Spinal neuropeptide expression and neuropathic behavior in the acute and chronic phases after spinal cord injury: Effects of progesterone administration.

Patients with spinal cord injury (SCI) develop chronic pain that severely compromises their quality of life. We have previously reported that progesterone (PG), a neuroprotective steroid, could offer a promising therapeutic strategy for neuropathic pain. In the present study, we explored temporal changes in the expression of the neuropeptides galanin and tyrosine (NPY) and their receptors (GalR1 and GalR2; Y1R and Y2R, respectively) in the injured spinal cord and evaluated the impact of PG administration on both neuropeptide systems and neuropathic behavior. Male rats were subjected to spinal cord hemisection at T13 level, received daily subcutaneous injections of PG or vehicle, and were evaluated for signs of mechanical and thermal allodynia. Real time PCR was used to determine relative mRNA levels of neuropeptides and receptors, both in the acute (1day) and chronic (28days) phases after injury. A significant increase in Y1R and Y2R expression, as well as a significant downregulation in GalR2 mRNA levels, was observed 1day after SCI. Interestingly, PG early treatment prevented Y1R upregulation and resulted in lower NPY, Y2R and GalR1 mRNA levels. In the chronic phase, injured rats showed well-established mechanical and cold allodynia and significant increases in galanin, NPY, GalR1 and Y1R mRNAs, while maintaining reduced GalR2 expression. Animals receiving PG treatment showed basal expression levels of galanin, NPY, GalR1 and Y1R, and reduced Y2R mRNA levels. Also, and in line with previously published observations, PG-treated animals did not develop mechanical allodynia and showed reduced sensitivity to cold stimulation. Altogether, we show that SCI leads to considerable changes in the spinal expression of galanin, NPY and their associated receptors, and that early and sustained PG administration prevents them. Moreover, our data suggest the participation of galaninergic and NPYergic systems in the plastic changes associated with SCI-induced neuropathic pain, and further supports the therapeutic potential of PG- or neuropeptide-based therapies to prevent and/or treat chronic pain after central injuries.

Galanin subtype 1 and subtype 2 receptors mediate opposite anxiety-like effects in the rat dorsal raphe nucleus.

About 40% of the dorsal raphe nucleus (DRN) neurons co-express serotonin (5-HT) and galanin. Serotonergic pathways from the DRN to the amygdala facilitate learned anxiety, while those from the DRN to the dorsal periaqueductal grey matter (DPAG) impair innate anxiety. Previously, we showed that galanin infusion in the DRN of rats induces anxiolytic effect by impairing inhibitory avoidance without changing escape behaviour in the elevated T-maze (ETM). Here, we evaluated: (1) which galanin receptors would be involved in the anxiolytic effect of galanin in the DRN of rats tested in the ETM; (2) the effects of galanin intra-DRN on panic-like behaviours evoked by electrical stimulation of the DPAG. The activation of DRN GAL1 receptors by M617 (1.0 and 3.0nmol) facilitated inhibitory avoidance, whereas the activation of GAL2 receptors by AR-M1896 (3.0nmol) impaired the inhibitory avoidance in the ETM, suggesting an anxiogenic and an anxiolytic-like effect respectively. Both agonists did not change escape behaviour in the ETM or locomotor activity in the open field. The anxiolytic effect of AR-M1896 was attenuated by the prior administration of WAY100635 (0.18nmol), a 5-HT1A antagonist. Galanin (0.3nmol) administered in the DRN increased discreetly flight behaviours induced by electrical stimulation of the DPAG, suggesting a panicolytic effect. Together, our results showed that galanin mediates opposite anxiety responses in the DRN by activation of GAL1 and GAL2 receptors. The anxiolytic effect induced by activation of Gal2 receptors may depend on serotonergic tone. Finally, the role of galanin in panic related behaviours remains uncertain.

Effects of bilateral adrenalectomy on systemic kainate-induced activation of the nucleus of the solitary tract. Regulation of blood pressure and local neurotransmitters.

