Distribution of galanin-immunoreactive nerve fibers in the rat nasal mucosa.

Galanin-like immunoreactivity was found in nerve fibers beneath and within the epithelium of the rat mucosa by the use of immunohistochemical techniques. Immunoreactive fibers were also noted close to blood vessels and seromucous glands in the lamina propria. Fast blue applied to the nasal mucosa underwent retrograde transport to some immunoreactive neurons of the trigeminal ganglion. Thus, the rat nasal mucosa was shown to be innervated by galanin-containing sensory nerves.     

VRL-1 immunoreactivity in the rat cranial autonomic ganglia.

Immunohistochemistry for VRL-1, a newly cloned capsaicin-receptor homologue, was performed on the rat cranial autonomic ganglia. The immunoreactivity (ir) was detected in the majority of neurones in the pterygopalatine (66%) and submandibular ganglia (68%). In the tongue and carotid body, parasympathetic neurones contained VRL-I ir. In the superior cervical ganglion, only 2% of postganglionic sympathetic neurones showed the immunoreactivity. VRL-1-ir nerve endings could not be detected in their peripheral tissues. These findings may suggest that VRL-1 has functions within neuronal cell bodies of the cranial autonomic ganglia.     

Origin of PACAP-immunoreactive nerve fibers innervating the subarachnoidal blood vessels of the rat brain.

The subarachnoidal cerebral blood vessels of the rat are innervated by nerve fibers containing different neuropeptides, e.g. pituitary adenylatecyclase activating polypeptide (PACAP). PACAP dilates brain arterioles and immunohistochemical studies of the rat have indicated that PACAP binds to a VPAC1-receptor in the cerebral vasculature of this species. We have investigated the perikaryal origin of the nerve fibers innervating the subarachnoidal blood vessels of the rat by combined retrograde tracing with Fluorogold and immunohistochemistry. The in vivo neuronal retrograde tracings were done by injection of 2% Fluorogold in water into the subarachnoidal space in the area of the middle cerebral artery. The retrograde transported tracer was detected by use of an antibody against Fluorogold. One week after the injections, the animals were vascularly perfused with Stephanini's fixative and labeled perikarya were found bilaterally in the trigeminal, sphenopalatine, and otic ganglia. The retrograde Fluorogold tracings were combined with immunohistochemistry for PACAP using a mouse monoclonal antibody and the biotinylated tyramide amplification system. Double labeled perikarya containing both Fluoro-gold and PACAP were found predominantly in the trigeminal ganglion, and only rarely in the otic and sphenopalatine ganglion. Summarizing, our retrograde tracings combined with immunohistochemistry indicate that the perikarya in the trigeminal ganglion are the main origin of PACAPergic nerve fibers projecting to the cerebral vasculature of the rat.     

Unmyelinated nerve fibers in sural nerve in pure autonomic failure

We examined sural nerve biopsy specimens from 7 patients with pure autonomic failure (PAF). The mean unmyelinated nerve fiber diensity in these patients was 40% less than in age-matched controls. Increased numbers of clusters of collagen pockets not containing unmyelinated axons were the most prominent finding in PAF. This appears to reflect recent dropout of a group of sympathetic efferents and suggests grouping of unmyelinated fibers by modality at the level of the sural nerve trunk.     

Neurofilament immunoreactivity in developing rat autonomic and sensory ganglia

Immunoreactivity to neurofilament (NF) antiserum appears early in the development of both the central and peripheral nervous systems of the rat fetus. In 10 somite embryos, positive cell bodies are present in the ventromedial part of anterior rhombencephalic and mesencephalic neural tube. From there the appearance of immunoreactivity spreads cranially to the prosencephalic anlage before closure of the anterior neuropore and caudally following the sequence of neural tube closure. Immunoreactivity increases rapidly in axon bundles of central and peripheral systems, but in immature cell bodies of sensory ganglia the NF material only forms a ring around the nucleus. At 16 days of gestation, some cell bodies are progressively loaded with NF-immunoreactive material as a thick perinuclear network first and then in more excentrically located aggregates. This category of neurons is mainly observed in the distal part of the trigeminal ganglion, in petrous and nodose ganglia and in cervical dorsal root ganglia. In adult ganglia large cell bodies and some small ones present high NF immunoreactivity. In autonomic cell bodies (in superior cervical ganglion and in parasympathetic cranial ganglia) the immunoreactive material only forms a perinuclear ring slowly transformed into a loose perinuciear meshwork at the end of gestation. Intensely reactive nerve fibers are observed in cranial sensory as well as in sympathetic and parasympathetic ganglia and nerves. No positive cell bodies and only a few NF-immunoreactive nerves are observed in the carotid bodies. The NF immunoreactivity is better visualized on sections of fresh frozen material, treated with acetone, than in fixed specimens.These results are compared to previous observations reported for other species and for developing dorsal root ganglia. This immunostaining may be used to detect differentiation of peripheral sensory and autonomic neurons under experimental conditions. The uneven distribution of NF immunoreactivity in sensory neurons from stage 16 days of gestation as specific for precise subpopulations of neurons is discussed.     

