Galanin-immunoreactive Cells Research Articles

Microinjection of galanin into the medial preoptic nucleus facilitates copulatory behavior in the male rat

The medial preoptic area (MPOA) is an important region for masculine sexual behavior. Because galanin (GAL) immunoreactive cells within the MPOA are affected by the gonadal steroid environment and GAL binding is apparent, GAL was microinjected site specifically in 0, 10, 50, 100, and 500 ng doses in order to determine effects on copulatory behavior. Unilateral microinjection of GAL within the medial preoptic nucleus facilitated copulatory behavior in a dose-responsive fashion, evidenced by an increase in the percentage of males that displayed sexual behaviors and a decrease in mount and intromission latencies. These effects required the presence of gonadal steroids, and were not due to general arousal as measured in open field testing. The techniques of survival analysis were used to display data and for statistical analysis of intromission and mount latencies; these approaches revealed significant effects that were not evident with more commonly used procedures. The results support the suggestion that sexually dimorphic galaninergic cell groups within the MPOA are involved in gonadal steroid-induced masculine sexual behavior.

Distribution of galanin-immunoreactive cells within sexually dimorphic components of the medial preoptic area of the male and female rat

A high percentage of galanin-immunoreactive (GAL-I) cells within sexually dimorphic components of the medial preoptic area (MPOA) of the rat also concentrate estrogen and GAL microinjected within the medial preoptic nucleus (MPN) facilitates masculine sexual behavior after testosterone priming. Thus, we determined the distribution of GAL-I cells within the MPOA and their response to gonadal steroids. We report significantly greater numbers of GAL-I cells within the central division of the medial preoptic nucleus (MPNc) and fewer within the anteroventral periventricular nucleus (AVPv), of the gonadectomized male than the gonadectomized female; that GAL-I cell numbers and densities within the AVPv are increased significantly in the intact, testosterone- or estrogen-treated male compared to the gonadectomized male and that GAL-I cell numbers and densities within the MPNc and GAL-I cell densities within the medial division of the MPN (MPNm), are increased significantly by gonadal steroids in rats of both sexez. The results suggest an involvement of galaninergic cells within the MPOA in the regulation of sexually dimorphic, gonadal steroid-sensitive functions.

Identification of galanin-immunoreactive cells in the anterior pituitary of male monkeys (Macaca fascicularis).

The localization of galanin (GAL) was studied in the anterior pituitary of adult male macaque monkeys immunohistochemically. GAL-immunoreactive fiber bundles were noted in the posterior lobe of the pituitary. Cells immunoreactive for GAL were observed in the anterior lobe, and colocalization studies revealed that GAL-like immunoreactivity was present in gonadotrophs and thyrotrophs. The differences among the monkey, human and rat in GAL-immunoreactive localization may indicate that the regulation of GAL in the three species differs.

Estrogen-concentrating cells within cell groups of the medial preoptic area: sex differences and co-localization with galanin-immunoreactive cells

Male and female rats have approximately equal numbers of estrogen(E)-concentrating cells within the medial preoptic area (MPOA). Several cell groups within this brain region are sexually dimorphic, however, and these groups may have sexually different numbers of E-containing cells; this, in turn, may reflect sex differences in neural-regulated functions. In order to study this possibility, the distribution of E-concentrating cells was determined using estrogen autoradiography. Except for the lateral portion of the medial preoptic nucleus (MPNm), the density of E-concentrating cells was 3–5-times higher within the most medially situated cell groups of the female than the male, i.e., within the anteroventral periventricular nucleus (AVPv), periventricular preoptic area (PVPO), medial portion of the medial preoptic nucleus (MPNm), and its central portion (MPNc). In addition, we determined whether E-concentrating cells also express the neuropeptide, galanin. An average of 13% of the E-concentrating cells were galanin positive, which represented 15% of the galanin-immunoreactive population. These results demonstrate a frank and dramatic sex difference in the distribution of E-concentrating cells within sexually dimorphic regions of the MPOA, and also suggest that an interaction between galanin and gonadal steroids may be an important means by which cells within the MPOA regulate reproductive function.

