Immunocytochemical distribution of met-enkephalin-Arg6-Gly7-Leu8 in the rat lower brainstem

The distribution of methionine-enkephalin-Arg6-Gly7-Leu8, a unique peptide derived from proenkephalin A in the rat brainstem, was studied immunocytochemically by using a highly specific antiserum to this octapeptide sequence. Immunoreactive perikarya with various shapes and sizes were detected in many regions of the rat brainstem. Dense accumulation of immunoreactive perikarya and fibers was seen in the nuclei associated with special sensory and visceral functions, such as the interpeduncular nucleus, the parabrachial nucleus, the nucleus of the solitary tract, and the nucleus of the spinal tract of the trigeminal nerve. Clusters of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya and fibers were observed in certain areas considered to play a role in nociception and analgesia, such as the central gray of the midbrain central gray and the raphe magnus nucleus. Some methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactive perikarya were distributed in the lateral reticular nucleus, the nucleus of the solitary tract, and the raphe magnus nucleus, where monoaminergic neurons were also detected. In addition to the previously reported enkephalinergic cells, we found many methionine-enkephalin-Arg6-Gly7-Leu8 containing neurons; the rostral and caudal linear nucleus of raphe, the median raphe nucleus, entire length of the raphe magnus nucleus, the medial longitudinal fasciculus, the cuneate nucleus, the external cuneate nucleus, the gracile nucleus, and the area postrema. The wide distribution of this octapeptide-like immunoreactivity reflected neurons expressing the preproenkephalin A gene distributed more widely than previously reported and that innervated many regions.     

An immunohistochemical study of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity-containing neurons in the parasympathetic preganglionic regions of the rat spinal cord

The localization of the methionine-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu)-like immunoreactivity-containing neurons in the rat lumbosacral spinal cord was immunohistochemically examined by an antiserum very specific to Met-Enk-Arg-Gly-Leu. The immunoreactive neurons occupied the positions corresponding to the parasympathetic preganglionic nuclei determined by the previous horseradish peroxidase (HRP)-tracing experiments. The present study suggests that the parasympathetic preganglionic neurons in the rat lumbosacral spinal cord produce preproenkephalin A and its related peptides.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in rat duodenum

The distribution of Met-enkephalin-Arg6-Gly7-Leu8 (Met-ENK-Arg6-Gly7-Leu8) in the rat duodenum was determined using specific antibodies against Met-ENK-Arg6-Gly7-Leu8 and the immunofluorescence microscope technique. Met-ENK-Arg6-Gly7-Leu8 immunoreactive perikarya have been detected in the myenteric plexus. These neuronal cell bodies were large in diameter and round in shape. Met-ENK-Arg6-Gly7-Leu8 immunostained nerve fibres were noted in both the circular muscle layer and, more abundantly, in interconnecting myenteric plexus nerve fibre bundles. These findings might indicate that Met-ENK-Arg6-Gly7-Leu8 has important physiological roles as neurotransmitter and/or neuromodulator in the human and mammalian gastrointestinal tract.     

Met5-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscopic study

By using both light and electron microscopic immunocytochemical methods, Met5-Enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive structures were detected in the pelvic ganglion of male rats. Denervation studies were carried out to determine the origin of these immunoreactive fibers and the projection of immunoreactive neurons within the pelvic ganglion. MEAGL-like immunoreactivity was found in numerous axon boutons, some small, intensely fluorescent (SIF) cells, and a few principal ganglion neurons. Most of the immunoreactive nerve fibers formed pericellular plexuses surrounding the ganglion cells. In addition, there were a few scattered varicose fibers. These fiber plexuses could be classified into two types: type I (approximately 90% of fibers), which consisted of 80-120 small boutons that synapsed on either the dendrites (80% of cases) or somata (20% of cases) of principal neurons; and type II (approximately 10% of fibers), which consisted of 20-40 larger boutons that formed axodendritic synapses exclusively. After transection of the hypogastric and pelvic nerves, virtually all of the pericellular fiber plexuses disappeared, whereas the scattered varicose fibers remained. According to their ultrastructure, these remaining fibers were considered to arise from SIF cells. Following the injection of Fast Blue into the bladder wall, some of the MEAGL-like immunoreactive principal neurons were retrogradely labeled. The results of this study indicate that there are two origins for the MEAGL-like immunoreactive fibers detected in the pelvic ganglion: most arise from preganglionic neurons in the spinal cord, and a small proportion may originate from intraganglionic MEAGL-like immunoreactive SIF cells or principal neurons. Some MEAGL-like immunoreactive principal neurons may project to the urinary bladder.     

