Ultrastructural localization of neurotensin-like immunoreactivity within dense core vesicles in perikarya, but not terminals, colocalizing tyrosine hydroxylase in the rat ventral tegmental area.

Within the rat ventral tegmental area (VTA), the parabrachial pigmentosus and paranigral subdivisions are known to differ in their functional responses to injected neurotensin. These subdivisions also vary in their connections with other brain regions and in their number of neurotensin-containing perikarya as seen by light microscopy. In both subdivisions, there may be intracellular as well as synaptic relations between dopamine and neurotensin. Dopaminergic neurons are known to be physiologically activated by neurotensin (NT) and may also contain this peptide. To characterize further the cellular relationships in each subdivision, we examined the ultrastructural immunocytochemical localization of a rat antiserum against NT and a rabbit antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections. The NT antiserum was raised against the entire peptide sequence. Immunoblots showed that the antiserum recognized the original antigen as well as the related peptides neuromedin N and lysine 8- arginine 9- neurotensin 10-13 (LANT-6). In both the parabrachial pigmentosus and paranigral subdivisions, neurotensin-like immunoreactivity (NTLI) was localized predominantly in the large (80-100 nm) dense core vesicles using the peroxidase anti-peroxidase (PAP) method. In tissue labeled for NT by the PAP method and for TH by immunoautoradiography, serial section analysis revealed that all perikarya containing NTLI (n = 19) were also TH-positive. Three times as many perikarya colocalized NTLI and TH in the parabrachial pigmentosus subdivision (n = 15) as in the paranigral subdivision (n = 4). Occasionally, a perikaryon containing TH and NTLI could be found in direct apposition to a TH-labeled perikaryon without glial separation. In contrast to perikarya and dendrites, terminals showing NTLI (38 in parabrachial pigmentosus, 29 in paranigral) lacked detectable TH labeling. Of the terminals containing NTLI whose synaptic junctions could be identified, 48% were symmetric and 10% were asymmetric. The targets of these terminals included perikarya and dendrites lacking detectable immunoreactivity (69% in parabrachial pigmentosus, 55% in paranigral), immunolabeled for TH (26% in parabrachial pigmentosus, 38% in paranigral) or containing both NTLI and TH (5% in parabrachial pigmentosus, 7% in paranigral). Single terminals containing NTLI sometimes contacted more than one neuronal target, some of which were apposed to each other without glial separation. TH-labeled terminals synapsed onto double-labeled perikarya in the paranigral subdivision, but were not observed to do so in the parabrachial pigmentosus subdivision.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunohistochemical localization of substance P and enkephalin in the nucleus tractus solitarii of the rhesus monkey, Macaca mulatta

The nucleus tractus solitarii in the monkey Macaca mulatta was found to have several subdivisions based upon cytoarchitectonics and immunohistochemistry. Subdivisions that could be identified included commissural, medial, parvicellular, dorsolateral, ventrolateral, intermediate, and interstitial. Substance P and enkephalin immunoreactivity was localized within discrete regions of the nucleus tractus solitarii, by means of the peroxidase-antiperoxidase technique. Substance P immunoreactivity occurred most frequently in the interstitial subdivision of the nucleus tractus solitarii. Moderate accumulations of substance P immunoreactivity were present in the commissural, medial, parvicellular, dorsolateral, and intermediate subdivisions, but very little was present in the ventrolateral subdivision. Enkephalin immunoreactivity followed the staining patterns of substance P; however, the amounts of enkephalin immunoreactivity were less than amounts for substance P. Following colchicine treatment, large numbers of enkephalin-immunoreactive neurons were distributed throughout all subdivisions, many being located in the parvicellular and medial subdivisions. The few substance P-immunoreactive neurons found were restricted to the parvicellular subdivision. The distribution of substance P and enkephalin immunoreactivity in M. mulatta is very similar to that described in the cat and rat. In addition, the extensive overlap of the distribution of these two putative neurotransmitters provides morphological evidence for their possible participation in the autonomic regulation within the nucleus tractus solitarii.     

