Dopamine Beta-hydroxylase Immunoreactivity Research Articles

Noradrenergic system of the zebra finch brain: Immunocytochemical study of dopamine-?-hydroxylase

Oscine birds are among the few animal groups that have vocal learning, and their brains contain a specialized system for song learning and production. We describe here the immunocytochemical distribution of dopamine-beta-hydroxylase (DBH), a noradrenergic marker, in the brain of an oscine, the zebra finch (Taeniopygia guttata). DBH-positive cells were seen in the locus coeruleus, the nucleus subcoeruleus ventralis, the nucleus of the solitary tract, and the caudolateral medulla. Immunoreactive fibers and varicosities had a much wider brain distribution. They were particularly abundant in the hippocampus, septum, hypothalamus, area ventralis of Tsai, and substantia nigra, where they formed dense pericellular arrangements. Significant immunoreactivity was observed in auditory nuclei, including the nucleus mesencephalicus lateralis pars dorsalis, the thalamic nucleus ovoidalis, field L, the shelf of the high vocal center (HVC), and the cup of the nucleus robustus archistriatalis (RA), as well as in song control nuclei, including the HVC, RA, the lateral magnocellular nucleus of the anterior neostriatum, and the dorsomedial nucleus (DM) of the intercollicular complex. Except for the DM, DBH immunoreactivity within song nuclei was comparable to that of surrounding tissues. Conspicuously negative were the lobus paraolfactorius, including song nucleus area X, and the paleostriatum. Our results are in agreement with previous studies of the noradrenergic system performed in nonoscines. More importantly, they provide direct evidence for a noradrenergic innervation of auditory and song control nuclei involved in song perception and production, supporting the notion that noradrenaline is involved in vocal communication and learning in oscines.

Distribution of dopamine beta-hydroxylase-like immunoreactive fibers within the shell subregion of the nucleus accumbens.

The nucleus accumbens (Acb) can be divided into distinct subfields, delineated on the basis of histochemical markers as well as by afferent and efferent projection patterns. The shell subregion has reciprocal relationships with a variety of limbic areas and brainstem autonomic structures, and has been suggested to participate in motivation-related processes, including reward, stress, and arousal. The locus coeruleus (LC)-noradrenergic system has similarly been implicated in the modulation of behavioral state and stress-related processes, and previous studies have demonstrated reciprocal projections between the locus coeruleus and Acb shell. To better understand the anatomical substrate through which LC could influence activity within Acb shell, immunohistochemical methods were used to visualize the extent and the distribution of noradrenergic axons within this structure. Coronal sections of rat brain were processed to visualize immunoreactivity for the norepinephrine synthetic enzyme dopamine beta-hydroxylase (DBH), a specific marker for noradrenergic processes. In some cases, alternate sections were processed for immunohistochemical localization of substance P, in order to delineate core, shell, and pallidal compartments. Moderate-to-dense DBH-like immunoreactivity (DBHir) was found in approximately the caudal half of the shell subregion, particularly in caudalmost (septal pole) and ventral zones. The innervation of the septal pole was contiguous with a dense innervation of the bed nucleus of the stria terminalis. Few immunoreactive fibers were observed in the caudate-putamen, Acb core, or rostral Acb shell. Many DBHir fibers within the shell region were highly arborized with numerous varicosities, features indicative of terminal fields. These observations suggest noradrenergic systems might modulate certain processes associated with stress, behavioral state, or reinforcement via actions within the Acb shell.

Co-expression of GAP-43 with dopamine-beta-hydroxylase in adult rat spinal cord.

Dual colour immunofluorescence was used to compare the distribution of dopamine-beta-hydroxylase (DBH) and GAP-43 in the spinal cord of the adult rat. GAP-43 immunostaining was observed in all spinal cord regions containing DBH immunoreactivity. DBH and GAP-43 double-labelled fibres and varicosities were present in the lateral and ventral funiculi of the white matter and in the ventral horn where they were most prominent around motoneurones. In the dorsal horn and around the central canal, GAP-43 immunoreactivity was too abundant to determine whether DBH immunoreactive fibres were double labelled. We conclude that the noradrenergic system is one of a small number of spinal cord systems that express high basal levels of GAP-43 in the adult.

