Non-varicose Fibres Research Articles

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.

Immunocytochemical localization of the dopamine transporter in human brain.

The dopamine transporter (DAT) was localized in normal human brain tissue by light microscopic immunocytochemistry by using highly specific monoclonal antibodies. Regional distribution of DAT was found in areas with established dopaminergic circuitry, e.g., mesostriatal, mesolimbic, and mesocortical pathways. Mesencephalic DAT-immunoreactivity was enriched in the dendrites and cell bodies of neurons in the substantia nigra pars compacta and ventral tegmental area. Staining in the striatum and nucleus accumbens was dense and heterogeneous. Mesocortical DAT immunoreactivity in motor, premotor, anterior cingulate, prefrontal, entorhinal/perirhinal, insular, and visual cortices was detected in scattered varicose and a few nonvaricose fibers. Varicose fibers were relatively enriched in the basolateral and central subnuclei of amygdala, with sparser fibers in lateral and basomedial subnuclei. Double-labeling studies combining DAT and tyrosine hydroxylase (TH) immunostaining in the ventral mesencephalon showed two subpopulations of dopaminergic neurons differentiated by the presence or absence of DAT-immunoreactivity in the A9 and A10 cell groups. In other dopaminergic cell groups (All, A13-A15), TH-positive hypothalamic neurons showed no detectable DAT-immunoreactivity. However, fine DAT-immunoreactive axons were scattered throughout the hypothalamus, particularly concentrated along the medial border, with more coarse axons present along the lateral border. These findings demonstrate that most mesotelencephalic dopamine neurons of human brain express high levels of DAT throughout their entire somatodendritic and axonal domains, whereas a smaller subpopulation of mesencephalic dopamine cells and all hypothalamic dopamine cell groups examined express little or no DAT. These data indicate that different subpopulations of dopaminergic neurons use different mechanisms to regulate their extracellular dopamine levels.

The distribution of nitric oxide synthase-, adenosine deaminase- and neuropeptide Y-immunoreactivity through the entire rat nucleus tractus solitarius: Effect of unilateral nodose ganglionectomy

The present study has employed immunocytochemistry on free-floating sections of adult rat medulla oblongata to characterise the distribution of nitric oxide synthase- (NOS), adenosine deaminase- (ADA) and neuropeptide Y- (NPY) immunoreactivity (IR) throughout the entire rostro–caudal axis of the nucleus tractus solitarius (NTS). In addition, unilateral nodose ganglionectomy was performed in a group of rats to determine whether any observed immunoreactivity was associated with central vagal afferent terminals. NOS-IR was found throughout the entire NTS, in cells, and both varicose and non-varicose fibres. Furthermore, unilateral nodose ganglionectomy resulted in a clear reduction in NOS-IR (visualised with diaminobenzidine) in a highly restricted portion of the ipsilateral medial NTS. Similarly, ADA- and NPY-containing cells, fibres and terminals were also found throughout the adult rat NTS. However, following unilateral nodose ganglionectomy, there was no apparent reduction in either ADA-IR or NPY-IR on the denervated side of the NTS. These data indicate a role for nitric oxide, purines and neuropeptide Y as neuromodulators within the rat NTS, although only nitric oxide appears to be primarily associated with vagal afferent input. Adenosine deaminase and neuropeptide Y-containing neurons appear to be predominantly postsynaptic to vagal input, although their possible association with vagal afferents cannot be completely excluded.

Distribution of FMRFamide-like immunoreactivity in the nervous system of Lumbricus terrestris.

The distribution of FMRFamide-like immunoreactive cell bodies and fibers in the nervous system of the earthworm Lumbricus terrestris has been studied by means of immunocytochemistry. The cerebral ganglion contains 150-170 immunoreactive nerve cells that are organized into six major groups in the rostral and five major groups in the caudal part of the ganglion; 160-180 immunoreactive nerve cells are present in the subesophageal ganglion, and 80-90 in the ventral cord ganglia. Immunoreactive neurons of the subesophageal and the ventral cord ganglia show similar distributions, in that FMRFamide-like immunoreactive cells form a ventromedial and a lateral cell group. Neuropil in all parts of the central nervous system shows intensively stained varicose and non-varicose fibers. Each segmental nerve contains FMRFamide-like immunoreactive fibers that can partly be traced to the two muscle layers of the body wall, and a fine immunoreactive network lies among the muscle fibers. A similar network is found in the wall of the alimentary canal. Immunopositive perikarya and fibers have been detected in the prostomial nerves, in the stomatogastric system. Some epithelial cells of the body wall are also immunopositive. The morphological characteristics and localization of FMRFamide immunoreactive neurons suggest that they may be involved in: (1) central integratory processes; (2) neuromuscular regulation in both the body wall and enteric system; (3) sensory processes.

