Enkephalin-immunoreactive Fibers Research Articles

Postnatal maturation of endogenous opioid systems within the periaqueductal grey and spinal dorsal horn of the rat

Significant opioid-dependent changes occur during the fourth postnatal week in supraspinal sites (rostroventral medulla [RVM], periaqueductal grey [PAG]) that are involved in the descending control of spinal excitability via the dorsal horn (DH). Here we report developmentally regulated changes in the opioidergic signalling within the PAG and DH, which further increase our understanding of pain processing during early life. Microinjection of the μ-opioid receptor (MOR) agonist DAMGO (30ng) into the PAG of Sprague-Dawley rats increased spinal excitability and lowered mechanical threshold to noxious stimuli in postnatal day (P)21 rats, but had inhibitory effects in adults and lacked efficacy in P10 pups. A tonic opioidergic tone within the PAG was revealed in adult rats by intra-PAG microinjection of CTOP (120ng, MOR antagonist), which lowered mechanical thresholds and increased spinal reflex excitability. Spinal adminstration of DAMGO inhibited spinal excitability in all ages, yet the magnitude of this was greater in younger animals than in adults. The expression of MOR and related peptides were also investigated using TaqMan real-time polymerase chain reaction and immunohistochemistry. We found that pro-opiomelanocortin peaked at P21 in the ventral PAG, and MOR increased significantly in the DH as the animals aged. Enkephalin mRNA transcripts preceded the increase in enkephalin immunoreactive fibres in the superficial dorsal horn from P21 onwards. These results illustrate that profound differences in the endogenous opioidergic signalling system occur throughout postnatal development.

Neurochemical architecture of the filum terminale in the rat

Contrary to the widespread assumption, the filum terminale in the rat possesses a precise glial and neuronal organization. The processes of glial fibrillary acidic protein-stained astrocytes form a rich, three dimensional array. The crescent shaped white matter could be outlined with antibody detecting oligodendrocytes. The neurons in the filum terminale, labeled with neuron-specific nuclear protein, are distributed in a small midline group (dorsal nucleus) dorsal to and in two symmetrical clusters at both sides of the central canal (lateral nuclei). Nitric oxide synthase-, calretinin-, choline acetyltransferase-, substance P- and neurokinin receptor-1-immunoreactive neurons were detected in the lateral nuclei. Axons were classified based on their course and termination. Small number of calcitonin gene-related peptide-immunoreactive fibers was found exclusively in the dorsal nucleus. Nitric oxide synthase-, substance P-, and neurokinin receptor-1-stained axon arborizations were detected mainly in the lateral nucleus. A dense array of extremely fine vesicular glutamate transporter 2- and fine, synaptophysin-immunoreactive varicosities covered densely the lateral nuclei. Fine glycine-transporter 2-immunoreactive axon arborization like structures were seen also in the lateral nucleus. Vesicular glutamate transporter 1- and choline acetyltransferase-immunoreactive axons arborized in the entire gray matter. Serotonin- and enkephalin-immunoreactive fibers congregated in the dorsolateral portion of the white matter, called “shoulder region”, while calretinin- and thick, varicose neurokinin receptor-1-stained axons were also seen in the same area of the white matter. Synaptophysin-immunoreactive fine varicosities colocalized only with vesicular glutamate transporter 2 immunoreaction. Substance P and glycine-transporter 2-immunoreactive puncta were found in close contact with neurokinin receptor-1-immunostained perikarya and dendrites.

Opposing regulation of the locus coeruleus by corticotropin-releasing factor and opioids

Substantial clinical and preclinical findings support an association between stress and opiate abuse. To understand the mechanisms underlying this association, it is important to identify substrates of the stress response and endogenous opioid systems that interact and specific points at which stress circuits and endogenous opioid systems intersect. This review focuses on corticotropin-releasing factor (CRF), a critical substrate of the stress response, and its potential for interactions with endogenous opioid systems within the pontine nucleus, locus coeruleus (LC), a brain region that has been implicated as a target in response to stress and opiates. Evidence is reviewed supporting the hypothesis that CRF and endogenous opioids interact to co-regulate the LC. Thus, CRF- and enkephalin-immunoreactive fibers innervating LC dendritic fields overlap, and some axon terminals in this region co-localize CRF and enkephalin. CRF and opioids have opposing effects on LC neuronal discharge and on intracellular signaling mechanisms within LC neurons. Finally, a history of stress or opiate use induces plasticity in CRF-LC or opiate-LC interactions, respectively. Disruptions in the CRF/opioid balance as a result of this plasticity are proposed to result in hyperactivity or hyperresponsiveness of the LC-norepinephrine (NE) system. Co-regulation of the LC-NE system by CRF and opioids may be important in acute adaptation to stress. Potential clinical consequences of an imbalance in this regulation as a result of prior stress include increased risk of opiate self administration and decreased sensitivity to opiates used in clinical settings. Conversely, chronic exposure to opiates may predispose individuals to stress-related psychiatric disorders.

