Immunoreactive Nerve Terminals Research Articles

NPY Y1 receptor like immunoreactivity exists in a subpopulation of β-endorphin immunoreactive nerve cells in the arcuate nucleus: a double immunolabelling analysis in the rat

Double immunolabelling immunohistochemistry in the arcuate nucleus of the rat demonstrates that neuropeptide Y (NPY) Y1 receptor like immunoreactivity of β-endorphin is strongly present in a subpopulation β-endorphin immunoreactive nerve cell bodies, while the small NPY immunoreactive nerve cell bodies lie in an arcuate area rich in NPY immunoreactive nerve terminals forming an uniform plexus. It is postulated that NPY Y1 receptors in β-endorphin neurons may mediate some actions of NPY on motivational processes and pain control as well as on hypophyseal hormone secretion, involving at least in part a regulation of the tubero-infundibular dopamine (DA) neurons.

Ultrastructure of bombesin-like immunoreactive nerve terminals in the nucleus of the solitary tract and the dorsal motor nucleus

Bombesin (gastrin-releasing peptide 14–27) inhibits gastric function and feeding when microinjected into the nucleus of the solitary tract (NTS) / dorsal motor nucleus of the vagus (DMV) complex. We performed a preembedding immunoelectron microscopic study in rats to describe the bombesin containing nerve terminals and to characterize their postsynaptic structures. 228 bombesin-LI nerve terminals which made synaptic contacts in the NTS/DMV complex were studied. Labeling was heaviest over dense core vesicles and lighter over small clear vesicles. The dense core vesicles were typically located along the plasmalemma away from the synaptic face, a finding that is typical of neuropeptide containing nerve terminals. The postsynaptic structures were most often medium sized dendrites (56%) and small sized dendrites (27%), with similar percentages in the NTS and DMV. In the DMV, synapses on cell bodies (8%) were more frequent than in the NTS (1%). In the NTS, synapses on dendritic spines (10%) were more frequent than in the DMV (4%). Only a single axo-axonal contact was identified. These findings add to the increasing body of evidence that bombesin is a neurotransmitter/neuromodulator in the NTS/DMV complex. Bombesin rarely makes presynaptic (axo-axonal) contacts that might inhibit the release of excitatory neurotransmitters, but rather makes postsynaptic contacts potentially effecting vagal motoneurons.

Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco–lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12–T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.

Melatonin is protective against MPTP-induced striatal and hippocampal lesions.

The in vivo effect of melatonin on MPTP-induced neurotoxicity in mouse brain was studied. Melatonin (10 mg/kg) or saline was administered intraperitoneally (i.p.) to mice 30 min prior to a s.c. injection of MPTP (20 mg/kg). After MPTP treatment, the animals received melatonin or saline injections every hour for three hours. Mice were killed 4 hours after the MPTP injection. Regionally-specific increases in lipid peroxidation were observed in corpus striatum and hippocampus (71% and 58%, respectively), but not in cerebral cortex, cerebellum or midbrain. Treatment with melatonin completely reversed the rises in lipid peroxidation products. MPTP-treated mice showed a significant decrease in the striatal tyrosine hydroxylase immunoreactive nerve terminals, an effect that was also prevented by melatonin. These data show that melatonin is neuroprotective in this MPTP model of Parkinson's disease and suggest that melatonin, an endogenous antioxidant and nontoxic compound, may have potential beneficial effects for this neurodegenerative disorder.

Synaptic interactions of retrogradely labeled hypoglossal motoneurons with substance P-like immunoreactive nerve terminals in the cat: a dual-labeling electron microscopic study.

This study has investigated the synaptic interactions between hypoglossal motoneurons and substance P (SP)-immunoreactive terminals. Cholera toxin B conjugated to horseradish peroxidase was injected into the tip of the tongue on the right side of six ketamine-anesthetized cats. Two to five days later, the animals were killed. Cells containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. TMB forms crystalline reaction products that are very distinct at the electron microscopic level. The tissues were then processed for immunocytochemistry using an antiserum against SP. The chromogen used in this case, diaminobenzidine, yields amorphous reaction products. At the light microscopic level, labeled cells were observed primarily ipsilaterally in both intermediate and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were seen at the level of obex. At the electron microscopic level, both asymmetric and symmetric synapses were observed. SP-immunoreactive nerve terminals formed asymmetric synapses with labeled dendrites and symmetric synapses with labeled perikarya. SP-labeled terminals also synapsed on unlabeled dendrites and somata. These are the first ultrastructural studies demonstrating synaptic interactions between hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons that innervate intrinsic tongue muscles are modulated by SP and that SP may play a role in the control of fine movements of the tongue.