Immunohistochemical study on the distribution of neuropeptides within the pontine tegmentum—Particularly the parabrachial nuclei and the locus coeruleus of the human brain

The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in human parabrachial nuclei and the pontine tegmentum with immunohistochemical stainings. Brains of seven adult human subjects of 35–72 years were fixed within 2 h post mortem. Serial sections were immunostained by antisera of 14 different neuropeptides—oxytocin, vasopressin, thyrotropin-releasing hormone, angiotensin II, calcitonin gene-related peptide, β-endorphin, dynorphin A, dynorphin B, leucine-enkephalin, α-melanocyte stimulating hormone, substance P, neuropeptide Y, cholecystokinin and galanin—alternately. All of these peptides were found to be present in nerve fibers and terminals, but only two, angiotensin II and dynorphin B, in cell bodies of the parabrachial nuclei. Calcitonin gene-related peptide-, neuropeptide Y-, cholecystokinin- and galanin-immunoreactive cells were present in other areas of the pontine tegmentum, like the motor trigeminal nucleus, locus coeruleus, periventricular gray matter but not in the parabrachial nuclei. Peptidergic fibers were distributed unevenly throughout the pontine tegmentum having unique, individual distribution patterns. In the parabrachial nuclei, substance P, neuropeptide Y, cholecystokinin and galanin showed the highest density of immunoreactive neuronal networks. Moderate to low concentrations of immunoreactive processes were detected by calcitonin gene-related peptide, α-melanocyte stimulating hormone, dynorphin B, thyrotropin releasing hormone, leucine-enkephalin, dynorphin A, angiotensin II, β-endorphin, vasopressin and oxytocin antisera, respectively. Other pontine tegmental areas, like the locus coeruleus, dorsal tegmental, pontine raphe and motor trigeminal nuclei as well as the centrat gray of the tegmental region exhibited a varying assortment of neuropeptides with distinct, individual localization patterns. Their detailed topographical distributions are mapped and given in coronal sections.

Origin of galanin-immunoreactive nerve fibers in the rat paracervical autonomic ganglia and uterine cervix

Retrograde axonal tracing with fluorogold in conjunction with immunohistochemistry was used to examine the source of galanin-immunoreactive nerve fibers in the paracervical ganglia and uterine cervix of the female rat. Immunohistochemistry revealed galanin-immunoreactive neuron somata in lumbosacral dorsal root ganglia and around the central canal of the lumbosacral spinal cord (lamina X). Injection of fluorogold into the paracervical ganglia resulted in labelled cells in dorsal root ganglia and the sacral parasympathetic nucleus of the spinal cord; but fluorogold-labelled, galanin-immunoreactive cells were found only in dorsal root ganglia. Injection of the tracer in the cervix resulted in labelled cells in the paracervical ganglia and dorsal root ganglia; however, fluorogold-labelled, galanin-immunoreactive cells were again evident only in dorsal root ganglia. It is suggested that the galanin-immunoreactive nerve fibers and varicosities in the paracervical ganglia and uterine cervix are sensory fibers from spinal dorsal root ganglia. The galanin-immunoreactive varicosities in the ganglia could play a role in the modulation of pelvic visceral activity, while those in the musculature of the cervix could influence contractility.

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled complementary (c)RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III-VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular, supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.

Hypothalamic galanin‐immunoreactive neurons projecting to the posterior lobe of the rat pituitary: A combined retrograde tracing and immunohistochemical study

To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin-enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin-immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cells/number of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cells/number of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 51-56% also contained galaninlike immunoreactivity. (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity.