Sprouting of central noradrenergic fibers in the dentate gyrus following combined lesions of its entorhinal and septal afferents

Virtually all of the afferents to the hippocampal formation undergo collateral sprouting after removal of adjacent afferent systems. However, the central noradrenergic (NA) afferents, which demonstrate a remarkable propensity for regeneration and sprouting in other regions of the brain, have not been found to sprout in the denervated hippocampal formation. The present study was designed to determine if the pattern of innervation by NA fibers in the dentate gyrus of adult rats can be altered by interruption of the other major afferents. The innervation pattern of NA fibers was examined in the dentate gyrus 4 weeks after removal of the ipsilateral and/or contralateral entorhinal afferents and/or transection of the fimbria-fornix and supracallosal stria. The noradrenergic identity of the fibers was indicated by immunoreactivity for dopamine beta hydroxylase (DBH) and peripheral sympathetic fibers were demonstrated by immunoreactivity for nerve growth factor receptor (NGFr), which did not stain cholinergic fibers in this application. In control brains, the noradrenergic innervation of the dentate molecular layer was light and uniform across the width of the layer. Transection of the perforant path (ipsilateral entorhinal afferents) or ventral hippocampal commissure (contralateral entorhinal afferents) resulted in a significant increase in innervation density in the outer half of the molecular layer, and the combination of these two lesions produced the greatest increase. In those brains with transection of the ipsilateral and contralateral entorhinal afferents, the denervated dentate gyrus had a nearly twofold increase in density of DBH-immunoreactive fibers within the outer half of the molecular layer. These fibers tended to course parallel to the pial surface rather that perpendicular as in control sections. Transection of the fimbria-fornix alone had no affect on the innervation pattern of DBH-ir fibers in the molecular layer. When the fimbria-fornix was transected in combination with both of the other lesions, an overall increase in innervation density occurred, but there was no further increase in the difference between the inner and outer halves of the molecular layer. No NGFr-immunoreactive fibers were observed in the molecular layer in any of the brains, indicating that the DBH-immunoreactive fibers in this region were not of peripheral origin. It is concluded that removal of the ipsi- and contralateral entorhinal afferents to the dentate gyrus results in the sprouting of central NA fibers in the outer half of the molecular layer.

Immunohistochemical and histochemical evidence for the presence of noradrenaline, serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body.

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.

Immunocytochemical study of tyrosine hydroxylase and dopamine beta-hydroxylase immunoreactivities in the rat pancreas.

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory activity of the acinar cells, duct cells and endocrine cells.

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes, neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland.

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intra-adrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Uptake of 5-hydroxydopamine into non-sympathetic nerves of guinea-pig uterine artery in late pregnancy.

Perivascular nerve fibres of the uterine artery of virgin and late pregnant guinea-pigs were examined under the electron microscope following loading with 5-hydroxydopamine, a marker for catecholamine uptake, and immunohistochemistry for dopamine beta hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Varicosities, loaded with 5-hydroxydopamine labelled vesicles, and immunoreactive axons were counted in whole transverse sections of uterine arteries. Localization of the immunoreactivities in 5-hydroxydopamine-labelled vesicles was also studied. Colocalization of substance P and dopamine beta hydroxylase immunoreactivities was investigated at the light microscopic level. Both total and relative number of varicosities with 5-hydroxydopamine-labelled vesicles in a whole section of the artery increased in late pregnancy (61.2 +/- 10.2 versus 24.5 +/- 3.2 in virgin, representing 35% and 27% respectively, of all varicosities). Also the number of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons increased, but their relative proportion remained unchanged. In virgin guinea-pigs only calcitonin gene-related peptide and neuropeptide Y immunoreactivities were associated with varicosities loaded with small dense-cored vesicles, while in late pregnancy 5-hydroxydopamine-labelled vesicles were also seen in a number of vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons. Double immunolabelling for dopamine beta hydroxylase and substance P immunoreactivity showed that substance P immunoreactivity was not present in dopamine beta hydroxylase-immunoreactive axons of the uterine artery, of neither virgin nor late pregnant guinea-pigs. It is concluded that vascular hypertrophy of the uterine artery in late pregnancy is associated with an increase in the number of perivascular nerve fibres, that involves many, if not all of the subpopulations of neurons supplying the uterine artery. Also 5-hydroxydopamine-labelled varicosities were increased, but the results of the present study indicate that some of the nerve fibres that are able to take up 5-hydroxydopamine in late pregnancy are not sympathetic (i.e. are sensory and/or parasympathetic in origin). The relevance of these findings in pregnancy is discussed.

