 0�25 mM [Ca²⁺]_o). The continuous line is described by a hyperbolic relation (see text) with half-saturating concentration 0�15 mM and a relative maximum at 117 %.

The curve described above differed significantly from some earlier studies of synaptic transmission (see Discussion). Since Mg²⁺ may interfere with Ca²⁺ channels and synaptic function, the lack of Mg²⁺ in the extracellular solution was considered as a possible reason for this difference. However, experiments with Mg²⁺ added to the external solution did not provide support for a significant influence of Mg²⁺ on the obtained responses. With 1 mM Mg²⁺ added to the extracellular solution, the KCl-evoked peak current reached an amplitude of 101 � 3 % (mean � S.D., n = 3) of the amplitude obtained in standard Mg²⁺-free external solution. Also when [Ca²⁺]_o was reduced in two cells (to 0�5 and 0�25 mM, respectively), the KCl-evoked synaptic responses obtained with 1 mM Mg²⁺ in the extracellular solution (crosses in Fig. 6) did not differ significantly from those in Mg²⁺-free solution. These results also suggest that the change in surface potential, expected from the addition of Mg²⁺, did not significantly influence the synaptic responses.

Voltage dependence of KCl-evoked synaptic currents

In order to evaluate the effects of presynaptic membrane potential on the release of GABA, KCl was applied in different concentrations, expected to depolarize the presynaptic terminals to different levels. Under the assumptions that the presynaptic membrane potential is determined largely by the K⁺ permeability and that the intraterminal K⁺ concentration is near 140 mM (see Methods for details), 140 mM external KCl is expected to depolarize the terminals to near 0 mV. When 23 mM KCl, expected to give a presynaptic depolarization to near -40 mV, was applied, no GABA-mediated postsynaptic current was evoked (n = 2). With KCl concentrations in the range 38-92 mM, postsynaptic currents were elicited, although of smaller amplitude than with 140 mM KCl (Fig. 7). Thus, 38, 60, and 92 mM KCl, expected to depolarize the synaptic terminals to -30, -20 and -10 mV (see Methods), resulted in postsynaptic currents with an average peak amplitude of (mean � S.E.M.) 6 � 3 % (n = 8), 60 � 6 % (n = 8) and 86 � 11 % (n = 4), respectively, of the control amplitude obtained with 140 mM KCl. Application of 208 mM (n = 5) or 308 mM (n = 5) KCl (expected to give presynaptic potentials of +10 and +20 mV; see Methods) did not result in currents significantly differing from those obtained with 140 mM KCl. It was concluded that transmitter release is most probably triggered in a voltage-dependent manner, at a minimal presynaptic potential near -30 mV and with maximal release obtained near 0 mV. This is consistent with a release triggered by high-threshold Ca²⁺ channels.

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Figure 7. Synaptic currents dependent on KCl concentration

Currents elicited by the application of 140 mM (A), 60 mM (B) and 23 mM (C) KCl, expected to depolarize presynaptic terminals to 0, -20 and -40 mV, respectively. Note that with 23 mM KCl only a very small inward current is elicited. Four superimposed records for each KCl concentration. (The KCl application starts at about time 70 ms and continues for the rest of the traces shown.) All records obtained from the same postsynaptic cell at -34 mV.

Effects of Cd²⁺ and Ni²⁺ on KCl-evoked synaptic currents

Further evidence concerning the channel types involved in the GABA release was obtained by the use of the inorganic Ca²⁺ channel blockers Cd²⁺ and Ni²⁺. Different Ca²⁺ channel types show different sensitivity to these blockers. Although the sensitivity varies among different preparations, low-threshold T-type Ca²⁺ channels, but also high-threshold R-type Ca²⁺ channels, often show a relatively high sensitivity to block by Ni²⁺, whereas most high-threshold Ca²⁺ channel types (L, N, P, Q and R) show a relatively high sensitivity to block by Cd²⁺ (Fox, Nowycky & Tsien, 1987; Zhang et al. 1993; Randall & Tsien, 1995; Huguenard, 1996; Reuter, 1996). When 200 µM Cd²⁺ was added to the external solution, the KCl-evoked synaptic currents were rapidly blocked (Fig. 8A). The block was complete, although easily reversible upon washout of Cd²⁺ (n = 7). When 200 µM Ni²⁺ was added to the external solution, the peak amplitude of the KCl-evoked synaptic current was reversibly reduced to 40 � 8 % (mean � S.E.M., n = 8) of control amplitude (Fig. 8B).