Distribution of NPY Y5-like immunoreactivity in the rat brain

The pharmacology and brain mRNA distribution of the neuropeptide Y (NPY) rat Y5 (rY5) receptor has led to the hypothesis that this receptor might mediate the hypothalamic feeding response to NPY in addition to many other physiologic functions. However, through the use of autoradiographic techniques, only very low levels of Y5-like immunoreactive (Y5-ir) binding are detected in the rat brain. To localize the Y5 protein in the rat brain, polyclonal antibodies were raised to the carboxyl terminus of the rY5 receptor. The resulting antisera were affinity purified and characterized by specific binding to HEK293 cells that had been stably transfected with the rY5 receptor. Utilizing immunohistochemical techniques, we found a discrete pattern of Y5-ir in the rat brain. In initial studies, very low levels of Y5-ir were detected, and TSA amplification was required to visualize the staining. Areas with the highest levels of expression in clude the piriform cortex, supraoptic nucleus, and hippocampus. Areas with moderate levels of expression include the lateral septum, amygdala, arcuate nucleus, paraventricular hypothalamic nucleus, locus coeruleus, and cerebellum. With several exceptions, this pattern of distribution is consistent with earlier reports of rY5 mRNA and receptor protein expression.     

Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in rat and human cerebrospinal fluid: influence of neuroleptic drugs and electroconvulsive shock

Met5-Enkephalin-Arg6-Gly7-Leu8 immunoreactivity was quantitated in both rat and human cerebrospinal fluid (CSF) by radioimmunoassay with a carboxy-terminal directed antiserum. The immunoreactivity in CSF was chromatographically characterized in both species and was found to consist almost exclusively of high molecular weight forms. In human CSF there was approximately 300 fmol/ml and in the rat 1,500 fmol/ml of immunoreactivity. The possibility of a rostro-caudal gradient was examined in the human by analyzing the first and the twenty-fifth ml of CSF drawn during a lumbar puncture: none was found. The immunoreactivity was fairly stable; no loss of immunoreactivity was observed after 24 h of incubation of rat CSF at 37 degrees C. Electroconvulsive shock (ECS) produced a significant elevation in CSF content but only after a course of chronic administration; a single acute ECS produced no increase. Human subjects with schizophrenia who were being treated with antipsychotic drugs had elevated levels of immunoreactivity in comparison to non-mediated patients and normals. The high levels of this immunoreactivity in CSF, its stability and the evidence that the content can change with physiological and pharmacological manipulation indicate that Met5-Enkephalin-Arg6-Gly7-Leu8 immunoreactivity can serve as a new and useful CSF marker for investigating the CNS enkephalin system in neurological or psychiatric disorders.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive peptides in rat brain: presence of multiple molecular forms

An antiserum to the opioid octapeptide met5-enk-arg6- gly7 -leu8 was used to measure the distribution and molecular weight heterogeneity of met5-enk-arg6- gly7 -leu8-immunoreactive (IR) peptides in rat brain. High concentrations of met5-enk-arg6- gly7 -leu8-IR peptides were found in the striatum and hypothalamus; low concentrations were observed in the cortex and cerebellum. Intermediate levels of immunoreactivity were found in other brain regions. Thus the distribution of met5-enk-arg6- gly7 -leu8-IR peptides closely parallels the distribution of met5-enkephalin. The immunoreactivity present in brain regions was characterized by gel filtration and reverse-phase high pressure liquid chromatography. In the striatum, more than 85% of the met5-enk-arg6- gly7 -leu8-IR peptides eluted from a Biogel P-30 column at the position of the authentic octapeptide; the identity of this material was further confirmed by HPLC. Gel filtration of extracts prepared from the medulla-pons resulted in two peaks of immunoreactive material of equal size; one eluted at approximately 8000 daltons and one eluted in the position of the octapeptide. Enzymatic digestion (trypsin + carboxypeptidase B) of the 8000 dalton-IR peptide resulted in the generation of met5-enkephalin-IR. Extracts prepared from other brain regions contained varying amounts of this high molecular weight form of met5-enk-arg6- gly7 -leu8.     