The neuropeptide calcitonin gene-related peptide stimulates insulin-like growth factor I production by primary fetal rat osteoblasts.

Calcitonin gene-related peptide (CGRP)-immunoreactive sensory nerve terminals infiltrate all tissues including bone, in which CGRP may play a local regulatory role. To initiate studies on the role of this neuropeptide in bone, osteoblasts were isolated from fetal rat calvariae, treated with CGRP, and analyzed for cAMP and insulin-like growth factor I (IGF-I) production. CGRP alpha and -beta induced a cAMP accumulation in osteoblastic cells, suggesting that they express functional receptors for CGRP. CGRP induced an increase in both IGF-I transcripts and immunoreactive polypeptide. In contrast to prostaglandin E2 (PGE2) treatment, this increase was not accompanied by an augmentation in IGF binding proteins. Although PGE2 induced a more significant increase in IGF-I transcripts than did CGRP, the concentration of IGF-I polypeptide produced by osteoblasts was similar in response to both treatments. It is concluded from this study that CGRP has potent anabolic effects on osteoblasts, an observation which opens possibilities to study the potential therapeutic role of CGRP in osteoporosis.

What are the roles of substance p and neurokinin-1 receptors in the control of negative chronotropic or negative dromotropic vagal motoneurons? a physiological and ultrastructural analysis

Recent data indicate that there is a cardiotopic organization of negative chronotropic and negative dromotropic neurons in the nucleus ambiguus (NA). Negative dromotropic neurons are found in the rostral ventrolateral NA (rNA-VL), negative chronotropic neurons are found in the caudal ventrolateral NA (cNA-VL), and both types of neurons are found in an intermediate level of the ventrolateral NA (iNA-VL). Substance P (SP) immunoreactive nerve terminals synapse upon negative chronotropic vagal motoneurons in the iNA-VL, and SP microinjections in the NA cause bradycardia. In the present report we have attempted to: (1) define the type of tachykinin receptor which mediates the negative chronotropic effect of SP microinjections into the iNA-VL; (2) define the physiological effect of microinjections of a selective SP agonist into the rNA-VL on atrioventricular (AV) conduction; and (3) find ultrastructural evidence for synaptic interactions of SP-immunoreactive nerve terminals with negative dromotropic vagal motoneurons in the rNA-VL. Microinjections of the excitatory amino acid glutamate (Glu) into the iNA-VL to activate all local vagal preganglionic neurons caused both bradycardia and a decrease in the rate of AV conduction. Injections of the selective neurokinin-1 (NK-1) receptor agonist drug GR-73632 also caused bradycardia, however the rapid onset of agonist induced desensitization prevented an evaluation of potential effects on AV conduction in the iNA-VL. These data suggest that the SP-induced bradycardia which can be elicited from the NA is mediated, at least in part, by NK-1 receptors. Microinjections of Glu into the rNA-VL caused a decrease in AV conduction without an effect on cardiac rate. On the other hand, GR-73632 microinjections into rNA-VL did not affect AV conduction. Following injections of the β subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the left atrial fat pad ganglion which selectively mediates changes in AV conduction, retrogradely labeled neurons were histochemically visualized in the rNA-VL. These tissues were subsequently processed for the simultaneous immunocytochemical visualization of SP, and examined by electron microflapy. Histochemically labeled neurons were large, multipolar, with abundant cytoplasm containing large masses of rough endoplasmic reticulum, and exhibited distinctive dendritic and somatic spines. Unlabeled nerve terminals were noted to form either asymmetric or symmetric synapses with dendrites, dendritic spines, and perikarya of histochemically labeled neurons. SP-immunoreactive nerve terminals were also detected in the rNA-VL. SP terminals typically contained numerous small pleomorphic vesicles, multiple large dense core vesicles, and several mitochondria, and they synapsed upon unlabeled dendritic profiles. A total of 154 SP-immunoreactive nerve terminals were observed on photomicrographs of tissues which also contained histochemically labeled profiles. None made an identifiable synapse with a retrogradely labeled profile on the sections examined. In summary, both physiological and ultrastructural data indicate that SP terminals in the iNA-VL do modify the output of negative chronotropic vagal motoneurons. This effect is mediated by NK-1 receptors. On the other hand both physiological and ultrastructural data indicate that SP terminals in the rNA-VL do not modify the output of negative dromotropic vagal motoneurons. Therefore different mechanisms (neurotransmitters or receptors) mediate the central vagal control of cardiac rate and AV conduction.