Distribution of cholecystokinin-like immunoreactivity in the rat main olfactory bulb

The anatomical localization of cholecystokinin-like immunoreactivity (CCK-I) within the rat main olfactory bulb was analyzed by using the peroxidase-antiperoxidase immunocytochemical technique. Neurons or neuronal processes containing CCK-I were localized within all laminae of the olfactory bulb except the olfactory nerve fiber layer. A large population of CCK-I neurons, with morphology, size, and distribution corresponding to that of the middle and external tufted cells, was observed within a zone extending from the deep periglomerular region through the superficial one-half to one-third of the external plexiform layer. A smaller number of immunoreactive perikarya were found in the deep external plexiform layer, the glomerular layer, and rarely within the inner plexiform layer. These CCK-I neurons appeared to correspond to internal tufted cells, periglomerular cells, and deep short-axon cells, respectively. Dense CCK-I staining of fibers and terminals was present within the internal plexiform layer and, less densely, within the neuropil of the granule cell layer. In addition, terminal-like CCK-I was localized within layer 1A of the anterior olfactory nucleus, the olfactory tubercle, and the most rostral piriform cortex. This observation provides corroboration for the identification of the principal CCK-I neuron in the rat olfactory bulb as the centrally projecting middle tufted cell. The present results, demonstrating the localization of CCK-I to both local circuit and projection neurons of the olfactory bulb and to terminal-like puncta in the internal plexiform and granule cell layers, suggest that CCK may be significantly involved in olfactory processing at several levels.     

Substance P,neurotensin, enkephalin,and catecholamine-synthesizing enzymes: Light microscopic localizations compared with autoradiographic label in solitary efferents to the rat parabrachial region

The immunocytochemical localizations of substance P, neurotensin, enkephalin and the catecholamine-synthesizing enzymes tyrosine hydroxylase and dopamine-β-hydroxylase were examined in the rat parabrachial region. The immunoreactivity for each marker was compared with the distribution of superimposed autoradiographic labeling of parabrachial afferents after unilateral injection of ³H-amino acids into the caudal portion of the medial nucleus of the solitary tract (m-NTS). Substance-P- and neurotensinlike immunoreactivity (SPLI and NTLI, respectively) were localized primarily in varicose processes in the ventrolateral quadrant of the parabrachial region. The SPLI and NTLI were differentially localized with respect to each other; however, both peptides were detected in regions of the parabrachial containing dense autoradiographic label. In contrast, enkephalinlike immunoreactivity (ELI), tyrosine hydroxylase, and dopamine-β-hydroxylase were detected in processes and a few perikarya located outside the ventrolateral parabrachial region. The ELI was primarily in the dorsolateral, and the catecholamine-synthesizing enzymes were primarily in the medial parabrachial regions which contained sparse autoradiographic labeling of transported amino acids. We conclude that in the rat parabrachial region, SPLI and NTLI are contained within two distinct populations of afferents which may originate from perikarya in the caudal m-NTS, whereas ELI and the cate-cholamines are more likely to be found in other afferents or possibly in intrinsic neurons.     

Neurotensin terminals form synapses primarily with neurons lacking detectable tyrosine hydroxylase immunoreactivity in the rat substantia nigra and ventral tegmental area.

A light and electron microscopic double antigen localization technique was employed to examine the fine structural relationship between neurotensin-containing axon terminals and dopaminergic neurons in the substantia nigra and ventral tegmental area of the rat. At the light microscopic level, neurotensin-immunoreactive terminals were densely distributed throughout the substantia nigra pars compacta and ventral tegmental area in close proximity to tyrosine hydroxylase-immunoreactive somata and dendrites. On electron microscopic examination, direct synaptic connections were identified between neurotensin-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive perikarya and dendrites. However, only 8.2% and 8.8% of the neurotensin-immunoreactive axonal profiles detected in the substantia nigra and ventral tegmental area, respectively, were found in direct apposition with tyrosine hydroxylase-immunostained elements. In turn, only 9.3% and 10.0% of tyrosine hydroxylase immunoreactive dendrites sampled from the substantia nigra and ventral tegmental area, respectively, were seen in contact with neurotensin immunopositive axon terminals. However, neurotensin-immunoreactive and tyrosine hydroxylase-immunolabelled elements were frequently identified in close anatomical proximity (less than 5 microns) to one another. These results are interpreted in light of the selective association of neurotensin receptors with dopaminergic neurons in the substantia nigra and ventral tegmental area to suggest a predominantly parasynaptic mechanism of action for neurotensin in the ventral midbrain.     

