Enkephalin-like Immunoreactivity Research Articles

Light and electron microscopic analysis of enkephalin-like immunoreactivity in the basolateral amygdala, including evidence for convergence of enkephalin-containing axon terminals and norepinephrine transporter-containing axon terminals onto common targets

Modulatory interactions of opioids and norepinephrine (NE) in the anterior subdivision of the basolateral nucleus of the amygdala (BLa) are critical for the consolidation of memories of emotionally arousing experiences. Although there have been several studies of the noradrenergic system in the amygdalar basolateral nuclear complex (BLC), little is known about the chemical neuroanatomy of opioid systems in this region. To address this knowledge gap the present study first examined the distribution of met-enkephalin-like immunoreactivity (ENK-ir) in the BLC at the light microscopic level, and then utilized dual-labeling immunocytochemistry combined with electron microscopy to investigate the extent of convergence of NE and ENK terminals onto common structures in the BLa. Antibodies to ENK and the norepinephrine transporter (NET) were used in these studies. Light microscopic examination revealed that a subpopulation of small nonpyramidal neurons expressed ENK-ir in all nuclei of the BLC. In addition, the somata of some pyramidal cells exhibited light to moderate ENK-ir. ENK+ axon terminals were also observed. Ultrastructural analysis confined to the BLa revealed that most ENK+ axon terminals formed asymmetrical synapses that mainly contacted spines and shafts of thin dendrites. ENK+ terminals forming symmetrical synapses mainly contacted dendritic shafts. Approximately 20% of NET+ terminals contacted a structure that was also contacted by an ENK+ terminal and 6% of NET+ terminals contacted an ENK+ terminal. These findings suggest that ENK and NE terminals in the BLa may interact by targeting common dendrites and by direct interactions between the two types of terminals.

Segregation of met–enkephalin from vesicular acetylcholine transporter and choline acetyltransferase in sympathetic preganglionic varicosities mostly lacking synaptophysin and synaptotagmin

Sympathetic preganglionic neurons (SPN) coexpress the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase and different peptides in their cell bodies, but can express them independently in separate varicosities, indicating that SPN segregate transmitters to different synapses. Consequently, there are populations of preganglionic varicosities (peptidergic and noncholinergic) that store peptides but not ACh. We studied in the cell bodies and axon processes of the rat SPN the expression and the proportional coexpression of the vesicular ACh transporter-like immunoreactivity (VAChT), a specific marker of cholinergic synaptic vesicles or ChAT-like immunoreactivity (ChAT), and the peptide methionine enkephalin-like immunoreactivity (mENK), and confirmed the presence of a population of SPN peptidergic, noncholinergic varicosities. We characterized these varicosities by exploring the occurrence of synaptophysin-like immunoreactivity (Syn), a marker of small clear vesicles, and synaptotagmin-like immunoreactivity (Syt), a preferential marker of large dense core vesicles. We found that (i) VAChT and mENK, like ChAT–mENK, were coexpressed in only 59% of the mENK-containing varicosities, although they colocalized in the SPN cell bodies; and (ii) almost 60% of the population of mENK-containing varicosities did not express Syn or Syt, and over 80% of the mENK-containing varicosities negative for VAChT also lacked Syn. These data prove that SPN segregate mENK from VAChT and ChAT, and show that most of the subset of mENKergic varicosities negative for VAChT also does not express Syn, suggesting the presence of a different vesicular pattern in these sympathetic preganglionic varicosities.

