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3.1. INTRODUCTION

ACh is a major excitatory neurotransmitter in the insect CNS (Pitman, 1985; Sattelle, 1985). The majority of insect cholinergic receptors are nicotinic, in contrast to the vertebrate CNS, where most cholinergic receptors have a muscarinic pharmacology (Ben-Barak & Dudai, 1979; Salvatena & Foders, 1979). It is becoming apparent from work on Manduca (Trimmer & Weeks, 1989), locust (Baines & Bacon, 1994) and cockroach (David & Pitman, 1990; Lapied & Hue, 1991; Harrow & Sattelle, 1983), however, that nicotinic and muscarinic receptors can be colocalised on the same neurone. There is evidence to suggest that these muscarinic receptors modulate the excitability of the neurone (Trimmer & Weeks, 1989, 1993; Le Corronc & Hue, 1993; Baines & Bacon, 1994), thereby increasing the probability that a nicotinic receptor-mediated depolarisation will result in the propagation of an action potential.

In vertebrates, muscarinic cholinergic receptors can be grouped into subtypes according to their pharmacology. This was first proposed by Hammer et al (1980) on the basis that muscarinic receptors in rat brain could be blocked by pirenzepine (classed as M | receptors), whereas those located in the peripheral tissues were insensitive to the drug (M2 receptors). It soon became apparent that

the pirenzepine-insensitive receptors were not a homogeneous group and could be further classified according to their sensitivities to particular antagonists. Those in the heart could be blocked by AF-DX 116 (Giachetti et al, 1986) and methoctramine (Melchiorre et al, 1987), whereas those in smooth muscle were blocked by 4-DAMP (Barlow et al, 1976), HHSiD and p-F-HHSiD (Lambrecht et al, 1989). These three receptor subtypes were teiTned M^ (brain), M2 (cardiac) and

M3 (smooth muscle).

These antagonists were designed for vertebrate muscarinic receptors, but studies have demonstrated that they also show some selectivity for insect muscarinic receptors, although they are less specific. Pirenzepine-sensitive

muscarinic receptors have been found on isolated locust neurones (Benson, 1992), motoneurone Df of the cockroach (David & Pitman, 1993a), PPR motoneurone of

Manduca (Trimmer & Weeks, 1993) and cockroach giant interneurones (Le Corronc & Hue, 1993). However, HHSiD and methoctramine also decrease the response to exogenously applied ACh in motoneurone Df, but are less effective (David & Pitman, 1993a). HHSiD and 4-DAMP have similar potencies to pirenzepine in isolated locust neurones (Benson, 1992), whereas methoctramine has virtually no effect. There is a binding site in the brains of honey bee, housefly and cockroach with a relatively low affinity for pirenzepine and AF-DX 116 compared with 4-DAMP (Abdallah et al, 1991). 4-DAMP and methoctramine have no effect on the muscarinic receptor-mediated depolarisation of cockroach giant intemeurones (Le Corronc & Hue, 1993); these antagonists, as well as HHSiD and AF-DX 116, were weak antagonists at the muscarinic receptors of PPR motoneurone (Trimmer & Weeks, 1993).

a-BTX blocks most, but not all, of the response of the first basalar motoneurone to exogenous ACh (Leitch et al, 1993). The experiments presented here were performed to investigate this a-BTX-resistant component: the effects of muscarinic antagonists on the ACh-mediated depolarisation of the neurone were studied to determine whether the receptors have a muscarinic pharmacology; and the effect of the vertebrate M^ subtype-selective agonist McN-A-343 on the input resistance and spike threshold of the neurone was investigated to examine whether these receptors can modulate the excitability of the motoneurone.

3.2. RESULTS

The resting membrane potential of the first basalar motoneurone was generally between -50 and -60 mV, measured 15 min after impalement. The mean value of the resting potential was -54 0 mV (S.E.M. ± 0 93 mV, n=25). The input resistance of the neurone was measured in two-electrode current clamp: current was injected with one electrode to hyperpolarise the membrane by 10 mV, and the deflection of the membrane potential monitored with the second electrode. The input resistance varied between 3 and 10 MQ; the mean value was 4 3 MQ (S.E.M. ±0-31 M Q,n-25).

3.2.1. MUSCARINIC PHARMACOT.OGY

When ACh is pressure-applied onto the surface of the first basalar motoneurone, the cell is transiently depolarised. This response can be reduced by the addition of 10"^ M a-BTX (Figure 3.1; n=3). After 60 min, the amplitudes of the depolarisations were between 15 and 35 % of the control (mean 26-7%; S.E.M. ± 4*9 %, n=3). a-BTX was added to the preparations in the following experiments to block the nicotinic receptors on the cell in order to study the a- BTX-resistant response to ACh. The toxin was used as supplied by the manufacurer and was not further purified. The agonist was pressure-applied to the cell (10"2 M, 200-300 ms) once every 2 min. Figure 3.2 shows that applying ACh in this way does not cause the receptors to desensitise. The effect of more frequent application of ACh was not investigated.

There is the possibility that the a-BTX-resistant component of the ACh- induced depolarisation is the result of incomplete block of the nicotinic receptors. The effect of an increased concentration of a-BTX was not examined; however, the results of the experiments in this chapter suggest that the toxin-resistant component has a muscarinic pharmacology and is not due to partial antagonism of the nicotinic receptors.

Figure 3.1. The effect of lO'^M a-BTX on the response of the first basalar motoneurone to pressure applied ACh (lO'^M , 200 ms). The neurone was exposed to the toxin for 60 min, and the amplitude of the depolarisation has been reduced to approximately 30 % of the control.

Scale: vertical 2 mV horizontal 1 min

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