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hyperpolarization; upon termination o f the current pulse, a small membrane depolarization, termed a membrane 'rebound', was observed. In the example given, the resting membrane potential o f the isolated B A l motoneurone was -43mV. The injection o f hyperpolarizing current (bottom trace; InA, 200ms, approximately 0.04Hz) evoked approximately 7mV membrane hyperpolarizations which were associated with 3mV membrane 'rebounds' (top trace). Using Ohm's Law (see Section 2.2.4.5), the membrane input resistance o f this neurone was estimated to be 7MO.

B. Injection o f depolarizing current into the isolated B A l motoneurone soma at resting membrane potential evoked membrane oscillations; upon termination o f the current pulses, distinct membrane afterhyperpolarizations were seen. In the example shown the resting membrane potential o f the neurone was -59mV. The top trace shows that the amplitude o f the evoked membrane oscillations and the amplitude o f membrane afterhyperpolarization increased, as the amplitude o f the injected current was increased (bottom trace). As a single electrode recording technique was used and microelectrode resistance was not continuously monitored, the amplitude o f the injected current could not be accurately determined.

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Figure 2.3.IB shows the responses o f a different isolated B A l motoneurone preparation (resting membrane potential -59mV) to small depolarizing current pulses (400ms) o f increasing amplitude, measured using the single microelectrode recording technique. As shown, a viable isolated B A l motoneurone typically elicited membrane oscillations on injection o f depolarizing current pulses and on the termination o f each pulse, prominent transient membrane afterhyperpolarizations were observed (n = 30). The amplitude o f the membrane oscillations and afterhyperpolarizations increased as the amplitude o f the injected cunent increased (up to approximately 5.5nA; see Figure 2.3.IB).

2.3.2. The presence o f cholinergic receptors on isolated B A l motoneurones It has previously been shown that cholinergic receptors are present on B A l motoneurones in situ (Anderson, 1995). This population o f receptors include receptors with a distinct nicotinic pharmacology (i.e. are sensitive to a- bungarotoxin; see below) and those with a broadly muscarinic pharmacology. However, the data o f this study indicated that the latter type o f receptor had a 'mixed' (nicotinic/muscarinic) pharmacology. One o f the aims o f the current study was to investigate the pharmacological profile o f cholinergic receptors upon the soma membrane o f the B A l motoneurone. The series o f experiments described below demonstrate that functional cholinergic receptors are present on the soma membrane. Furthermore, these receptors consist o f two populations of acetylcholine receptor subtypes: nicotinic and a-bimgarotoxin-resistant (termed 'muscarinic') acetylcholine receptors.

2.3.2.1. The effects o f acetylcholine on isolated B A l motoneurones

The initial evidence for the presence o f acetylcholine (ACh) receptors on the isolated B A l motoneurone soma was obtained from experiments in which

ACh (lO'^M to 10"^M) was bath-applied to the isolated neurones. The bath-application o f this neui'otransmitter evoked membrane depolarization and an increase in membrane conductance in a dose-dependent manner (n = 29). The threshold for ACh to evoke a change in membrane potential was between lO'^M and lO'^M in isolated B A l motoneurones.

Figure 2.3.2A shows the dose-dependent nature o f the ACh-induced response on an isolated B A l motoneurone (single microelectrode recording, continuously perfused with locust saline). In this preparation the bath-application o f lO'^M ACh did not appear to evoke any response (Figure 2.3.2A1), however, lO'^^M ACh evoked a Im V membrane depolarization which was not associated with any apparent change in membrane input resistance (Figure 2.3.2AÜ). Following the bath-application o f lO'^M ACh, the membrane potential was depolarized by 4.5mV which was associated with a 15% increase in membrane conductance (Figure 2.3.2Aiii). Figure 2.3.2Aiv shows the effect o f the bath-application o f lO’^M ACh to this preparation. In this instance, ACh evoked a 13mV membrane depolarization with a 127% increase in membrane conductance.

The presence o f acetylcholine receptors on the B A l motoneurone soma was further suggested using the non-selective cholinergic agonist, carbachol (carbamylcholine chloride; CCh). Since it is a non-hydrolyzable analogue o f ACh, and, therefore, it is not affected by cholinesterase, it is a more potent cholingeric agonist than ACh on preparations which possess cholinesterase activity. This was demonstrated using isolated B A l motoneurone preparations. The bath-application o f CCh evoked a more pronounced membrane depolarization, which was associated with a greater increase in membrane conductance than that observed when the same concentration o f ACh was bath-applied to the neurone (n = 5). Using a different preparation to that used in Figure 2.3.2A (single microelectrode recording, not continuously perfused with locust saline), the responses o f the

Figure 2.3.2. The effects o f excitatory non-specific cholinergic agents upon isolated B A l motoneurones.

A. Acetylcholine (ACh) evokes a dose-dependent change in membrane potential, which at higher concentrations is associated with a detectable increase in membrane conductance (10‘^M to lO'^M; n = 29). From Ohm's Law, an increase in membrane conductance was seen as a decrease in membrane input resistance, which was monitored by the amplitude o f the voltage deflections evoked by small hyperpolarizing current pulses injected into the neurone (approximately 0.5Hz). The solid bars above each trace (and in subsequent figures) represents the duration o f drug application. The right-hand side end o f each bar signifies when washing commenced.