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Figure 8. Effects of Cd²⁺ and Ni²⁺ on synaptic current

A, currents elicited by the application of 140 mM KCl (at time 1000-1800 ms) after perfusion with control solution (upper trace) and after addition of 200 µM Cd²⁺ (lower trace). B, currents elicited as in A; control solution (upper trace) and after addition of 200 µM Ni²⁺ (lower trace). A and B from different cells. Holding potential, -14 mV in both cases.

Effects of organic Ca²⁺ channel blockers on KCl-evoked synaptic currents

Nifedipine

To obtain more specific information on the Ca²⁺ channel types involved, we used organic Ca²⁺ channel blockers. First, nifedipine, which is expected to selectively block L-type Ca²⁺ channels (Fox et al. 1987), was applied. However, 10 µM nifedipine, added to the external solution, caused no significant change in KCl-evoked synaptic currents within 20 min in six cells tested (Table 1). Thus, we found no support for a role of L-type channels in triggering GABA release onto MPN neurons.

Table 1. Effects of organic Ca²⁺ channel blockers

			Number of affected cells
Blocker	Conc.	Channel type	Major block (> 70 %)	Intermediate block (30-70 %)	Minor block (< 30 %)	Total number of cells
Nifedipine	10 µM	L	0	0	6	6
$omega$ -Conotoxin MVIIC	1�0 µM	N,P,Q	8	1	0	9
$omega$ -Conotoxin GVIA	1�0 µM	N	2	1	4	7
$omega$ -Agatoxin IVA	25 nM	P	4	2	3	9
$omega$ -Conotoxin MVIIC *	1�0 µM	Q	3	1	3	7

The data are numbers of cells showing major, intermediate or minor effects of organic Ca²⁺ channel blockers on the KCl-evoked synaptic current; the total number of cells tested with each blocker is given in the final column. The third column denotes the channel type (see Discussion) expected to be affected. * The degree of block represents the fraction of total current that was not affected by preapplication of 1�0 µM $omega$ -conotoxin GVIA + 25 nM $omega$ -agatoxin IVA but was blocked by 1�0 µM $omega$ -conotoxin MVIIC.

$omega$ -Conotoxin MVIIC

We proceeded to use $omega$ -conotoxin MVIIC, which is a blocker of N-, P- and Q-type Ca²⁺ channels (Zhang et al. 1993; Wheeler et al. 1994). (See Discussion for the criteria used for classification of channel types.) Application of 1�0 µM $omega$ -conotoxin MVIIC resulted in a complete or major ( > 80 %) block of the KCl-evoked synaptic current (peak amplitude) in eight of nine cells tested (Fig. 9) and about 50 % block in one cell (Table 1). The block onset occurred with a time constant of about 1�5-3 min (see lower curve in Fig. 12A below). The effect was, although to a variable extent in different cells, reversible (Fig. 9). Thus, the results suggest that N-, P- or Q-type channels may be involved in the transmitter release.

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Figure 9. Effects of $omega$ -conotoxin MVIIC on synaptic current

Synaptic currents evoked by 140 mM KCl (at 500-1300 ms). A, current in control solution. B, current 5 min after the addition of 1�0 µM $omega$ -conotoxin MVIIC to the external solution. C, current 15 min after washout of $omega$ -conotoxin MVIIC. Holding potential, -34 mV in A-C.