Cholecystokinin-, enkephalin-, and substance P-like immunoreactivity in the dentate area,hippocampus, and subiculum of the domestic pig

The distribution of cholecystokinin-like, enkephalin-like, and substance P-like immunoreactivities is described in the dentate area, hippocampus, and subiculum of the domestic pig (Sus scrofa domesticus) as a baseline for future experimental studies. The distributions in the pig are compared with previous observations in other species. Cholecystokinin-like immunoreactive nerve cell bodies were intensely stained and present in large numbers in all subfields studied. Cholecystokinin-like immunoreactive terminals appeared as stained puncta, whereas fibers were only rarely encounterd. The puncta were mainly seen in the dentate molecular layer and dentate granule cell layer, the pyramidal cell layer of the hippocampal regio inferior, stratum moleculare of the hippocampal regio superior, and in the subiculum. Enkephalin-like immunoreactive nerve cell bodies were faintly stained and generally present in very small numbers, except for some pyramidal cells in the subicular cell layer. Enkephalin-like immunoreactive fibers were few in number, whereas stained puncta appeared with variable densities. Puncta of particularly high densities were found in the dentate molecular layer, whereas they appeared of moderate density in the dentate hilus, stratum moleculare of the hippocampal regio superior, and in the subiculum. Substance P-like immunoreactive nerve cell bodies were few and very faintly stined. They primarily occurred in the dentate hilus, stratum oriens of the hippocampus, and in the subicular cell layer. Stained fibers were few in number, whereas stained puncta were present in abundant numbers corresponding to the mossy fiber projection in the dentate hilus and the layer of mossy fibers of the hippocampal regio inferior, and in moderate numbers in stratum moleculare of the hippocampal regio superior and in the subiculum. For all three neuropeptides there were consistent and very characteristic variations in the distribution of immunoreactivity along the septotemporal axis of the hippocampus. When viewed in a comparative perspective the distribution of enkephalin-like and substance P-like terminals in the domestic pig displayed striking differences from the basic pattern observed in other species. This contrasted with the distribution of cholecystokinin-like neurons and terminals, which resembled more closely these species. © 1993 Wiley-Liss, Inc.     

Ultrastructural localization of tyrosine hydroxylase immunoreactivity in the rat diagonal band of Broca.

The present study sought to establish the cellular basis for the catecholaminergic (i.e., noradrenaline and dopamine) modulation of neurons in the horizontal limb of the diagonal band of Broca (HDB) in the rat brain. The light and electron microscopic localization of antigenic sites for a polyclonal antibody directed against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), were examined in the HDB using a double-bridged, peroxidase-antiperoxidase method. By light microscopy, numerous punctate, varicose processes with intense TH-immunoreactivity (TH-I) were detected in the HDB. Additionally, a few small, bipolar, or multipolar TH-immunoreactive neurons were observed. Ultrastructural analysis of single sections revealed that the TH-labeled processes were axons and axon terminals. Axons (n = 134) with TH-I were primarily unmyelinated. Terminals with TH-I (n = 169) were 0.3-1.4 microns in diameter and contained many small, clear vesicles and 0-5 larger dense-core vesicles. The types of associations (i.e., asymmetric synapses, symmetric synapses, and appositions which lacked a membrane specialization in the plane of section analyzed) formed by the TH-labeled terminals were quantitatively evaluated. The TH-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (134 out of 169), (2) were closely apposed without glial intervention to unlabeled and TH-labeled terminals (11 out of 169), or (3) had no neuronal associations in the plane of section analyzed (24 out of 169). The relatively rare (n = 4) associations with unlabeled perikarya were mostly characterized by symmetric synaptic specializations. The majority of the TH-labeled terminals were associated with the shafts of small dendrites (66% of 134). Moreover, most of the associations on dendrites and dendritic spines were further characterized by asymmetric synaptic specializations; however, many were also appositions without any apparent glial intervention in the plane of section analyzed. Additionally, the TH-labeled terminals were often associated with only one dendrite, which, in the same plane of section, was sparsely innervated by other terminals. Astrocytic processes usually surrounded the portions of the terminals and dendrites not involved in the region of association. The TH-immunoreactive perikarya were small (7-12 microns), ovoid, and had an indented nucleus with some heterochromatin. Their scant cytoplasm contained mitochondria, Golgi complexes, and endoplasmic reticulum. A few immunoreactive dendrites, presumably derived from the local neurons, were also detected. Both TH-immunoreactive perikarya and dendrites were associated primarily with unlabeled terminals, although a few terminals with TH-I also contacted them.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of GABA-like immunoreactivity in the rat amygdaloid complex