Carotid sinus nerve terminals which are tyrosine hydroxylase immunoreactive are found in the commissural nucleus of the tractus solitarius.

Tyrosine hydroxylase immunoreactive sensory neurons in the petrosal ganglion selectively innervate the carotid body via the carotid sinus nerve. Central projections of the carotid sinus nerve were traced with horseradish peroxidase. The commissural nucleus of the tractus solitarius was examined by dual labelling light and electron microscopy. Dense bilateral labelling with horseradish peroxidase was found in the tractus solitarius and commissural nucleus of the tractus solitarius. Horseradish peroxidase was found in unmyelinated axons, myelinated axons, and nerve terminals. About 88% of horseradish peroxidase-labelled carotid sinus nerve axons were unmyelinated. Tyrosine hydroxylase immunoreactivity was identified in unmyelinated axons, myelinated axons, dendrites, perikarya, and nerve terminals. Most tyrosine hydroxylase immunoreactive axons (93%) in the commissural nucleus of the tractus solitarius were unmyelinated. Tyrosine hydroxylase immunoreactivity was simultaneously identified in carotid sinus nerve unmyelinated axons, myelinated axons, and nerve terminals. These double-labelled terminals comprised 28% of the number of tyrosine hydroxylase immunoreactive terminals in the commissural nucleus of the tractus solitarius, and 55% of transganglionically-labelled terminals. Therefore, there are both central and peripheral sources of tyrosine hydroxylase immunoreactive nerve terminals in the commissural nucleus of the tractus solitarius. These data support the hypothesis that peripheral tyrosine hydroxylase immunoreactive neurons are involved in the origination of the chemoreceptor reflex. Axo-axonic synapses between peripheral carotid sinus nerve afferent terminals and central terminals containing tyrosine hydroxylase immunoreactivity were observed in 22% of the axo-axonic synapses observed. Thus, central tyrosine hydroxylase immunoreactivity neurons are involved in the modulation of the chemo-and/or baroreceptor reflexes. Synaptic contacts were not observed between carotid sinus nerve afferents and tyrosine hydroxylase immunoreactive perikarya of dendrites. Catecholaminergic neurons are thus unlikely to be the second order neurons of either the chemo-or baroreceptor reflex in the commissural nucleus of the tractus solitarius.

Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support for this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

Ultrastructural analysis of tryptophan hydroxylase immunoreactive nerve terminals in the rat cerebral cortex and hippocampus: their associations with local blood vessels

