Alarin moderated myocardial hypertrophy via inhibiting cyclic adenosine monophosphate/protein kinase A signaling pathway to attenuate autophagy.

Alarin could alleviate myocardial infarction-induced heart failure. The present study was to explore whether alarin could alleviate myocardial hypertrophy via inhibiting cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway to attenuate autophagy. Myocardial hypertrophy was induced by angiotensin (Ang) II infusion in vivo in mice and by Ang II treatment of neonatal rat cardiomyocytes (NRCMs) in vitro. The Ang II-induced hypertrophy and fibrosis of the heart were alleviated after alarin administration in mice. The increased atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (ß-MHC), and the decreased alpha-myosin heavy chain (α-MHC) induced by Ang II were reversed by alarin treatment in NRCMs. Alarin inhibited the increases of cAMP and PKA in NRCMs. Treatment with cAMP or overexpression of PKA blocked the attenuating effects of alarin on Ang II-induced hypertrophy in NRCMs. Alarin reduced the Ang II-induced increases of LC3, Beclin 1, autophagy-related gene (Atg)3 and Atg5 in NRCMs. The overexpression of cAMP and PKA reversed the alleviating effects of alarin on the increased autophagy induced by Ang II in NRCMs. These results indicated that alarin could moderate cardiac remodeling. Alarin improved myocardial hypertrophy via inhibiting the cAMP/PKA signaling pathway to attenuate autophagy.

Treatment with celastrol protects against obesity through suppression of galanin-induced fat intake and activation of PGC-1α/GLUT4 axis-mediated glucose consumption.

Overweight and obesity may cause several metabolic complications, including type 2 diabetes mellitus and hyperlipidemia. Despite years of progress in medicine, there are no highly effective pharmacological treatments for obesity. The natural compound celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium Wilfordi (thunder god vine) plant, exerts various bioactivities including anti-diabetic and anti-obese effects. Although celastrol could decrease food intake and obesity, the detailed mechanism for celastrol is still unclear as yet. Herein, we intended to determine the effect of celastrol on obesity and the underlying mechanisms. In the present study, diet-induced obese mice were treated with 100 µg/kg/d celastrol for the last 21 days, and 3T3-L1 cells were treated with celastrol for 6 h. The present findings showed that celastrol suppresses fat intake, and leads to weight loss by inhibiting galanin and its receptor expression in the hypothalamus of mice fed a high-fat diet. More importantly, in addition to these direct anti-obesity activities, celastrol augmented the PGC-1α and GLUT4 expression in adipocytes and skeletal muscles to increase glucose uptake through AKT and P38 MAPK activation. Celastrol also inhibited gluconeogenic activity through a CREB/PGC-1α pathway. In conclusion, the weight-lowering effects of celastrol are driven by decreased galanin-induced food consumption. Thus, this study contributes to our understanding of the anti-obese role of celastrol, and provides a possibility of using celastrol to treat obesity in clinic.

Activation of galanin and cholecystokinin receptors in the lumbosacral spinal cord is required for ejaculation in male rats.

The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic and motor neurons in the lumbosacral spinal cord. LSt cells regulate ejaculatory reflexes by release of neuropeptides that are co-expressed in their axons, as previously demonstrated for gastrin-releasing peptide and enkephalin. Here, the role of two other neuropeptides co-expressed in LSt cells for ejaculatory reflexes is demonstrated: galanin and cholecystokinin (CCK). Adult male rats were anesthetized, spinalized, and received intrathecal infusions of galanin receptor antagonist Galantide (1 or 10 nmol) or CCK receptor antagonist proglumide (71 or 714 nmol). The dorsal penile nerve (DPN) was electrically stimulated to trigger ejaculatory reflexes and seminal vesicle pressure (SVP) and rhythmic contractions of the bulbocavernosus muscle (BCM) were analyzed as parameters of emission and expulsion respectively. Treatment with galanin or CCK antagonists significantly reduced SVP increases and BCM bursting, demonstrating that galanin and CCK are required for ejaculation. Next, anesthetized, spinalized males received intrathecal infusions of galanin (0.15 or 0.3 nmol) or CCK(26-33) (4.35 nmol) and effects on subthreshold DPN stimulations were determined. Intrathecal infusions of galanin or CCK facilitated ejaculatory reflexes induced by subthreshold DPN stimulation in all animals, but did not trigger ejaculatory reflexes in the absence of DPN stimulation. Together, these results demonstrate that galanin and CCK both act in the spinal ejaculation generator to regulate ejaculation. However, effects of galanin and CCK were dependent on DPN stimulation, suggesting that these neuropeptides may act in concert with other LSt co-expressed neuropeptides.

Effects of the Galanin Receptor Antagonist M40 on Cardiac Function and Remodeling in Rats with Heart Failure.