Origin and distribution of phrenic primary afferent nerve fibers in the spinal cord of the adult rat

Previous studies from this laboratory have localized and morphologically characterized phrenic motor neurons in the rat spinal cord at light and electron microscopic levels. The present investigation used a modification of the TMB method for the retrograde transport of horseradish peroxidase (HRP) to describe at light microscope levels the origin and distribution of phrenic primary afferent axons in the adult rat spinal cord. Dry HRP crystals were applied to the central stump of the transected phrenic nerve in the neck to label spinal ganglion cell bodies and thus determine the levels of origin of afferent axons in the phrenic nerve. Camera lucida drawings were then made from serial sections through the appropriate spinal cord levels to determine the specific distribution of transganglionically labeled phrenic central axonal processes within the spinal cord. HRP-labeled phrenic primary neurons were observed in the C3 to C7 spinal ganglia. The camera lucida studies indicated that the transganglionically labeled central processes of phrenic primary afferent axons distributed into the dorsal horn at the C4 and C5 levels of the spinal cord. Furthermore, central processes distributing to C5 were more numerous than those that distributed to C4. Afferent axons were never seen in the dorsal horn at C3, C6, or C7. As spinal ganglion cells were labeled at C3 above and C6 and C7 below, it follows that central processes of phrenic afferent fibers descend and ascend in the dorsal columns of the spinal cord before distributing into the dorsal horn. Specifically, the labeled primary afferent axons and their collateral branches were found in the fasciculus cuneatus, and in laminae I, II, III, and IV of the dorsomedial aspect of the dorsal horn. The function of these central axonal processes is unknown, but based on a comparison of our morphologic data with previous physiological and anatomical studies, we suggest that phrenic afferent fibers may arise from proprioceptors (muscle spindles and Golgi tendon organs), nociceptors, or rapidly adapting mechanoreceptors (Pacinian corpuscles) within the diaphragm.     

The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat.

It is known that the vagus nerve contains catecholaminergic fibers. However, the origin of these fibers has not been systematically examined. In this study, we addressed this issue using retrograde tracing from the subdiaphragmatic vagus nerve combined with immunocytochemistry. The cervical and thoracic sympathetic trunk ganglia, the nodose ganglia and the dorsal motor nucleus of the vagus nerve were examined following injection of Fluoro-Gold or cholera toxin horseradish peroxidase conjugate into the trunks of the subdiaphragmatic vagus nerve of rats. Numerous retrogradely labeled neurons were seen in the nodose ganglion and the dorsal motor nucleus of the vagus nerve. Very few labeled neurons were found in the sympathetic ganglia (less than 0.06% of the neurons in either superior cervical ganglion or cervicothoracic ganglion were retrogradely labeled). Double labeling with immunofluoresence for catecholamine synthesizing enzymes revealed that: (1) 92% of all Fluoro-Gold retrogradely labeled tyrosine hydroxylase immunoreactive neurons were found in parasympathetic sources (75% in the dorsal motor nucleus of the vagus nerve and 17% in the nodose ganglia), and only 8% in the cervicothoracic sympathetic ganglia; (2) 12% of the retrogradely labeled catecholaminergic neurons in the dorsal motor nucleus of the vagus nerve were also dopamine-beta-hydroxylase immunopositive neurons; (3) 70% of the retrogradely labeled neurons in the sympathetic ganglia were tyrosine hydroxylase immunopositive and 54% of these catecholaminergic neurons contained dopamine-beta-hydroxylase, while 30% of the retrogradely labeled neurons were non-catecholaminergic neurons. These results indicate that catecholaminergic fibers in the abdominal vagus nerve are primarily dopaminergic and of parasympathetic origin, and that only an extremely small number of these fibers, mostly noradrenergic in nature, arise from postganglionic sympathetic neurons.     