Expression of cholecystokinin, enkephalin, galanin and neuropeptide Y is markedly changed in the brain of the megencephaly mouse

Megencephaly, enlarged brain, is a major sign in several human neurological diseases. The mouse model for megencephaly ( mceph/mceph) has an enlarged brain, presumably due to brain cell hypertrophy, and exhibits neurological and motor disturbances with seizure-like activity, as well as disturbances in the insulin-like growth factor system. Here, we report that expression of the neuropeptides cholecystokinin, enkephalin, galanin and neuropeptide Y is dramatically changed in mceph/mceph brains compared to wild type, as revealed by in situ hybridization and immunohistochemistry. The changes were confined to discrete brain regions and occurred in a parallel fashion for peptides and their transcripts. For cholecystokinin, mceph/mceph brains had region-specific up- and down-regulations in several layers of the hippocampal formation and increased levels in, especially ventral, cortical regions. Enkephalin messenger RNA expression was up-regulated in the dentate gyrus granular layer and in ventral cortices, but down-regulated in the CA1 pyramidal layer. Enkephalin-like immunoreactivity was elevated in mossy fibers of the hippocampus and the ventral cortices. Galanin expression was increased in several layers and interneurons of the hippocampal formation, as well as in ventral cortices. Galanin-like immunoreactivity was reduced in nerve terminals in the forebrain. Neuropeptide Y expression was increased in the hippocampal formation and ventral cortices. Whether the mainly increased peptide levels contribute to the excessive growth of the brain or represent a consequence of this growth and/or of the neurological and motor disturbances remains to be elucidated.

Opioid peptide pharmacology and immunocytochemistry in an animal model of self-sustaining status epilepticus

In a model of self-sustaining status epilepticus induced in rats by 30 min intermittent stimulation of the perforant path through chronically implanted electrodes, a decrease in dynorphin-like immunoreactivity in the dentate gyrus and CA3 was observed 3 h and 24 h after the induction of status epilepticus. Enkephalin-like immunoreactivity decreased 3 h but not 24 h after perforant path stimulation. Injection into the hilus of the dentate gyrus 10 min prior to stimulation of the κ-receptor agonist dynorphin-A(1–13), the δ-receptor antagonists ICI-174864 and naltrindole, as well as i.p. injection of naloxone prevented the development of status epilepticus. Perihilar administration of the δ-agonist [ d-Ser 2]Leu-enkephalin-Thr 6 or the κ- antagonist nor-Binaltorphimine, but not of the μ-agonist [ d-Ala 2,N-Me-Phe 4,Gly-ol 5]-Enkephalin, facilitated the establishment of self-sustaining status epilepticus. Injection into the hilus of dynorphin-A(1–13) after the end of perforant path stimulation, stopped established status epilepticus, while administration of naloxone, naltrindole and ICI-174864 were ineffective. We conclude that κ-opioids in the hippocampus counteract initiation and maintenance of status epilepticus, while δ-opioids promote initiation, but not maintenance of seizure activity. These data are important for the understanding the mechanisms which underlie initiation and maintenance of status epilepticus and for the development of new approaches for its effective management.

Short- and Long-term Effects of Irradiation on Laryngeal Mucosa of the Rat

Although radiotherapy is often used to treat laryngeal carcinoma, there is little information on the effects of this treatment on laryngeal structures. Rats were irradiated to the head and neck region and the larynges were studied by light- and electron-microscopy and immunohistochemistry. Ten days after irradiation, a change in the ultrastructural appearance of the granules of the subglottic glands was observed. Substance P-, bombesin- and enkephalin-like immunoreactivity was increased in local ganglionic cells and glandular nerve fibres. The mast cells were reduced in number. At examination 4-6 months after irradiation, there were no obvious differences compared with controls concerning mast-cell numbers and neuropeptide expression. The ultrastructural changes seen in the subglottic glands remained to some extent. The results show that structural changes in the subglottic glands occur concomitantly with an increased expression of certain neuropeptides in the innervation of these glands, which implies a relationship between these two parameters. The mast cells respond drastically to irradiation, but in the long run, regeneration of these cells occurs.

Coexistence of enkephalin- and tyrosine hydroxylase-like immunoreactivities in nerve fibers of the temporomandibular joint capsule of the guinea pig.

