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Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inward-rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. Ih has relatively fast, voltage-dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium-dependent current that was poorly sensitive to TTX. In AH neurones, the post-action-potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω-conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω-agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N- but not of P/Q-type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch-clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea-pig duodenum are under the control of four major currents, IAHP, Ih, an N-type Ca2+ current and a slowly inactivating Na+ current.
The aim of the present paper was to determine the electrotonic properties of myenteric neurones, using patch-clamp recording from non-dissociated myenteric neurones, with emphasis on the identification and quantitation of the ionic currents that modulate the resting membrane potential of AH neurones. Recordings were made with a technique we have recently developed for patch-clamp recording from intact ganglia (Kunze et al. 2000).
The electrophysiological properties of myenteric neurones in intact ganglia from the small intestine of the guinea-pig have been investigated previously by intracellular recordings in myenteric plexus/longitudinal muscle preparations. The first intracellular recording studies were performed using duodenal (Hirst et al. 1974) and ileal tissue (Nishi & North, 1973). In both of these parts of the intestine, the studies separated the myenteric neurones into two groups, S and AH neurones, terms that were introduced in 1974 for duodenal neurones (Hirst et al. 1974). S neurones were so named because they received prominent fast synaptic inputs, while AH neurones did not. AH neurones were so named because the action potential is followed by a long-lasting after-hyperpolarization (AHP). It was later demonstrated that both cell types receive slow EPSPs (Wood & Meyer, 1978; Johnson et al. 1980, 1981; Bornstein et al. 1984).
Correlations between electrophysiological characteristics and cell morphology have been made by intracellular recording using micropipettes filled with fluorescent dyes (e.g. biocytin or neurobiotin) in the ileum (Hodgkiss & Lees, 1983; Iyer et al. 1988; Bornstein et al. 1991) and the duodenum (Clerc et al. 1998). These and other studies showed that AH neurones comprise a single population with Dogiel type II morphology, and were later shown to be sensory, while S neurones were uniaxonal neurones (Bornstein et al. 1994). Motor neurones to muscle, secretomotor neurones and interneurones, of which there are four types in the myenteric ganglia of the guinea-pig small intestine (Costa et al. 1996; Furness, 2000) are S neurones.
At the present time, two different ionic currents are known to modulate the resting membrane potential of AH neurones directly: a K+ inward rectifier current (IKir), which has been described in both AH and S neurones, and a slow AHP current (IAHP), which is typical of the AH neurones. A cationic current activated by hyperpolarization (Ih) has been described in AH neurones (Galligan et al. 1990) and in a subpopulation of S neurones, the filamentous interneurones in the ileum (Song et al. 1997). This current has been shown to contribute to the membrane potential in other types of neurone (Doan & Kunze, 1999). In addition, a high-voltage-activated (HVA) Ca2+ current is activated during the action potential of AH neurones (Hirst et al. 1974) and less prominently in some filamentous interneurones (Song et al. 1997; Clerc et al. 1998). In AH neurones, but not in the filamentous interneurones of the duodenum (Clerc et al. 1998), this Ca2+ current indirectly controls the membrane potential by activating the IAHP.
Although described in a sharp electrode study (Galligan et al. 1990), the Ih has not been recognized in a recent patch electrode study of dissociated myenteric neurones (Zholos et al. 1999) in which only IKir was identified. We have found both currents in AH neurones and, because Ih is known to activate at potentials between −45 and −60 mV in other neuronal types (Pape, 1996), we investigated its possible contribution to the resting membrane potential. We have also detected a slowly inactivating Na+ current that was poorly sensitive to TTX and might modulate the resting membrane potential of these neurones.
The activation of IAHP is triggered by the opening of HVA Ca2+ channels whose identity is controversial (Baidan et al. 1992b; Furness et al. 1998; Starodub & Wood, 1999; Vogalis et al. 2001). Intracellular studies (Kunze et al. 1994; Furness et al. 1998) as well as a recent patch-clamp study (Vogalis et al. 2001) suggest that N-type but not L-type channels are involved, because the hump on the falling phase of the action potential of AH neurones persisted in the presence of nicardipine, but was attenuated by the N-type Ca2+ channel blocker Ω-conotoxin GVIA (Ω-CgTX GVIA). According to patch-clamp studies performed in dissociated myenteric neurones, the HVA Ca2+ current is suppressed by this conotoxin (Baidan et al. 1992b). In the case of rat myenteric neurones in cell culture, both L- and N-type Ca2+ channels contributed to the HVA Ca2+ current (Franklin & Willard, 1993). However, on the basis of the effect of Ω-CgTX MVIIC, which is a blocker of P- and Q-type Ca2+ channels, and to a lesser extent of N-type channels (Uchitel, 1997), Starodub & Wood (1999) concluded that AH neurones express mainly P/Q-type channels. Therefore, we performed a systematic pharmacological investigation to characterize the HVA Ca2+ channel types. In addition, we evaluated the sensitivity of IAHP to [Ca2+]i.
