Striated Perineal Muscles: Location of Somatic and Autonomic Neurons Projecting to the Male Pig Ischiocavernous Muscle. Neurochemical Features of the Sympathetic Subset.

The location, number and size of the central and peripheral neurons innervating the ischiocavernous muscle (ICM) were studied in male pigs by means of Fast Blue (FB) retrograde neuronal tracing. Moreover the immunohistochemical properties of the sympathetic ganglia were investigated combining the double immunolabeling method. After injection of FB into the left ICM, a mean number of 245.3 ± 134.9 labeled neurons were found in the ipsilateral ventral horn of the S1-S3 segments of the spinal cord (SC), 129.7 ± 45.5 in the L6-S3 ipsilateral and S2-S3 contralateral spinal ganglia (SGs), 2279.3 ± 622.1 in the ipsilateral L2-S2 and contralateral L5-S2 sympathetic trunk ganglia (STGs), 541.7 ± 158 in the bilateral caudal mesenteric ganglia (CMGs), and 78.3 ± 35.8 in the microganglia of the pelvic plexus (PGs). The mean area of the ICM projecting neurons was 1217 ± 69.7 µm2 in the SC, 2737.5 ± 176.5 µm2 in the SGs, 982.8 ± 36.8 µm2 in the STGs, 865.9 ± 39.14 µm2 in the CMGs and 426.2 ± 24.72 µm2 in the PGs. The FB positive neurons of autonomic ganglia contained Dopamine ß hydroxylase, vesicular acetylcholine transporter, neuronal nitric oxyde sinthase, calcitonine gene related peptide, leu-enkephaline, neuropeptide Y, substance P, vasoactive intestinal polypeptide, and somatostatine often colocalized with tyrosine hydroxylase. The particular localization of the motor somatic nucleus, the abundant autonomic innervation and the qualitatively different content of ICM projecting sympathetic neurons suggest a complex regulation of this striated muscle involved in involuntary functions, such as the erection, ejaculation, micturition and defecation. Anat Rec, 301:837-848, 2018. © 2017 Wiley Periodicals, Inc.

Immunohistochemical Properties of the Peripheral Neurons Projecting to the Pig Bulbospongiosus Muscle.

Retrograde neuronal tracing and single labelling immunofluorescence methods were used to define the neurochemical content of the peripheral autonomic and sensitive neurons projecting to the male pig striated bulbospongiosus muscle (BSM). The retrograde fluorescent neuronal tracer Fast Blue (FB) was injected into the left bulbospongiosus muscle of four intact impuberal pigs. After a 10-day survival time, the ipsilateral sacral sympathetic trunk ganglia (STGs), the caudal mesenteric ganglion (CMG), and the sacral spinal ganglia (SGs) were collected from each animal. In FB+ neurons of these ganglia, the presence of cathecolamine- (tyrosine hydroxylase-TH), acetylcholine- (vesicular acetylcholine transporter-VChAT), or nitric oxide-synthesizing (neuronal Nitric Oxide Synthase-nNOS) enzymes and of some biologically active peptides (calcitonine gene-related peptide-CGRP, Leu-Enkephaline-LENK, Neuropeptide Y-NPY, Substance P-SP and Vasoactive Intestinal Peptide-VIP) were studied. The ipsilateral STGs FB+ neurons showed immunoreactivity principally for TH and NPY and in decreasing order for VIP, VChAT, SP, CGRP, nNOS, and LENK. The left CMG FB+ neurons were immunoreactive to TH and NPY, and in smaller proportions for VIP, LENK, VChAT, CGRP, nNOS, and SP. The ipsilateral SGs FB+ neurons resulted immunoractive for CGRP, LENK, NPY, nNOS, SP, and VChAT. The heterogeneous neurochemical content of the peripheral neurons projecting to the pig BSM allows us to hypothesize the involvement of autonomic ganglia in the activity of both blood vessels and striated fibers of the muscle and the involvement of sensory ganglia in the afferent transmission from the muscle to spinal cord and in antidromic mechanisms that causes the relaxation of the BSM blood vessels. Anat Rec, 299:1192-1202, 2016. © 2016 Wiley Periodicals, Inc.