Distribution of immunoreactive metorphamide (adrenorphin) in discrete regions of the rat brain: comparison with Met-enkephalin-Arg6-Gly7-Leu8

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (greater than 120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (less than 60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.     

Co-localization of substance P and Met-enkephalin-Arg6-Gly7-Leu8 in the intraspinal neurons of the rat, with special reference to the neurons in the substantia gelatinosa

A double-labeling immunofluorescence technique was employed to investigate the co-localization of the functionally antagonistic neuropeptides, substance P and enkephalins, within intraspinal neurons of the rat. Anti-Met-enkephalin-Arg6-Gly7-Leu8 (Enk-8) antiserum was used as a marker of the preproenkephalin A neuron system. The observations were focused on the lumbar spinal cord. Co-localization was most prominent within neurons in the substantia gelatinosa, in which more than 95% of substance P-like immunoreactivity neurons showed Enk-8-like immunoreactivity. These double-labeled cells corresponded to 45% of Enk-8-like immunoreactive neurons in the same area. This suggests that SP/Enk-8 interaction occurs at the axon terminals of the substantia gelatinosa neurons. In deeper layers of the dorsal horn (laminae III, IV), only 14% and 6% of SP-like immunoreactive and Enk-8-like immunoreactive neurons were double labeled, respectively. Co-localization was also observed in neurons located in the laminae I, V, VII and X, suggesting concomitant involvement of these peptides in a variety of spinal cord functions.     

Colocalization of proenkephalin peptides in rat brain neurons

A serial, thin-section immunocytochemical study of the anatomical distribution of Leu-enkephalin and BAM-22P (an adrenal proenkephalin peptide) demonstrated that both immunoreactivities occur within the same neurons throughout brain. However, neither peptide immunoreactivity could be observed in neurons containing dynorphin A immunoreactivity. These results are consistent with the possibility that the enkephalin precursor in brain is similar to that sequenced in adrenal, but fail to support the hypothesis that the dynorphin precursor is a major source of Leu-enkephalin in brain.     

The opioid octapeptide Met5-enkephalin-Arg6-Gly7-Leu8: characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met5-enkephalin-Arg6-Gly7-Leu8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met5-enkephalin-Arg6-Gly7-Leu8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immuno-reactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met5-enkephalin-Arg6-Gly7-Leu8 was further identified to be authentic Met5-enkephalin-Arg6-Gly7-Leu8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met5-enkephalin and Met5-enkephalin-Arg6-Phe7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Immunocytochemical analysis of [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive structures in the rat superior cervical ganglion

Indirect immunofluorescence and immunoelectron microscopy were employed to analyze the enkephalinergic systems in the rat superior cervical ganglion (SCG). These systems were identified using specific antiserum against [Met5]Enkephalin-Arg6-Gly7-Leu8 (ENK-8), a peptide which is derived only from proenkephalin A. Abundant ENK-8 like immunoreactive (ENK-8-LI) neurons and fibers were observed in the SCG, but their distribution patterns were heterogenous; ENK-8-LI neurons were localized preferentially in the caudal two-thirds of the SCG, while immunoreactive fibers were found to be distributed more densely in the rostral one-third than in the remaining part of the SCG. Most of the ENK-8-LI neurons were large and had ultrastructural features resembling those of principal cells, some were identified electron microscopically as small intensely fluorescent (SIF) cells. ENK-8-LI fibers were varicose in appearance and surrounded the perikarya of neurons. Since most of these fibers were not detected after experimental decentralization of the SCG and since ENK-8-LI terminals were seen to contain small lucent vesicles, most of the former were thought to be preganglionic fibers. Immunoreactive fibers mainly formed synaptic contacts with the dendrites of non-immunoreactive principal cells, but a small proportion of ENK-8-LI principal cells also received synaptic input from them. Occasionally, immunoreactive fibers formed synapses with the processes or the soma of both ENK-8-LI and non-immunoreactive SIF cells. On the basis of these findings, we conclude that: (1) preganglionic ENK-8-LI fibers terminate mainly on the principal cells, which are devoid of ENK-8-LI structures; (2) the majority of ENK-8-LI neurons are principal cells, while the remainder are SIF cells; (3) inputs to these cells mainly involve structures lacking ENK-8 immunoreactivity; and (4) there are, however, a small number of ENK-8-LI preganglionic fibers which terminate on ENK-8-LI principal cells and SIF cells.     

