The galanin signaling cascade is a candidate pathway regulating oncogenesis in human squamous cell carcinoma

To identify putative biomarkers in squamous cell carcinoma (SCC), a survey of parallel chromosomal alterations and gene expression studies in 10 SCC cell lines were performed using array-comparative genomic hybridization (CGH) and oligo-microarray techniques. The most frequent changes were gains of 11q13.1-13.3 and losses of 18q12.1-23 in SCC. Furthermore, the expression levels of the sets of genes at both these loci in SCC were measured using microarray analysis. By combining the array-CGH with the microarray data, 10 genes at 11q13.1-13.3 and 6 genes at 18q12.1-23 whose expression correlated with chromosomal alterations were identified. To verify the expression levels of the identified genes, we used expression analysis data derived from our earlier study of clinical specimens. In clinical samples, six genes (GAL, GSTP1, MRPL11, MRPL21, SF3B2, and YIF1A) at 11q13.1-13.3 and one gene (GALR1) at 18q23 showed a significant difference between normal and tumor samples. GAL, coding for the neuropeptide galanin, and GALR1, a galanin receptor, were identified as candidate genes of oncogenesis in SCC. The expression levels of GAL, GALR1, GALR2, and GALR3 were confirmed by real-time PCR. The expression ratio between GAL and GALR1 showed a significant negative correlation. GALR1 is a G-protein-coupled receptor that activates GTP-binding proteins to trigger signaling cascades such as the mitogen-activated protein kinase pathway, and is a well-established mitogenic pathway. This further supports the hypothesis that the genes involved in the GAL signaling cascade are candidates for regulation of oncogenesis in SCC.     

Increased galanin receptor occupancy in Alzheimer's disease

Increased galanin (GAL) may be associated with the cognitive deficits characteristic of Alzheimer’s disease (AD). However, both increased and decreased GAL receptor density has been reported in AD brain. Previous studies indicate pre-treatment with guanine nucleotides displaces endogenous GAL from GAL receptors (GALR), providing an indirect measurement of GALR occupancy. In addition, pre-treatment with guanine nucleotides may provide a more accurate measurement of GALR density since it would avoid the masking of GALRs by residual binding of endogenous GAL. Thus, in the present study, we examined the influence of pre-treatment with guanine nucleotides on ¹²⁵I-GAL binding in multiple regions of normal and AD brain. Our results indicate that GTP pre-treatment enhances GAL binding in specific regions in normal and AD brain. In addition, our results suggest an increase in the number of GALRs occupied by endogenous GAL in the deep layers of the frontal cortex and the lateral hypothalamus of AD subjects compared to normal subjects. The regional differences in GALR density and receptor occupancy between normal and AD subjects may play a role in the cognitive disturbances associated with the disease.     

Involvement of galanin receptors 1 and 2 in the modulation of mouse vagal afferent mechanosensitivity

It is established that the gut peptide galanin reduces neuronal excitability via galanin receptor subtypes GALR1 and GALR3 and increases excitability via subtype GALR2. We have previously shown that galanin potently reduces mechanosensitivity in the majority of gastro-oesophageal vagal afferents, and potentiates sensitivity in a minority. These actions may have implications for therapeutic inhibition of gut afferent signalling. Here we investigated which galanin receptors are likely to mediate these effects. We performed quantitative RT-PCR on RNA from vagal (nodose) sensory ganglia, which indicated that all three GALR subtypes were expressed at similar levels. The responses of mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of galanin receptor ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors which respond only to mucosal stroking. Galanin induced potent inhibition of mechanosensitivity in both types of afferents. This effect was totally lost in mice with targeted deletion of Galr1 . The GALR1/2 agonist AR-M961 caused inhibition of mechanosensitivity in Galr1 +/+ mice, but this was reversed to potentiation in Galr1 −/− mice, indicating a minor role for GALR2 in potentiation of vagal afferents. We observed no functional evidence of GALR3 involvement, despite its expression in nodose ganglia. The current study highlights the complex actions of galanin at different receptor subtypes exhibiting parallels with the function of galanin in other systems.     