$omega$ -Conotoxin GVIA

To further specify the presynaptic Ca²⁺ channels involved, we applied $omega$ -conotoxin GVIA, which is a more selective blocker of N-type Ca²⁺ channels (although it may to some degree affect R-type Ca²⁺ channels from a marine ray, IC₅₀ = 2 µM; Zhang et al. 1993). When 1�0 µM $omega$ -conotoxin GVIA was applied, a marked heterogeneity in the effect on the KCl-evoked synaptic current recorded from seven different cells was observed. A complete or major ( > 70 %) block of the peak current was obtained in two cells (Fig. 10A) within 15 min of toxin application. However, it had no or a minor effect (< 20 %), within a longer recording period ( > 20 min), on four cells (Fig. 10B), and blocked about 40 % of the current in one cell (Table 1). It should be noted that, for this toxin, as well as for those described below, a lack of effect was accepted only after a duration of toxin application that was longer than the duration that caused a major block in recordings from other cells. The time course of block onset was somewhat slower compared with the effect of $omega$ -conotoxin MVIIC: the time constant was about 4-8 min (see Fig. 12A). The block was not reversible within 10 min of washout of the toxin. The results obtained with $omega$ -conotoxin GVIA thus suggest that N-type channels are necessary for GABA release from some synaptic terminals, but play no crucial role in others.

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Figure 10. Effects of $omega$ -conotoxin GVIA on synaptic current

Synaptic currents evoked by 140 mM KCl (at 1000-1700 ms) in two different cells. A, current in control solution (upper trace) and after 13 min in 1�0 µM $omega$ -conotoxin GVIA (lower trace). Note that, in the control situation, there is a steady outward current component, between the transient components: this steady component is also blocked by the toxin. Holding potential, -14 mV. B, current in control solution (upper trace) and after > 18 min in 1�0 µM $omega$ -conotoxin GVIA (lower trace), in a different cell than in A. Holding potential, -24 mV.

$omega$ -Agatoxin IVA

The possible involvement of P-type channels was tested by the application of 25 nM $omega$ -agatoxin IVA, at this concentration mainly affecting P-type channels (Zhang et al. 1993). This resulted in a major block ( > 70 %) of peak current within 20 min in four of nine cells tested (Fig. 11A), and in a more moderate block (50-70 %) in two cells, but no or a minor block (< 10 %) in three cells (Table 1). The marked differences in effect among cells were clearly noted although the time course of block onset often was considerably slower than for the toxins described above: time constants were in the range 5-15 min (Fig. 12A). A partial reversibility was observed, after washout of the toxin in two cells, but was not quantified in detail. These results suggest that P-type channels, like N-type channels, are necessary for GABA release from some synaptic terminals, but are without a crucial role in others.

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Figure 11. Blocking effects of $omega$ -agatoxin IVA and of $omega$ -conotoxin MVIIC

A, currents evoked by 140 mM KCl; control solution (upper trace) and in the presence of 25 nM $omega$ -agatoxin IVA (lower trace). Holding potential, -34 mV. B, currents evoked by 140 mM KCl in the presence of 1 µM $omega$ -conotoxin GVIA + 25 nM $omega$ -agatoxin IVA (upper trace) and, from the same cell at a later time, in the presence of 1 µM $omega$ -conotoxin MVIIC (lower trace). Holding potential, -34 mV. Different cells in A and B.

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Figure 12. Time course of block onset

Time course of toxin effects on KCl-evoked synaptic currents. A, the different symbols denote recordings from different postsynaptic cells. Application of 1�0 µM $omega$ -conotoxin MVIIC ( squf ), 1�0 µM $omega$ -conotoxin GVIA ( cir and square ) and 25 nM $omega$ -agatoxin IVA ( fullcir ) started at time = 0. The variability of blocking effect is exemplified for $omega$ -conotoxin GVIA with a cell with only minor block () and another with major block ( cir ). The three lower continuous curves are described by a single exponential function fitted to the data. The time constants were 1�9 min ( squf ), 6�3 min ( cir ) and 14 min ( fullcir ). The data indicated by open squares were fitted by a straight line. B, blocking effects of combined application, at time = 0, of 1�0 µM $omega$ -conotoxin GVIA and 25 nM $omega$ -agatoxin IVA to two different cells. In one cell, in which no block was seen, an external solution containing 1�0 µM $omega$ -conotoxin MVIIC was applied after 22 min ( square ).