Neurochemical features of the autonomic neurons projecting to the cremaster muscle of the boar.

The cremaster muscle (CM) is a striated muscle showing some unusual features for ordinary striated muscles, in fact it receives, besides somatic innervation, a conspicuous autonomic sympathetic innervation. The autonomic neurons associated with the CM of 4 male intact pigs were typified combining the retrograde nontrans-synaptic fluorescent tracer Fast Blue (FB) and double labeling immunohistochemical methods. We collected the L4 sympathetic trunk ganglion (STG), that our preliminary studies proved to contain the highest number (575.5 ± 152.93; mean ± S.E.M., n = 4) of FB+ sympathetic neurons projecting to CM. About half of the CM projecting neurons of this ganglion were catecholaminergic and showed the colocalization of Tyrosine Hydroxylase (TH) with Neuropeptide Y (NPY), Leu-Enkephaline (LENK), Vasoactive Intestinal Polypeptide (VIP), Calcitonine Gene Related Peptide (CGRP), Substance P (SP), neuronal Nitric Oxyde Sinthase (n-NOS), and Vesicular Acetylcholine Transporter (VAChT). The noncatecholaminergic neurons were immunoreactive for all the other markers tested, even if in small percentages. The conspicuous and heterogeneous contribution of the sympathetic autonomic neurons to the muscle innervation is consistent with the hypothesis of a possible origin of the CM fibers by transdifferentiation of the smooth muscle-like gubernaculum mesenchyma into striated myotubes, suggesting that the cremaster myogenesis is independent from that of the abdominal muscles.

Localization and neurochemical features of the sympathetic trunk ganglia neurons projecting to the urethral muscle. An experimental study in a porcine animal model.

The striated perineal urethral muscle (UM) is involved in the voluntary control of the micturition requiring complex interactions between afferent and efferent (autonomic and somatic) pathways to store and periodically eliminate urine. Our aim was to define the site, cross sectional area and phenotype of sympathetic trunk ganglia (STG) neurons projecting to the porcine UM, combining retrograde neuronal tracer Fast Blue (FB) and double immunohistochemical labelling methods. The research was carried out on 3 male intact pigs, in which we counted a total number of 4992.67 ± 834.35 (mean ± S.E.M., n = 3) FB+ neurons distributed in the bilateral T12-S3 STG. These neurons were significantly larger in lumbar STG than in the sacral ones. Moreover we highlighted the presence of Dopamine ß hydroxylase (DßH), Vesicular Acetylcholine Transporter (VAChT), neuronal Nitric Oxyde Sinthase (n-NOS), Calcitonine Gene Related Peptide (CGRP), Leu-Enkephaline (LENK), Neuropeptide Y (NPY), Substance P (SP), Vasoactive Intestinal Polypeptide (VIP) and Somatostatine (SOM) and their eventual co-existence with Tyrosine Hydroxylase(TH) in both lumbar and sacral FB+ neurons. In particular, lumbar and sacral STG neurons expressed similar percentages of immunoreactivity for TH, SP and CGRP, but showed significantly different levels of immunoreactivity for NPY, VIP, VAChT, LENK, nNOS, DßH and SOM. Taken together, these data indicate a different contribution of lumbar and sacral pathways in the sympathetic transmission to the boar UM.

Double labelling immunohistochemistry on the sympathetic trunk ganglia neurons projecting to the extrinsic penile smooth musculature of the pig: an experimental study on the retractor penis muscle.