Electron microscopic examination of the catecholaminergic innervation of neurophysin- or vasopressin-containing neurons in the rat hypothalamus

The ultrastructural relationships between neurophysin (NP)- or vasopressin (VP)-containing neurons and catecholamine terminals in the paraventricular nucleus and supraoptic nucleus of the rat were observed by means of a technique combining immunocytochemistry using NP I/II or VP antiserum with autoradiography after [3H]noradrenaline (NA) injection or 5-hydroxydopamine (5-OHDA) uptake. NP- or VP-like immunoreactive nerve cell bodies and fibers received synaptic contacts from a large number of immunonegative axon terminals. The presynaptic elements that innervate the neurosecretory neurons were studied. Axon terminals labeled with [3H]NA or 5-OHDA made synaptic contacts with NP- or VP-like immunoreactive nerve cell bodies and fibers. Furthermore, axodendritic and/or axo/axonic and axosomatic synapses occurred between the same NP- or VP-like immunoreactive neurons. These findings suggest that at least NA- and 5-OHDA-containing neurons play some important role in the control of neurosecretion in the NP- or VP-producing neurons of the rat hypothalamus and that the axon collaterals of NP- and VP-containing neurons make synaptic contacts with the same kind of neurons to form a recurrent collateral circuit.     

Immunocytochemical survey of haloperidol-induced immunoreactive changes of [Met]enkephalin-Arg6-Gly7-Leu8 in the rat forebrain.

It has already been demonstrated that chronic treatment with the dopamine receptor blocker, haloperidol, results in an increase of proenkephalin-A-derived peptides in the caudate-putamen (CP). To examine this phenomenon at the cellular level, we used immunocytochemistry to investigate the effects of haloperidol on [Met]enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity in the rat forebrain. After daily haloperidol (5 mg/kg, IP, for 6 days) or haloperidol decanoate (70 mg/kg, IM, given once or twice) treatment, immunoreactive neurons appeared diffusely in the whole CP and in the core part of the nucleus accumbens (Acb) and less frequently in the outer shell part of the Acb and the cell-dense layer of the tuberculum olfactorium (TuO). Increase of MEAGL-immunoreactive fibers in the CP, Acb, and TuO was also detected after these treatments, a particularly prominent increase being found in the striopallidal terminals in the globus pallidus and ventral pallidum. Haloperidol or haloperidol decanoate had no effect on MEAGL immunoreactivity in the cerebral cortex, amygdala, or hypothalamus. Reserpine treatment (5 mg/kg, IP, for 6 days) caused similar effects on the dorsal and ventral striopallidal system, and the direct injection of 6-hydroxydopamine (10 micrograms/5 microliters) into the CP led to the appearance of MEAGL-immunoreactive neurons in accordance with the depleted dopaminergic terminal area. These findings suggest that haloperidol influences enkephalinergic neurons region specifically and that in the dorsal and ventral striopallidal enkephalinergic system haloperidol increases MEAGL immunoreactivity in cell bodies, fibers, and terminals by blocking intrastriatal dopaminergic neurotransmission.     

Cholecystokinin-8-like immunoreactivity in the rat anterior commissure revealed by immunocytochemistry

The present experimental immunohistochemical study demonstrated that one of the major components of the anterior commissure of the rat is the cholecystokinin-8-like immunoreactivity (CCKI)-containing fiber system. We further showed that these fibers reached the contralateral side and dissociated into CCKI fibers in the ipsi- and contralateral anterior limbs.     

Intragranular colocalization of CRF and Met-Enk-8 in nerve terminals in the rat median eminence

The ultrastructural localization of rat corticoliberin (rCRF) and methionine-enkephalin octapeptide (Met-Enk-8) in the external layer of the rat median eminence were examined by double-immunogold labeling with anti-rCRF and anti-Met-Enk-8 sera labeled with small-sized (Gs) and medium-sized (Gm) gold particles, respectively. Two types of immunolabeled terminals were distinguished: one type with small granules (70 nm) labeled with Gs, and the other with large granules (100 nm) labeled with Gm. In both types, however, some granules were labeled with both Gs and Gm.     