Suprachiasmatic nucleus and intergeniculate leaflet in the diurnal rodent Octodon degus: retinal projections and immunocytochemical characterization

The neural connections and neurotransmitter content of the suprachiasmatic nucleus and intergeniculate leaflet have been characterized thoroughly in only a few mammalian species, primarily nocturnal rodents. Few data are available about the neural circadian timing system in diurnal mammals, particularly those for which the formal characteristics of circadian rhythms have been investigated. This paper describes the circadian timing system in the diurnal rodent Octodon degus, a species that manifests robust circadian responses to photic and non-photic (social) zeitgebers. Specifically, this report details: (i) the distribution of six neurotransmitters commonly found in the suprachiasmatic nucleus and intergeniculate leaflet; (ii) the retinohypothalamic tract; (iii) the geniculohypothalamic tract; and (iv) retinogeniculate projections in O. degus. Using immunocytochemistry, neuropeptide Y-immunoreactive, serotonin-immunoreactive and [Met]enkephalin-immunoreactive fibers and terminals were detected in and around the suprachiasmatic nucleus; vasopressin-immunoreactive cell bodies were found in the dorsomedial and ventral suprachiasmatic nucleus; vasoactive intestinal polypeptide-immunoreactive cell bodies were located in the ventral suprachiasmatic nucleus; [Met]enkephalin-immunoreactive cells were located sparsely throughout the suprachiasmatic nucleus; and substance P-immunoreactive fibers and terminals were detected in the rostral suprachiasmatic nucleus and surrounding the nucleus throughout its rostrocaudal dimension. Neuropeptide Y-immunoreactive and [Met]enkephalin-immunoreactive cells were identified in the intergeniculate leaflet and ventral lateral geniculate nucleus, as were neuropeptide Y-immunoreactive, [Met]enkephalin-immunoreactive, serotonin-immunoreactive and substance P-immunoreactive fibers and terminals. The retinohypothalamic tract innervated both suprachiasmatic nuclei equally; in contrast, retinal innervation to the lateral geniculate nucleus, including the intergeniculate leaflet, was almost exclusively contralateral. Bilateral electrolytic lesions that destroyed the intergeniculate leaflet depleted the suprachiasmatic nucleus of virtually all neuropeptide Y- and [Met]enkephalin-stained fibers and terminals, whereas unilateral lesions reduced fiber and terminal staining by approximately half. Thus, [Met]enkephalin-immunoreactive and neuropeptide Y-immunoreactive cells project equally and bilaterally from the intergeniculate leaflet to the suprachiasmatic nucleus via the geniculohypothalamic tract in degus. This is the first report examining the neural circadian system in a diurnal rodent for which formal circadian properties have been described. The data indicate that the neural organization of the circadian timing system in degus resembles that of the most commonly studied nocturnal rodents, golden hamsters and rats. Armed with such data, one can ascertain differences in the functional organization of the circadian system between diurnal and nocturnal mammals.

Nerve terminals containing neuropeptides decrease in number after massive proximal small bowel resection in the piglet.

The aim of this study was to evaluate possible changes in the neuropeptide innervation pattern of the remaining porcine ileum following 75% proximal resection of the small intestine. Three-month-old piglets were operated on and two months postoperatively full-thickness specimens of the proximal part of the distal ileum wall were taken. Age-matched 3- and 5-month-old unoperated piglets were used as controls. The number and intensity of VIP-, galanin-, enkephalin-, substance P-, and somatostatin-containing nerve fibers were estimated in sections processed for immunofluorescence microscopy and subjected to quantitative scoring. The VIP-, galanin-, and enkephalin-immunoreactive fibers of the circular muscle layer and villi were also quantitated by computer-assisted morphometry. The number and intensity of VIP-immunoreactive fibers in the mucosa and circular muscle layer markedly decreased after resection as compared to 3-month-old and 5-month-old controls (P < 0.05). The galanin immunoreactivity index decreased significantly after resection in the circular muscle layer as compared to both control groups (P < 0.05). The increase in the number of enkephalin-immunoreactive nerve fibers that normally occurred from 3 to 5 months of age was inhibited by the resection. We were not able to see any differences in somatostatin or substance P immunoreactivity between the groups. The results suggest that massive resection induces significant changes in the neuropeptide-containing innervation of the remaining small intestine. These findings are compatible with altered motor activity and mucosa function in the remain intestine.