Galanin-like innervation of rat submandibular and sublingual salivary glands: origin and effect on acinar cell membranes.

The distribution and source of a galanin-like innervation of rat salivary glands has been examined. Additionally, submandibular and sublingual acinar cell membrane responses to galanin or a cholinergic agonist were studied. Galanin-immunoreactive fibers were observed throughout the submandibular and sublingual glands in association with ducts and acini. A subset of submandibular ganglion cells expresses galanin immunoreactivity. Parasympathectomy resulted in a marked decrease in galanin immunoreactivity in the glands. Sympathectomy resulted in marked reduction of dopamine beta-hydroxylase immunoreactivity with no appreciable change in galanin immunoreactivity. Retrograde labeling experiments demonstrated that galanin-immunoreactive sensory neurons in the trigeminal ganglion do not innervate the submandibular or sublingual gland. These results indicate that the galanin-like innervation of rat salivary glands is derived from parasympathetic nerves to the glands. Since rat sublingual glands contain largely mucous acini while rat submandibular gland acini are seromucous, electrophysiological responses to galanin and the muscarinic agonist, bethanechol, were compared. Agonist-induced voltage shifts varied between the two glands. The galanin-induced response at the level of the resting membrane potential in submandibular acinar cells was a hyperpolarization, while that in sublingual acinar cells was a depolarization. There was also a greater voltage dependence to the galanin-induced submandibular response than to the sublingual response. Differences were also noted in the acinar cell response to cholinergic stimulation between these glands. These results demonstrate the existence of a galanin-like innervation to salivary glands that may be functionally relevant. Moreover, the results challenge the idea that agonist-induced membrane responses are similar among acinar cells of different glands.

Soluble and membrane-bound forms of dopamine beta-hydroxylase are encoded by the same mRNA.

A full length cDNA clone for bovine dopamine beta-hydroxylase was expressed in rat pheochromocytoma PC12 cells by stable transformation of this cell line with a plasmid expression vector. The recombinant protein exhibited dopamine beta-hydroxylase enzyme activity and was found in both the soluble and membrane fractions of the secretory vesicle. Immunoprecipitation of cell extracts from recombinant cell lines with dopamine beta-hydroxylase antisera followed by fractionation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two subunits, which migrated to relative molecular masses of 76 and 78 kDa. The recombinant protein co-fractionated with neurotransmitter when subcellular structures were separated by sucrose gradient density centrifugation, suggesting that the protein was routed to the secretory vesicles. Dopamine beta-hydroxylase immunoreactivity in those sucrose gradient fractions presumed to contain secretory vesicles was resistant to treatment with trypsin unless the nonionic detergent Triton X-100 was also present to disrupt membrane structure. The 76- and 78-kDa isoform were each found in both the membrane and soluble fractions of the secretory vesicle. Treatment of cultured cells with nerve growth factor or 8-(4-chlorophenylthio)-cyclic AMP alters the relative distribution of the subunits such that the 76-kDa form predominates. The subcellular distribution of a dopamine beta-hydroxylase cDNA clone lacking the first 16 nucleotide residues was also determined. The predicted amino acid sequence of the protein encoded by this cDNA would be deleted of the first 13 residues of the signal sequence, which were reported to be present in the membrane-bound form, but not the soluble form, of native dopamine beta-hydroxylase (Taljanidisz, J., Stewart, L., Smith, A. J., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061). Immunoprecipitable dopamine beta-hydroxylase derived from expression of the deleted cDNA was found in both the membrane-bound and soluble fractions of the secretory vesicle. These experiments demonstrate that the membrane-bound and soluble forms of dopamine beta-hydroxylase are derived from one primary translation product, which is also sufficient to produce enzyme activity. In addition, the amino-terminal amino acids encoding residues 1-13, which compose the hydrophilic region of the signal sequence, are not necessary for the biogenesis of membrane-bound dopamine beta-hydroxylase.