Glutamatergic transmission through metabotropic and ionotropic receptors, including kainate receptors, plays an important role in the nucleus of the solitary tract (NTS) functions. Glutamate system may interact with several other neurotransmitter systems which might also be influenced by steroid hormones. In the present study we analyzed the ability of systemic kainate to stimulate rat NTS neurons, which was evaluated by c-Fos as a marker of neuronal activation, and also to change the levels of NTS neurotransmitters such as GABA, NPY, CGRP, GAL, NT and NO by means of quantitative immunohistichemistry combined with image analysis. The analysis was also performed in adrenalectomized and kainate stimulated rats in order to evaluate a possible role of adrenal hormones on NTS neurotransmission. Male Wistar rats (3 month-old) were used in the present study. A group of 15 rats was submitted either to bilateral adrenalectomy or sham operation. Forty-eight hours after the surgeries, adrenalectomized rats received a single intraperitoneal injection of kainate (12 mg/kg) and the sham-operated rats were injected either with saline or kainate and sacrificed 8 hours later. The same experimental design was applied in a group of rats in order to register the arterial blood pressure. Systemic kainate decreased the basal values of mean arterial blood pressure (35%) and heart rate (22%) of sham-operated rats, reduction that were maintained in adrenalectomized rats. Kainate triggered a marked elevation of c-Fos positive neurons in the NTS which was 54% counteracted by adrenalectomy. The kainate activated NTS showed changes in the immunoreactive levels of GABA (143% of elevation) and NPY (36% of decrease), which were not modified by previous ablation of adrenal glands. Modulation in the levels of CGRP, GAL and NT immunoreactivities were only observed after kainate in the adrenalectomized rats. Treatments did not alter NOS labeling. It is possible that modulatory function among neurotransmitter systems in the NTS might be influenced by steroid hormones and the implications for central regulation of blood pressure or other visceral regulatory mechanisms control should be further investigated.

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve.

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.

Morphological and biochemical characterisation of sensory neurons infected in vitro with rabies virus.

This work was aimed at the morphological and biochemical characterisation of the most susceptible neuronal subpopulation to rabies virus (RABV) infection. Adult mouse DRG cultures were infected with RABV and double-processed for viral antigen detection and neuropeptides: calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). It was found that 56% of the neurons in culture were small (diameter < 20 microm) but, in spite of this, 69% of the infected neurons had intermediate and large diameters (> or = 20 microm). More than 50% of infected neurons expressed NPY, VIP or SP, whereas no association was found between infected neurons and the presence of CGRP or GAL. Despite SP having been shown to be a small neuron marker, it was found that RABV infects medium and large-sized SP positive cells. RABV preference for larger neurons could explain part of the neuropathogenesis since it can be suggested that, following a rabid accident, the virus uses large neurons (mainly innervating muscle and joints) in vivo to be transported later on to the central nervous system.

Decreases in the expression of CGRP and galanin mRNA in central and peripheral neurons related to the control of blood pressure following experimental hypertension in rats.

Calcitonin gene-related peptide (alpha CGRP) and galanin (GAL) are peptides known to participate in central mechanisms of blood pressure control. Nonetheless, variations in the synthesis of the peptides in response to a hypertensive challenge are not well described, specially using a model, which allows acute and chronic analyses. In this study, we have employed in situ hybridization to analyse changes in mRNA expression of alpha CGRP and GAL in the nucleus tractus solitarii (NTS), hypothalamic paraventricular nucleus (PVN) as well as petrosal and nodose ganglia after aortic coarctation-induced hypertension in rats. Acute (2h) and chronic (3 and 7 days) analyses were performed in order to evaluate the involvement of both peptides in different periods of hypertension. The analysis of relative mRNA levels showed significant differences between sham-operated and aortic coarcted hypertensive rats. alpha CGRP mRNA expression was decreased 2h (40%) and 3 days (42%) in nodose and petrosal ganglia, respectively, after coarctation. No changes in CGRP mRNA signal were seen in the NTS and PVN in the analysed periods. GAL mRNA expression was decreased in the NTS (19%) and PVN (55%), 3 and 7 days, respectively, after coarctation-induced hypertension. No changes in GAL mRNA expression were observed in petrosal and nodose ganglia following aortic coarctation. Data suggest that alpha CGRP and GAL may participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes might be involved with the adaptation to the hypertensive state, while changes at the chronic phase might be related to counteraction of hypertension.