Origins and projections of peptide-immunoreactive nerves in the male rat genitofemoral nerve

Following injection of retrograde tracer to one genitofemoral nerve of male rats, motoneurones and dorsal root ganglion cells of segmental levels L 1/L 2 were labelled ipsilaterally. Many labelled motoneurones were calcitonin gene-related peptide- (CGRP) immunoreactive. In the ganglia, a proportion of the labelled cells were CGRP-, tachykinin- or galanin-immunoreactive (10%: 6%: 53% respectively). In comparison with ganglia of the contralateral side, galanin-immunoreactive cells were significantly increased in the ipsilateral ganglia. Unilateral genitofemoral nerve section induced a loss of CGRP- or tachykinin- and an increase of galanin-immunoreactive cells in the ipsilateral L 1/L 2 ganglia. In the ipsilateral L 1/L 2 dorsal horn, CGRP and tachykinin, but not galanin, immunoreactivity was reduced. In the cremaster muscle and scrotal skin of either side galanin-immunoreactive fibres were not visible and CGRP- and tachykinin-immunoreactive fibres were depleted in the ipsilateral side. Capsaicin induced a loss of CGRP- and tachykinin-immunoreactive ganglion cells and of CGRP, tachykinin or galanin immunoreactivity from the dorsal horn. In the scrotal skin, CGRP- and tachykinin-immunoreactive fibres were depleted. By contrast in the muscle, a few CGRP-immunoreactive fibres persisted. The data demonstrate that (i) the genitofemoral nerve originates at segmental levels L 1/L 2; (ii) CGRP- and tachykinin-immunoreactive sensory and CGRP-immunoreactive motor neurones project to the cremaster muscle and scrotal skin; and (iii) nerve pertubation induces an increase of galanin-immunoreactive sensory neurones, the significance of which remains to be elucidated.

Distribution of galanin-like immunoreactivity in the human brain

The distribution of galanin-like immunoreactivity has been studied in complete coronal sections taken from 9 normal human brains (4 male, 5 female mean age 54, range 42–72 years). Galanin-immunoreactive cells were restricted largely to the basal nucleus of Meynert and to the supraoptic, ventromedial and posterior areas of the hypothalamus. Fibre staining was more widespread, seen throughout the hypothalamus and in the diagonal band, septum, amygdala, hippocampus and scattered throughout the cortex. This distribution may indicate a role for galanin in the modulation of hypothalamic and basal forebrain function in man.

Age-related loss of galanin-immunoreactive cells in the rat septal area

We have examined the distribution of galanin-like immunoreactive (LI) cell bodies in the medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (nDBB) of young (3 months) and aged (25–30 months) rats, and assessed their respective contribution to the septohippocampal pathway. Immunohistochemical techniques were used alone or combined with the retrograde transport of a protein-gold complex injected into the dorsal hippocampus. In both groups, galanin-LI cells were observed in the MS and the nDBB. In aged rats, a significant decrease in both the staining intensity and the number of galanin-LI perikarya throughout the MS-nDBB complex was observed. Some immunoreactive cells appeared shrunken. The reduction in cell number ranged from 30 to 85%. There was also a decrease in the proportion of septohippocampal neurons containing galanin in aged rats (13% vs 20% in young animals) which however did not reach statistical significance. These results suggest that galanin-positive cells in the medial septal area undergo alterations with aging in the rat.

Galanin-like immunoreactivity within amygdaloid and hypothalamic neurons that project to the midbrain central grey in rat

The possibility that galanin-immunoreactive cells within the central nucleus of the amygdala (Ce) and the lateral part of the bed nucleus of the stria terminalis (BSTL) project to the midbrain central grey in the rat was investigated using the combined immunofluorescence-retrograde tracing technique. Injections of Fluoro-Gold tracer were made into the ventrolateral midbrain central grey and the brain tissue was processed for galanin-like immunoreactivity using the avidin-biotin Texas red immunofluorescence technique. Cells containing both galanin-like immunoreactivity and fluorescent retrograde tracer were identified within the medial Ce and in the ventral part of the BSTL. Retrogradely labeled galanin-like immunoreactive cells were also observed within the medial preoptic area and to a lesser extent within the dorsomedial and paraventricular hypothalamic nuclei. The results of this study demonstrate the presence of long descending pathways containing galanin-like immunoreactivity that originate from discrete regions of the amygdala and hypothalamus and terminate within the midbrain central grey.