Neuropeptide Y and enkephalin immunoreactivity in retinorecipient nuclei of the hamster pretectum and thalamus.

This investigation was stimulated by the historical confusion concerning the identity of certain pretectal nuclei and by large differences reported between species with respect to which nuclei receive retinal innervation. Subcortical visual nuclei were studied using immunohistochemistry to identify retinal projections labeled following intraocular injection of cholera toxin, b fragment. In addition, neuropeptide Y (NPY) or enkephalin (ENK) immunoreactive cells and fibers were also evaluated in the retinorecipient pretectal and thalamic areas. The results confirm the established view that the retina directly innervates the nucleus of the optic tract (NOT), posterior (PPT), and olivary pretectal (OPT) nuclei. However, the retina also innervates the hamster medial (MPT) and anterior (APT; dorsal division) pretectal nuclei, results not previously reported in rodents. A commissural pretectal area (CPT) sparsely innervated by retina is also described. The data show for the first time that the posterior limitans nucleus (PLi) receives a moderately dense, direct retinal input. The PLi does not project to the cortex and appears to be a pretectal, rather than thalamic, nucleus. All retinal projections are bilateral, although predominantly contralateral. The PLi contains a moderately dense plexus of NPY- and ENK-IR fibers and terminals. However, peptidergic fibers also traverse the ATP and connect with the dorsomedial pretectium. The OPT contains ENK- and NPY-IR neurons and fibers, but is specifically identifiable by a moderately dense plexus of ENK-IR terminals. Numerous ENK-IR neurons are found in the NOT and PPT. The latter also has moderate numbers of ENK-IR fibers and terminals, but few NPY-IR neurons or fibers. The MPT contains modest numbers of ENK-IR fibers. The APT has no NPY-IR neurons or terminals, but an occasional ENK-IR neuron is seen and there is sparse ENK-IR innervation. Peptidergic innervation of the visual nuclei does not appear to be derived from the retina. The results show a set of retinally innervated, contiguous nuclei extending from the thalamic ventrolateral geniculate nucleus dorsomedially to the midbrain CPT. These nuclei plus the superior colliculus comprise a dorsal "visual shell" embracing a central core of caudal thalamus and rostral midbrain.

Enkephalin innervation of the paraventricular nucleus of the hypothalamus: distribution of fibers and origins of input

The opioid peptide enkephalin emerges as a major neuromodulator in the regulation and integration of the physiologic response in stressful conditions. The paraventricular nucleus of the hypothalamus is a coordinating center of neuroendocrine and autonomic functions. However, the detailed distribution of the enkephalin fibers and terminals in the paraventricular nucleus and the sources of enkephalinergic innervation are not well defined. In the present study, we used immunocytochemistry for the proenkephalin-derived octapeptide met-arg 6-gly 7-leu 8 enkephalin to determine the distribution of enkephalin-immunoreactive fibers and somata within paraventricular nucleus. Without colchicine pretreatment, enkephalinergic fibers were prominent mainly in the ventromedial part of the parvicellular subdivision of the paraventricular nucleus, appearing in coronal sections as a dense collection of short segments of enkephalin-immunoreactive fibers. In the periventricular portion of the paraventricular nucleus, enkephalin-immunoreactive fibers produced a moderate plexus of short enkephalin-immunoreactive fibers dorsoventrally oriented. With colchicine treatment, a dense cluster of enkephalin-immunoreactive cell bodies was located in the dorsomedial and the dorsal parts of the parvicellular subdivisions. These enkephalin-immunoreactive neurons were small (< 10 μm) to medium sized (10–15 μm), with round and elongated shapes. Retrograde transport of wheat germ-conjugated gold particles. WGA-apoHRP-Au, from the paraventricular nucleus, combined with immunocytochemistry for enkephalin revealed that the major sources of extrahypothalamic enkephalin afferents to the paraventricular nucleus are provided by enkephalin neurons neurons in the lateral reticular nucleus and the paragigantocellularis reticular nucleus of the medulla (∼20% of retrogradely labeled neurons within this nucleus were double labeled) and in the nucleus solitary tract (∼10% of retrogradely labeled neurons within thisnucleus were double labeled). Retrogradely labeled enkephalin neurons were also observed in the medial preoptic area, median preoptic nucleus, dorsomedial hypothalamic nucleus, lateral septum and hypothalamic arcuate nucleus. These enkephalinergic pathways from the medulla and the forebrain could represent an anatomical substrate underlying the opioid effects on paraventricular neurons during physiological processes, such as a cardiovascular regulation, feeding or stress responses.