$omega$ -Conotoxin MVIIC after preapplication of $omega$ -conotoxin GVIA and $omega$ -agatoxin IVA

To investigate if Q-type Ca²⁺ channels were also involved in the GABA release, we used $omega$ -conotoxin MVIIC. However, since this toxin also may block N- and P-type channels, we first applied a combination of 1�0 µM $omega$ -conotoxin GVIA and 25 nM $omega$ -agatoxin IVA. As expected from the above experiments, in seven cells tested, this treatment resulted in a complete or major block ( > 80 %) of the KCl-evoked synaptic current (peak amplitude) in three cells, about 50 % block in one cell, but no or a minor effect (< 10 %) within recording periods of > 20 min in three cells. The peptide $omega$ -conotoxin MVIIC was subsequently applied to the latter three cells, where the combination of $omega$ -conotoxin GVIA and $omega$ -agatoxin IVA was ineffective, as well as to the cell where these two toxins were only partially effective. After application of 1�0 µM $omega$ -conotoxin MVIIC to these four cells, a major block ( > 70 %) of the KCl-evoked synaptic current was achieved within 15 min (Figs 11B and 12B; see also Table 1). Thus, it may be concluded that Q-type channels also play a major role in GABA release from some synaptic terminals, but are without any crucial role in others.

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

In the present work, a preparation of dissociated neurons with adherent functional synaptic terminals was used to characterize the Ca²⁺-mediated control of GABA release onto medial preoptic neurons. This preparation has the advantage of a minimum of presynaptic structures and thereby eliminates the indirect influence of presynaptic elements that are not in the vicinity of the release sites (e.g. relatively remote presynaptic Ca²⁺ channels). Furthermore, the limited length of remaining postsynaptic dendrites implies that all synapses were close to the recording site on the cell body. Thus, synaptic currents could be recorded under voltage-clamp control, and any influence of dendritic Ca²⁺ channels (see e.g. Usowicz, Sugimori, Cherksey & Llinás, 1992; Magee & Johnston, 1995), which could have affected postsynaptic potentials in other studies, was also excluded. The possibility that synaptic terminals may adhere to dissociated neurons has been previously reported (Drewe et al. 1988; Akaike et al. 1992).

GABA_A receptor-mediated spontaneous synaptic currents

The spontaneous synaptic currents recorded had a time course similar to GABAergic 'miniature' currents recorded in other preparations, including slices with medial preoptic neurons (Hoffman, Wuarin & Dudek, 1994b). The unimodal amplitude distribution was similar to the broad, skewed distributions of miniature postsynaptic currents in several other preparations (see e.g. Bekkers & Stevens, 1995). From this and the additional evidence obtained from the reversal potential and the sensitivity to bicuculline, it seems most likely that the currents were GABA_Areceptor-mediated Cl^- currents activated by synaptic release of GABA. This is also consistent with the recent findings of GABA-evoked currents in medial preoptic neurons (Karlsson, Haage & Johansson, 1997). The unimodal amplitude distibution and the relatively low frequency of events over time support the notion that most currents were caused by the release of a single transmitter quantum.