Production and immunocytochemical application of a highly sensitive and specific monoclonal antibody against rat dopamine-?-hydroxylase

A highly sensitive and specific monoclonal antibody against the enzyme dopamine beta-hydroxylase (DBH) from rat was produced and coded DBH 41. The generated hybridoma secreted immunoglobulins of mouse IgG1 subtype, as determined by radial immunodiffusion. This antibody, characterized by immunoblotting against a crude rat DBH preparation, was found to specifically recognize two bands of molecular weight 70 and 75 kDa corresponding to the soluble and membrane bound forms of the enzyme, respectively. With regard to species specificity, the anti-DBH antibody recognizes only the rat DBH molecule as it exhibits no cross-reactivity with either mouse, human, rabbit, guinea pig, cat or bovine DBH. Comparative immunocytochemical localization of DBH and TOH immunoreactivity was performed in different brain regions and we found that the DBH 41 antibody specifically stained DBH-containing neurons and fibers in the rat central nervous system (CNS). The high sensitivity of the DBH 41 antibody permitted us to detect immunologically the presence of the enzyme even in areas where only scattered DBH-containing fibers were present.

Clinical, Autonomic and Therapeutic Observations in Two Siblings With Postural Hypotension and Sympathetic Failure due to an Inability to Synthesize Noradrenaline From Dopamine Because of a Deficiency of Dopamine Beta Hydroxylase

A brother and sister with long-standing symptoms of postural hypotension are described. They were considerably worse in the morning, after exercise and in warm weather. In the male, erection was unaffected but ejaculation was prolonged or absent. Both had nocturia, but there were no urinary bladder, bowel or sweating abnormalities. Autonomic function tests confirmed sympathetic adrenergic failure with spared sympathetic cholinergic and intact parasympathetic function. There were no other neurological abnormalities. Noradrenaline and adrenaline were undetectable in the plasma, but plasma dopamine was elevated. Urinary levels of noradrenaline and adrenaline metabolites were below detection limits, but dopamine metabolites were normal or elevated. Dopamine beta-hydroxylase activity was undetectable in the plasma. Immunohistochemical studies of perivascular cutaneous tissue confirmed normal peptidergic and tyrosine hydroxylase immunoreactivity, with absent dopamine beta-hydroxylase immunoreactivity. The findings were consistent with an enzymatic deficit in the conversion of dopamine to noradrenaline. The parents were clinically and biochemically normal. Treatment of both patients with the synthetic amino acid, d-l-threo-dihydroxyphenylserine, which contains a hydroxyl group and is converted to noradrenaline by dopa-decarboxylase, reduced symptoms and signs of postural hypotension and increased levels of plasma noradrenaline and its urinary metabolites. In the male, ejaculation became possible. Behavioural changes included a feeling of confidence and optimism, with a tendency to be argumentative. The laevo isomer also raised blood pressure and plasma noradrenaline levels. The drug had no direct pressor effects, as its actions were prevented by the dopa-decarboxylase inhibitor, carbidopa.

Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus.

Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine-beta-hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF

In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C6 glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C6 glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64-82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histo-fluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.

Catecholamine innervation of the rat hypoglossal nucleus

The catecholamine innervation of the hypoglossal nucleus (XII) was investigated immunocytochemically by comparing the distribution patterns of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the rat. Numerous TH- and DBH-positive profiles were found throughout XII, while only occasional PNMT immunoreactivity was observed. Significantly, the distribution patterns of TH and DBH immunoreactivity were coextensive with the most intense staining found ventromedially along the caudal half of XII. We conclude, therefore, that the catecholamine innervation of XII is largely noradrenergic, and that motoneurons innervating the genioglossi muscles, the principal protrusors of the tongue, are the primary targets of this input.