Relationships between catecholaminergic neurons and cholecystokinin-containing neurons in the caudal part of the dorsomedial medulla oblongata of the rat: light and electron microscopic observations by the ‘mirror technique’

The ‘mirror technique’ was used in immuno-electron microscopy to examine the synaptic relationships between neuronal structures containing catecholamines and cholecystokinin (CCK) in the caudal part of the dorsomedial medulla oblongata in the rat, using antisera against tyrosine hydroxylase and CCK. CCK-immunoreactive axon terminals made synaptic contacts with catecholaminergic neurons. Some of these catecholaminergic neurons contained CCK.     

Neuropeptide Y innervation of the hippocampal region in the rat and monkey brain

Using antibodies to neuropeptide Y (NPY) in combination with immunohistochemical techniques we have studied the distribution of cell bodies and nerve terminals containing NPY immunoreactivity (-i) in the hippocampal region of rats and monkeys (cynomolgus). In colchicine-pretreated rats a large number of NPY-positive cells are present in all areas of the hippocampal region. The NPY-i cells range in size from small (diameter across soma: 10-15 micron) to large (approximately 20 micron). Most of the NPY-i cells are situated in the hilus, in the subgranular zone of the area dentata, and in the stratum oriens of Ammon's horn. A majority of these are polymorphic cells but cells of different morphology are present in these layers as well. These include small spheroid cells and dentate pyramidal basket cells that are distinct from the polymorphic cells in the subgranular zone. The subicular complex (e.g., the subiculum, pre-, and parasubiculum) and the entorhinal area contain fewer NPY-i cells than the rest of the hippocampal region. In the dorsal parts of the pre- and parasubiculum numerous small cells are scattered throughout all layers, while in the entorhinal area the NPY-stained cells are situated primarily in the deep layers (V and VI). In the ventral part of the lateral entorhinal area large multipolar and bitufted cells are found in layers II-VI. In the untreated monkey brain NPY-positive cells are found in the hilus of the area dentata and in the deep (IV through VI) layers of both the medial and lateral entorhinal area. Fewer NPY-stained cells are present in the subicular complex and in the entorhinal area. In the monkey as well as in the rat, NPY-stained cells are present in the angular bundle and in the alveus. A dense network of NPY-i fibers innervates the entire hippocampal region in both the rat and the monkey. The hippocampal NPY-i preterminal processes are present primarily in stratum moleculare of Ammon's horn and in the outer one-third of this layer in the area dentata. The NPY-positive innervation of the dentate molecular layer is far more prominent in the monkey than in the rat brain. Numerous NPY-stained fibers are scattered in other areas as well. In all retrohippocampal structures, and in particular the entorhinal area, the NPY-i fibers form a massive network that innervates all layers to about the same extent, with the exception of the molecular layer, which is more densely innervated than the other layers.     

GABAergic neurons containing CCK-8-like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus

The coexistence of cholecystokinin-octapeptide-like (CCK-L) and/or vasoactive-intestinal-polypeptide-like immunoreactive (VIP-LI) materials and glutamate decarboxylase (GAD) was studied in the rat hippocampus and dentate gyrus by means of immunohistochemistry. Consecutive 40-micron-thick sections were incubated in different antisera and those cells which were bisected by the plane of sectioning so as to be included at the paired surfaces of two adjacent sections were identified. The coexistence of the immunoreactivities for these peptides and GAD in the same cell could thus be determined by observing the immunoreactivity of the two halves of the cell, incubated in two different antisera. Almost all of the CCK-LI neurons were also GAD immunoreactive, whereas only about 10% of the GAD-immunoreactive neurons were CCK-LI. The percentages of GAD-immunoreactive neurons which were also immunoreactive for CCK were dependent on the laminar area in which they were found: i.e., 15-20% in the stratum radiatum of the hippocampus, about 10% in the stratum pyramidale, and about 6% in the stratum oriens. In contrast to the CCK-LI neurons, only about 40% of the VIP-LI neurons were identified to be also GAD immunoreactive, which might correspond to only part of the GAD-immunoreactive neurons. Furthermore the coexistence of VIP-LI and CCK-LI materials was recognized in about 10% of the CCK-LI neurons or about 35% of the VIP-LI neurons, indicating that some GABAergic neurons (presumably about 1%) in the rat hippocampus and dentate gyrus may contain both CCK-LI and VIP-LI materials.