The innervation of joints has been suggested to play an important role for their normal function as well as for the pathogenesis of chronic arthritic conditions. It is therefore important to elucidate the functional anatomy of this innervation. The expression of methionine enkephalin-like immunoreactivity (ENK-LI) and tyrosine hydroxylase (TH)-LI as well as their possible colocalization were examined in the temporomandibular joint of the guinea pig: Nerve fibers with ENK-LI were demonstrated in the synovium of the guinea pig temporomandibular joint capsule with the use of indirect immunofluorescence. The ENK+ fibers were found close to the surface of the synovial membrane, deeper in the synovium, and at the walls of blood vessels. Coexistence of ENK- and TH-LI was observed frequently in the synovial nerve fibers. After removal of the superior cervical ganglion (SCG), the ENK-containing nerve fibers were reduced substantially in number on the operated side or were completely absent if the cervical sympathetic trunk was also excised. The findings indicate that the majority of fibers with ENK-LI derive from the SCG ENK may act as a neuromodulator on the catecholaminergic nerves and may also have an analgesic effect in the joint.

Coexistence of enkephalin‐ and tyrosine hydroxylase‐like immunoreactivities in nerve fibers of the temporomandibular joint capsule of the guinea pig

Background The innervation of joints has been suggested to play an important role for their normal function as well as for the pathogenesis of chronic arthritic conditions. It is therefore important to elucidate the functional anatomy of this innervation. Methods The expression of methionine enkephalin-like immunoreactivity (ENK-LI) and tyrosine hydroxylase (TH)-LI as well as their possible colocalization were examined in the temporomandibular joint of the guinea pig. Results Nerve fibers with ENK-LI were demonstrated in the synovium of the guinea pig temporomandibular joint capsule with the use of indirect immunofluorescence. The ENK+ fibers were found close to the surface of the synovial membrane, deeper in the synovium, and at the walls of blood vessels. Coexistence of ENK- and TH-LI was observed frequently in the synovial nerve fibers. After removal of the superior cervical ganglion (SCG), the ENK-containing nerve fibers were reduced substantially in number on the operated side or were completely absent if the cervical sympathetic trunk was also excised. Conclusions The findings indicate that the majority of fibers with ENK-LI derive from the SCG. ENK may act as a neuromodulator on the catecholaminergic nerves and may also have an analgesic effect in the joint. Anat. Rec. 251:226–229, 1998. © 1998 Wiley-Liss, Inc.

Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.

Development of the enkephalin-, neurotensin- and somatostatin-like (ENSLI) amacrine cells in the chicken retina

The development of the enkephalin-, neurotensin- and somatostatin-like immunoreactive (ENSLI) amacrine cells in the chicken retina has been investigated by radioimmunoassay (RIA) and immunocytochemistry (ICC). By RIA, enkephalin-like immunoreactivity (ENK-LI) was detected at embryonic day (E) 5 at only very low levels, which gradually increased until E17. From E18 to E21, there was a relatively rapid increase in ENK-LI levels, and just after hatching, there was a very steep rise. By ICC, the cell bodies of the ENSLI amacrine cells were first detected in the inner nuclear layer on E18, with no immunostaining in the inner plexiform layer (IPL). On E21, more cells were detected and processes in the IPL were visible, but detailed arborisations were not clear. On postnatal day (P) 1, the ENSLI amacrine cells showed a morphology similar to that in mature retina in both the density of cell bodies and the ramification pattern of processes. Antibodies to neurotensin and somatostatin revealed a similar developmental pattern. Thus, the three peptides appear to follow a similar developmental pattern in the ENSLI amacrine cells, suggesting that the three peptides respond similarly to developmental stimuli, just as they are released in parallel in response to physiological stimulation from mature ENSLI amacrine cells. After hatching, higher levels of ENK-LI were detected by RIA and more ENSLI amacrine cell bodies and processes were detected by ICC in animals kept in the light than in those kept in the dark. In retinas kept in the light for 12 h, it was found that immunoreactive processes in the IPL formed strongly stained patches, but this was not observed in retinas kept in the dark for 12 h.

Neurochemical compartments in the human forebrain: evidence for a high density of secretoneurin-like immunoreactivity in the extended amygdala.