The segmental distribution of afferent fibers from the vaginal cervix and hypogastric nerve in rats.

Injections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) into the walls of the vagina and cervix (vaginocervical injections) of rats resulted in labeling of dorsal root ganglia (DRG) cells located at T11-L4 and L6-S2. In a second group of animals, exposure of the hypogastric nerve to HRP-WGA resulted in a similar bimodal distribution of labeled cells as compared to vaginocervical injections. In a third group, unilateral hypogastric nerve transection prior to injection of HRP-WGA into the vaginocervical walls resulted in a significant reduction in DRG cells labeled at T13, L1, L2, L6 and S1. Bilateral transection of the hypogastric nerves prior to vaginocervical injections eliminated labeled DRG cells at thoracolumbar levels but not at L6 and S1. Bilateral pelvic neurectomy reduced, but did not eliminate labeled DRG cells at L6 and S1 following vaginocervical injections. These results indicate that the hypogastric nerve constitutes a major sensory pathway from the vaginocervical walls to thoracic, lumbar and sacral levels of the spinal cord. The hypogastric nerve may subserve the transmission of noxious input from the vaginocervical walls as well as the activation of ascending spinal pathways involved in neuroendocrine reflexes during parturition.     

Age-related changes in galanin-immunoreactive cells of the rat medial septal area.

Age-related changes in the cholinergic cells have been reported in the rat medial septal area. The neuropeptide galanin is colocalized with acetylcholine in the majority of the medial septal neurons. To assess possible age-related changes in the galanin-containing septal cells, we have examined, with immunohistochemical methods, the distribution pattern, density, and morphological features of galanin-containing cells in the rat medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (DBB) in 1, 3-6, 9-12, 16-18, 24-27, and 28-30 month-old rats. A morphometric computerized analysis was also performed. In addition, the intensity of the immunolabelling was measured by densitometry. Galanin-like immunoreactivity (galanin-LI) was present in both the MS and the DBB. Our results clearly indicate a progressive age-related decrease in the number of galanin-positive cells throughout the MS-DBB complex. Our quantitative study revealed a significant loss of galanin-positive cells in the MS-DBB complex of 16-18 (50.4%), 24-27 (52.3%), and 28-30 (52.4%) month-old rats compared to 3-6 month-old animals. A non-significant reduction (28.6%) in galanin-LI cell number was observed in 3-6 month-old rats compared to 1 month-old animals. The morphometric analysis demonstrated a significant reduction (18%) in the surface of galanin-positive cells remaining in the 28-30 month-old group. Furthermore, a significant decrease in the immunolabelling intensity was consistently observed in animals of 16 month-old and older. To determine whether changes in galanin-positive cells were associated with cholinergic changes, the number of cells stained for acetylcholinesterase (AChE) was estimated in 3-6, 9-12, 16-18, and 24-27 month-old rats. There was a 43% decrease in the number of AChE-positive cells and a 71% loss of galanin-positive cells in 24-27 month-old rats compared to 3-6 month-old. The galanin-cell loss in the medial septal area was therefore associated with a parallel, although smaller, cholinergic septal cell loss.     

Peripheral nervous system in the larynx. An anatomical study of the motor, sensory and autonomic nerve fibers.

The laryngeal peripheral nervous system is presented on the basis of our results in the cat, following 10 years of investigation using mainly tracer techniques. The present paper focused on the localization of each laryngeal motoneuron, the myotopical arrangements of motoneurons innervating the pharyngeal and esophageal striated muscles within the nucleus ambiguus in the motor nerve supply, and also on the location of neurons, the distribution and density of nerve fibers, the area and laterality of the innervation, and the pathway to the larynx in the sensory and sympathetic nerve supplies. Regarding the parasympathetic nerve supply, the neural ganglia and the ganglionic cells in and around the laryngeal nerves and in the laryngeal framework are demonstrated. Most of this innervation, however, is still unclear. Discussions from the literature are also reported.     