Retrograde neuronal tracing and double labelling immunofluorescence methods were used to define the neurochemical content of sympathetic trunk ganglia neurons projecting to the pig retractor penis muscle, which was taken as an experimental model of the male genital smooth musculature. After the injection of Fast Blue into the bulbo-penile portion of the retractor penis muscle, the eventual co-existence of the catecholaminergic marker tyrosine hydroxylase with calcitonine gene related peptide, leu-enkephalin, neuropeptide Y, neuronal nitric oxide synthase, substance P, vasoactive intestinal polypeptide or vesicular acetylcholine transporter was studied in the ipsilateral S1 sympathetic trunk ganglia, which resulted to contain the greatest number of autonomic retractor penis muscle projecting cells. The observation of Fast Blue positive neurons under the fluorescent microscope allowed the identification of different subpopulations of catecholaminergic and non-catecholaminergic retractor penis muscle-projecting neurons. The majority of catecholaminergic cells contained tyrosine hydroxylase alone, while the remaining part showed co-localization of tyrosine hydroxylase with all the other tested markers. These last neurons were immunoreactive, in decreasing percentages, for neuropeptide Y, leu-enkephalin, neuronal nitric oxide synthase, substance P, calcitonine gene related peptide, vasoactive intestinal polypeptide and vesicular acetylcholine transporter. The majority of non-catecholaminergic neurons were immunonegative for all the tested markers. The remaining non-catecholaminergic cells contained, in decreasing percentages, neuropeptide Y, neuronal nitric oxide synthase, leu-enkephalin, vasoactive intestinal polypeptide, vesicular acetylcholine transporter, substance P and calcitonine gene related peptide. Our findings documented the complexity of the neurochemical interactions that regulate both the motor functions of RPM and the blood flow through the muscle.

Sensory and autonomic neurons project both to the smooth retractor penis and to the striated bulbospongiosus muscles. Neurochemical features of the sympathetic subset.

Aim of the present study was to verify, by means of double retrograde neuronal tracers technique, the hypothesis that a subpopulation of sensory and autonomic neurons send collateral axons to both smooth and striated genital muscles. We also wanted to define the neurochemical content of the eventually retrogradelly double labeled (RDL) neurons in the sympathetic trunk ganglia (STG). We used six intact pigs and we injected the tracer Diamidino Yellow (DY) in the smooth left retractor penis muscle (RPM) and the tracer Fast Blue (FB) in the striated left bulbospongiosus muscle (BSM). Rare (2 ± 0.6) RDL neurons were found in the ipsilateral S2 spinal ganglion (SG), 220 ± 42 in the ipsilateral STGs, from L3 to S3, 19 ± 15 in the contralateral S1-S2 ones and 22 ± 5 in the bilateral caudal mesenteric ganglia (CMG). The RDL neurons of the STG were IR for TH (85 ± 13%), DßH (69 ± 17%), NPY (69 ± 23%), nNOS (60 ± 11%), LENK (54 ± 19%), VIP (53±26%), SOM (40 ± 8%), CGRP (34 ± 12%), SP (31 ± 16%), and VAChT (28 ± 3%). Our research highlights the presence of sensory and sympathetic neurons with qualitatively different neurochemical content sending axons both to the smooth RPM and to the striated BSM of the pig. These RDL neurons are likely to project to the smooth vasal musculature to create the ideal physiological conditions in which these muscles can optimize the erectile function.

Immunohistochemical characteristics of the nerve fibres of sow retractor clitoridis muscle.

The occurrence of several biologically active neuropeptides (calcitonine gene-related peptide, leu-enkephaline, neuropeptide Y, substance P, and vasoactive intestinal peptide) or nitric oxide-synthesizing enzymes (neuronal nitric oxide synthase), tyrosine hydroxylase, vesicular acetylcholine transporter, and their co-localization with tyrosine hydroxylase were investigated by immunohistochemistry in the retractor clitoridis muscle of slaughtered sows. Single immunolabelling revealed that tyrosine hydroxylase and neuropeptide Y immunoreactive nerve fibres were the most numerous, followed by the neuronal nitric oxide synthase and calcitonine gene-related peptide immunoreactive ones, the vasoactive intestinal peptide, substance P and leu-enkephaline immunoreactive nerve fibres were few and vesicular acetylcholine transporter immunoreactivity were observed only in single fibres. Double immunolabelling revealed the only co-localization of tyrosyne hydroxylase with neuropeptide Y. The most reliable labelling of nerve fibres of the retractor clitoridis muscle was observed around blood vessels, followed by non-vascular smooth muscles. The present data indicate that the sow retractor clitoridis muscle receives nerve fibres that exhibit different chemical codes and, likely, differences in their chemical coding depend on the target-structure.

Localization of the autonomic, somatic and sensory neurons innervating the cranial tibial muscle of the pig.