Hyperalgesia and increased neuropathic pain-like response in mice lacking galanin receptor 1 receptors

The neuropeptide galanin may have a role in modulation of nociception, particularly after peripheral nerve injury. The effect of galanin is mediated by at least three subtypes of receptors. In the present study, we assessed the nociceptive sensitivity in mice lacking the galanin receptor 1 gene (Galr1) and the development of neuropathic pain-like behaviours after photochemically induced partial sciatic nerve ischaemic injury. Under basal condition, Galr1 knock-out (Galr1(-/-)) mice had shortened response latency on the hot plate, but not tail flick and paw radiant heat, tests. The mechanical sensitivity was not different between Galr1(-/-) and wild type (Galr1(+/+)) mice, whereas the cold response was moderately enhanced in Galr1(-/-) mice. Both Galr1(-/-) mice and Galr1(+/+) controls developed mechanical and heat hypersensitivity after partial sciatic nerve injury. The duration of such pain-like behaviours was significantly increased in Galr1(-/-). The Galr1(-/-) mice and Galr1(+/+) mice did not differ in their recovery from deficits in toe-spread after sciatic nerve crush.The results provide some evidence for an inhibitory function for the neuropeptide galanin acting on galanin receptor 1 (GALR1) in nociception and neuropathic pain after peripheral nerve injury in mice.     

Distribution and regulation of galanin receptor 1 messenger RNA in the forebrain of wild type and galanin-transgenic mice

To learn more about molecular alterations in the brain that occur as a consequence of either the chronic excess or absence of peptide neurotransmitters, we examined the impact of genetically manipulating the neuropeptide galanin on the expression of one of its cognate receptors, galanin receptor 1. First, we examined the distribution of galanin receptor 1 messenger RNA in the mouse forebrain, and found it to be abundantly expressed in many brain regions, including in numerous hypothalamic and other forebrain regions associated with neuroendocrine function. The distribution of galanin receptor 1 messenger RNA in the mouse was similar to previous reports in the rat, with additional expression noted in the caudate putamen and in several midbrain regions. Next, using quantitative in situ hybridization, we measured cellular levels of galanin receptor 1 messenger RNA in the brains of mice that either overexpress galanin (galanin transgenic) or lack a functional galanin gene (galanin knockout). We report that relative to wild-type controls, the expression of galanin receptor 1 messenger RNA was increased in discrete areas of the brain in galanin-transgenic mice, but that depletion of galanin/noradrenergic innervation to the hypothalamus with the neurotoxin 6-hydroxydopamine did not alter levels of galanin receptor 1 messenger RNA. We also report that levels of galanin receptor 1 messenger RNA were not different between galanin-knockout and wild-type mice. These results suggest that compensatory adjustments in the expression of cognate receptors represent one mechanism by which the developing nervous system attempts to maintain homeostasis in response to overexpression of a peptidergic transmitter. However, the lack of significant changes in galanin receptor 1 messenger RNA in galanin-knockout mice suggests that developmentally programmed levels of receptor expression are maintained even in the complete absence of ligand.     

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.     

A novel, systemically active, selective galanin receptor type-3 ligand exhibits antidepressant-like activity in preclinical tests

The neuropeptide galanin is widely expressed in limbic nuclei in the brain, and plays an important role in the regulation of homeostatic and affective behaviors, in part through its modulation of central monoamine pathways. Recent evidence suggests that galanin and its receptors may be involved in the efficacy of various modalities of antidepressant treatments. We have previously demonstrated that systemically active, non-peptide galanin receptor type-1/2 agonists exhibit antidepressant-like effects in the rat forced swim test. Here we evaluate a novel galanin receptor type-3 (GalR3) antagonist in preclinical tests of anxiety and depression. At multiple doses, the compound displayed no effects in the elevated plus maze in mice. By contrast, the compound decreased time spent immobile in the tail suspension test by mice. Additionally, the GalR3 drug decreased time spent immobile in the forced swim test in rats, similarly to the effects of desipramine, yet did not increase locomotor activity in an open field test. These combined data from two species indicate that GalR3 receptor antagonists may exhibit antidepressant-like effects.     