Distributions of tyrosine hydroxylase-, dopamine-beta-hydroxylase-, and phenylethanolamine-N-methyltransferase-immunoreactive neurons in the brain of the hamster (Mesocricetus auratus).

Antibodies to the catecholamine synthetic enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) were used in an immunohistochemical analysis of the brain of the golden hamster. The distributions and morphological characteristics of neurons displaying immunoreactivity to these enzymes were examined in sets of adjacent sections. Various novel groups of TH-immunoreactive neurons were found. A distinct feature observed in the hamster brain was the presence of a population of magnocellular multipolar neurons in the basal forebrain which displayed intense TH immunoreactivity. These cells were found predominantly in the vertical and horizontal limbs of the nucleus of the diagonal band of Broca and in the lateral preoptic area. Many small TH-positive cells were also found scattered in the deeper layers of the cortex in the hamster. The pericentral divisions of the inferior colliculus contained a large number of TH-immunoreactive neurons, and a few small bipolar cells in the lateral superior olive were also stained. A major cell group was found in the lateral parabrachial nucleus at the level of the locus ceruleus that displayed TH but not DBH immunoreactivity and was obviously separate from the TH- and DBH-positive cells of the locus ceruleus. Additional TH-positive cell groups were found along the seventh nerve, within the medial longitudinal fasiculus, in the nucleus raphe pallidus, and in the pars caudalis of the spinal trigeminal nucleus. The various catecholamine cell groups described by many people in the rat by use of histochemical and immunohistochemical techniques were also present in the hamster brain. These included the noradrenergic, TH- and DBH-immunoreactive cell groups of the pons and medulla. The hamster also displayed groups of medullary neurons displaying immunoreactivity to TH, DBH, and PNMT. These appeared similar in distribution and morphology to the adrenaline cell groups described in the rat. TH-immunoreactive cell groups in the olfactory bulb, hypothalamus, substantia nigra, and ventral tegmental area of the hamster appeared to correspond to the dopaminergic cells groups described in the rat and other species. In addition, as in the rat and cat, numerous TH-positive cells were found in the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, and the area postrema. These observations suggest that catechols may be present in neurons in the cortex, basal forebrain, auditory brainstem, and the parabrachial nucleus of the hamster. These studies also emphasize the need for caution in making generalizations regarding transmitter distributions across species.

Immunoreactive dopamine beta-hydroxylase in neuronal groups in the goldfish brain.

Dopamine beta-hydroxylase immunoreactivity is visualized by immunocytochemical techniques for the first time in brainstem neurons of a teleost, the goldfish, Carassius auratus. Three anatomically separate populations of immunoreactive cells are elucidated: one group of moderately stained cells is identified in the isthmal tegmentum; a second group forms a continuous column of densely stained cells, lateral or dorsolateral to the medial longitudinal fasciculus from the midmedulla to the medullospinal transition zone, and a third group of densely stained cells is located dorsomedially in the postobecular region of the medulla. The location of these dopamine-beta-hydroxylase-immunoreactive neurons is similar to that in mammalian brains, i.e. in the locus coeruleus, lateral medullary tegmentum and area postrema. The presence of norepinephrine-synthesizing neurons in the above-listed brainstem regions of the goldfish provides further evidence for homologizing these nuclei to the same-named regions in mammalian brains.

The distribution of tyrosine hydroxylase-immunoreactive fibers in primate neocortex is widespread but regionally specific