The location of sympathetic, somatic and sensory neurons projecting to the cranial tibial muscle of the pig hindlimb was studied with the neuronal non-transynaptic tracer Fast Blue. Additionally, the number and the size of these neurons were determinated. The Fast blue, randomly applied to the cranial tibial muscle belly of 3 pigs, labelled sympathetic neurons in the ipsilateral L5-S3 and contralateral S1 sympathetic trunk ganglia and in the prevertebral caudal mesenteric ganglia of both sides. The somatic motoneurons were identified in the ipsilateral ventral horn of the S1 segment of spinal cord, while the sensory neurons were located in the ipsilateral L7-S1 spinal ganglia. The diameter of the multipolar sympathetic neurons oscillated between 26 and 46 microm in the sympathetic trunk ganglia and between 18 and 42 microm in the caudal mesenteric ganglia. The size of the multipolar spinal motoneurons oscillated between 33 and 102 microm. The size of the pseudounipolar sensory neurons oscillated between 23 and 67 microm. In all ganglia, the labelled neurons were localized at random and did not show a somatotopic distribution. Our results document a conspicuous autonomic innervation projecting to the "classic" skeletal cranial tibial muscle. Probably this innervation is destined to the muscle vessels.

Striated perineal muscles: location of autonomic, sensory, and somatic neurons projecting to the male pig bulbospongiosus muscle.

The location, number, and size of the neurons innervating the bulbospongiosus muscle (BSM) were studied in male pigs, by means of Fast Blue (FB) retrograde transport. After injection of FB into the left BSM, labeled neurons were found bilaterally in the L2-S4 sympathetic trunk ganglia (STGs), in the caudal mesenteric ganglia (CMGs), in the microganglia of the pelvic plexus (PGs), in a dorsolateral area with respect to the central canal of S1-S3 segments of the spinal cord (SC) and in the S1-S4 ipsilateral and S2-S3 contralateral spinal ganglia (SGs). The mean number of labeled FB cells was 3,122 +/- 1,968 in STGs, 979 +/- 667 in CMGs, 108 +/- 104 in PGs, 89 +/- 39 in SC and 77 +/- 23 in SGs. The area of the multipolar neurons was 852 +/- 22 microm(2) in the STGs, 878 +/- 23 microm(2) in the CMGs and 922 +/- 31 microm(2) in the PGs. The multipolar SC neurons had an area of 1,057 +/- 38 microm(2), while pseudounipolar SG cells had dimensions of 2,281 +/- 129 microm(2). Our research enables us to highlight two peculiarities regarding the innervation of the boar BSM: the very high number of labeled autonomic neurons and the particular localization of the motor somatic nucleus.

Experimental study to localize the neurons projecting to the lamb retractor penis muscle.

Aim of the present study was to locate the neurons projecting to the lamb retractor penis muscle, a smooth muscle associated to the penis. The retrograde neuronal tracer Fast Blue was injected into the bulbopenile portion of the left retractor penis muscle. Labelled cells were found bilaterally in the S2-S4 spinal ganglia, from the last two lumbar (L5-L6 or L6-L7) to S4 sympathetic trunk ganglia and in the hypogastric and pelvic plexuses. Fast blue-positive (FB+) neurons were also found in the intermediate gray substance in the S1-S4 segments of the spinal cord. Our research enables us to describe the organization of the innervation of the lamb retractor penis muscle, highlighting the site of the primary afferent, postganglionic efferent and presumably preganglionic parasympathetic neurons projecting to the muscle.

Peripheral neurons innervating the extrinsic smooth penile musculature of the pig: experimental study by retrograde transport and immunohistochemistry.