Galanin type 1 receptor knockout mice show altered responses to high-fat diet and glucose challenge

Galanin, a brain and pancreatic peptide with three receptor subtypes (GALR1, GALR2, and GALR3), is hypothesized to participate in energy homeostasis and glucoregulation. Hypothalamic galanin expression is induced by dietary fat, and intra-hypothalamic galanin administration has orexigenic/anabolic properties. Systemic galanin infusion alters glucoregulation in non-human species, partly through direct actions on pancreatic islets. However, the physiologic significance of endogenous galanin-GALR signaling is unclear. The present studies tested the hypotheses that GALR1 deficiency alters food intake and feed efficiency following switches to high-fat diet and that GALR1 deficiency alters whole-body glucose homeostasis. Adult, male GALR1 knockout (-/-), heterozygote (+/-), and C57BL/6J control (+/+) mice were studied. GALR1 deficiency impaired adaptation to a 3-day high-fat diet challenge, leading to increased food intake, feed efficiency and weight gain. However, during the following 2 weeks, GALR1 knockout mice decreased intake, consuming less daily energy than while maintained on low-fat diet and also than heterozygote littermates. Chow-maintained GALR1 knockout mice showed relative hyperglycemia in fed and d-glucose (i.p. 1.5 g/kg)-challenged states. GALR1 knockout mice showed normal food intake, feed efficiency and weight accrual on low-fat diets, normal fasted glucose levels, and normal glucose sensitivity to porcine insulin (i.p. 1 IU/kg) in vivo. The results support the hypotheses that galanin-GALR1 systems help adapt food intake and metabolism to changes in dietary fat and modulate glucose disposition in mice.     

Bidirectional regulation of stress responses by galanin in mice: Involvement of galanin receptor subtype 1

The neuropeptide galanin has been shown to play a role in psychiatric disorders as well as in other biological processes including regulation of pain threshold through interactions with three G-protein coupled receptors, galanin receptor subtypes 1–3 (GalR1−3). While most of the pharmacological studies on galanin in stress-related disorders have been done with rats, the continuous development of genetically engineered mice involving galanin or its receptor subtype(s) validates the importance of mouse pharmacological studies. The present study on mice examined the homeostatic, endocrinological and neuroanatomical effects of the galanin, injected intracerebroventricularly (i.c.v.), in regulation of stress responses after restraint stress. Furthermore, the roles of GalR1 on these effects were studied using GalR1 knockout (KO) mice. The core body temperature and the locomotor activity were monitored with radio telemetry devices. Galanin (i.c.v.) decreased locomotor activity and exerted a bidirectional effect on the restraint stress–induced hyperthermia; a high dose of galanin significantly attenuated the stress-induced hyperthermic response, while a low dose of galanin moderately enhanced this response. The bidirectional effect of galanin was correlated with changes in stress hormone levels (adrenocorticotropic hormone and corticosterone). To neuroanatomically localize the effects of galanin on stress response, cFos immunoreactivity was assessed in galanin receptor rich areas; paraventricular nucleus (PVN) of the hypothalamus and the locus coeruleus (LC), respectively. A high dose of galanin significantly induced cFos activity in the LC but not in the PVN. In GalR1KO mice, a high dose of galanin failed to induce any of the above effects, suggesting the pivotal role of GalR1 in decreased locomotor activity and stress-resistant effects caused by galanin i.c.v. injection studied here.     

Increase of galanin mRNA in lumbar dorsal root ganglion neurons of adult rats after partial sciatic nerve ligation

Partial sciatic nerve ligation (PSNL) is a widely used model for the study of neuropathic pain. However, there is little information on neuropeptide expression in primary sensory neurons after PSNL. We examined galanin (GAL) mRNA expression in L4 and L5 dorsal root ganglion (DRG) neurons of adult rats after PSNL. We found that 4 and 14 days after PSNL the percentages of GAL mRNA positive neurons were significantly increased in the ipsilateral DRG compared to the contralateral side. Using combined retrograde fluorescent dye tracing and in situ hybridization, we found that 47% of the injured neurons and 10% of the spared neurons were GAL mRNA positive. Since only 2-3% of neurons in the contralateral uninjured DRG were GAL mRNA positive, PSNL induced up-regulation of GAL mRNA in both injured and spared DRG neurons.     

