**4. Location of avian muscarinic receptors**

In birds, fundamental pharmacological studies indicate that mAChRs located in the CNS are associated with vision, navigation, metabolism, and central thermoregulation [35]. However, the role of each of the mAChRs subtypes has not been established in either the CNS or peripheral nervous system [36]. Muscarinic receptor subtypes are expressed in cochlear neurons, supporting cells, central auditory neurons [37], avian vestibular hair cells, nerve terminals, ganglion cells, [38] and also in the avian retina [39]. **Table 4** narrates the important locations of muscarinic receptors in birds.

#### **4.1 Avian muscarinic receptors in the central nervous system**

Even though the information on muscarinic receptor subtype M1 present in avians is few, some studies have given proof for the presence of central muscarinic M1 receptor subtype. A study [20] has reported the role of muscarinic receptor subtypes M1 and M3 involved in carbachol-induced hypophagia in neonatal broiler chicken. Moreover, the study provided first evidence for muscarinic receptor mediated hypophagic effect in domestic fowl and that the hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors, which were similar to previous reports in mammals [40, 41]. In this study, M2 and M4 receptors had no role in feeding behavior in neonatal broiler chicken, but both M2 and M4 receptors had a prominent role in feeding behavior in rat [42]. It is a well-known fact that there is a significant difference on the role of neurotransmitters in the feeding behavior between avian and mammals [20].

Another study [16] was conducted to assess the functional role of muscarinic acetylcholine receptors in chick brain slices using whole-cell patch-clamp recordings of neurons in the lateral spiriform nucleus. The lateral spiriform nucleus (SpL) forms part of the avian basal ganglia system. It receives cholinergic innervation from the nucleus semilunaris, as well as glutamatergic input from the ansa lenticularis subthalamus, GABAergic input from the paleostriatum primitivum and both GABAergic and possibly dopaminergic inputs from the substantia nigra [43]. But their functional roles in influencing motor behavior remain largely unknown, even after depicting the anatomical pathways. Possibly, the ACh released from cholinergic nerve terminals in or close to the SpL enhances the release of GABA by means of nicotinic AChR activation, which is located on GABAergic axon terminals [44]. Similarly, ACh might act simultaneously on muscarinic AChRs also.

Results of their study revealed that bath application of carbachol, a muscarinic agonist, enhanced the frequency of spontaneous postsynaptic currents and produced

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

a pronounced postsynaptic inward current in normal ACSF (Artificial Cerebrospinal Fluid) with a slower onset but more prolonged action. These effects may be due to the possible muscarinic AChRs belonging to the M3 subtype category and are located some distance from release sites, requiring activation of voltage-dependent sodium channels and N-type voltage-dependent calcium channels (VDCCs) to trigger enhanced GABA release. To determine the muscarinic receptor subtype that mediated enhancement of spontaneous GABAergic IPSCs (Inhibitory Postsynaptic Currents), several muscarinic antagonists were used for testing, of which 4- DAMP mustard alone completely blocked muscarine's effect. As per existing data on 4-DAMP mustard, it exhibits a high affinity for the M3 receptor, whereas its affinity for other subtypes is significantly lower [45]. Moreover, the pharmacological profile suggested that M3 receptors predominantly contributed to the muscarinic enhancement of GABA release, thus giving evidence for the same.

Another study reported a cDNA that encodes a chicken protein that is homologous to mammalian prion protein (PrPc). PrPc in mammals is an altered isoform of the infectious particle prion (PrPsc) thought to be responsible for spongiform encephalopathies in humans and animals. It is a cellular protein of unknown function in mammals. Chicken prion-like protein (ch-PrLP) is expressed in embryos as early as day 6 in the central nervous system, mostly in the motor neurons. It was found that this protein is abundant in preparations of an acetylcholine receptor-inducing activity based on its ability to synthesize nicotinic receptors in cultured myotubes. Hence, according to the study, it is likely that they serve normally in the neuromuscular junction and central nervous system to regulate chemoreceptor numbers [46].