Physiological evidence has indicated that serotonin (5-hydroxytryptamine) could be a regulator of cerebral blood flow in various regions of the brain. In the present study, tryptophan hydroxylase immunocytochemistry was used to characterize, both at the light and electron microscopic levels, serotonergic nerve terminals and primarily their relationships with intraparenchymal microarterioles and capillaries in the rat frontoparietal cortex, entorhinal cortex and hippocampus. Irrespective of the brain area, serotonergic varicosities were primarily apposed to either dendrites or nerve terminals, were on average 0.37 micron2 in surface area (0.69 micron calculated diameter) and 12-22% of them engaged in synaptic junctions, mostly with dendritic elements. Perivascular terminals (defined as immunolabelled varicosities located within a 3 micron perimeter around the vessel basal lamina) in the frontoparietal cortex represented 8-11% of all immunoreactive terminals counted, as determined by light and electron microscopy, respectively. In the entorhinal cortex and hippocampus, the proportion of perivascular terminals was only determined at the ultrastructural level and corresponded to 10% and 4%, respectively. In the frontoparietal cortex, serotonergic varicosities were located significantly closer (n = 250, 0.98 +/- 0.05 micron; P < 0.001) to the blood vessels than those of the entorhinal cortex (n = 116, 1.41 +/- 0.08 microns) or hippocampus (n = 105, 1.31 +/- 0.08 microns). Of all perivascular serotonergic terminals in the frontoparietal cortex, 26% were in the immediate vicinity (0-0.25 micron) of the vessel wall, with 2.8% directly abutting on the basement membrane, while 11.6% were separated from it only by a thin astrocytic leaflet. This situation contrasts with that observed in the entorhinal cortex and hippocampus, where no immunoreactive varicosity was ever seen directly contacting the vessel basal lamina and with only 10-13% of the terminals being within 0.25 micron from the vessels. The surface area of perivascular serotonergic terminals was comparable in all regions studied and corresponded to 0.22 micron2; these virtually never engaged in synaptic contacts with adjacent neuronal structures. Our results indicate that tryptophan hydroxylase-immunolabelled terminals are identical to previously characterized serotonin-containing varicosities. Furthermore, the present data show intimate associations between serotonergic terminals and microvessels in the three regions examined. However, perivascular terminals in the frontoparietal cortex were more frequent and/or located much closer to local microvessels than those in the other regions, and might be more directly involved in neurogenic control of local cerebral blood flow.

The fetal expression of Proenkephalin mRNAs and Met-enkephalin immunoreactivity in the hypothalamoseptal tract and adjacent hypothalamic areas of the guinea pig brain

The development of the enkephalinergic hypothalamoseptal tract in the guinea pig brain was studied from embryonic day 30 until birth. Proenkephalin (PE) mRNAs were detected in the hypothalamic magnocellular dorsal nucleus (MDN) by in situ hybridization with a synthetic 35S-labeled oligonucleotide. The Met-enkephalin-like immunoreactivity (Met-enk-LI) in the MDN and the lateral septum (LS) was detected with antibodies against Met-enkephalin, on adjacent cryostat sections. At the same time, an immunohistochemical study of the arrangement of enkephalinergic axon terminals in the LS at birth was performed at the electron microscopic level. PE mRNAs were first found to be expressed in the MDN at embryonic day 32 (E32) and increased to reach a maximal level at E48. Met-enk-LI was consistently detectable from E38 in numerous perikarya of the MDN as well as in nerve terminals of the LS. The number of Met-enk-LI cells of the MDN decreased after this stage until birth, whereas positive nerve endings in the LS increased. At the electron microscopic level, numerous cell bodies of the LS at birth were consistently surrounded by Met-enk immunoreactive nerve terminals. Cells expressing the PE gene and Met-enk-LI were also observed from E38 to E44 in the periventricular area. Some of these cells were found double-labeled with Met-enkephalin and Somatostatin antisera. The enkephalinergic system of the hypothalamoseptal tract appears at early embryonic stages and may be essential in regulating septal neuronal functions early in gestation. Differing ontogenic onsets of the enkephalinergic hypothalamoseptal and periventricular-median eminence tracts suggest possible developmental and functional differences.

Peptidergic innervation of the bovine vagina and uterus

The distribution of neuropeptide Y, substance P, vasoactive intestinal polypeptide, Leu5-enkephalin, bombesin/gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin, cholecystokinin and catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase was studied immunohistochemically in nerve fibres supplying the bovine vagina and uterus. The nerves containing tyrosine hydroxylase or dopamine-beta-hydroxylase and neuropeptide Y-immunoreactivity were particularly numerous in both organs. Substance P, vasoactive intestinal polypeptide and Leu5-enkephalin-containing nerves were less numerous whereas somatostatin and calcitonin gene-related peptide-immunoreactive nerves occurred occasionally. Bombesin/gastrin-releasing peptide and cholecystokinin immunoreactivities were not present in nervous fibers of the bovine uterus and vagina. Generally, the immunoreactive nerve terminals, fibers, networks or nerve bundles were present below the serous membrane, between smooth muscle cells of muscular layers, around blood vessels, in the submucosal layer and below the luminal epithelium of the uterus and cervix.