Distribution of peptide-containing nerve fibres in achalasia of the oesophagus.

In this study the innervation of the normal human oesophagus was compared with samples taken from 12 patients undergoing Heller's cardiomyotomy for achalasia. The distribution of all nerve fibres in the oesophageal wall was revealed by immunoreactivity to neuron specific enolase and subpopulations of nerve fibres were revealed by immunoreactivity to vasoactive intestinal peptide, neuropeptide Y, enkephalin and substance P. In healthy oesophagus, many nerve fibres immunoreactive for vasoactive intestinal peptide and neuropeptide Y were present in the circular and longitudinal muscle layers of the oesophageal wall and in the cardia of the stomach, whereas fibres immunoreactive for enkephalin and substance P were uncommon. Neuropeptide Y-reactive fibres were commonly seen around blood vessels. In the myenteric plexus cell bodies reactive for vasoactive intestinal peptide and neuropeptide Y were prevalent, as were varicose and non-varicose fibres. In contrast, samples from patients with achalasia revealed few nerve fibres immunoreactive for vasoactive intestinal peptide or neuropeptide Y in either circular or longitudinal muscle, suggesting damage to the inhibitory motor neurons to the muscle layers. Very few fibres were found that were reactive for neuron-specific enolase, indicating that other fibre population (e.g. excitatory cholinergic motor neurons) are also damaged in achalasia. These abnormalities were observed in biopsies from both the constricted and dilated portions of the oesophagus, but the pattern of innervation in the gastric cardia was normal. Myenteric ganglion cells were seen in the oesophagus in only two patients and varicose nerve fibres in the myenteric plexus were uncommon. Neuropeptide Y-reactive perivascular nerve fibres were still found in achalasia as well as non-varicose nerve fibres in the myenteric plexus. These findings indicate damage to all intrinsic neurons in the oesophageal wall in achalasia; however, extrinsic nerve fibres appear to be intact.

Immunocytochemical localization of tyrosine hydroxylase and gamma-aminobutyric acid in the mesencephalic trigeminal nucleus of the cat: a light and electron microscopic study.

Recent studies conducted on the rat have demonstrated that the mesencephalic trigeminal nucleus (MTN) neurons, involved in the proprioceptive transmission, contain some neuroactive substances, including classical and amino acid neurotransmitters. In addition, there is evidence that serotonin could not act as a neurotransmitter at the first synaptic relay in the cat MTN. In the present study, we aimed to examine two other possible neurotransmitter systems, i.e., catecholamines and gamma-aminobutyric acid (GABA), and the relationships between GABA-immunoreactive (IR) neurons and tyrosine hydroxylase (TH)-IR axonal varicosities in the MTN of the cat. To ensure the localization of immunoreactive structures, the experiments were carried out at the light and electron microscopic level using single immunostaining for TH and GABA alone. The correlation between GABA-IR cell bodies and TH-IR fibers was investigated by means of double-labeling immunogold and peroxidase technique for GABA and TH. Light microscopically, a few GABA-IR neurons were observed in the cat MTN. These small-size, labeled cells, most likely interneurons, were apposed to unstained large mesencephalic trigeminal cells. Most of the large nonreactive MTN neurons were closely surrounded by fine TH-IR varicose or nonvaricose fibers and dot-like structures, presumably nerve terminals. Under the electron microscope, TH-IR fibers were not seen in synaptic contact and only rarely appeared to be in close proximity to neuronal profiles of small GABAergic cells, which contained gold particles. Taken together with earlier studies from other laboratories, the present findings suggest that GABAergic system might play an indirect role in the proprioceptive information processing in the cat MTN by interactions of GABA-immunoreactive neurons with the systems that control the transmission of selected sensory information. In contrast, the presence of TH-IR fibers in direct apposition to the majority of MTN neurons provide further evidence that presumed GABAergic interneurons have extensive interactions with catecholamine varicosities and raise the possibility that catecholamines could modify the transmission on these neurons.

