Despite the wide variety of effects associated with the activation of muscarinic receptors on different peripheral organs it appeared that they were either identical or very similar because known antagonists, like atropine, were equally effective against all muscarinic responses. A decade ago, one drug, pirenzepine, was found to be a hundredfold more active against ACh-induced gastric acid secretion than against other peripheral muscarinic effects. The receptors blocked by pirenzapine became known as M1 and all the others as M2. Recently some differences between muscarinic M2 receptors on heart (inhibitory) and those on exocrine glands (generally excitatory) became apparent through slight (fivefold) differences in the binding of some antagonist drugs (tools) such as AF-DX-116 and 4-DAMP. The former was more active on the receptors in the heart, accepted as M2 receptors, while the glandular ones, blocked preferentially by 4-DAMP, became M3. Molecular biology has since confirmed the existence of these three receptors and revealed (at the time of printing) two more — M4 and M5. The M1 receptor mediates most of the central postsynaptic muscarinic effects of ACh while the M2 is predominantly a presynaptic autoreceptor.
The structure of the muscarinic receptor is very different from that of the nicotinic. They are single-subunit proteins which belong to the group of seven transmembrane receptors (like adreno and dopamine receptors) typically associated with second messenger systems. The major difference between muscarinic receptors is in the long cytoplasmic linkage connecting the fifth and sixth transmembrane domain, suggesting different G-protein connections and functions. Thus M1, M3 and M5 receptors are structurally similar and their activation causes stimulation of guanylate cyclase and an increase in cyclic GMP as well as inosotal triphosphate hydrolysis through an increase in G-protein (Gp) (Fig. 6.4).
The M2 and M4 receptors also show structural similarities. Through G-protein (G1) they inhibit cyclic AMP production and open K+ channels while activation of another G-protein (G0) closes Ca2+ channels. The latter effect will cause membrane hyperpolarisation as will the G1-induced increase in K+ efflux. The reduction in cAMP production, although possibly leading to depolarisation, is more likely to explain the presynaptic reduction in ACh release associated with the M2 receptor.
Cholinergic receptors should obviously be found where ACh is concentrated and cholinergic pathways terminate. Autoradiography with appropriately labelled ligands does in fact show M1 receptors to be predominantly in the neocortex and hippocampus (where pathways terminate) and in the striatum where ACh is released from intrinsic neurons. By contrast, M2 receptors are found more in the basal forebrain where ascending cholinergic pathways originate. Such a distribution is in keeping with the postsynaptic action of the M1 receptor and the presynaptic cell body (autoinhibition) mediated effects of its M2 counterpart. Unfortunately the ligands available for labelling are not sufficiently specific to use this technique to reliably distinguish M1 from M3 and M5 receptors or M2 from M4. In situ hybridisation studies of receptor mRNA, which detects cell body receptors, is more sensitive and confirms the M1 dominance in the neocortex, hippocampus and striatum with M2 again in subcortical areas. Receptor mRNA for the M3 is, like that for M1, in the cortex and hippocampus but not in the striatum while that for M4 is highest in the striatum and low in the cortex. Elucidation of the precise functional significance of such a distribution awaits the arrival of much more specific ligands for the receptor subsets. In their absence a more detailed analysis of the distribution of muscarinic and nicotinic receptors is not justified here but see Hersch et al. (1994), Levey et al. (1991), Wall et al. (1991) and Wess (1996).
Nicotinic receptors have been found and studied predominantly in the hippocampus, cerebral cortex and viatral tegmented area (VTA).
Activation of nicotinic receptors causes the rapid opening of Na+ channels and membrane depolarisation. This is a feature of cholinergic transmission at peripheral neuromuscular junctions and autonomic ganglia but while it is found in the CNS, it is not widely observed. Exogenously applied nicotinic agonists have been shown to directly excite neurons through somato-dendritic receptors in various brain regions while the excitatory response of GABA interneurons in the hippocampus and dopamine neurons in the VTA following some afferent stimulation is reduced by nicotinic antagonists (see Jones, Sudweeks and Yakel 1999). Nicotinic receptors also mediate the fast response of ACh released at the endings of collaterals from motoneuron axons to adjacent inhibitory interneurons (Renshaw cells) in the ventral horn of the spinal cord (see below).