Gender differences in the expression of galanin and vasoactive intestinal peptide in oestrogen-induced prolactinomas of Fischer 344 rats.

We have previously described a sexual dimorphism in oestrogen-induced anterior pituitary tumorigenesis in Fischer 344 rats, with female tumours averaging twice the size of those of males. Neonatal androgenization of female Fischer 344 rats with 100 micro g of testosterone propionate reverted that effect, causing a 'male-like' phenotype. The peptides galanin and vasoactive intestinal peptide (VIP) are possible mediators of oestrogen effects on the anterior pituitary, including hyperprolactinemia and lactotroph proliferation. To further extend our previous findings, we investigated the expression of galanin and VIP in the anterior pituitary of control and oestrogenized male, female and neonatally androgenized female Fischer 344 rats. At 3 months of age, rats were deprived of their gonads and divided into control and diethylstilbestrol (DES)-treated groups. In the anterior pituitary of control rats, galanin and VIP immunoreactive cells were absent. However, in DES-treated rats, pituitaries from normal ovariectomized females showed higher number of galanin and VIP positive cells than pituitaries from neonatally androgenized ovariectomized females and gonadectomized males. This pattern correlated with changes in anterior pituitary weight and serum prolactin. Our study suggests that sexual differences in oestrogen-induced pituitary tumorigenesis could be due to the differential expression of galanin and VIP. Furthermore, our data support the fact that neonatal exposure to androgens, as in normal males and androgenized females, may condition the response of the pituitary gland to oestrogens in adult life.

Coexistence of neuropeptides and their possible relation to neuritic regeneration in primary cultures of magnocellular neurons isolated from adult rat supraoptic nuclei.

The coexistence of vasopressin (VP), oxytocin (OXY), galanin (GAL) and cholecystokinin (CCK) and the synthesis of GAL and CCK during neuritic regeneration was investigated in cultured magnocellular neurons, isolated from adult rat supraoptic nuclei. Double-labelling immunofluorescence was performed after 7 days of culture using primary antibodies for VP, OXY, GAL and CCK (paired in all possible combinations) and secondary antibodies labelled with either fluorescein or rhodamine. Confocal laser scanning microscopy revealed the coexistence of the mentioned peptides in all possible combinations, an unexpected result considering that the only combinations observed in tissue sections are VP-GAL and OXY-CCK. Freshly dispersed cells were devoid of any neuritic processes and showed a very poor immunocytochemical staining reaction for GAL and CCK. In contrast, neurons cultured for 7, 12 and 21 days showed many neurites and a strong immunoreactivity for GAL and CCK indicative of an increased synthesis of both peptides in the regenerating neurons. This increased synthetic activity is consistent with transient upregulation of these peptides observed in situ after hypophysectomy by other authors. The results suggest that the upregulation of GAL and CCK is functionally related to the neuronal regeneration processes observed during culture and that the 'uncommon' coexistences as well as the prolonged sythesis of GAL and CCK may be due to the lack of environmental inputs, which normally regulate the expression and up- and downregulation of these peptides in vivo.

An immunohistochemical study of temperature-related changes in galanin and nitric oxide synthase immunoreactivity in the hypothalamus of the toad.

Galanin (GAL) and nitric oxide synthase (NOS) have been implicated in the control of thermogenesis in mammals. An experimental protocol was designed to determine whether or not the expression of these molecules in the hypothalamus of the toad could be related to environmental temperature changes. Exposure of the animals to low temperature increased the number and intensity of NOS-positive neurons in the magnocellular hypothalamic region, in contrast to a weak immunoreactivity observed in control animals kept in a natural environment at a spring-summer temperature (23-27 degrees C). Also a significantly higher number of GAL-immunoreactive (-IR) cells was observed in the preoptic area as compared to that observed in controls, while no difference in the intensity of GAL immunostaining intensity was detected. These results show a temperature-related expression of GAL and NOS in the hypothalamus and preoptic area of the toad. The results suggest a possible role of GAL and NOS in the regulation of hibernation in these animals.