Enkephalin-immunoreactive fastigial neurons in the rat cerebellum project to upper cervical cord segments

By using enkephalin immunohistochemisal combined with retrograde fluorescent labelling, a great majority of neurons in the rat cerebellum sending their axons to the spinal cord were shown to contain enkephalin immunoreactivity. These neurons were numerous and clustered in the fastigialnucleus but far less abundant in other cerebellar nuclei. Enkephalin-immunoreactive fibers present in the ventral horn and the central cervical nucleus of upper cervical cord segments almost completely disappeared contralaterally following kainic acid-induced cell loss in the fastigial nucleus. The results indicate that fastigial and some other cerebellar nucleus neurons provide enkephalin-containing projections toward these spinal sites.

Localization of enkephalinergic neurons in the central nervous system of the salmon ( Salmo salar L.) by in situ hybridization and immunocytochemistry

The distribution of neurons expressing preproenkephalin (PPE) mRNA in the brain of the salmon was investigated by means of non-radioactive in situ hybridization, and directly compared with the distribution of enkephalin-immunoreactive (ENKir) neurons. This approach, utilized here for the first time in a non-mammalian vertebrate for the identification of neurons containing opioid peptides, permitted a detailed analysis of the distribution of putative enkephalinergic neurons in the salmon brain. Several cell groups containing neurons that express PPE mRNA also contain ENKir neurons. Such cell groups are located in the ventral telencephalic area, the nucleus of the rostral mesencephalic tegmentum and another nucleus immediately dorsal to it, the torus semicircularis, the valvula cerebelli and the corpus cerebelli. These cell groups consistently contain larger numbers of PPE mRNA expressing cells than ENKir ones. Some cell groups express PPE mRNA, but do not contain ENKir neurons. These cell groups are located in the dorsal telencephalic area, the inferior lobes of the hypothalamus, the pretectal area, the magnocellular superficial pretectal nucleus, the optic tectum, the oculomotor nucleus, the trochlear nucleus, the magnocellular vestibular nucleus, the secondary gustatory nucleus, the superior and medial reticular nuclei, the motor nucleus of the vagus and the ventral horn of the spinal cord. Moreover, some cell groups contain ENKir neurons, but no PPE mRNA expressing neurons. These cell groups are located in the ventromedial thalamic nucleus, the lateral tuberal nucleus, the nucleus of the lateral recess and the nucleus of the posterior recess. The majority of these periventricular ENKir neurons were of the cerebrospinal fluid-contacting type. ENKir neurons were also located in the dorsal lateral tegmental nucleus and in area B9. The results also permitted a tentative identification of enkephalinergic neurons afferent to the optic tectum, that have previously not been identified with immunocytochemistry, located in the dorsal telencephalic area, as well as enkephalinergic neurons intrinsic to the tectum that may contribute to the laminar arrangement of ENKir fibers in the optic tectum.

Comparison of immunohistochemical localization of[Met 5]enkephalin-Arg 6-Gly 7-Leu 8, [Met 5]enkephalin, neuropeptide Y and vasoactive intestinal polypeptide in the superior cervical ganglion of the rat