The distribution of GABA-like (GABA-Li) immunoreactivity in the rat amygdaloid complex was studied by using an anti-GABA antibody. GABA-Li positive neurons and processes were present in every nucleus of the complex. Three patterns of immunoreactivity were revealed: (1) the intercalated masses and the lateral olfactory tract nucleus exhibited the most intense staining of the neuropil, and virtually every neuron was labeled, (2) the central and medial nuclei contained intensely labeled neuropil and moderately labeled neurons, and (3) in the remaining nuclei, the neuropil was weakly labeled, and relatively numerous GABA-Li neurons were present. Our results suggest that: (1) the intercalated masses and lateral olfactory tract nucleus consist of large aggregates of GABA-Li immunoreactive neurons, and (2) the lateral, basal dorsal, and the posterior cortical nuclei may constitute a significant source of GABAergic connections to other amygdaloid nuclei, in particular to the medial and central nuclei.     

Distribution of substance P-like, leucine-enkephalin-like, and bombesine-like immunoreactivity and acetylcholinesterase activity in the visual system of salamanders

In the urodele species Salamandra salamandra and Batrachoseps attenuatus, the distribution of the neuropeptides substance P (SP), leucine-enkephalin (LENK), and bombesine (BOMB) was investigated by means of immunohistochemistry in brain areas containing retinofugal projection sites (tectum mesencephali, praetectum, thalamus) as well as in brain regions postsynaptic to the tectum. The activity of acetylcholinesterase (AChE) was studied histochemically. Despite its simplified, two-layered morphology, the salamander tectum shows a high degree of neurochemical differentiation, characterized by a laminar organization of neuropeptide-like immunoreactivity and AChE-activity comparable to that found in the anuran tectum, which has a multi-layered morphology. SP-like immunoreactivity constituted four tectal laminae, two of them occurring in the stratum opticum. LENK-like immunoreactivity formed three laminae, one in the stratum opticum. BOMB-like immunoreactivity formed one lamina within the stratum opticum and one in the tectal efferent layers. Layers 1 and 2 of the stratum opticum revealed high AChE-activity, whereas low activity was found in deep fiber layers containing tectal efferents. The outer cellular layer also revealed AChE-activity. After enucleation of one eye, the contralateral tectum lacked neuropeptide-like immunoreactivity and AChE-activity in the layers containing retinofugal projection sites. No reduction of immunoreactivity was found in nuclei postsynaptic to the tectum. Our experiments suggest that the secondary simplification that has taken place in salamanders with respect to tectal morphology did not affect the neurochemical differentiation of the tectum.     

Species differences in the distribution of substance P and tyrosine hydroxylase immunoreactivity in the olfactory bulb