#### *4.1.1 Muscarinic receptors in pigeon brain*

The avian nidopallium caudolaterale (NCL), situated in the caudal telencephalon, serves comparable functions to the mammalian prefrontal cortex, although both are not homologous structures. In the last decades, assumed homologies between avian and mammalian brain components has been studied [47]. It assumes that mammalian and avian pallia share a homologous pallial identity that may be derived from a common ancestry [48]. However, this does not imply that cortical or subcortical pallial areas must be exactly homologous to pallial components in birds.

Various researchers have proved that the mammalian prefrontal cortex (PFC) and the avian NCL share several anatomical neurochemicals [49], electrophysiological and functional [50] characteristics. Hence, the similarities between NCL and PFC may likely do not result from common ancestry, but may be due to an evolutionary convergence. In a quantitative analysis of different receptor binding sites, using autoradiography, by labelling the muscarinic cholinergic M1 receptor with pirenzepine and the muscarinic cholinergic M2 receptor with oxotremorine, the study compared the receptor fingerprints of NCL with those of frontal areas in mammals.

ACh is an essential regulator of cortical excitability and plays important roles for arousal, attention, and cognitive processes [51, 52]. These functions are mediated by muscarinic and nicotinic ACh receptors. Cholinergic M1 receptors were highest in humans if compared to macaque monkey, rhesus monkey, rat and pigeon, while M2 and nicotinic receptors showed equal densities [53]. However, pigeons showed an inverted pattern of M1/M2 binding in the NCL compared to other species which suggests an increased inhibitory control on local circuits, this may be a compensating mechanism for the shift to glutamatergic processing which was at highest concentration in the avian nidopallium [54].

#### *4.1.2 Muscarinic receptors in avian nerve fibers*

DRG (dorsal root ganglia) is a collection of bipolar cell bodies of neurons, formed when the dorsal sensory root of spinal nerves exits the neural foramina, surrounded by layers of satellite glial cells (SGCs) [55]. DRG neurons are pseudo-unipolar cells. They give rise to one fiber from which both central and peripheral projections derive, forming peripheral and central sensory branches. These branches contain both myelinated and unmyelinated fibers differing in size, conduction velocity, and perception specification, e. g. nociceptive and thermal sensory neurons [56].

Immunocytochemical studies conducted in the chick DRGs has shown that muscarinic receptors are present in almost all neurons of DRG with a Kd value for [3 H]QNB (H-3-quinuclidinyl benzilate) comparable to that reported for mAChRs in other tissue with cholinergic innervation. Hence, DRG neurons not only express cholinergic neurotransmission markers, a high-affinity choline uptake system (HACU) [57], but are also muscarinic cholinoceptive [58]. Functional studies using d-tubocurarine in chick dorsal root ganglia have shown that only about 50% of DRG neurons are responsive to acetylcholine and are sensitive to dtubocurarine suggesting their nicotinic nature [59]. Thus, the divergent distribution of both muscarinic and nicotinic acetylcholine receptor types may indicate their different role in DRG neurons.

A similar study has also identified the presence of muscarinic cholinergic receptors and their microanatomical localization in chicken dorsal root ganglia. They used pirenzepine in competition binding experiments, which showed high affinity for the cloned M1 receptor and labels the M1 receptor of the pharmacological classification [3]. Though the absolute specificity of pirenzepine was questioned by many studies for M1 muscarinic cholinergic receptors [60], the sensitivity of [3 H]QNB binding to the compound suggests the expression of the M1 receptor subtype in chick DRG.