NPY-like immunoreactivity in sensory nerve fibers in rat sciatic neuroma.

Neuropeptide Y (NPY)-like immunoreactivity was examined by fluorescence immunohistochemistry in rat sciatic neuromas, L5 dorsal root ganglias (DRGs) and L5 dorsal roots 1-3 weeks after chronic nerve injury. Anterograde tracing demonstrated that a large number of NPY-positive neuroma fibers were sensory. These fibers were mostly large diameter axons, in line with the finding that a majority of NPY-immunoreactive neurons in the DRG were medium- to large-sized neurons which showed immunoreactivity to the neurofilament antibody RT 97. In dorsal roots NPY immunoreactivity was strong after sciatic neuroma formation. Dorsal rhizotomy and ligation, on the other hand, did not induce NPY immunoreactivity at any of the sites examined.     

Origin of small primary afferent substance P-immunoreactive nerve fibers in the guinea-pig heart

Primary afferent and substance P (SP)-immunoreactive nerve fibers of the guinea pig and rat heart were investigated by physiological and immunohistochemical methods. Immunohistochemistry revealed abundant SP-positive fibers in the guinea pig atria, with fewer in the ventricles. Only an occasional fiber was seen in the rat atrium or ventricle. Sectioning the vagus nerve did not noticeably influence the supply of SP-immunoreactive nerve fibers in the guinea pig heart. When the atria or ventricles were stimulated, afferent nerve fiber activity was recorded from the second and third thoracic dorsal roots. In guinea pig atria 3 types of fibers were identified on the basis of conduction velocities: A delta 1, A delta 2 and C fibers. Only A delta fibers were identified in the ventricle. By vagal recordings, A delta fibers were demonstrated but a C fiber response could not be shown in this nerve. SP-immunoreactivity in primary afferent fibers was depleted by the neurotoxin capsaicin. Capsaicin treatment also caused a reduction in the conduction velocity of small diameter myelinated A delta 2 (by 29%) and unmyelinated C fibers (by 46%). In the rat heart, evidence for A delta 2 or C fibers was not found. These results indicate that primary afferent and SP-immunoreactive fibers are numerous in guinea pig heart, but few in the rat. It is concluded that most of these fibers have their cell bodies of origin in the dorsal root ganglia.     

Differentially expressed genes in rat dorsal root ganglia following peripheral nerve injury.

Ordered differential display PCR was used to identify differentially expressed genes in rat dorsal root ganglia at 7 days following chronic constriction injury (CCI) of the sciatic nerve. Fourteen differentially displayed cDNA bands were isolated, cloned and verified by RT-PCR. The four mRNAs were increased, which included mRNAs encoding heat shock protein 27, fatty acid binding protein, apolipoprotein D and one novel gene. Six down-regulated clones were microtubule-associated protein 1B, protein tyrosine phosphatase alpha, Kv1.2 channel, myelin protein SR13, medium-sized neurofilament protein, and one novel gene. Our results show that many differentially regulated genes after CCI may play a role in nerve degeneration and/or regeneration and provide a molecular framework for understanding the peripheral mechanism underlying neuropathic pain.     

Calcitonin gene-related peptide containing autonomic efferent pathways to the pelvic ganglia of the rat

Indirect immunofluorescence method was employed to investigate the involvement of calcitonin gene-related peptide (CGRP) in the autonomic efferent innervations of the pelvic visceral organs of the rat. Cells labeled with Fast blue (FB) injected into the pelvic ganglia were observed in the sacral parasympathetic nucleus; about 30% of these neurons showed CGRP-like immunoreactivity. These CGRP-like immunoreactive neurons were located in the dorsomedial part of the sacral parasympathetic nucleus, extending their dendrites mediolaterally. FB-labeled cells were also found in the upper lumbar level (L1, L2) of the spinal cord. Some of these neurons also showed CGRP-like immunoreactivity. CGRP-like immunoreactive varicose fibers were seen in the pelvic ganglia surrounding individual ganglion cells. Considerable amount of these fibers were not affected by sensory deafferentation, so they probably originated from autonomic efferent neurons.