Secretoneurin is a 33-amino acid neuropeptide produced by endoproteolytic processing from secretogranin II, which is a member of the chromogranin/ secretogranin family. In this immunocytochemical study we investigated the localization of secretoneurin-like immunoreactivity in the human substantia innominata in relation to the ventral striatopallidal system, the bed nucleus-amygdala complex and the basal nucleus of Meynert. A high density of secretoneurin immunostaining was found in the medial part of the nucleus accumbens. All subdivisions of the bed nucleus of the stria terminalis displayed a very prominent immunostaining for secretoneurin, whereas substance P and enkephalin showed a more restricted distribution. A high concentration of secretoneurin immunoreactivity was also observed in the central and medial amygdaloid nuclei. In the lateral bed nucleus of the stria terminalis and the sublenticular substantia innominata, the appearance of secretoneurin immunoreactivity was very similar to that of enkephalin-like immunoreactivity, exhibiting mostly peridendritic and perisomatic staining. The ventral pallidum and the inner pallidal segment displayed strong secretoneurin immunostaining. Secretoneurin did not label cholinergic neurons in the basal forebrain. This study demonstrates that secretoneurin-like immunoreactivity is prominent in the bed nucleus-amygdala complex, referred to as extended amygdala. The distribution of secretoneurin-like immunoreactivity in comparison with that of other neuroanatomical markers suggests that this forebrain system is a discret compartment in the human forebrain.

Distribution of enkephalin-like immunoreactivity in the cat digestive tract

Immunohistochemical investigations were carried out to determine the pattern of distribution of methionine- and leucine-enkephalin-like materials in the cat pylorus, duodenum, ileum and proximal and distal colon. The present results indicate that leucine-enkephalin-like materials are less densely distributed than methionine-enkephalin-like materials, but that the two patterns of distribution show some similarities. Considerable regional differences exist however in the distribution of these enkephalin-like materials in the muscular layers. In the duodenum, ileum and proximal colon, the immunoreactivity was mainly confined to the myenteric plexus and the circular muscle layer, where it was present in nerve cell bodies and in numerous fibres. In the longitudinal muscle and submucous layers, a few immunoreactive fibres were observed which sometimes surrounded blood vessels. In the pylorus and the distal colon, however, numerous immunoreactive fibres were observed in the longitudinal and circular muscle layers; the immunoreactivity was detected in the cell bodies of numerous myenteric plexus neurons but those of only a few submucous plexus neurons. In addition, the pylorus tissues contained immunoreactive plexi which were localized either within the longitudinal muscle or between the serosa and the longitudinal muscle layer. These plexi were connected to the myenteric plexus by immunoreactive nerve strands. In all the small intestinal segments studied, numerous immunoreactive varicosities were present in the deep muscular plexus, in the inner part of the circular muscle layer. Our results suggest that in cats, the nervous control of external muscular layers mediated by enkephalins shows regional differences. In the pylorus and the distal colon, it involves both the longitudinal and circular muscle layers, whereas in other intestinal segments, only the circular muscle layer is involved.

Enkephalin-like immunoreactivity in human skin is found selectively in a fraction of CD68-positive dermal cells: increase in enkephalin-positive cells in lesional psoriasis.

Opioid peptides are synthesized in neurons, endocrine cells, monocytes/macrophages and B and T lymphocytes. They interact with opioid receptors located on immune cells and nociceptive nerve terminals. Because opioid peptides might be of importance in inflammatory skin diseases, for example psoriasis, sections of skin from psoriatic patients were immunohistochemically stained with antisera against methionine and leucine enkephalin, CD68 (KP1, PG-M1), calprotectin (M747), M130 (Ber-MAC3), CD1a and CD3. Enkephalin-like activity was detected selectively in dermal CD68-positive macrophages/monocytes. The activity showed no association with the activation markers M747 and Ber-MAC3. There was a statistically significant increase in enkephalin-positive cells in involved psoriatic skin compared with uninvolved and normal skin. These results were confirmed by radioimmunoassay which showed elevated levels in extracts from involved psoriatic skin compared with uninvolved skin (81%) and normal skin (204%). Furthermore, preproenkephalin mRNA of an expected size was detected in involved psoriatic skin. If the increased levels of enkephalins present in monocytes/macrophages in psoriatic skin lesions reach the threshold for biological activity, they may play a role in the regulation of the inflammatory processes seen in this skin disease.