Immunolocalization of putative neurotransmitters innervating autonomic regulating neurones of cat ventral medulla

This study investigated possible sites of contact of nerve fibers containing a range of putative neurotransmitter substances onto neurons in the cat ventral medulla oblongata concerned with autonomic, particularly cardiovascular, regulation. The parasympathetic preganglionic neurons of the nucleus ambiguus were identified by retrograde horseradish peroxldase tracing from the vagus nerve, and the groups of neurons in the Al and C1 cell areas and the raphe nucleus by cafscholamine enzyme or 5-hydroxytryptamins (5-HT) inurnxuohistoduemistry, respectively. Immunoreactive (-ir) nerve fibers and terminals in the vicinity of these neurons were visualized by subjecting the sections to a dual-staining technique using a brown peromddase-diaminobenzidine reaction product and a blue alkaline phosphatese-Fast blue reaction product: By employing monochrome photography with combinations of blue and orange-red filters, it was possible to discriminate neural elements displaying one or the other reaction product, or colocalization of reaction products. The results revealed the presence of calcitonin gene-related peptide (CORP) and galanin (GAL)ir in some motonsurons of the nucleus ambiguus, but not in those innervating the heart via the cardiac vagus nerve. The latter group of parasympathetic efferent neurons were found to be densely innervated by fibers innnrunoreactive for dopamine β-hydroxylase (DBH, indicating noradrenalineL ghrcine (GLY), γ-aminobutyric acid (GABA), 5-HT, enkephalin (ENK, neuropeptide Y (NPY), substance P (SP), and i hormone (TRK and, to a lesser extent, by other netropep ide-ir fibers. The came cells of the rostal C1 and caudal A1 groups showed a broadly similar pattern of innervation, most noticeably by fibers immurwreactive for DBH, GABA, 5-HT, cholecystokinin (CCIQ, CGRP, ENK, GAL, NPY, and SP. The 5-HT-ir neurons of the raphe nucleus, some also containing SP, TRH, ENK, or aorticotropin-releasing factor (CRF)-ir, were most prominently innervated by terminals containing DBH, GABA, CCK, ENK, NPY, TRH, somatostatin (SRIF), and vasoactive intestinal polypeptide (VIP)-ir. Although the proof that these groups of neurons receive functional synaptic contacts from the immunoreactive fibers awaits furtis ultrastructural studies, the results do suggest that a wide range of putative transmitters may influence the activity of efferent neurons in the cat medulla controlling autonomic functions.

Distribution of FMRFamide-like immunoreactivity in the brain of the lungfish Protopterus annectens

The distribution of FMRFamide-like immunoreactive peptides was studied in the brain of the African lungfish, Protopterus annectens, using the indirect immunofluorescence technique. The main populations of FMRFamide-positive cell bodies were detected in the forebrain and in the mesencephalic tegmentum. In the telencephalon, only a small number of FMRFamide-immunoreactive neurons was localized at the level of the subpallium, in the nucleus septi medialis. The diencephalon contained two prominent groups of FMRFamide-positive cell bodies located in the preoptic and periventricular preoptic nuclei. The thalamus exhibited only scattered FMRFamide-immunoreactive perikarya in its ventral part. In the mesencephalon, a group of positive cell bodies was identified in the caudal region of the tegmentum. A strong immunoreaction was also detected in the nervus terminalis. In the pituitary, most of the cells of the intermediate lobe were brightly stained. FMRFamide-like immunoreactive fibers and nerve terminals were widely distributed in the brain. In the telecephalon, numerous fibers were observed in several regions of the pallium and subpallium. A dense plexus of fibers was found in the hypothalamus and the thalamus. Immunoreactive fibers were seen coursing along the ventral wall of the infundibular cavity and terminating in the pars nervosa of the pituitary. The tectum and the ventral mesencephalon were also densely innervated. In contrast, the caudal brainstem only showed scarce immunoreactive processes. Coexistence of FMRFamide- and neuropeptide Y-like immunoreactivity was observed in the preoptic nucleus and in the nervus terminalis. The widespread distribution of FMRFamide-immunoreactive neurons in the brain and pituitary of P. annectens suggests that the peptide may exert both neuromodulator and neuroendocrine functions. The similarity between the distribution patterns of FMRFamide and neuropeptide Y in the brain of lungfish and amphibians supports the concept of a close phylogenetic link between these two groups.