Effects of pentylenetetrazole-induced kindling on thyrotropin-releasing hormone biosynthesis and receptors in rat brain

It has been postulated that changes in thyrotropin-releasing hormone biosynthesis may be involved in the mechanism of kindling--an animal model of epileptogenesis. To test this hypothesis, a time-course study was carried out to investigate the effects of pentylenetetrazole kindling (40 mg/kg i.p., daily for eight days) on the expression of gene coding for preprothyrotropin-releasing hormone, the thyrotropin-releasing hormone tissue level and thyrotropin-releasing hormone receptor parameters in rat brain. As shown by an in situ hybridization study, a single, convulsant dose of pentylenetetrazole (70 mg/kg i.p.) increased the preprothyrotropin-releasing hormone messenger RNA level in the dentate gyrus of the hippocampal formation and piriform cortex after 3 h and, to a greater extent, after 24 h. Those changes were accompanied with increases in the thyrotropin-releasing hormone level in the striatum, hippocampus, amygdala and piriform cortex. Seven days after single pentylenetetrazole administration, the thyrotropin-releasing hormone level was still significantly elevated in the piriform cortex and striatum. Acute pentylenetetrazole decreased the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum after 3 and 24 h, and increased that density in the piriform cortex and amygdala after 24 h and seven days, respectively. The thyrotropin-releasing hormone receptor affinity (Kd) was decreased in the striatum and increased in the amygdala after only 3 h. Kindled rats showed a moderate increase in the preprothyrotropin-releasing hormone messenger RNA content in the dentate gyrus of the hippocampal formation and piriform cortex after 3 and 24 h; however, a significant decrease in those parameters was found after 14 days. After 3 and 24 h, pentylenetetrazole kindling also elevated the thyrotropin-releasing hormone content in the hippocampus, piriform cortex, and striatum (in the latter structure after 24 h only), whereas in the septum the thyrotropin-releasing hormone level was decreased. After seven days, the thyrotropin-releasing hormone level was still elevated in the hippocampus and piriform cortex of kindled rats, but after 14 days it was significantly lowered in the hippocampus. The kindled rats also showed a significant decrease in the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum (after 24 h, seven and 14 days), and an increase in the piriform cortex (after seven days). The thyrotropin-releasing hormone receptor affinity (Kd) value was increased in the hippocampus after seven and 14 days, and in the piriform cortex after seven days. These results indicate that pentylenetetrazole kindling induces long-lasting alterations in the thyrotropin-releasing hormone biosynthesis and thyrotropin-releasing hormone receptor affinity in discrete regions of rat brain. These region-specific changes, in particular down-regulation of the thyrotropin-releasing hormone biosynthesis in the hippocampus, may be involved in chronic neuronal hyperexcitability associated with kindling.     

The neuropeptide galanin as an in vivo modulator of brain 5-HT1A receptors: possible relevance for affective disorders

The neuropeptide galanin is widely distributed throughout the central nervous system with multiple and diverse biological functions mediated by different receptor subtypes. In the rat, galanin-like immunoreactivity is expressed in a population of 5-hydroxytryptamine (5-HT, serotonin) neurons in the dorsal raphe with extensive projections to the forebrain areas, e.g., hippocampus. This review summarizes results from experimental studies in rodents showing that in vivo galanin is a potent modulator of brain 5-HT transmission, and in particular 5-HT1A receptor-mediated functions. Galanin, given intracerebroventricular (i.c.v.), was demonstrated to have strong inhibitory interactions with 5-HT1A receptor functions, particularly in the dorsal raphe but also in the hippocampus. Since pre- and postsynaptic 5-HT1A receptors in the dorsal raphe and hippocampus are implicated in the action of antidepressant drugs and in depressive disorders, it is suggested that galanin receptors may be an important target for development of novel antidepressant drugs. This view is supported by a recent study in the rat showing that the galanin antagonist M35, given i.c.v., could block the depression-like behavior in the forced swim test induced by galanin, while M35 produced an antidepressant-like effect on its own.     