Some of the results presented here have been published in abstract form (Clerc et al. 2000).
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In this work we have evaluated the electrophysiological properties of myenteric neurones using the patch-clamp method applied to non-dissociated neurones within intact ganglia of a myenteric plexus/longitudinal muscle preparation.
AH neurones had relatively broad action potentials with a mean half width of 2.8 ms. This figure compares well with those obtained with sharp electrodes: 2.8 ms in the duodenum (Clerc et al. 1998) and 2.1–2.7 ms in the ileum (Hirst et al. 1985b; Iyer et al. 1988; Brookes et al. 1995). The falling phase of the action potential consistently exhibited a hump (Clerc et al. 1998). Two inward currents have been shown to underlie the action potential in the soma, INaT and a Ca2+ current (Hirst et al. 1974; Hirst et al. 1985a). A significant difference from the published values, which were obtained with intracellular electrodes, concerns the action potential amplitude measured from the resting potential, 102 mV in this work compared to 72 mV in the duodenum (Clerc et al. 1998) and 71–87 mV in the ileum (Hirst et al. 1985b; Iyer et al. 1988; Brookes et al. 1995).
A similar observation applied to S neurones. The mean action potential amplitude we measured from the resting potential was 73 mV. Values derived from intracellular recordings ranged from 56 to 65 mV in the duodenum (Clerc et al. 1998) and 68 mV in the ileum (Iyer et al. 1988).
In view of the greater spike amplitude and of the larger Rin (see below), it was expected that patch electrodes would record a more hyperpolarized potential than sharp electrodes. The voltage recorded with patch electrodes is affected by a liquid-junction potential that is due to the low ionic strength of the pipette saline. According to Barry & Lynch (1991), the theoretical junction potential derived from absolute ionic mobilities of the pipette saline would be +4 to +5 mV. This evaluation underestimates the actual shift in voltage when the membrane patch is ruptured, since the cell interior contains large immobile anions. A direct comparison between the recordings of sympathetic neurones performed simultaneously with both types of electrode revealed a junction potential of +9 mV (Gola & Niel, 1993). Substraction of this value gives a mean resting potential of −66 mV for AH and −56 mV for S neurones.
Rin was substantially greater with patch electrodes compared to sharp electrodes, which confirms direct comparisons made in isolated myenteric neurones by Baidan et al. (1992a), who found that the Rin of a mixed sample of AH and S neurones was 1010 MΩ with patch electrodes and 111 MΩ with sharp electrodes. In AH neurones in intact ganglia we recorded an Rin of 502 MΩ, compared with average values of 92–190 MΩ with sharp electrodes in intact ganglia (Hodgkiss & Lees, 1983; Iyer et al. 1988; Christofi & Wood, 1994; Kunze et al. 1994; Brookes et al. 1995; Smith et al. 1999). Average values for S neurones in the guinea pig small intestine, recorded with intracellular electrodes, range from about 150 to 350 MΩ (Hodgkiss & Lees, 1983; Iyer et al. 1988; Bornstein et al. 1991; Kunze et al. 1994; Smith et al. 1999), although particular subgroups of neurones have different values, for example, longitudinal muscle motor neurones, which have very small cell bodies, have an Rin of over 500 MΩ (Smith et al. 1999). The value we obtained as an average over all S neurones, 713 MΩ, is 2–3 times the values measured with intracellular electrodes.
Ih was originally observed in guinea-pig AH myenteric neurones by Galligan et al. (1990) using a single-electrode voltage-clamp device. These authors found a half-activation at −85 mV and a voltage sensitivity of 10 mV. These values compare well with our data (half activation, −72 mV and voltage sensitivity, 8.2 mV) when the junction potential mentioned above is taken into consideration. The kinetics of activation and deactivation of Ih are strongly voltage dependent. Compared to the h-type currents described in several other cell types (Pape, 1996), Ih in AH neurones has relatively fast on/off kinetics.
In cultured myenteric neurones, Zholos et al. (1999) observed a time-dependent current activated by hyperpolarization, which closely resembles the Ih described here, particularly with regard to its kinetics. They ascribed this current to the activation of an inwardly rectifying K+ conductance. However, IKir are instantaneously activated by hyperpolarizing pulses. In addition, they are fully blocked by 2 mm Ba2+, whereas the current observed by Zholos et al. (1999) persisted in 5 mm Ba2+, although it was slightly decreased (see Pape, 1996, as well as our data for the effect of Ba2+ on Ih). The following observations support our identification of the current as Ih. First, Ih was present at the cell resting potential (i.e. at voltages much more positive than EK; −90 mV in standard conditions: see IAHP section). Second, reducing [K+]o from 5 to 2 mm did not affect the location of the activation curve, although it greatly reduced the size of the slowly activating component. Third, the sag in the voltage response to small current pulses appeared at voltages positive to EK, which could not be accounted for by a Kir current. Together, these data lead us to conclude that in addition to IKir, in situ as well as in culture, AH neurones do express Ih.