Presence of calcitonin gene-related peptide in intraepithelial nerve fibers and motor end-plates of the cat esophagus: a light and electron microscopic study

The morphology and distribution of the motor end-plates in the striated muscle and the terminal nerve fibers in the epithelium of the wall of the esophagus, which contain calcitonin gene-related peptide, were studied by light and electron microscopic immunocytochemistry. Varicose immunoreactive nerve fibers arising from the subepithelial plexus were seen to penetrate into the epithelium where they ended in terminal boutons. These nerve fibers lost their Schwann cells just at the point of penetration into the epithelium. Characteristically, the epithelial cells of the spinous layer showed prominent tonofilaments in the part of the cytoplasm in contact with the immunoreactive nerve varicosities, but membrane specializations between these structures were not observed. In the striated muscle of the esophageal wall there were small, elliptical, immunoreactive motor end-plates, which contained a small number of axonal clear vesicles and mitochondria. They were associated with relatively short and rarely branched junctional folds, reduced postjunctional surfaces and few organelles in the underlying sarcoplasm, features characteristic of the neuromuscular junctions of slow-fatiguing red muscle fibers. The two types of immunoreactive nerve endings, epithelial and muscular, presumably participate in afferent and efferent limbs respectively of the neural control of esophageal motility. The relationship between immunoreactive nerve terminals and epithelial cells in the spinous layer exhibiting prominent tonofilaments allowed us to speculate about the existence of two different patterns of reception to sensory stimuli. The intraepithelial fibers that end in the middle layer of the epithelium could be related to mechanoreceptor reflexes, while those that end in the upper layer may be related to thermoreceptor reflexes or facilitate information about the chemical and other characteristics of foods.

Visualization of PGP 9.5 immunoreactive nerve terminals in the mouse snout epidermis.

Intraepidermal free nerve endings were investigated in the mouse snout skin by means of an immunohistochemical procedure using a rabbit antiserum against protein gene product 9.5 (PGP 9.5). Immunoperoxidase reactivity was detected in different subtypes of intraepidermal nerves and cells. The great majority of axons observed in the stratified epithelium were varicose; a small percentage was either smooth (non-varicose) or irregularly shaped. Intraepidermal nerves ended at different levels within the epidermis, often with a terminal knob-like swelling. Various patterns of intraepidermal innervation could be distinguished. Most fibres entering the epidermis originated from large bundles running a horizontal course below the dermo-epidermal junction. Such fibres ascended vertically through the stratified epithelium in a "candelabrum-like" fashion, without emitting collaterals. Other fibres branched profusely and ended in complex intraepidermal neural networks. Less frequently, intraepidermal fibres terminated with large irregularly shaped expansions of different morphologies. Some of these were the intraepidermal continuations of axons within Meissner's corpuscles. Some fibres appeared to come into contact with PGP 9.5-immunoreactive cells (which closely resembled Merkel cells) located in the stratum basale. Rare suprabasal dendritic cells (Langerhans cells?) also became visible.

Chronic continuous infusion of nicotine increases the disappearance of choline acetyltranferase immunoreactivity in the cholinergic cell bodies of the medial septal nucleus following a partial unilateral transection of the fimbria fornix

Previous studies have demonstrated that chronic continuous nicotine treatment via minipumps partially protects against mechanically induced degeneration of the nigrostriatal dopamine neurons in the male Sprague-Dawley rat. In the present study we investigated how a 4-week continuous infusion with (-)-nicotine via minipumps implanted subcutaneously in the male Sprague-Dawley rat (0.125 mg/kg-1 h-1) influences the anterograde and retrograde changes occurring in the septohippocampal cholinergic neurons following a unilateral transection of the fimbria fornix. Choline acetyltransferase and acetylcholinesterase immunocytochemistry was performed in combination with computer-assisted morphometry and microdensitometry. Measurements of choline acetyltransferase enzyme activity was performed in the dorsal hippocampus. The chronic nicotine infusion significantly increased the disappearance of the choline acetyltransferase immunoreactive nerve cell area within the medial septal nucleus of the lesioned side. However, the disappearance of the acetylcholinesterase immunoreactive nerve terminals within the dentate gyrus (molecular layer) and of choline acetyltransferase enzyme activity within the dorsal hippocampus was not found to be influenced by the chronic nicotine infusion. Thus, chronic infusion of (-)-nicotine does not appear to exert any protective activity on mechanically injured septohippocampal cholinergic neurons but may instead increase their dysfunction. In comparison with the dopaminergic neurons it may therefore be that the continuous chronic nicotine exposure does not lead to sufficient desensitization of the nicotinic cholinoceptors of the cholinergic neurons to reduce the chronic influx of sodium and calcium ions via the nicotinic ion channels and thus intraneuronal calcium levels and energy demands.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous morphine is stored in specific brain neurons