Galanin receptor over-expression within the amygdala in early Alzheimer's disease: an in vitro autoradiographic analysis

Galanin receptor (GALR) expression is increased in various areas of the limbic system in end stage Alzheimer's disease (AD). The amygdaloid complex is a key component of the limbic circuit, is involved in homeostatic and cognitive functions, is impacted in AD and contains the peptide and receptor for galanin. Although GALR expression occurs in the amygdala in end stage AD, it remains to be determined whether a plasticity response occurs early or late in the disease. Therefore, we analyzed the distribution and associated changes in GALR binding in the amygdala during the progression of AD using an in vitro receptor autoradiographic method. Human galanin ([125I]hGAL) receptor binding was performed on brain sections from early and late AD cases, as well as normal age-matched control subjects. In aged controls, densities of [125I]hGAL binding sites were found in the central and the corticomedial nuclei. Relative to controls, possible/early AD cases displayed significantly greater numbers of [125I]hGAL binding sites in the central nucleus and cortico-amygdaloid transition area. In contrast, we found a decrease in the number of binding sites for [125I]hGAL in late as compared to early AD cases. The over-expression of GALRs in subfields of the amygdaloid early in AD suggests that galaninergic systems play a key role in limbic related behavioral changes during the disease process.     

Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence

The role of the neuropeptide galanin (GAL) in rat hypothalamus has been studied in different experimental models. Thus, the effect of GAL on potassium-induced dopamine release was analyzed in vitro, and the localization of GAL and GAL binding sites was studied with immunohistochemistry and receptor autoradiography, respectively. In the median eminence GAL and presumably dopamine were found to coexist in nerve endings and this area contained a high density of 125I-GAL binding sites. In vitro experiments revealed that GAL inhibited the release of [3H]dopamine in a dose-dependent manner (IC50 = 7-10 nM), possibly via a presynaptic receptor.     

Ethanol intake increases galanin mRNA in the hypothalamus and withdrawal decreases it

Alcoholism can be viewed as a motivational disorder that results from alterations in brain systems for ingestive behavior. Therefore, it was hypothesized that alcohol intake might alter the expression of hypothalamic peptides that stimulate feeding. Earlier studies showed that hypothalamic injection of the feeding-stimulatory peptide, galanin (GAL), increases the release of dopamine (DA) in the nucleus accumbens (NAc), as does systemic alcohol, leading to a focus on GAL. Results of this study demonstrate the following: (1). Ethanol, injected daily (0.8 g/kg 10% v/v) for 7 days in male rats, markedly increased the expression of GAL but not of neuropeptide Y (NPY). This occurred in specific hypothalamic nuclei, namely the dorsomedial nucleus (DMN), paraventricular nucleus (PVN) and perifornical lateral hypothalamus (PLH). (2). Rats induced to drink ethanol ad libitum, by gradually increasing the concentration from 1% to 9% v/v without adding sugar or flavoring, exhibited a similar stimulation of GAL mRNA in the PVN and GAL immunoreactivity in the DMN and PVN. (3). Rats given increasing ethanol concentrations, with 12 h access starting 4 h into the dark cycle, had a mean blood alcohol concentration of 18 mg/dl and exhibited a similar increase in GAL expression in the DMN and PVN. (4) Withdrawal from the opioid effects of 9% ethanol, produced by injection of naloxone (3 mg/kg sc), reversed this ethanol effect by significantly reducing GAL expression in the DMN and PLH below baseline levels. These studies suggest a possible role for hypothalamic GAL in alcohol abuse.     

Evidence for a role of the neuropeptide galanin in spatial learning.

The neuropeptide galanin coexists with acetylcholine (ACh) in the basal forebrain cholinergic neurons and modulates cholinergic activity in the forebrain. The cholinergic forebrain neurons appear to play a significant role in learning and memory, as suggested by a severe loss of these neurons in Alzheimer's disease. The involvement of endogenous galanin in learning is demonstrated here by the use of the recently synthesized high-affinity galanin antagonist M35 [galanin(1-13)-bradykinin(2-9) amide] (Kd = 0.1 nM). Intracerebroventricular (i.c.v.) administration of M35 (6 but not 3 nmol) produced a significant (P < 0.025) facilitation of acquisition in a spatial learning test (Morris swim maze) without any increase in swim speed. Thus, M35 (6 nmol) shortened the escape latency, reduced the number of failures to reach the platform, and shortened the path length to reach the hidden platform. M35 (3 and 6 nmol) tended to enhance retention performance seven days after the last training session. Receptor autoradiographic studies on the distribution of [125I]M35 following i.c.v. administration show that it binds preferentially in the periventricular regions including the hippocampus. These results suggest that galanin may modulate spatial learning and memory and that galanin antagonists may provide a new principle in the treatment of Alzheimer's disease.