KCl-evoked synaptic currents mediated by GABA release

Dependence of GABA release on external Ca²⁺ concentration

The dependence of the KCl-evoked synaptic current on external Ca²⁺ concentration, [Ca²⁺]_o, (i.e. the hyperbolic relation with half-saturation at 0�15 mM; Fig. 6) differed dramatically from other preparations where the synaptic response depends on the third or fourth power of [Ca²⁺]_o (e.g. Dodge & Rahamimoff, 1967). It has been suggested that KCl-evoked depolarizations, that are usually much longer in duration than action potentials, may produce larger Ca²⁺ influx and thus cause the release system to operate nearer saturation (Wheeler, Randall & Tsien, 1996). Although we limited the Ca²⁺ influx by omitting Ca²⁺ from the depolarizing KCl solution, it seems possible that a large Ca²⁺ influx per depolarization may have contributed to the obtained hyperbolic relation. However, it should be noted that the obtained relation depends on the relation between [Ca²⁺]_o and Ca²⁺ current (I_Ca) as well as on the relation between I_Ca and transmitter release. Although the relation between [Ca²⁺]_o and I_Ca is often assumed to be linear (e.g. Wheeler et al. 1996), hyperbolic relations with half-saturating concentrations in the near-millimolar range have been recorded from rat basal forebrain neurons (Allen, Sim & Brown, 1993; see also Mintz et al. 1995). Whether the relation between I_Ca and [Ca²⁺]_o is linear or hyperbolic, it does not seem likely that the postsynaptic response is dependent on the third or fourth power of I_Ca under the experimental conditions used here. Thus, a major block of the postsynaptic current was most probably caused by a major block of the presynaptic I_Ca. (For example, with a linear relation between I_Ca and [Ca²⁺]_o, a 70 % reduction of the postsynaptic current would imply roughly 95 % reduction of I_Ca; see Fig. 6.)

Ca²⁺ channel types triggering GABA release

The dependence on external Ca²⁺, and on KCl concentration suggested that the synaptic currents were mediated by high-threshold Ca²⁺ channels in the presynaptic terminals. The pharmacological analysis in the present work showed that Ca²⁺ channels sensitive to $omega$ -conotoxin GVIA, channels sensitive to low concentrations of $omega$ -agatoxin IVA, as well as channels insensitive to $omega$ -conotoxin GVIA and $omega$ -agatoxin IVA but sensitive to $omega$ -conotoxin MVIIC, were all involved in GABA release onto the medial preoptic neurons. According to the pharmacological criteria that have mainly been used to classify high-threshold Ca²⁺ channels in other preparations (Fox et al. 1987; Zhang et al. 1993; Wheeler et al. 1994; Randall & Tsien, 1995; Reuter, 1996), these channels correspond to N-, P- and Q-type Ca²⁺ channels. Thus, for simplicity, we have used the same terminology in the present study.

According to a majority of earlier studies, transmitter release onto central neurons is mediated by presynaptic N- and/or P-type Ca²⁺ channels (Luebke et al. 1993; Takahashi & Momiyama, 1993; Ohno-Shosaku et al. 1994; Regehr & Mintz, 1994; Mintz et al. 1995; Tsien, Lipscombe, Madison, Bley & Fox, 1995; Poncer et al. 1997), although roles for Q- and L-type channels also have been reported for hippocampal presynaptic terminals (Wheeler et al. 1994; Reuter, 1995). The toxin concentrations used in some of the earlier reports, however, did not allow for a distinction of P- and Q-type channels. Furthermore, in many of these reports, it seems possible that relatively remote (presynaptic or dendritic) Ca²⁺ channels may have contributed to the results and/or that the use of cultured cells or synaptosomes may have introduced differences from more physiological situations.

Distribution of Ca²⁺ channel types and of synaptic terminals

In the present work, a prominent finding was the heterogeneity of toxin effects observed in different postsynaptic cells. This is most easily interpreted as differences in the contribution of presynaptic N-, P- and Q-type Ca²⁺ channels to the GABA release onto the different cells. (As discussed above, a major postsynaptic block by the toxins, was most probably caused by a major block of presynaptic I_Ca). Thus, our results imply that each of the three channel types (N, P and Q) alone contributes to the majority of GABA release from synaptic terminals on some cells, but is without a crucial role at others. This observation provides information on how the different channel types are distributed in individual synaptic terminals as well as on how different synaptic terminals are distributed on different postsynaptic cells.