An antiserum directed against tyrosine hydroxylase (TH), an enzyme involved in dopamine and norepinephrine synthesis, was used to visualize axons immunohistochemically in monkey neocortex. Labeled fibers were distributed throughout the entire neocortex, but they had striking patterns of regional and laminar specialization. For example, primary motor cortex contained the greatest density of TH-labeled fibers, whereas primary sensory regions were sparsely innervated. Marked heterogeneity of fiber density was also present among the association regions of the frontal, parietal, and temporal lobes. In addition, the laminar pattern of innervation in a given region was correlated with its fiber density. Sparsely innervated regions had labeled fibers only in layer I and sometimes layer VI. In regions of intermediate density, labeled fibers tended to be located in layers I-superficial III and layers V-VI, whereas in densely innervated motor cortex TH-immunoreactive fibers were present in all cortical layers. Comparison of these distribution patterns with those produced by an antiserum directed against dopamine-beta-hydroxylase (DBH), a specific marker of neocortical noradrenergic axons, revealed marked differences. DBH-immunoreactive fibers were observed in some cortical locations where few or no TH-labeled fibers were present. In other regions, the density of TH-immunoreactive processes far exceeded that of DBH-labeled fibers. These findings indicate that nearly all of the immunoreactive fibers revealed by this anti-TH antiserum are dopaminergic. This interpretation was further supported by lesions of the ascending noradrenergic fibers in the brain stem, which reduced DBH immunoreactivity, but not TH immunoreactivity, in neocortex. The distinctive innervation patterns of TH-immunoreactive fibers suggest a functional specialization of the dopaminergic projections to primate neocortex.

Neuropeptide Y localization in the rat amygdaloid complex

Neuropeptide Y (NPY)-, avian pancreatic polypeptide (APP)-, and molluscan cardioexcitatory peptide (FMRF)-like immunoreactivity in the amygdaloid complex of the rat was investigated immunohistochemically. The distribution of each of these peptides within the amygdala is identical and cross-blocking studies indicate that all three antisera recognize the NPY antigen. Morphologically distinct populations of NPY immunoreactive neurons are differentially distributed in the medial amygdaloid nucleus and at the base of the stria terminalis. Dense plexuses of immunoreactive axons are present in the medial third of the central nucleus and in the dorsal half of the medial nucleus, with light to moderate fiber plexuses present in the lateral and basolateral nuclei and scattered axons present throughout the remainder of the amygdala. The distribution and appearance of NPY immunoreactive plexuses in the amygdala is similar to that described previously for noradrenergic axons arising from brainstem cell groups (Fallon, Koziell, and Moore: J. Comp. Neurol. 180:509-532, '78). However, injections of the noradrenergic neurotoxin 6-hydroxydopamine into the amygdala result in a complete loss of dopamine-beta-hydroxylase (DBH) immunoreactivity in the amygdala and surrounding cortex but leave much of the NPY immunoreactive plexus intact. Similarly, lesions of the locus coeruleus deplete DBH immunoreactivity, leaving NPY-like immunoreactivity in the amygdala unaffected. These results indicate that much of the NPY immunoreactive plexus observed in the amygdala does not arise from brainstem sources in which NPY and noradrenaline are colocalized. Lesions of the stria terminalis or medial nucleus have no observable effect on the density or distribution of NPY immunoreactive terminal fields in the basal forebrain and hypothalamus, suggesting that immunoreactive neurons in the amygdaloid complex do not contribute significantly to this innervation.

Postnatal development of dopamine-β-hydroxylase-immunoreactive fibers of the spinal cord of the rat

The development of dopamine-beta-hydroxylase (DBH)-immunoreactive fibers of the rat spinal cord was studied in the cervical, thoracic and lumbar regions at birth, 6, 14, 30 and 90 days of age. The adult pattern of distribution of DBH-immunoreactive fibers is similar to that of catecholaminergic histofluorescence. Our results suggest that DBH immunoreactivity is more sensitive than glyoxylic acid methods in early development, since the immunocytochemical method demonstrates stained fibers at an earlier age than the histofluorescence method. At birth noradrenergic innervation was already established; DBH immunoreactivity was notable in the ventral horn of the lumbar region and in the zona intermedia (ZI); the new finding was that not only the intermediolateral cell column but also the dorsal autonomic nucleus and the transverse connections between them were distinctly stained and were periodically organized. With increasing age, the general rule is that the intensity of the staining increased simultaneously in all regions. In the gray matter, networks of stained fibers became more complex. In contrast to earlier workers, we found no evidence for 'selective destruction' of noradrenergic fibers within the ZI in the postnatal period. The exceptions to the rule are DBH-immunoreactive fibers in the ventral white matter; these fibers increased in number up to day 14 and appeared to decrease thereafter; they were most numerous in the cervical region and diminished in number caudally; these fibers were diffuse in the cervical region but as they coursed caudally they became concentrated in a more ventral marginal location.