The localization of [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 and [Met 5]enkephalin immunoreactivities was studied in the rat superior cervical ganglion. The distribution of these enkephalin-containing peptides in the ganglion was correlated to that of neuropeptide Y and vasoactive intestinal polypeptide. Three different populations of peptide-containing postganglionic neurons were demonstrated. (1) A minor population (10–20%) of principal neurons was immunoreactive for [Met 5]enkephalin-Arg 6-Gly 7-Leu 8 but not immunoreactive for neuropeptide Y nor vasoactive intestinal polypeptide. (2) The major population (about 50–70%) was immunoreactive for neuropeptide Y but not for [Met 5]enkephalin-Arg 6-Gly 7-Leu 8. (3) Few vasoactive intestinal polypeptide-immunoreactive principal neurons (less than 2% of all principal neurons) were observed in the ganglion. All vasoactive intestinal polypeptide-immunoreactive neurons were also immunoreactive for neuropeptide Y but not for [Met 5]enkephalin-Arg 6-Gly 7-Leu 8. [Met 5]enkephalin-Arg 6-Gly 7-Leu 8- and [Met 5]enkephalin-immunoreactive nerve fibers had a similar distribution. These enkephalin immunoreactive nerve fibers were seen to enclose both neuropeptide Y-containing principal neurons and neurons devoid of neuropeptide Y immunoreactivity. Furthermore, there were enkephalin-immunoreactive fiber baskets around vasoactive intestinal polypeptide-immunoreactive neurons and sometimes also around solitary enkephalin-immunoreactive neurons. Previously reported diverse role of enkephalins in the rat superior cervical ganglion is supported by this study.

Immunologically induced sympathectomy of preganglionic nerves by antibodies against acetylcholinesterase: Increased levels of peptides and their messenger rnas in rat adrenal chromaffin cells

Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within l h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.

Distribution of peptide-containing nerve fibers in the gastric musculature of patients undergoing surgery for gastroesophageal reflux.

The distribution of nerve fibers and cell bodies reactive for the peptides enkephalin, neuropeptide Y, substance P, and vasoactive intestinal peptide were studied in biopsies of external muscle taken from the gastric body and antrum of 17 patients undergoing surgery for gastroesophageal reflux, and in regions of healthy stomach resected as part of gastric cancer operations. The results were correlated with preoperative measurements of liquid and solid emptying from the stomach in the patients with gastro-esophageal reflux. In three cases, no delay was detected in either liquid or solid emptying, and the distribution of peptide immunoreactive fibers in the external muscle was similar to that of healthy muscle. In 14 cases, the emptying of either liquids or solids or both was delayed, and in eight of these clearcut changes in the distribution of peptide-immunoreactive fibers occurred. In six cases, the number of enkephalin-immunoreactive fibers was fewer than normal in the biopsy from the gastric body (in one of these samples the number of substance P-immunoreactive fibers was also reduced). In another cae, the number of substance P-immunoreactive fibers in the antrum was reduced, and in another the number of vasoactive intestinal peptide and neuropeptide Y-immunoreactive fibers in the antral biopsy was reduced. It is concluded that in patients with gastroesophageal reflux who have delayed gastric emptying, a proportion demonstrate abnormalities of the peptide-immunoreactive fibers that innervate the gastric external muscle.

Enkephalin and serotonin innervation of somatostatin-immunoreactive medullary reticular formation neurones

Somatostatin cells in the rat medullary reticular formation (Md) have been studied using peroxidase-antiperoxidase immunocytochemistry. Large fusiform and multipolar somatostatin immunoreactive cells were observed in the ventral subnucleus (MdV) running in a band close to the border with the dorsal subnucleus (MdD). In the same region somatostatin-, serotonin- and enkephalin-immunoreactive fibres occur and double staining revealed that these all contact the somatostatin-immunoreactive cells, with enkephalin making a particularly dense innervation.

Partial down-regulation at post-transcriptional level of the gene expression for preproenkephalin in the superior cervical ganglion of the maturing rat

In rats at the postnatal week 3 (P3W), enkephalin-immunoreactivity was detected in subsets of principal ganglion cells in the superior cervical ganglion and nerve fibers within the submandibular gland, one of the targets for the ganglion, whereas it disappeared from them at P8W. Enkephalin-immunoreactive ganglion cells and intraglandular fibers were detected again after the colchicine-pretreatment at P8W. By in situ hybridization the population density of ganglion cells expressing mRNA for preproenkephalin and the expression intensity were similar in both ganglia at P3W and P8W. These findings suggest that the post-transcriptional down-regulation of gene expression for preproenkephalin is involved in the disappearance of enkephalin-immunoreactivity in the adult ganglion.