Peripheral autonomic and sensitive neurons projecting to the extrinsic smooth penile musculature of the pig were studied by means of retrograde tracing and single-labelling immunofluorescence methods. The fluorescent retrograde tracer Fast Blue was injected into the left retractor penis muscle, that was taken as an experimental model of the male genital smooth musculature, of 4 castrated pigs. After a 7 day survival time, the ipsilateral paravertebral ganglion S1, the caudal mesenteric ganglion and the dorsal root ganglion S2 were collected. In these ganglia, the presence and the distribution of immunoreactivities to cathecolamine- (Tyrosine Hydroxylase), acetylcholine- (Vesicular Acetylcholine Transporter), or nitric oxide-synthesizing (neuronal Nitric Oxide Synthase) enzymes and to some biologically active peptides (Calcitonine Gene-Related Peptide, Leu-Enkephaline, Neuropeptide Y, Substance P and Vasoactive Intestinal Peptide) were studied. In paravertebral ganglion S1, Tyrosine Hydroxylase and Neuropeptide Y were the most frequently present substances. Also Leu-Enkephaline and neuronal Nitric Oxide were present quite frequently, while there was scarce immunoreactivity for the other antisera (in decreasing order Substance P, Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Calcitonine Gene-Related Peptide). In caudal mesenteric ganglion, in addition to Tyrosine Hydroxylase- and Neuropeptide Y-immunoreactivity, Substance P-, Vesicular Acetylcholine Transporter-, Vasoactive Intestinal Peptide-, Leu-Enkephaline- immunoreactivity were also frequently present, followed by neuronal Nitric Oxide- and Calcitonine Gene-Related Peptide- immunoreactivity. In dorsal root ganglion S2, Calcitonine Gene-Related Peptide and neuronal Nitric Oxide resulted to be the most frequently present neurotransmitters, followed by Vasoactive Intestinal Peptide, Vesicular Acetylcholine Transporter, Leu-Enkephaline, Substance P, Tyrosine Hydroxylase and Neuropeptide Y.

Sensory, motor somatic, and autonomic neurons projecting to the porcine cremaster muscle.

The location of sensory, somatic, and autonomic neurons projecting to the pig cremaster muscle (CM) was studied by means of the retrograde neuronal tracer Fast Blue (FB) technique. FB was randomly injected in the left CM of four impuberal pigs and serial sections of sensory and autonomic ganglia and spinal cord were examined under a fluorescence microscope. Additionally, some indications about the number and size of labeled neurons were given. Sensory pseudounipolar somata were located ipsilaterally in the L2-L6 and S1-S2 dorsal root ganglia, their total number ranging between 125 and 194, their mean diameter between 24 and 89 microm. Somatic multipolar motoneurons were located ipsilaterally in the L2-L4 neuromeres of the spinal cord, their total number ranging between 53 and 169, their mean diameter between 29 and 53 microm. Autonomic multipolar paravertebral ganglia neurons were located ipsilaterally from L1 to S4 and contralaterally from L2 to S2. Their total number ranged from 2,015 to 3,067 and their mean diameter between 25 and 55 microm. The multipolar caudal mesenteric ganglia neurons were located bilaterally, their total number ranging between 14 and 1,408 and their diameter from 22 to 39 microm. In two subjects only, multipolar neurons were also found ipsilaterally in the microganglia of pelvic plexus (2 and 13 neurons). Their mean diameter ranged between 28 and 54 microm. Our study documented that the CM-projecting neurons were located at different neural levels, with a predominance in the autonomic ganglia.

Localization of neurons projecting into the extrinsic penile smooth musculature of the pig: an experimental study on the retractor penis muscle.

The aim of this study was to locate in male pigs the sensory and autonomic ganglia innervating the retractor penis muscle (RPM), which was taken as an experimental model of the genital smooth musculature. The retrograde neuronal tracers horseradish peroxidase (HRP), Fast Blue (FB), and diamidino yellow (DY) were injected into the bulbopenile portion of the left RPM. The tracers highlighted a different affinity for the neuronal structures, although labelled cells supplying the RPM were generally found in bilateral dorsal root ganglia (DRGs, S1-S3), in bilateral paravertebral ganglia (PaGs, L2-S3), and in the left and right caudal mesenteric ganglia (CMGs). The mean number of labelled FB cells was 795 (range, 645-952) in DRGs, 16046.25 (range, 10226-18742) in PaGs, and 635.25 (range, 333-786) in CMGs. The mean diameter of pseudounipolar DRG cells was 60-75 microm, while the multipolar neurons of PaGs and CMGs had dimensions varying between 20-50 microm and 20-30 microm, respectively.