These studies document species differences in the distribution of the peptide substance P and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) within a central nervous system region of a number of mammalian species including the mouse, rat, guinea pig, rabbit, cat, and two species of hamster (Chinese and Syrian). Substance P-containing neuronal perikarya were observed in the main olfactory bulb (MOB) of both species of the hamster, but not in the MOB of the other species examined. In the accessory olfactory bulb (AOB), however, neuronal staining was observed in all species except the mouse. The number of stained somata and their intensity varied such that label was most prominent in the rat followed in decreasing order by the rabbit, guinea pig, cat, and hamster. The mouse displayed no perikaryal staining. Stained somata in AOB were found in the internal granule cell layer with dendritic processes ramifying through the internal plexiform layer to arborize within the mitral cell layer. The distribution of substance P-stained neurons in the MOB also differed between the two hamster strains. In the Syrian hamster, neurons were primarily juxtaglomerular. In the Chinese hamster, labeled perikarya were found in both the juxtaglomerular region and within the superficial aspect of the external plexiform layer (EPL). The mean longest diameter of the majority of substance P-labeled neurons in both species was greater than 10 micron, suggesting that they were tufted cells. Those in the EPL of the Chinese hamster were the largest (17 micron). Species differences also were observed in the distribution of substance P-positive axons and terminals within the MOB. Label was distributed primarily in the internal granule cell layer of the Syrian hamster and the internal plexiform layer of the Chinese hamster. Tyrosine hydroxylase staining was similar among species with the exception of the Syrian hamster. In the latter species, an additional large population of neurons was found within the external plexiform layer. In all other species, TH-stained neurons were found scattered throughout the MOB and occasionally the AOB but were not numerous in the EPL. Although most TH neurons were larger than 10 microns, in all species a population of smaller TH cells was observed primarily in the glomerular layer, suggesting that most neurons labeled with TH are tufted cells but that some may be periglomerular cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electron microscopic immunocytochemical localization of substance P in the area postrema of rat

Electron microscopic immunocytochemistry was employed to examine the ultrastructure of processes containing substance P-like immunoreactivity (SP-LI) in the area postrema of rat. SP-LI was present within axons and axon terminals which formed synapses with dendrites of intrinsic neurons in the rat area postrema. Specialized contacts between labeled processes and perivascular or ependymal glia cells were not observed. These data suggested that substance P may act as a neurotransmitter involved primarily with neuronal (axodendritic) interactions within the area postrema.     

Compartmental organization of the ventral striatum of the rat: immunohistochemical distribution of enkephalin, substance P, dopamine, and calcium-binding protein

In the caudate-putamen of the rat a patch/matrix organization can be recognized on the basis of the immunohistochemical distribution of several markers, which include enkephalin, substance P, dopamine, and calcium-binding protein. In the present experiments the distributional relations of these markers were investigated in the nucleus accumbens. The distribution of enkephalin fibers shows different inhomogeneities according to their location in the nucleus. Rostrally, heavily labeled areas stand out against a moderately stained background, whereas caudally, in medial and ventral parts of the nucleus, lightly stained areas delineate regions in the moderately stained neuropil. In the distribution of substance P, areas with high staining intensity were observed in the medial and ventral parts of the nucleus accumbens. Inhomogeneities in the distribution of strong dopamine immunoreactivity consist of weakly immunoreactive areas throughout the rostrocaudal extent of the nucleus accumbens and extremely heavily labeled areas in the medial and ventral parts of the nucleus. Calcium-binding protein immunoreactivity can only be detected in dorsal parts of the nucleus. The generally intense immunostaining for calcium-binding protein is interspersed with "blanks" of weak immunoreactivity. The heavily and moderately labeled enkephalin areas each maintain specific relations with inhomogeneities in the distribution of substance P, dopamine, and calcium-binding protein. Rostrally, the heavily labeled enkephalin areas coincide with areas strongly immunostained for calcium-binding protein and with lightly stained areas in the dopamine and substance P immunoreactivity patterns. In the same region lightly stained areas in the enkephalin distribution match heavily labeled substance P areas. Caudally, in the border region of the nucleus accumbens and the caudate-putamen, the heavily labeled enkephalin areas are either related to "blanks" or to the intense staining regions in the calcium-binding protein immunoreactivity distribution. The moderately labeled enkephalin areas caudomedially in the nucleus accumbens are in register with the heavily labeled regions in the distribution of substance P and with the extremely heavily labeled regions in the distribution of dopamine. Relations with connectivity are discussed and the inhomogeneities are compared to those in the caudate-putamen. It is concluded that in the ventral striatum either one patch and one matrix compartment exist with different immunohistochemical relationships or there are several compartments with different immunohistochemical characteristics and different input-output relations.     

