In O.v. nAChRs-activation (ACh and nicotine) leads to a short singing after a very short latency (~0,5 sec), while the mAChRs-activation (muscarine and pilocarpine) induces prolonged singing (3-60 min) (Heinrich et al., 1997).
Repetitive ACh stimulations, every 15 sec, cause arousal of the duration of stridulation. The latter additional excitation has been blocked by scopolamine, inhibitor of mAChRs (Heinrich et al., 1997), suggesting that the nAChRs-activation may initiate, while mAChRs-nAChRs-activation maintains and potentiates the stridulation. It remains to be demonstrated, whether the blockade of nAChRs can affect muscarine-stimulated stridulation in O.v. In addition, nothing is known about the role of AChE modulation in the initiation and maintenance of stridulation in this species.
In contrast to O.v., in Ch.b. ACh failed to trigger stridulation under any stimulation conditions tested, including repetitive injections every 15 sec, demonstrated by Furthermore, ACh applications to the same sites in the brain, where proctolin triggered singing also failed to initiate any song sequences. Co-injected with proctolin ACh had a suppressing effect, leading to decrease of the duration of stridulation (section 3.4.6). By examining the influence of AChRs-activation on proctolin-stimulated stridulation, only the blockade of nAChRs and not of mAChRs affected proctolin-stimulated singing. Under different stimulation conditions the blockade of nAChRs caused either inhibition or potentiation of the proctolin-induced stridulation (section 3.4.6.1). The blockade of nAChRs also potentiated muscarine-stimulated stridulation, suggesting a different role for nAChRs-activation in the cephalic control of of stridulation in Ch.b., than originally described role in O.v. It is proposed that nAChRs-activation may have a role in the modulation and not in the initiation of the stridulation in Ch.b.
In addition, modulation of AChE activity clearly affected – long potentiation, preceding by inhibition – both muscarinic and proctolinic responses. Nevertheless this pharmacological approach cannot offer information about the localization of mAChRs, the fact that AChE blockade inhibited the muscarinic responses (section 3.4.6.2), possible presynaptically localization of mAChRs within the neuronal circuit, controlling the stridulation in Ch.b., cannot be excluded.
Brought together these data suggests that fine-tune modulation of AChE activity may influence the initiation and maintenance of the stridulation.
It has been postulated that nAChRs-mediating excitation may trigger single sequences in Ch.b., because similar mechanisms have been described in O.v. (Heinrich et al., 1997; Wenzel et al., 2000, 2002). The received results do not support this proposal. The data support previous report, that exclusively activation of nAChRs in Ch.b. and other species with more complex rhythms of stridulatory hindleg movements than it is in O.v., cannot elicit stridulation (Heinrich et al., 2001). These species may require a stronger contribution of basal excitation, mediated by muscarinic, proctolin, glutamate and probably additional metabotropic receptor systems.
In the experiments where AChE activity was blocked through co-injection of eserine with proctolin no silent pauses were observed and a prolonged artificial singing was induced (section 3.4.6.2). The blockade of AChRs includes a prolonged ACh signal on AChRs. This might cause a specific desensitization of nAChRs and indirect effects on the time courses of proctolin-stimulated singing.
To clarify principal mechanisms underlying receptor desensitization, mechanisms of nAChRs-desensitization and its modulation are presented in more details.
Desensitization and modulation of nAChRs nAChRs progressively desensitize by prolonged activation through ACh. Recordings of a nicotinic receptor channel with the patch clamp technique (whole cell configuration) show a progressive diminution of the total current IACh in the presence of a high and constant concentration of ACh. (Fig. 4-1, from Clapham et. al., 1984).
The process of desensitization appears slowly and is slowly reversible. This is an intrinsic property of the receptor protein. The rate of desensitization of the nicotinic receptor seems to be related to its state of phophorylation. In fact, studies of the ionic flux through nicotinic receptors incorporated into liposomes have
shown that an increase in the level of phosphorylation of the receptors by cAMP can augment the Fig. 4-1 Desensitization of nAChR by prolonged activation of ACh (patch-clamp whole cell configuration). (from Clapham et al., 1984).
desensitization rate of these receptors. In the neuromuscular junctions a peptide present in the motoneurons is co-released with ACh. This peptide, CGRP (calcitonin gene-related peptide), is capable of increasing the level of cAMP in cultured
embryonic muscle cells, consequently increasing the number of phosphorylated nicotinic receptors (Fig. 4-2)(Hammond, 2001).
However, nothing is known, whether similar modulation of nAChRs-desensitization through neuropeptides may exist in CNS of vertebrates and in insects.
conformational states, even in the absence of ligand:
R R*
D1
D2
(R*, AR*, A2R* - open states; R, AR, A2R – closed states; D1, AD1, A2D1 and D2, AD2, A2D2 – desensitized states; A – ACh or any nicotinic agonist)
• The affinity and activity of the stereospecific sites carried by the nAChR differ between these four states
• Gating of the nAChR cannot be viewed solely as a ligand-triggered process but as reflecting an intrinsic structural transition of the receptor molecule, which may even occur in the absence of ligand. Moreover, at low concentrations, desensitized states can be stabilized under conditions of negligible channel opening.
• Upon two molecules ACh binding, each nAChR undergos fast activation leading to an open-channel state (in msec range), and slow desensitization reaction leading to a close-channel state reflectory to activation. Fast desensitization occurs in the 0,1 sec range and slow desensitization in the minute range (Hammond, 2001).
Fig. 4-2 Modulation of nAChR desensitization by CGRP (peptide) in a cultured muscle cell (from Mulle et al., 1988).