**5. Avian muscarinic receptors in sensory structures**

#### **5.1 Muscarinic receptors in avian vestibular end organs**

Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells has been conducted based on the patch clamp recordings from pigeon native hair cells with carbachol, a cholinergic agonist, resulted in reduction of the current through the inward rectifier pKir2.1 channel [32]. The cilia on the hair cells and the associated structures are important during sensory transduction.

They cloned and sequenced pigeon mAChR subtypes M2–M5 in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa's) ganglion and studied the expression of all five mAChR subtypes (M1–M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). In the study, vestibular hair cells, nerve fibers and ganglion cells expressed all five (M1–M5) mAChR subtypes. mAChRs M1 and M5 were found on the nerve terminals, supporting cells, and cilia of hair cells. And mAChRs M1, M3 and M5 were expressed on cuticular plates, myelin sheaths and Schwann cells. M2 and M4 mAChRs were seen on the nerve terminals. M2 was also present on the cuticular plates and supporting cells [38].

Immunohistochemistry and Immunocytochemistry results were consistent with results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia. It is clear from the study that the neuronal components of the labyrinth exhibit significant co-expression of the subtypes. Even though the study does not give data on quantitative expression of M1–M5 but do indicate that the mAChRs are widely present and co-expressed on elements in the vestibular peripheral system. The additional possibility of mAChR expression on efferent fibers and terminals forming autoreceptors should be analyzed using further research in this area.

Acetylcholine receptors often perform autocrine and neuronal activities [69]. Control of cell growth and proliferation and release of chemical mediators [70, 71] are the major autocrine functions. These functions are important in vestibular hair cell regeneration or maintenance and replenishment of endolymph and perilymph. Non-neuronal cholinergic function is attributed by the expression of mAChR subtypes on the supporting cells and Schwann cells [72].

#### **5.2 Muscarinic receptors involved in chick myopia**

Various studies conducted in the chick model of myopia has proved that the most effective anti-muscarinic agents for myopia includes the non-selective agents like atropine [73] and oxyphenonium [74], followed by the partially selective muscarinic antagonists M1 and M4 selective pirenzepine [75] and the M2 and M4 selective himbacine [76]. In a study using the selective M4 muscarinic receptor antagonist MT-3, it was effective in inhibiting form-deprivation myopia in the chick by means of inhibition of vitreous chamber elongation, the major structural cause of myopia, associated with inhibition of choroidal thinning in myopic chicks. While in certain studies using muscarinic antagonists such as atropine and pirenzepine, they also produced similar efficacy in reducing myopia, at doses substantially higher than that would be considered necessary for a muscarinic receptor-based mechanism, namely at micromolar concentrations.

But in this particular study, since the muscarinic antagonists applied were highly selective for the M4 and M1 receptors, the doses were calculated to be at nanomolar concentrations at the receptor level. Similarly, upon applying the highly selective M1 muscarinic antagonist MT-7, it had no inhibitory effect on form-deprivation myopia in chick, deriving a conclusion that the chick lacks an M1 receptor, as supported by their findings [26].

In a similar experiment involving the chick myopia model, attempts were made to isolate and process RNA and genomic DNA for the evidence of a functional M1 muscarinic receptor in chicken. However, the results produced no evidence for the same,

#### *Avian Muscarinic Receptors: An Update DOI: http://dx.doi.org/10.5772/intechopen.111720*

which itself indicates that the mRNA template necessary for the production of the M1 receptor protein is unlikely to be present in chick. Furthermore, it could be concluded that the chick lacks a gene or promoter sequence for the M1 receptor. Moreover, the study was consistent with previous report, which demonstrates a tenfold affinity of pirenzepine for the chick M2 receptor subtype than its mammalian counterpart [77]. Hence, the study suggests the possibility that pirenzepine inhibits myopia progression via the M2 receptor in birds and via the M1 receptor in mammals. Furthermore, it also suggests the possibility that muscarinic antagonists which prevents myopia in chicks mediates its action through another muscarinic subtype, probably the M4 subtype or through non-specific or non-receptor mediated mechanisms [25].

Moreover, this can be consistent with another finding which localized the presence of G protein-coupled receptor kinases (GRKs) in the avian retina [39]. GRKs are enzymes that are involved in the phosphorylation of serine/threonine residues in the carboxy-terminal of various of agonist-occupied G protein-coupled receptors [78, 79]. Retinal morphology and electrophysiology are relatively well characterized, since it is an excellent model for neurochemical studies of the nervous system since its development. At least six enzymes have been cloned and were extensively characterized from mammals [80]. Of these, mammalian retinal rods and cones expressed GRK1 [81]. In the chicken retina, two different types of photoreceptors expressed a novel GRK1 [82]. However, not only muscarinic receptors act via GPCR mediated mechanism but also dopaminergic D1, D2, D4 and D5 receptors [83], adenosine A1 and A2 receptors [84] and metabotropic glutamate receptors [85], among others, were characterized in this tissue.

In the study, G protein-coupled receptor kinases 2, 3 and 5 were expressed in different regions and cell populations of the chick retina. While immunoreactivity of GRK2 was found over all types of neurons of the retina and over both plexiform layers, immunolabeling for GRK3 was restricted to the inner portion of the retina, over the inner plexiform layer and amacrine and ganglion cell bodies. However, immunoreactivity for GRK5 was only found in amacrine and Muller glial cells bodies and processes [39].