In this preparation (resting membrane potential was -56mV; preparation continuously perfused with locust saline), the threshold for ACh to induce membrane depolarization was between lO-^M and 10“*M; the bath-application o f 10'^M ACh (i) did not appear to evoke a detectable change in membrane potential or membrane input resistance. However, lO'^M ACh evoked a Im V membrane depolarization (no detectable change in membrane input resistance was observed). Increasing the concentration o f ACh (lO'^M: (iii) and lO'^M; (iv)) applied to the motoneurone resulted in greater membrane depolarization which was associated with a dose-dependent decrease in input resistance. The effects o f ACh upon the B A l motoneurone were seen to reverse with washing.

B. Carbachol (carbamylcholine chloride; CCh), a non-selective, non-hydrolyzable cholinergic agonist, evoked membrane depolarization which was associated with an increase in membrane conductance; this response was dose-dependent (not shown). The effects o f this agonist were more pronounced than those observed using the same concentration o f ACh (n = 5).

Using a different preparation to that used in Figure 2.3.2A (resting membrane potential was -59mV; preparation not continuously perfused with locust saline), the effects o f bath-applied lO'^M and lO’^M ACh are shown (Bai and Baii, respectively). ACh evoked membrane depolarization associated with an increase in membrane conductance in both experiments. The bath-application o f CCh (10‘^M; Bb) evoked membrane depolarization and an increase in membrane conductance o f similar magnitude to that evoked by lO'^M ACh. Furthermore, in each experiment there was a prominent increase in the amplitude o f after­ hyperpolarization membrane 'rebounds', demonstrating the agonist-induced increase in membrane excitability.

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isolated neurone to the bath-application o f lO'^^M and lO'^M ACh are shown in Figures 2.3.2Bai and 2.3.2Baii, respectively, lO'^M ACh evoked a 4mV membrane depolarization which was associated with a 46% increase in membrane conductance, whilst lO'^M ACh evoked an 1 Im V membrane depolarization with a 100% increase in membrane conductance. However, the bath-application o f lO-^^M CCh to this preparation evoked a membrane depolarization o f lOmV, which was associated with a 125% increase in membrane conductance (Figure 2.3.2Bb). In each o f these examples and Figure 2.3.2Aiv, it was noted that the responses evoked by the cholinergic agonists were accompanied with an increase in neuronal excitability. This was shown by the increase in amplitude o f the membrane 'rebound's that occurred on the termination o f the hyperpolarizing current pulses, used to monitor membrane input resistance.

2.3.2.2. The effects o f nicotine on isolated B A l motoneurones

After it had been established that cholinergic receptors were present on the B A l motoneurone soma, the nature of these receptors was investigated. The bath- application o f the nicotinic acetylcholine receptor agonist, nicotine (NIC), confirmed the presence o f such receptors on the B A l soma membrane. That is, following its application to isolated B A l motoneurones, NIC (lO'^M to 5 X 10‘^M; 11 = 43) evoked a dose-dependent membrane depolarization and an

increase in membrane conductance. This agent was found to be the most potent cholinergic agonist tested on the B A l motoneurone soma.

An example o f the dose-dependent responses evoked by NIC on an isolated B A l motoneurone (resting membrane potential -48mV; not continuously perfused with locust saline) are shown in Figure 2.3.3A. Whilst no response was apparent following the bath-application o f lO’^M NIC (Figure 2.3.3A i), 10‘^M NIC evoked a 2mV membrane depolarization with a 18% increase in membrane

Figure 2.3.3. The effects o f the nicotinic acetylcholine receptor agonist, nicotine (NIC), on isolated B A l motoneurones.

A. The bath-application o f NIC evoked a dose-dependent membrane depolarization in isolated B A l motoneurones which was associated with an increase in membrane conductance (lO'^M to 5 x lO’^M, n = 43). In this preparation (resting membrane potential -48mV; not continuously perfused with locust saline) no response was apparent following the bath-application o f IQ-'^M NIC (Ai), however, as the concentration o f NIC was increased (10‘^M: A ii, 10‘^M: A iii and lO-'^M: Aiv), the agonist induced membrane depolarization associated with an increase in membrane conductance, the magnitude o f which increased as the concentration o f bath-applied NIC increased.

The ability o f nicotinic receptors to desensitize is illustrated in the above traces (A ii to Aiv). That is, in the absence o f washing (i.e. in the continued presence o f NIC), the NIC-induced effects appear to reverse with time (for further details regarding receptor desensitization see Discussion).

B, The nicotinic response o f the isolated B A l motoneurone is irreversibly blocked by the irreversible vertebrate nicotinic antagonist, a-bungarotoxin (a- BTX). Using a different preparation to that used in Figure 2.3.3A (resting membrane potential -45mV; continuously perfused with locust saline), the bath- application o f NIC (lO'^M) alone evoked membrane depolarization which was associated with an increase in membrane conductance, and the effects o f nicotine were shown to be reversible with washing (Bi). Following incubation with a-B TX (BTX: 10‘^M; 50 minutes), NIC (lO'^M) did not evoke a change in membrane potential or input resistance (Bii). Furthermore, no response was observed when the concentration o f NIC was increased to 10‘ *M (B iii), indicating the receptors mediating the response were completely blocked by the toxin.

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