We considered three different simplified models of synaptic terminals with respect to their Ca²⁺ channel composition and their distribution on postsynaptic cells (Fig. 13). If the proportion of channel types are similar in all terminals (model 1, Fig. 13A), then the effect of the different blockers should be similar for each postsynaptic cell studied. As this was contrary to our observations, with heterogeneous effects, we could reject model 1. If the synaptic terminals differ from each other with respect to the prevailing type of Ca²⁺ channel, but the different terminal types are distributed in similar proportions on each postsynaptic cell (model 2, Fig. 13B), then the blockers should also have similar effects for each postsynaptic cell. Thus, model 2 was also rejected. If synaptic terminals differ from each other and are also differentially distributed on the postsynaptic cells (model 3, Fig. 13C), however, the above observations may be explained. The study thus favours a model where synaptic terminals that are mainly influenced by a single specific type of Ca²⁺ channel (either N, P or Q) cluster on the same postsynaptic cell.

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Figure 13. Models of Ca²⁺ channel distribution in synaptic terminals on MPN neurons

Three schematically drawn models of postsynaptic cells with surrounding terminals. The main Ca²⁺ channel types triggering GABA release from the different terminals are indicated by shading. A, in model 1 several channel types are present in each terminal and the proportions of channel types are roughly similar in all terminals. B, in model 2 each terminal is influenced mainly by a single channel type, but the distribution of terminals with different channels is roughly similar for all postsynaptic cells. C, in model 3 each terminal is influenced mainly by a single channel type and the distribution of terminals with different channels is heterogeneous with respect to postsynaptic cells, although the terminals on individual postsynaptic cells are similar.

However, it must be noted that the above model is a simplification. Thus, model 3 should not be taken to mean that only one type of Ca²⁺ channel is present in all terminals on a postsynaptic cell, but rather that one type of channel often seems to exert a dominant influence on GABA release from most of these terminals. Furthermore, postsynaptic cells with terminals significantly influenced by more than one type of calcium channel were also observed in the present study. Thus, more complex models with variable proportions of channel types may have to be invoked to explain the results obtained in some recordings.

The findings above, and model 3, thus imply a heterogeneity among presynaptic terminals in the MPN. Although most earlier reports on central neurotransmission did not reveal similar findings, several recent studies of hippocampal neurons have emphasized a heterogeneity of presynaptic Ca²⁺ channel distribution (Reuter, 1995; Poncer et al. 1997; Reid et al. 1997). Heterogeneous release properties of individual hippocampal synapses have also been reported (Murthy et al. 1997).

The reasons for the relative homogeneity of presynaptic terminals on many of the individual postsynaptic cells in the present study (model 3) are unclear. Interestingly, Murthy et al. (1997), who reported heterogeneous release properties of individual synapses, also found that neighbouring synapses tended to have similar release properties. A speculative possibility is that the postsynaptic cell influences the channel composition in the contacting presynaptic terminals.

The existence of a variety of presynaptic Ca²⁺ channels, as suggested in the present study, implies a possibility for differential modulation. It is known that different high-threshold Ca²⁺ channel types in other preparations are affected by different modulating systems (see e.g. Tsien, Lipscombe, Madison, Bley & Fox, 1988). A differential modulation seems to be of more obvious advantage if the presynaptic terminals are heterogeneous with respect to channel population, as indicated here, than if all terminals have similar channel populations.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

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[Medline]

Acknowledgements

This work was supported by the Swedish Medical Research Council (Project No. 11202), the Royal Swedish Academy of Sciences, Magn. Bergvalls Stiftelse and Umeå University.

Corresponding author

S. Johansson: Department of Physiology, Umeå University, S-901 87 Umeå, Sweden.

Email: staffan.johansson{at}physiol.umu.se

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