Double labelling immunohistochemical characterization of autonomic sympathetic neurons innervating the sow retractor clitoridis muscle

Abstract

Retrograde neuronal tracing and immunohistochemical methods were used to define the neurochemical content of sympathetic neurons projecting to the sow retractor clitoridis muscle (RCM). Differently from the other smooth muscles of genital organs, the RCM is an isolated muscle that is tonically contracted in the rest phase and relaxed in the active phase. This peculiarity makes it an interesting experimental model. The fluorescent tracer fast blue was injected into the RCM of three 50 kg subjects. After a one-week survival period, the ipsilateral paravertebral ganglion S1, that in a preliminary study showed the greatest number of cells projecting to the muscle, was collected from each animal. The co-existence of tyrosine hydroxylase with choline acetyltransferase, neuronal nitric oxide synthase, calcitonin gene-related peptide, leuenkephalin, neuropeptide Y, substance P and vasoactive intestinal polypeptide was studied under a fluorescent microscope on cryostat sections. Tyrosine hydroxylase was present in about 58% of the neurons projecting to the muscle and was found to be co-localized with each of the other tested substances. Within fast blue-labelled cells negative to the adrenergic marker, small populations of neurons singularly containing each of the other enzymatic markers or peptides were also observed. The present study documents the complexity of the neurochemical interactions that regulate the activity of the smooth myocytes of the RCM and their vascular components.

Sensory and Autonomic Neurons Project Both to the Smooth Retractor Penis and to the Striated Bulbospongiosus Muscles. Neurochemical Features of the Sympathetic Subset

“…This peptide has been already found in the STG of the sow (Panu et al, 2001;Ragionieri et al, 2008) and cat (Lindh et al, 1989) and in the RPM and BSM. (a, c, e, g, i, m, o, q, s, and u): DYþ/FBþ multipolar neurons of the left, ipsilateral, L7 or S1 STG.…”

“…This peptide is commonly considered a marker of afferent pathways (Majewski et al, 1995;Kaleczyc et al, 1997;Czaja, 2000), therefore, its presence within sympathetic neurons is an unforeseen finding. However, CGRP has been also shown in the STG neurons of the boar (Hill and Elde, 1989;H€ app€ ol€ a et al, 1993;Lakomy et al, 1994), sow (Panu et al, 2001;Ragionieri et al, 2008) cat (Kummer and Heym, 1988;Lindh et al, 1989) and horse (Nasu et al, 2003). Moreover, as there is some evidence that a minor proportion of CGRP-containing nerve fibers supply human genital organs (Jen et al, 1997), we think they might play a functional role in the modulation of neurotransmission in the target organs of the STG neurons (H€ app€ ol€ a et al, 1993).…”

“…Cholinergic marker has been documented in the STG of the sow (Panu et al, 2001;Ragionieri et al, 2008), cat (Lindh et al, 1989), rat (Morales et al, 1995;Schafer et al, 1997Schafer et al, , 1998, guinea-pig (Elfvin et al, 1993) and man (Jarvi et al, 1989;Masliukov and Timmermans, 2004). The principal function of cholinergic innervation in the uro-genital system is the presynaptic inhibition of the adrenergic excitatory neurotransmission (Sj€ ostrand, 1965;Sann et al, 1995;Anderson et al, 1997;Saenz de Tejada et al, 2000).…”

Localization of peripheral autonomic neurons innervating the boar urinary bladder trigone and neurochemical features of the sympathetic component

“…23,68 Another reason could be that, in many species, including the pig, choline acetyltranferase- (ChAT-) and VAChT-immunoreactivity are present in a number of endothelial cells 69 and in non-neuronal cells within the urothelium, 70,71 suggesting that there may be other sources of acetylcholine with a role in some regulatory mechanisms. The co-localization of cholinergic and catecholaminergic markers has already been observed in STG neurons projecting to the pig bulbospongiosus muscle, 72 to the retractor penis muscle in the male pig 73 , and to the sow retractor clitoridis muscle 74 . Moreover, ChAT and DßH were found co-localized in the cranial cervical ganglion of fetal pig 75 .…”

Neurochemical Features of the Autonomic Neurons Projecting to the Cremaster Muscle of the Boar

“…NPY is a polypeptide widespread in the central and peripheral nervous system, in particular in the postganglionic sympathetic neurons (Gazza et al, 2003;Botti et al, 2006b,,;Ragionieri et al, 2008), implicated in the modulation of the vasoconstrictor response triggered by catecholaminergic neurons. The NPY found in our study, in large percentages colocalized with TH, could be involved in blood flow regulation of the pig CM, increasing the effects of noradrenaline on the contraction of vasal musculature (Ekblad et al, 1984;Pernow et al, 1988).…”