More recently much interest has been directed towards presynaptic nicotinic receptors that have been shown to enhance the release of a number of NTs, i.e. ACh, DA, NA, glutamate and GABA, in perfused synaptosomes or slices from various brain regions, as well as DA into microdialysates of the striatum in vivo. Thus they can be hetero- and not just autoreceptors (see Wannacott 1997). Since activation of these receptors can actually evoke, and not just facilitate, NT release, they probably work directly on nerve terminals to increase Na+ influx and initiate sufficient depolarisation to activate voltage-sensitive Ca2+ channels, although there could also be an influx of Ca2+ itself through the nicotinic gated channel. In fact the high permeability of some neuronal nicotinic receptors to Ca2+ ions provides an obvious mechanism for increasing transmitter release. Differences in the sensitivity of the presynaptic receptors to various agonists and antagonists indicate some heterogeneity but their relatively low affinity for nicotine (EC50 about 1 ^M) and the absence of clear evidence for their innervation means that their physiological role remains uncertain. Their activation by exogenous agonists could, however, have interesting therapeutic applications such as an increase in ACh release in Alzheimer's disease and mAChRs have been found to be reduced in the cortex and hippocampus of such patients.
Although ACh does not have a primary excitatory role like glutamate in the CNS, it does increase neuronal excitability and responsiveness, through activation of muscarinic receptors. It achieves this in two ways, both of which involve closure of K+ channels (see Chapter 2 and Brown 1983; Brown et al. 1996). The first is a voltage-dependent K+ conductance called the M conductance, Gm or Im. It is activated by any attempt to depolarise the neuron, when the opening of the M-channel and the consequent efflux of K+ counteracts the depolarisation and limits the generation of spikes. This current is inhibited by activation of muscarinic receptors and so ACh will tend to keep the neuron partially depolarised and facilitate repetitive firing and bust spiking. This slow cholinergic excitation in hippocampal neurons is shown in Fig. 6.5.
Figure 6.5 Illustrations of the slow excitatory effect of ACh. (a) Electrical stimulation (left-hand traces) of presumed cholinergic fibres (striatum oriens) in the rat hippocampal slice preparation (20 Hz for 0.5 s) induced a short latency epsp followed by an ipsp and a later slow epsp, recorded intracellularly in pyramidal neurons (control). The slow epsp was selectively potentiated by the anticholinesterase drug eserine (2 p,M) with the generation of action potentials. This firing and the slow epsp, but not the fast epsp or ipsp, were eliminated by the muscarinic antagonist atropine (0.1 p,M). The iontophoretic application of ACh (right-hand traces) in the presence of eserine only produced the slow epsp and superimposed firing which were also antagonised by atropine (resting membrane potentials 57-60 mV). These recordings show that the slow but not the fast epsp is cholinergic. (b) Extracellulary recorded multiunit response in layer V of the guinea pig anterior cingulate cortex slice preparation. Micropipette application of glutamate (10 ms of 1 mM) caused a rapid generation of action potentials up to 100 p.V in amplitude while ACh (40 ms of 1 mM) only generated smaller (10-20 p.V) potentials more slowly (50 ms) with larger ones (30-50 p.V) appearing later. These results again demonstrate the slow excitatory effect produced by ACh compared with the larger and more rapid primary depolarisation of glutamate. ((a) reproduced with kind permission from the Journal of Physiology and from Cole and Nicoll 1984 and (b) from McCormick and Prince 1986a)
Studies of the hippocampal neurons have shown a second K+ current which mediates the long-lasting after-hyperpolarisation following spiking. This is not voltage activated but is switched on by Ca2+ entry through channels opened during the initial depolarisation. It is inhibited by activation of muscarinic receptors and so its reduction will also lead to repetitive firing. ACh in fact seems to dampen the inbuilt brakes on cell firing (see also Chapter 2 and Figs 2.5 and 2.6).
Mi and M3 receptors mediate the excitatory effects and since this postspike hyperpolarisation is blocked by phorbol esters and is therefore presumably dependent on IP3 production, one would expect it to be mediated through M1 receptors (see above), especially as these are located postsynaptically. Unfortunately it does not appear to be affected by pirenzapine, the M1 antagonist. By contrast, muscarinic inhibition of the M current is reduced by the M1 antagonist but as it is not affected by phorbol esters is not likely to be linked to IP3 production, an M1 effect.
ACh can sometimes inhibit neurons by increasing K+ conductance and although it has been found to hyperpolarise thalamic neurons, which would normally reduce firing, strong depolarisation may still make the cell fire even more rapidly than normal. This appears to be because the hyperpolarisation counters the inactivation of a low-threshold Ca2+ current which is then activated by the depolarisation to give a burst of action potentials (McCormick and Prince 1986b).
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