Immunocytochemical localization and development of multiple kinds of neuropeptides and neuroendocrine proteins in the chick ultimobranchial gland

The ultimobranchial gland is an endocrine organ consisting of C cell groups. In chickens, the glands are richly supplied by nerve fibers immunoreactive for neurofilaments. It was found by immunocytochemical staining that C cells of chick ultimobranchial glands showed immunoreactivities for multiple kinds of neuropeptides and neuroendocrine proteins in addition to calcitonin, i.e., calcitonin gene-related peptide (CGRP), somatostatin, neurotensin, chromogranin A, and tyrosine hydroxylase. Furthermore, enkephalin-immunoreactive cells that showed long cytoplasmic processes and large cell bodies, being distinct from the C cell feature, were detected. The densities of these cells per unit area of ultimobranchial gland were assessed using computer-assisted image analysis system; calcitonin cells were 42.9 +/- 10.0%; CGRP cells 26.9 +/- 5.6%; neurotensin cells 8.6 +/- 6.9%; somatostatin cells 3.1 +/- 1.4%; chromogranin A cells 11.8 +/- 1.8%; tyrosine hydroxylase cells 10.0 +/- 5.2%; enkephalin cells 2.9 +/- 1.3%. Dense distributions of peptidergic nerve fibers were also detected in chick ultimobranchial glands. Numerous varicose fibers immunoreactive for substance P were distributed in the close vicinity to C cell clusters and blood vessels. Enkephalin-immunoreactive fibers were also prominent around C cell clusters. Galanin-, vasoactive intestinal peptide (VIP)-, and tyrosine hydroxylase-immunoreactive fibers were distributed around blood vessels only. Subsequently, the ontogeny of these neuropeptides, neuroendocrine proteins, and peptidergic innervations was examined in chickens at various developmental stages. In 10-day-old embryos, weak to moderately intense immunoreactivity for calcitonin was already present in almost all C cells. Immunoreactivities for somatostatin, CGRP, and tyrosine hydroxylase began to appear at this age. At 12 days of incubation, substance P-immunoreactive fibers were first detected in the parenchyma of ultimobranchial glands. Considerable numbers of enkephalin-immunoreactive fibers and cells were also observed. At 14 days of incubation, the largest populations of somatostatin- and enkephalin-immunoreactive cells were attained; the densities of somatostatin- and enkephalin-immunoreactive cells per unit area were 21.2 +/- 3.2% and 12.9 +/- 3.1%, respectively. Substance P-immunoreactive fibers became numerous throughout the gland at this age. Thereafter, calcitonin-, CGRP-, tyrosine hydroxylase-immunoreactive cells progressively increased in number with embryonic age, whereas somatostatin- and enkephalin-immunoreactive cells started to decrease. Chromogranin A- and neurotensin-immunoreactive cells began to appear at 16 days and 18 days of incubation, respectively. Galanin-, VIP-, and tyrosine hydroxylase-immunoreactive fibers were inconspicuous during embryonic life.

Human fetal spinal cord xenografts survive in the eye of athymic nude rat hosts.

Human fetal spinal cord tissue was recovered from elective abortions and grafted to the anterior chamber of the eye of adult athymic nude rats. The transplants slowly became vascularized from the host iris during the first months. There was a clear cut stage-dependent survival and growth along a more "human" time-table. Fetal spinal cord tissue from embryos younger than gestational week 8 showed a much better survival and growth than tissue from older stages. Using laminin immunohistochemistry blood vessels could be visualized in the grafts. The pattern of vascularization was, however, clearly abnormal; there were fewer vessels which had abnormally thick walls as compared to those in the normal spinal cord. Similar to rat spinal cord allografts the human spinal cord xenografts displayed a relative gliosis and were surrounded by a glial layer visualized with antibodies against glial fibrillary acidic protein. Neurofilament-immunoreactive fibres were found inside the glial layer. A variety of neurons were found including large polygonal motoneuron-shaped cells, albeit with CGRP and AChE negative cell bodies. Both Substance P and enkephalin-immunoreactive cells and fibres were found. It is concluded that xenografted fetal human spinal cord survives, grows and may provide a useful model for experimental studies of human spinal cord development and connectivity.