Blockade of Ih did not affect the resting membrane potential of AH neurones. This is in agreement with the data of Kilb & Luhmann (2000) obtained in the rat neocortex. As suggested by Galligan et al. (1990), the Ih contribution to the input conductance of AH neurones probably accounts for the relatively limited hyperpolarization upon activation of the slow IAHP. In our experiments, this is supported by the fact that even when a large IAHP was generated under voltage-clamp conditions, the AHP of the unclamped cells barely reached −80 mV, although the reversal potential of K+ ions was at −90 mV.
Our data provide evidence for the presence of an inward current activated from −60 mV in the myenteric sensory neurones. This current appears to be Na+ dependent because it was suppressed in the presence of NMDG. In addition, it was partly blocked by high concentrations of TTX (500 nm to 2 μm). The remaining current was not Ca2+ dependent because it persisted in the presence of Cd2+. In rat suprachiasmatic neurones, a low-threshold, partially TTX-resistant, inward current has also been described (Pennartz et al. 1997). The reduction in this low-threshold component of Na+ current when using slow instead of fast ramps suggests the involvement of a slowly inactivating rather than a persistent Na+ current (see Pennartz et al. 1997). Further experiments are needed to investigate the kinetics of this current. Persistent TTX-sensitive Na+ currents with voltage dependence similar to that described here have been observed in several preparations (Crill, 1996). Interestingly, as we observed, they were enhanced or presumably unmasked by increasing [K+]o (Somjen & Müller, 2000). It is possible that the low-threshold Na+ current identified in the present study is modulated by second-messenger systems, which might therefore affect neuronal excitability. Recently, such a modulation has been demonstrated to operate on a persistent Na+ current that was observed in the pyramidal neurones of the rat neocortex (Franceschetti et al. 2000).
The physiological significance of the inward rectifier observed in both S and AH neurones is not straightforward to deduce. Myenteric S neurones have a low resting potential, and receive mainly excitatory synaptic inputs. Therefore, S neurones have a low probability to enter the voltage region (around −90 mV) in which the inward rectifier may operate. This region corresponds to the limited window, positive to EK, which allows K+ ions to leave the cell. In the case of AH neurones, it can be hypothesized that IKir may act in synergy with IAHP to clamp the cell at a large polarization. This requires, however, a parallel reduction of Ih. Under these conditions, AH neurones would generate a long-lasting, almost permanent AHP, contributing to the low excitability state that AH neurones may enter. Although this idea has been put forward by Zholos et al. (1999), it still needs to be evaluated experimentally.
IAHP was highly variable in size and duration. A direct modulation of calcium-dependent K+ channels is possible. However, this variation is probably due to variations in [Ca2+]. We have shown directly that the IAHP amplitude progressively increased with the release of Ca2+ from intracellular stores by dialysing the neurone with 1 mm caffeine in the presence of a high concentration of BAPTA. In addition, a mechanism underlying the modulation of IAHP expression might be the blockade of Ca2+ channels upon synaptic activation, as has been suggested by Grafe et al. (1980).
Our data provide an unequivocal identification of HVA Ca2+ channels that activate the calcium-dependent K+ conductance that underlies the AHP. The absence of an effect of Ω-aga IVA on the action potential shows that these channels are not of the P/Q type, although Starodub & Wood (1999) suggested the existence of a P/Q-type current in cultured myenteric neurones. The strong decrease in duration of the action potential in the presence of Ω-CgTX GVIA and MVIIA, which have a high affinity for N-type channels, was never accentuated by adding Ω-CgTX MVIIC, which blocks both N- and P/Q-type channels. However, a small Ca2+ component of the action potential was resistant to all of the toxins. This suggests strongly that most of the HVA channels in guinea-pig myenteric sensory neurones belong to the N type. Abolition of the AHP with Ω-CgTX MVIIA demonstrated that N-type channels are the unique HVA Ca2+ channels, whose opening triggers the AHP, although a minor contribution of other HVA Ca2+ channels was suggested by previous data (Vogalis et al. 2001). This conclusion is strengthened by the fact that, in our experiments, the toxin was capable of blocking an AHP that was exaggerated by using 10 mm TEA to increase Ca2+ influx during the action potential. Our conclusion is consistent with the observation that myenteric sensory neurones express a high level of immunoreactivity to the α1 subunits of class B (N-type) but not of class A channels (P/Q-type; Kirchgessner & Liu, 1999).
In conclusion, our results extend knowledge of the types of currents expressed in the myenteric sensory neurones of the guinea-pig. We have shown that Ih is a major conductance of myenteric sensory neurones, despite the fact that this conductance was not recognized in a recent patch study. In addition, we have discovered the presence of a low-threshold sodium-dependent current that is poorly sensitive to TTX. Finally, we have provided unambiguous evidence that N-type Ca2+ channels are the unique HVA channels that trigger the AHP.