Distribution and origin of serotoninergic afferents to guinea pig cochlear nucleus.

The distribution of serotoninergic fibers in the guinea pig cochlear nucleus was studied with serotonin immunohistochemistry. In addition, the origin of the serotoninergic fibers was determined by combining the retrograde transport of wheat germ agglutinin-apohorseradish peroxidase (gold conjugated) with serotonin immunohistochemistry. Immunoreactivity was present in varicose and nonvaricose fibers that were unevenly distributed throughout the cochlear nucleus. The fibers were most prominent in the superficial layers of the dorsal cochlear nucleus and the anterior spherical cell area of the anteroventral cochlear nucleus. Although less prominent, serotonin-positive fibers were also present in the remaining part of the anteroventral cochlear nucleus and the posteroventral cochlear nucleus. A few positive fibers were present in the auditory nerve root and the dorsal and intermediate acoustic striae. Double-labeled cells were found throughout the rostral-caudal extent of the serotoninergic system from the caudal linear nucleus to the nucleus raphe pallidus. However, most were confined to the dorsal (52%) and median (18%) raphe nuclei. Some serotoninergic cell groups contained retrogradely labeled cells that were not serotonin immunoreactive, indicating nonauditory afferents to cochlear nucleus containing other neurotransmitter substances. Serotonin may tonically modulate auditory processing within the cochlear nucleus as well as influence certain ascending auditory pathways. Most of the serotonin in the cochlear nucleus comes from superior raphe nuclei that also project to basal ganglia motor systems and limbic structures. Therefore, the effect of serotonin on the cochlear nucleus may be related to level of arousal or behavioral state.

Serotonin 2/1 C receptor activation causes a localized expression of the immediate-early gene c-fos in rat brain: evidence for involvement of dorsal raphe nucleus projection fibres

Immunocytochemistry has been used to monitor the expression of the immediate-early gene c-fos in rat brain following administration of the serotonin 2/1 C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. At parenteral doses of 2 or 8 mg/kg the drug caused a highly localized expression of the Fos protein in frontal, parietal, cingulate and piriform cortex as well as in claustrum, mamillary bodies, globus pallidus, amygdala, nucleus accumbens and dorsomedial striatum. In particular, the location of heavy Fos immunoreactivity in the primary somatosensory cortex corresponds precisely to that region (layer Va) shown in other reports to receive a dense input of fine, non-varicose fibres which may arise from the dorsal raphe nucleus. All of the Fos-positive brain regions in the present study have been previously demonstrated to contain serotonin 2 receptor ligand binding sites. Interestingly, no Fos-positive cells were found in the hippocampus, another brain region known to contain serotonin 2 receptors. Pretreatment of animals with the serotonin 2/1 C receptor antagonist ritanserin (0.4 mg/kg) markedly attenuated Fos expression in all reactive areas of the brain. Counts of reactive cells indicated that this antagonism of the Fos response was statistically significant in these brain regions. Spiperone (1 mg/kg), a mixed serotonin 2 and dopamine D 2 antagonist, also attenuated the Fos response in the same regions, but had the effect of inducing Fos expression on its own in other extrapyramidal brain regions. Double labelling of reactive cells with different antisera recognizing Fos and neuron-specific enolase, and lack of double labelling with a glial fibrillary acidic protein antiserum, indicated that the Fos expression was in neurons within the brain nuclei examined. The results indicate that the c-fos immunocytochemical technique is a valuable tool for studying the anatomy and pharmacology of serotonin receptors in experimental animal brain.

Similarities between aberrant serotonergic fibers in the aged and 5,7-DHT denervated young adult rat brain.

Recent morphological observations have suggested neurotransmitter specific degeneration of amongst others, the serotonergic system in the aged rat brain. However, morphological studies can only give a static picture of the events that take place over a period of several months. In the present study we used an experimental model in which degeneration of the serotonergic system in the young adult rat brain was produced on a short time scale. Morphological changes were studied 2 h and 1 or 14 days after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT). Non-specific damage and severe depletion of serotonergic fibers was observed in the immediate surroundings of the injection site, representing the effects of high local concentrations of 5,7-DHT. Sometime after injection swollen varicosities and dilated non-varicose fibers were observed. Fourteen days after the 5,7-DHT treatment cluster-like fibers appeared. It is argued that these swollen and crumpled fiber knots are slowly degenerating fibers. A comparison is made with the abnormal serotonergic fibers in the aged rat brain and it is concluded that these aged abnormal fibers represent axonal degeneration of the serotonergic system in the senescent rat brain.