Development and distribution of enkephalin-immunoreactive elements in the chicken spinal cord

The development and distribution of methionine-enkephalin-immunoreactive elements were studied in the chicken spinal cord with the indirect immunofluorescence method. Methionine-enkephalin-like immunoreactivity was first detected in the chick spinal cord at embryonic stages 29–30 (incubation day 6). Before stage 35 (day 9). it was mainly observed in fibres almost throughout the while matter. Subsequently, fibres containing the peptide appeared in the ventral half of the gray matter, but mostly in the lateral portion of the neck of the dorsal horn. From stage 40 (day 13 or 14), fibres were especially noticed in laminae 1 and 2. and in the area dorsal to the central canal. In particular, many enkephalin-immunoreactive perikarya were observed in several spinal areas during this period. Such a distribution of both enkephalin-immunoreactive fibres and perikarya remained visible at later embryonic stages, but labelled cells gradually decreased in number and disappeared after hatching. With colchicine treatment, however, a similar distribution of the peptide was found in the spinal cord of adult chickens. As in the embryo, enkephalin-immunoreactive perikarya were mainly observed in the lateral portion of the neck of the dorsal horn, in lamina 1, and in the nucleus of the dorsolateral funiculus throughout the spinal eord. At the thoracic level, many were also located ventral to the central canal. Enkephalin-immunoreactive fibres increased notably in the gray matter of adult chickens. They mainly occurred in laminae 1 and 2, in the lateral portion of the neck of the dorsal horn, and in the area around, especially dorsal to. the central canal. In contrast, enkephalin-immunoreactive fibres decreased in the white matter and they were mainly observed in the dorsolateral funiculus, in Lissauer's tract, and in the lateral funiculus adjacent to the gray. The distribution of enkephalin-immunoreactive fibres was generally comparable at all spinal levels examined. In addition, examination of post-hatched chickens showed virtually the same results as in the adult.

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

The gross morphology and growth patterns of substance P, enkephalin-, somatostatin- and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P- and enkephalin-immunoreactive neurons were more abundant than somatostatin- and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.

Megacolon in Myotonic Dystrophy Caused by a Degenerative Neuropathy of the Myenteric Plexus

A 32-yr-old man with myotonic dystrophy had a left hemicolectomy performed because of a megacolon. The colonic mucosa, smooth muscle, and connective tissue appeared normal by hematoxylin and eosin and trichrome stains and transmission electron microscopy. In contrast, the myenteric plexus had markedly fewer neurons than normal on the hematoxylin and eosin stains. Silver staining of the plexus revealed degeneration and decreased numbers of argyrophilic neurons, which were smaller and had fewer processes and a more uneven staining quality than controls. Many axons were fragmented, and increased numbers of glial cell nuclei were present in the plexus. Degenerative changes in the neurons were present in a patchy distribution on transmission electron microscopy. Immunohistochemistry revealed a decrease of the substance Pand enkephalin-immunoreactive fibers in the muscularis externa. This suggests that colonic motor dysfunction associated with myotonic dystrophy may be caused by a visceral neuropathy that involves the substance P- and enkephalin-immunoreactive fibers of the smooth muscle.

Distribution and coexistence of peptides in nerve fibers of the external muscle of the human gastrointestinal tract

The nerve fibers that supply the external muscle of the human gastrointestinal tract were examined for their immunoreactivity to the neuropeptides enkephalin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal peptide, for tyrosine hydroxylase (a catecholamine-synthesizing enzyme), and for coexistence between immunoreactivities in nerve fibers. Studies on coexistence revealed that the majority of reactive nerve fibers could be placed in one of two classes: (a) those fibers with reactivity to enkephalin or substance P, or both, and (b) fibers containing one or both of the peptides neuropeptide Y and vasoactive intestinal peptide. Many fibers immunoreactive for vasoactive intestinal peptide or neuropeptide Y, or both, were found throughout the external smooth muscle of the gastrointestinal tract, but neuropeptide Y-reactive fibers were less common in the small and large intestines than in the stomach and esophagus. Fibers immunoreactive for enkephalin or substance P, or both, were sparse in the esophagus, increased in numbers to reach maximal frequency in the pylorus, and maintained a similar frequency in the small and large intestines. Fibers with somatostatin or tyrosine hydroxylase immunoreactivity were rare. In general, sphincter regions were similar to nonsphincter regions in peptide-immunoreactive fiber numbers and types, except that the internal anal sphincter had no enkephalin-immunoreactive fibers and very few substance P-reactive fibers. Moderate numbers of fibers reactive for neuropeptide Y and vasoactive intestinal peptide were found in the internal anal sphincter. It is suggested that enkephalin and substance P are in excitatory fibers and that vasoactive intestinal peptide and neuropeptide Y are in fibers inhibitory to the external muscle.