Distribution of galanin immunoreactivity in the respiratory tract of pig, guinea pig, rat, and dog.

Galanin, a newly discovered peptide isolated from porcine intestine, is known to cause contraction in rat smooth muscle preparations and to induce hyperglycaemia in dogs. By the use of radioimmunoassay and immunohistochemical techniques the concentration and distribution of galanin immunoreactivity were determined in several areas of the respiratory tract of five dogs, five guinea pigs, five rats, and two pigs. Antibodies were raised in rabbits to whole unconjugated natural porcine galanin. The highest galanin concentrations were found in the bronchus and the trachea of the dog, guinea pig, rat (2 pmol/g in each case), and pig (less than 1 pmol/g). The lowest galanin concentrations were found in the lung parenchyma. Gel chromatographic analysis in the pig showed one molecular form of galanin coeluting with the porcine galanin standard. By means of the indirect immunofluorescence technique on sections of tissues fixed in benzoquinone solution, galanin was found to be confined to nerve fibres in different regions of the respiratory tract. In the nasal mucosa of the pig nerve fibres containing galanin were distributed around seromucous glands and blood vessels and beneath the epithelium. In the trachea, bronchus, and major intrapulmonary airways of the pig, dog, and guinea pig galanin immunoreactive fibres were detected predominantly in smooth muscle, as well as around seromucous glands and in the adventitia of blood vessels. Rarely, galanin immunoreactive nerve fibres were found in the lung parenchyma. A few galanin immunoreactive ganglion cells also containing vasoactive intestinal polypeptide were found in the adventitia of the tracheobronchial wall of the pig and dog. The distribution of galanin suggests that it may have some influence on airway, vascular, and secretory functions in the mammalian respiratory tract.

Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species.

The distribution of galanin-immunoreactive (GAL-IR) neurons was mapped in detail in the gastro-intestinal tract of the rat, mouse, guinea-pig and pig by use of the indirect immunofluorescence technique. GAL-IR cell bodies were found in both the submucous and the myenteric plexus, with considerably higher numbers in the former ganglia. The largest number of GAL-IR perikarya was seen in the duodenal submucous plexus of the pig. With some (single) exceptions, GAL-IR cell somata were not observed in the myenteric plexus of the pig and guinea-pig, and in the submucous plexus of the esophagus and the stomach of the guinea-pig. GAL-IR fibers occurred in most parts of the gastro-intestinal tract. In the lamina propria a few non-varicose, weakly fluorescent fibers were noted in the mouse and rat, whereas in the pig and guinea-pig were large numbers of GAL-IR fibers with a varicose appearance was observed. These fibers were in all species most numerous in the distal portion of the intestinal tract. In the submucosa GAL-IR fibers were detected in all four species, and in the pig and guinea-pig some fibers surrounded blood vessels. A large number of GAL-IR fibers was generally seen in the circular smooth muscle layer, except in the guinea-pig, which only seemed to contain a few fibers. In the longitudinal muscle layer only single fibers could be detected. However, the gastric fundus region of the pig contained a moderate number of fibers in the longitudinally and obliquely oriented layers. In general, in the rat, mouse and pig, the submucous and myenteric plexus contained moderate or large numbers of GAL-IR fibers. In the guinea-pig, no or only single fibers were observed in the plexus of the upper gastro-intestinal tract and the rectum, while moderate numbers were seen in the ileum and colon. Thin adjacent sections stained for vasoactive intestinal polypeptide (VIP) and GAL revealed the coexistence of these two peptides in cell bodies of the myenteric plexus in the pig duodenum and guinea-pig colon. In these two species the GAL- and VIP-nerve fiber networks also exhibited marked similarities. However, in the rat and mouse VIP- and GAL-distribution patterns were in general different. The present findings indicate the presence of yet another neuropeptide or peptide family in the gastro-intestinal tract of several rodents and the pig.