In another study to establish muscarinic receptors modulate intracellular calcium levels in sensory neurons of chicks, E18 embryonic chicks were treated with muscarinic agonists such as muscarine and oxotremorine which resulted in an increase of intracellular calcium levels in fura-2 AM (fluorescent calcium indicator) loaded DRG neurons. This effect was antagonized by treatment with atropine and not with the same concentration of mecamylamine indicating that the increase in calcium concentration was due to muscarinic receptor activation. To substantiate the above findings, selective antagonists of muscarinic receptor subtypes were tested, and it also indicated that M1 to a greater extent and to a lesser extent M3 receptor subtypes were responsible for the observed intracellular calcium mobilization. These findings suggest a functional role for acetylcholine and muscarinic receptors in sensory transduction [17]. Moreover, second messengers such as cGMP and cAMPs muscarinic modulation have already been reported in DRG neurons by demonstrating the presence of M1 and possibly M3 subtype in the chick dorsal root ganglia [58].

Another study conducted to investigate the presence of mAChRs by immunolabeling neurons, nerve fibers, Schwann cells, and satellite cells in chicken also suggests the presence of mAChR subtypes. It showed a consistent presence of mAChRs in the neuronal plasma membrane, which suggests a probable role for mAChRs during neuronal differentiation ad exchange of information between neurons [61]. In the nerve fibers, mAChR was detected in the initial segment of emerging neuronal fibers at E12, but the unmyelinated axons of both peripheral and central branches were devoid of an immunoreaction product. This indicates mAChRs may not be involved in the transduction of sensory stimuli [62] in early life. Later, in young chicks several unmyelinated fibers, both central and peripheral, become immunopositive. In young

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

chicks, the immunoreaction product was detected in the axoplasm of numerous unmyelinated central axons, suggesting transport of mAChRs towards the nerve endings in the spinal cord.

In the satellite and Schwann cells, at E12 numerous perineuronal satellite cells surrounding the soma of immunopositive and immunonegative neurons were strongly labeled for mAChRs. Reciprocal communication between neurons and glial cells is well established [63]. Various neuroactive substances synthesized and released by glial cells are involved in neuronal differentiation and growth. Similarly, interaction between Schwann cells and axons are also important for the maintenance of structural integrity and functioning of axons. All these suggest a correlation that at early developmental stages of the avian DRG, the mAChRs expressed by satellite and Schwann cells is important to control the morphogenesis of neurons that take part in sensory functions as well as their axons.

#### *4.1.3 Muscarinic receptors in avian Edinger-Westphal nucleus*

Choroid in birds is extensively innervated by ciliary ganglion [64]. Edinger-Westphal (EW) nucleus is the source of the parasympathetic preganglionic input to the ciliary ganglion, upon electrical stimulation, increases the choroidal blood flow in pigeons [65]. Various pharmacological investigations suggest that NO (Nitric Oxide) mediates the EW-evoked vasodilatory response [66]. Earlier studies have identified M3 receptors in avian choroid [29], which mediates the tone of vascular beds in the avian choroid probably by the cholinergic-dependent release of endothelium-derived relaxing factor (presumably NO) [67, 68].

The study examined the role and the type of muscarinic receptors within the choroid that is involved in the increases in choroidal blood flow, using electrical stimulation of the nucleus of Edinger-Westphal (EW) nucleus to activate the ciliary ganglion input to choroid in pigeons. M3 receptors were blocked using a selective antagonist, 4-diphenyl-acetoxy-N-methylpiperedine (4-DAMP), it reduced the baseline choroidal blood flow. Simultaneously, atropine, a non-selective antagonist of muscarinic receptors, decreased the EW-evoked responses to a lesser extent than 4-DAMP.

The results of the study suggested a major role of M3 type muscarinic receptors in the EW evoked increases in choroidal blood flow in pigeons [30]. Based on another finding that the input of ciliary ganglion to choroid does not synthesize NO, but inhibitors of NO production do block EW-evoked choroidal vasodilation [66], it seems likely that the M3 receptors acted on by 4-DAMP are present on choroidal endothelial cells and mediate choroidal vasodilation via stimulation of endothelial release of nitric oxide.