Subcellular analysis of Tyr-aminopeptidase activities in the developing rat cerebellum

The endogenous opioid system seems to play important roles in the developing cerebellum. The first opioid peptide isolated, Met-enkephalin, is expressed transiently in this brain area. In the present study, several enzyme activities capable of hydrolyzing enkephalins are measured during the first month of cerebellar development, using Tyr- β-naphthylamyde as substrate and puromycin as inhibitor of one of the membrane-bound aminopeptidases. Puromycin-sensitive soluble and membrane-bound aminopeptidase activities decrease in the synaptosomal and mitochondrial fractions at the end of the first month of life, just when enkephalin-like immunoreactivity decreases in the cerebellum. Membrane-bound enzyme also decreases in the myelinic fraction. Synaptosomal activity increases after birth, coinciding with decreases in the activity in the microsomal fraction. Puromycin-insensitive and membrane-bound aminopeptidase shows less significant developmental changes and they occur mainly in the first week of life, coinciding with the axonal and dendrite growth. These results could suggest a possible role of these enzymes, together with the rest of the opioid system, in cerebellar development.

Plasma methionine enkephalin-like immunoreactivity in patients with methamphetamine psychosis.

Plasma methionine enkephalin-like immunoreactivity in patients with methamphetamine psychosis.

Immunohistochemical mapping of nitric oxide synthase in the rat hypothalamus and colocalization with neuropeptides

The localization and distribution of nitric oxide synthase in the hypothalamus have been studied with an immunohistochemical technique using antibodies to neuronal rat nitric oxide synthase. Subsequent double-labeling experiments examined the colocalization patterns of nitric oxide synthase and several peptides. Our results demonstrate a widespread occurrence of nitric oxide synthase-immunoreactive nerve cell bodies and processes throughout the hypothalamus, especially in various parts of the preoptic region, in the supraoptic and paraventricular nuclei, the lateral hypothalamic area, the ventromedial and dorsomedial nuclei, the arcuate nucleus and various parts of the mammillary region. Double labeling experiments showed that nitric oxide synthase-like immunoreactivity coexists with substance P-like immunoreactivity in the medial preoptic area, with oxytocin-, cholecystokinin- and galanin message-associated peptide-like immunoreactivity in the supraoptic nucleus, with enkephalin-, oxytocin- and corticotropin releasing factor-like immunoreactivity in the paraventricular nucleus and with enkephalin-like immunoreactivity in the arcuate nucleus. Furthermore, in the ventromedial nucleus, nitric oxide synthase-like immunoreactivity coexisted with enkephalin-, substance P-, and somatostatin-like immunoreactivity, and in the dorsomedial nucleus with enkephalin-, galanin message-associated peptide- and substance P-like immunoreactivity. In the mammillary region nitric oxide synthase-like immunoreactivity coexisted with enkephalin-, cholecystokinin-, and substance P-like immunoreactivity. Among these neuropeptides, enkephalin and substance P were most frequently found in nitric oxide synthase-immunoreactive neurons. We conclude that nitric oxide synthase-immunoreactive neurons contain neuropeptides in various parts of the hypothalamus, and that nitric oxide in the hypothalamus may be involved in a variety of neuroendocrine and autonomic functions.