Distribution of GABAAreceptor alpha 1 subunit-like immunoreactivity in comparision with that of enkephalin and substance P in the rat forebrain

The γ-aminobutyric acid-A receptor consists of several subunits. In this immunohistochemical study we investigated the regional distribution of the α1 subunit with an antibody directed against a specific amino acid sequence (1-9) of the α1 subunit. We compared the distribution pattern of the α1 subunit-like immunoreactivity with that of substance P- and enkephalin-like immunoreactivities in adjacent sections of the rat forebrain. α1 subunit-like immunoreactivity appeared in the form of varicosities and fibers. A band-like terminal staining pattern (woolly fibers) that has been shown by others for substance P- and enkephalin-like immunoreactivity is also observed for α1 subunit-like immunoreactivity. In contrast to substance P and enkephalin, numerous α1 subunit-like immunoreactive perikarya were found. The highest density of α1 subunit-like immunoreactive fibers and perikarya was found in the pallidal areas and the substantia nigra pars reticulata whereas the nucleus accumbens and the caudate putamen displayed a low density. α1 subunit-like immunoreactive neurons resembled typical pallidal neurons. Some of these neurons were pericellularly stained with enkephalin-like immunoreactive varicosities in the dorsal pallidum. The distribution pattern of α1 subunit-like immunoreactivity reflects a partial overlap with the substance P and enkephalin system although a differential distribution to each of these peptides was observed for cell bodies, fibers, and axon terminals. © 1995 Wiley-Liss, Inc.     

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish,scyliorhinus canicula L

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat α-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution. © 1995 Wiley-Liss, Inc.     

Galanin-like immunoreactivity in the brain of teleosts: distribution and relation to substance P, vasotocin, and isotocin in the Atlantic salmon (Salmo salar).

The presence of galanin-like substances and their relation to substance P-, vasotocin-, and isotocin-immunoreactive neurons and fibers in the brain of teleosts was investigated with immunohistochemical methods. Two specific antisera against synthetic porcine galanin (GAL) revealed cell bodies and fibers in the brain of four different teleost species (Salmo salar, Carassius carassius, Gasterosteus aculeatus, and Anguilla anguilla). In all four species the main location of galanin immunoreactivity was in the hypothalamo-pituitary region. A detailed study of the distribution of galanin immunoreactivity in S. salar showed that galanin immunoreactive (GALir) perikarya were present in the nucleus preopticus periventricularis, an area that may be compared to the supraoptic nucleus in mammals, and in the nucleus lateralis tuberis, a nucleus involved in pituitary control in fishes that may be compared with the arcuate nucleus in mammals. GALir perikarya were found also in the nucleus recessus lateralis and in the nucleus recessus posterior. Numerous GALir fibers were present in the telencephalon and diencephalon, whereas only small numbers of fibers were found in the brainstem. In contrast to the situation in mammals, no GALir perikarya were observed in the brainstem areas corresponding to the noradrenergic locus coeruleus and serotonergic raphe nuclei in S. salar. We did not find any coexistence of GALir substances with arginine vasotocin or isotocin in neurosecretory neurons, as has been shown for galanin with the mammalian counterparts vasopressin and oxytocin. Also, the galanin-like substance(s) and their structurally closest related peptide family, the tachykinins, belong to separate neuronal systems in teleosts. The presence of GALir neurons in brain areas known to be involved in pituitary control, and a massive GALir innervation of the pituitary, strongly indicate a role for galanin-like substances in pituitary control also in teleosts. Furthermore, the presence of extrahypothalamic GALir fibers suggests involvement of galanin-like substances in other brain functions in teleosts. In conclusion, there are general similarities between teleosts and mammals concerning the distribution of galanin-like substances. However, there seem to be substantial differences in their distribution relative to functionally related peptides within the hypothalamo-pituitary system. Whereas galanin appears to be colocalized and released together with vasopressin and oxytocin in mammals, in teleosts the homologous substances are contained within different sets of neurons that innervate the same target, the pituitary.     