Growth hormone inhibits the hypophysectomy-induced expression of galanin in hypothalamic neurons of the toad (Bufo arenarum hensel).

The expression of the neuropeptide galanin was analyzed by immunohistochemistry in magnocellular and preoptic hypothalamic neurons of toads following hypophysectomy (HPX) and pars distalectomy (PDX). There was a marked increase in the galanin-like immunoreactive expression in magnocellular hypothalamic cells 3 days after HPX, followed by a decrease to normal levels after 7 days. No changes in the expression of galanin were detected after PDX in these neurons when compared to controls. Moreover, 7 days after HPX or PDX the number of cells expressing galanin was significantly increased in the preoptic area, where numerous intraependymal cells were intensely immunoreactive. The hypophysis grafts into the hind limb in HPX or PDX animals prevented increased galanin-like immunoreactivity in preoptic cells but not in magnocellular neurons. Similarly, PDX toads given growth hormone showed no GAL-LI in the intraependymal preoptic cells. These results suggest the presence of a region regulation of galanin expression in the preoptic area by hypophyseal hormones, in particular growth hormone.

Galanin-like immunoreactivity in the sea cucumber Holothuria glaberrima.

Galanin, a neuropeptide of 29 amino acids originally purified from porcine small intestine, has been found in most vertebrate groups, where it is present in both the central and peripheral nervous systems. In this study, galanin-like immunoreactivity was detected in an invertebrate, the sea cucumber Holothuria glaberrima. In this organism, fibers and cells expressing galanin-like immunoreactivity were found in the enteric nerve plexuses of the esophagus, and large and small intestine, particularly in the serosal layer. Immunoreactivity was also found in the ectoneural portion of the radial nerve and in nervous elements within the body wall. Preabsorbtion tests indicated that the observed immunoreactivity was not due to cross-reactivity of the antibody with other peptides also found in the intestinal plexuses. The immunoreactivity to galanin was quantified in various tissues using radioimmunoassay and partially characterized with high performance liquid chromatography. These results show that although the holothurian peptide shows common immunological determinants with porcine galanin, it differs with regard to certain biochemical properties.

[Galanin, a new neuropeptide. Review]. / Galanina, un nuevo neuropéptido. Revisión.

Galanin is a 29 amino-acid peptide originally isolated from porcine intestine. It is synthesized as part of a large precursor peptide the preprogalanin. Immunological studies has showed that there is interspecies conservation of the N terminal portion although the C-terminal portions has a little of immunoreactivity. Galanin has a number of pharmacological properties in whole animals and isolated tissues. Galanin contracts isolated preparation from rat fundus, ileum, colon and urinary bladder. Direct administration of galanin (pGal) into the rat third ventricule stimulates food intake, increases plasma growth hormone and prolactin levels and decrease dopamine levels in the median eminence. Intravenous infusion in dog and humans induce a hyperglycemia and glucose intolerance and inhibits the insulin, somatostatin and pancreatic polipeptide secretion from pancreas. Galanin is a estrogen-stimulated peptide. Estrogens increase dramatically the synthesis of their mRNA and the peptide in the rat pituitary. Galanin-like immunoreactivity is widely distributed in several mamalian species including humans. In the central nervous system it was found in medium emminence, hypothalamus, arcuate nucleus etc. Its localization in neurosecretory granules suggest that galanin functions as a neurotransmitter. The detection of a Gal-immunorectivity in the plasma after 17 beta estradiol stimulation suggests that galanin has a distal target and therefore, may be an additional anterior pituitary hormone. Galanin has been localized in reproductive tissues and this suggests that it may play an estrogen mediated role in the hypothalamic and pituitary function. However, the molecular mechanisms involved in their function remain to be studied.

Galanin-like immunoreactive expression in the central nervous system of the toad.

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.

Galanin-like immunoreactive expression in the central nervous system of the toad

The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.(AU)