Evidence for the presence of substance P-like immunoreactivity in the human cerebellum

Preliminary results on the localization of substance P-like immunoreactivity in the human cerebellum are presented. Cerebella from newborn and adult subjects were examined. While only sporadic positive fibres were detected in the adult tissue, the immunoreactive material appeared more abundant in the cerebella from newborn subjects. Varicose and non-varicose fibres and dot-like nerve terminals were present with different density in various regions. The paucity of immunoreactive perikarya suggests that most of the cerebellar substance P-like immunoreactive material has an extrinsic origin.

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammilary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammilary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.

Fluorescence histochemical study of Auerbach’s plexus of the ileum of 6-hydroxydopamine-treated guinea pig

In the present investigation the action of 6-hydroxydopamine (6-OHDA) on Auerbach's plexus of the ileum of the guinea pig has been studied with Falck and Hillarp's histochemical technique. A singel dose of 15-25 mg/kg of 6-OHDA causes degeneration of some of the adrenergic nerve fibres after 6 h of treatment. 200-250 mg/kg of 6-OHDA is responsible for the total disappearance of nerve fibres in the ganglia and internodal strands within 24 h. 6 h after 100 mg/kg of 6-OHDA treatment, the intervaricose fibres have mostly disappeared and a few nerve fibres are swollen. The pre-terminal fibres are not much affected. After 24 and 48 h of 100 mg/kg treatment with 6-OHDA, most of the varicose and non-varicose fibres have disappeared and very few fibres are left, these having become intensely fluorescent and much swollen, probably due to the accumulation of storage granules transported to this site by axoplasmic flow. After 6-8 days of treatment with 6-OHDA all the fluorescen fibres and cell bodies have disappeared. A few faintly fluorescent green and yellow cell bodies could also be observed; however, their chemical nature could not be determined.

Histochemical mapping of catecholamine neurons and fiber pathways in the pontine tegmentum of the dog

The distribution of catecholamine-containing neurons and fiber pathways were studied in the dog's pontine tegmentum by the Falck-Hillarp method. There are fundamental similarities between the dog and other mammals. Most catecholamine fluorescent cell bodies in the dog are located within the nucleus locus coeruleus and subcoeruleus in the pontine tegmentum. They are more diffuse and extensive than in the rat, monkey and human, and rather similar to those of the cat. In this study, catecholamine fiber pathways were clearly traced without using the lesion-degeneration technique. The dorsal pathway from the locus coeruleus consists of more compact bundles of non-varicose fibers than the rat, cat and monkey. It runs parallel to the mesencephalic tract of the trigeminal nerve in pons and midbrain. The ventral pathway courses as loosely arranged varicose fibers in the midreticular formation of the pons and sends axon collaterals to the caudal midbrain to form a dense network of varicose fibers. There are fluorescent plexi of fine varicose fibers at the ventromedial and dorsolateral aspect of the superior cerebellar peduncle. Cells of the medial and lateral parabrachial nucleus, in addition to those of locus coeruleus, send fluorescent fibers to this peduncle. No fluorescent fibers are found in other peduncles.

The identification of dopamine in the rabbit's carotid body.

Application of the Falck & Hillarp histochemical technique to the rabbit carotid body reveals three fluorescent structures: brilliantly fluorescent Type I cells, varicose perivascular nerves, and weakly fluorescent non-varicose fibres. The Type I cell fluorescence is similar to that of a dopamine model system and has the appropriate activation and emission maxima. A catecholamine, identified as dopamine, has been extracted from homogenized carotid bodies, and estimated by the trihydroxyindole procedure. The concentration of the dopamine in the carotid body is estimated to be 20 to 40μg/g. This is very much greater than that of the noradrenaline present, of which there is about 1·5μg/g. The fluorescence of the Type I cells is attributed to the dopamine and it is suggested that the amine may be granule-bound. The unusually high concentration of dopamine could imply that it is not merely a metabolic intermediate in the carotid body.