Morphine immunoreactive-like compounds were detected - by immunohistochemical techniques- inside the cell body, fibers and terminals of neurons of different brain areas. Immunoreactivity sometimes was situated in dots around the perikarion in a ring-like fashion. The antisera revealed: numerous nerve cell bodies with variable morphology throughout the cerebral cortex; granule cells of the dentate gyrus, and pyramidal cells of the hippocampus; numerous granule cells in the granular layer and small neurons and fibers in the molecular layer of the cerebellum; cell bodies in the dorsal horn and around the central canal of the spinal cord. In the anterior horn immunoreactive nerve fibers and terminals were observed around the perikaria of some motor neurons

Immunofluorescent identification of a delta (delta)-opioid receptor on primary afferent nerve terminals.

Antisera were produced against synthetic peptides predicted from the recent cloning of a delta opioid receptor, DOR-1. Confocal microscopic examination of immunostained spinal cord sections revealed that DOR-1 immunoreactive (-ir) nerve fibers and terminals form a moderately dense plexus within the superficial dorsal horn of rats and mice. These fibers decreased dramatically following dorsal rhizotomy and consistent with these observations a population of small diameter neurons in ganglia exhibited DOR-1-ir. DOR-1-ir ganglion neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), and their terminals in the spinal cord contained both CGRP- and DOR-1-ir, the latter presumably located as a 'presynaptic' receptor. Interestingly, terminals containing DOR-1-ir appeared to be closely apposed by fibers and terminals containing enkephalin (ENK)-ir, which suggests that ENK may be a physiologically relevant ligand for the receptor encoded by DOR-1, and that DOR-1 may act to regulate the release of transmitters from small diameter primary afferent neurons.

Large calibre primary afferent neurons projecting to the gracile nucleus express neuropeptide Y after sciatic nerve lesions: an immunohistochemical and in situ hybridization study in rats.

Using immunohistochemistry and in situ hybridization, we studied changes in expression of some neuropeptides in large and medium-sized neurons in lumbar 4 and 5 rat dorsal root ganglia projecting to the gracile nucleus, in response to peripheral axotomy. Fourteen days after unilateral sciatic nerve transection, many large neurons and some medium-sized neurons in ipsilateral dorsal root ganglia were strongly neuropeptide Y-positive. Galanin-, vasoactive intestinal polypeptide (VIP)- and peptide histidine-isoleucine (PHI)-like immunoreactivities coexisted with neuropeptide Y-like immunoreactivity in some of these neurons. After axotomy numerous large and medium-sized cells contained neuropeptide Y mRNA in the ipsilateral ganglia, whereas no hybridization was seen in the contralateral or control ganglia. Cross-sectioned, large neuropeptide Y-positive fibres were observed in a somatotopically appropriate zone within the ipsilateral gracile fasciculus. A dense network of neuropeptide Y-immunoreactive, large nerve fibres and terminals was seen in the ipsilateral gracile nucleus. A small number of galanin- and VIP/PHI-like immunoreactive nerve fibres and terminals were also observed in adjacent sections. Neuropeptide Y-like immunoreactivity colocalized with galanin- or VIP/PHI-like immunoreactivity in some nerve fibres. None of these neuropeptide immunoreactivities could be detected in nerve fibres and terminals in the control or contralateral gracile nucleus. These findings suggest that neuropeptides, in addition to their role in small dorsal root ganglion neurons, may have a function in large and medium-sized dorsal root ganglion neurons projecting to laminae III and IV in the dorsal horn as well as to the gracile nuclei, as a part of their response to peripheral axotomy.