Distribution of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem

The activities of the three major catecholamines synthesizing enzymes were determined on supernatants from microcubes dissected out from 500 microns thick coronal sections of the rat lower brainstem. The microcubes exhibiting significant or high enzymatic activities represented only one-third of the microcubes analyzed and were generally localized either in the dorso-medial or in the ventro-lateral region of the lower brainstem. In the ventro-lateral region, referred to as the A1-C1 region, the rostro-caudal distribution of the enzymatic activities exhibited a small difference between the maximal TH and DBH activities and the maximum PNMT activity located 500 microns more rostrally. Conversely, in the dorso-medial region, referred to as the A2-C2 region, there was a much larger rostro-caudal difference (about 1500 microns) between the peaks of TH and DBH activities and the more rostral peak of PNMT activity. Such a distribution suggests a separation between the adrenaline (A) and the noradrenaline (NA) containing neurons within the dorso-medial region. According to the results of this study, a preferential microdissection of the A versus NA neurons seems possible in the A2-C2 region of the rat medulla oblongata.     

Regional and subcellular compartmentation of the dopamine transporter and tyrosine hydroxylase in the rat ventral pallidum

The ventral pallidum (VP) is a major intermediary in the prefrontal cortical circuitry regulating sensorimotor gating and locomotor behavior, both of which are potently modulated by catecholamines. The VP catecholaminergic innervation is derived from midbrain dopaminergic neurons that differ in expression levels of the dopamine transporter (DAT) and from brainstem noradrenergic neurons without DAT. The preferentially low level of DAT in dopaminergic terminals in the prefrontal cortex and in striatal regions projecting more extensively to the VP medial (VPm) compared with VP lateral (VPl) compartment suggests possible region-specific differences in VP axonal distribution of DAT. To test this hypothesis, we examined the electron microscopic localization of DAT and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the VPm and VPl of rat brain. In both regions, DAT and TH were localized primarily in small unmyelinated axons and morphologically heterogeneous axon terminals. DAT-immunogold particles were few in number, but mostly located on the plasma membrane. In contrast, TH immunoreactivity was distributed in the cytoplasm of individual profiles, many of which were without detectable DAT. In comparison with TH, the mean area density of DAT-labeled axons was low throughout the VP. The mean area density of DAT-immunogold axon terminals, however, was significantly higher in VPl than in VPm, whereas that of TH-labeled axons was higher in VPm than in VPl. This dissociation suggests that, compared to the VPl, the VPm receives the greatest input from catecholaminergic afferents that are either nondopaminergic or characterized by having low levels or less terminal distributions of DAT.     

Distribution of myomodulin-like immunoreactivity in the brain and retrocerebral complex of the locust,schistocerca gregaria

The distribution of myomodulin-like immunoreactivity is described for the brain and retrocerebral complex of an insect, the locust, Schistocerca gregaria. The locust brain contains 70–100 neuronal cell bodies and numerous neuropilar processes exhibiting myomodulin-like immunoreactivity. The most marked feature of the staining is a group of lateral tritocerebral neurones that form a highly immunoreactive tract that gives rise to a complex neuropile of stained processes in the dorsal tritocerebrum. This tract continues dorsally and bifurcates into a major branch that exits the brain via nervi corpora cardiaca 1 (NCC1) to innervate the corpora cardiaca and the corpora allata. A minor branch, consisting of several individual axons, combines with immunoreactive processes from the ventral nerve cord and generates a complex immunoreactive neuropile in the anterior and posterior regions of the protocerebrum. Immunoreactive processes are also found in the structured neuropile of the central body complex. Immunoreactive cell bodies are also found in the antennal lobes, in the lateral margins of the protocerebrum, in the optic lobes, and in a few cells in the pars intercerebralis. The results suggest that myomodulin-like neuropeptides may play roles as central neurotransmitters or neuromodulators in insects as well as being released into the circulation as neurohormones or acting as releasing agents for neurohormones in neurohaemal areas. They also further strengthen the idea that myomodulins, which were first identified in molluscs, may represent another interphyletic family of neuropeptides. © 1995 Wiley-Liss, Inc.