Localization of enkephalin immunoreactivity in the spinal cord of the long‐tailed ray Himantura fai

Enkephalin-like immunoreactivity (ENK-LI) was found throughout the spinal cord of the long-tailed ray Himantura fai. The densest ENK-LI was in the superficial portion of lamina A of the dorsal horn. Lamina B and the deeper parts of lamina A contained radially oriented, labelled fibres. Laminae C, D, and E contained many longitudinally orientated fascicles which were surrounded by a reticulum of transversely orientated, labelled fibres, some of which projected into the ventral and lateral funiculi. Labelled fibres were found in the dorsal commissure and around the central canal, but the later did not cross the midline. One-third of all enkephalinergic cells were found throughout laminae A and B, while two-thirds were located in the medial half of C, D, and E. Occasionally a labelled cell was located in the lateral funiculus. The ventral horn (laminae F and G) contained many enkephalinergic fibres but no labelled nuclei. A few dorsal column axons contained ENK-LI. In the lateral funiculus there were two groups of labelled axons, a superficial, dorsolateral group, and a deeper group, occupying a crescent-shaped region. The ventral funiculus also contained many labelled axons. The central projection of the dorsal root passed through the substantia gelatinosa and divided into rostrally and caudally projecting fascicles within lamina C. The root, and these fascicles, both lacked ENK-LI. In contrast, the fascicles in laminae D and E did contain enkephalinergic fibres. The origin of the various fibre systems and the role of enkephalin in the regulation of sensory processing and motor output are discussed. © 1996 Wiley-Liss, Inc.

Localization of enkephalin immunoreactivity in the spinal cord of the long-tailed ray Himantura fai.

Enkephalin-like immunoreactivity (ENK-LI) was found throughout the spinal cord of the long-tailed ray Himantura fai. The densest ENK-LI was in the superficial portion of lamina A of the dorsal horn. Lamina B and the deeper parts of lamina A contained radially oriented, labelled fibres. Laminae C, D, and E contained many longitudinally oriented fascicles which were surrounded by a reticulum of transversely oriented, labelled fibres, some of which projected into the ventral and lateral funiculi. Labelled fibres were found in the dorsal commissure and around the central canal, but the later did not cross the midline. One-third of all enkephalinergic cells were found throughout laminae A and B, while two-thirds were located in the medial half of C, D, and E. Occasionally a labelled cell was located in the lateral funiculus. The ventral horn (laminae F and G) contained many enkephalinergic fibres but no labelled nuclei. A few dorsal column axons contained ENK-LI. In the lateral funiculus there were two groups of labelled axons, a superficial, dorsolateral group, and a deeper group, occupying a crescent-shaped region. The ventral funiculus also contained many labelled axons. The central projection of the dorsal root passed through the substantia gelatinosa and divided into rostrally and caudally projecting fascicles within lamina C. The root, and these fascicles, both lacked ENK-LI. In contrast, the fascicles in laminae D and E did contain enkephalinergic fibres. The origin of the various fibre systems and the role of enkephalin in the regulation of sensory processing and motor output are discussed.

Changes in enkephalin and neuropeptide Y-like immunoreactivity in rabbit chromaffin tissues during perinatal development

Enkephalin-like immunoreactivity (ENK-LI), neuropeptide Y (NPY)-LI and dopamine-β-hydroxylase (DBH)-LI were found within the chromaffin cells of both the paraaortic body and the adrenal medulla of the newborn rabbit using immunohistochemistry. Cells positive to DBH-LI were abundant in both the paraaortic body and the adrenal medulla. ENK-LI positive cells were frequent in the paraaortic body, but more sparse in the adrenal medulla. A few cells staining for NPY-LI could be detected in both organs. Some nerve fibers within these organs also contained substance P-LI and calcitonin-gene related peptide-LI. The tissue contents of ENK-LI and NPY-LI, as measured by radioimmunoassay, increased after birth in the adrenal glands and were significantly higher than the fetal levels from 1 week of age. In the paraaortic body the lowest content of ENK-LI was found around birth, whereas the content of NPY-LI was highest at that time. With advancing postnatal age, the content of ENK-LI increased, whereas the content of NPY-LI decreased. At each age, there was a higher content of ENK-LI as compared to NPY-LI in both organs. This indicates that the synthesis of ENK-LI and NPY-LI in the paraaortic body is differently regulated during perinatal development.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizure-susceptible El mice play an important role in the pathogenesis of seizures.