BACKGROUND: Sympathetic activation and parasympathetic withdrawal are important characteristics of heart failure. Recent studies demonstrate that galanin reduces the discharge of acetylcholine and inhibits vagal bradycardia by acting on galanin receptor type 1 (GalR1). We speculated that blocking GalR1 is beneficial for heart failure. METHODS: Rats with heart failure were induced by myocardial infarction. The rats were injected intraperitoneally with galanin receptor antagonist M40 solution (30 nmol/kg) or saline for 4 weeks. Cardiac function was assessed by echocardiography and brain natriuretic peptide (BNP) in plasma. The ratio of heart weight to body weight (HW/BW), hematoxylin-eosin (HE), and Masson trichrome stain was used to evaluate cardiac remodeling. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) in plasma, and sarco(endo)plasmic reticulum Ca(2+) -ATPase (SERCA2) in heart tissue were detected to confirm the mechanism of the cardioprotection effect. RESULTS: Compared with rats injected with saline, M40 effectively improved cardiac function of contraction; decreased BNP, IL-6, and HW/BW (all P < 0.05); attenuated cardiac fibrosis; and upregulated SERCA2 (P < 0.05). CONCLUSION: M40 improves cardiac function and attenuates remodeling, suggesting that galanin receptor antagonist may be a potential therapeutic agent for HF.

Modification of Anxious Behavior after Psychogenic Trauma and Treatment with Galanin Receptor Antagonist.

Effects of blockage of central galanin receptors on anxiety manifestations were studied in rats with psychogenic trauma. Psychogenic trauma was modeled by exposure of a group of rats to the situation when the partner was killed by a predator. Antagonist of galanin receptors was intranasally administered before stress exposure. Animal behavior was evaluated using the elevated-plus maze test, free exploratory paradigm, and open-field test. Psychogenic trauma was followed by an increase in anxiety level and appearance of agitated behavior. Blockage of galanin receptors aggravated behavioral impairment, which manifested in the pathological anxious reactions - manifestations of hypervigilance and hyperawareness. The results suggest that endogenous pool of galanin is involved into prevention of excessive CNS response to stressful stimuli typical of posttraumatic stress disorder.

Effects of galanin subchronic treatment on memory and muscarinic receptors.

The cholinergic pathways, which originate in the basal forebrain and are responsible for the control of different cognitive processes including learning and memory, are also regulated by some neuropeptides. One of these neuropeptides, galanin (GAL), is involved in both neurotrophic and neuroprotective actions. The present study has evaluated in rats the effects on cognition induced by a subchronic treatment with GAL by analyzing the passive avoidance response, and the modulation of muscarinic cholinergic receptor densities and activities. [(3)H]-N-methyl-scopolamine, [(3)H]-oxotremorine, and [(3)H]-pirenzepine were used to quantify the density of muscarinic receptors (MRs) and the stimulation of the binding of guanosine 5'-(γ-[(35)S]thio)triphosphate by the muscarinic agonist, carbachol, to determine their functionality. Some cognitive deficits that were induced by the administration of artificial cerebrospinal fluid (aCSF) (i.c.v. aCSF 2 µl/min, once a day for 6 days) were not observed in the animals also treated with GAL (i.c.v. 1.5 mmol in aCSF, 2 µl/min, once a day for 6 days). GAL modulates the changes in M1 and M2 MR densities observed in the rats treated with aCSF, and also increased their activity mediated by G(i/o) proteins in specific areas of the dorsal and ventral hippocampus. The subchronic administration of the vehicle was also accompanied by an increased number of positive fibers and cells for GAL around the cortical tract of the cannula used, but that was not the case in GAL-treated rats. In addition, the increase of GAL receptor density in the ventral hippocampus and entorhinal cortex in the aCSF group was avoided when GAL was administered. The number of acetylcholinesterase (AChE)-positive neurons was decreased in the nucleus basalis of Meynert of both GAL- and aCSF-treated animals. In summary, GAL improves memory-related abilities probably through the modulation of MR density and/or efficacy in hippocampal areas.

Effect of endogenous galanin on glucose transporter 4 expression in cardiac muscle of type 2 diabetic rats.

Although galanin has been shown to increase glucose transporter 4 (GLUT4) expression in skeletal muscle and adipocytes of rats, there is no literature available about the effect of galanin on GLUT4 expression in cardiac muscle of type 2 diabetic rats. In this study, we investigated the relationship between intracerebroventricular administration of M35, a galanin receptor antagonist, and GLUT4 expression in cardiac muscle of type 2 diabetic rats. The rats tested were divided into four groups: rats from healthy and type 2 diabetic drug groups were injected with 2 µM M35 for three weeks, while both control groups with 2 µl vehicle control. The euglycemic-hyperinsulinemic clamp test was conducted for an index of glucose infusion rates. The cardiac muscle was processed for determination of GLUT4 expression levels. The present study showed that the plasma insulin and retinol binding protein 4 (RBP4) levels were higher in both drug groups than controls respectively. Moreover, the results showed the inhibitive effect of central M35 treatment on glucose infusion rates in the euglycemic-hyperinsulinemic clamp test and GLUT4 expression levels in the cardiac muscle. These results demonstrate that endogenous galanin, acting through its central receptor, has an important attribute to increase GLUT4 expression, leading to enhance insulin sensitivity and glucose uptake in cardiac muscle of type 2 diabetic rats. Galanin and its fragment can play a significant role in regulation of glucose metabolic homeostasis in cardiac muscle and galanin is an important hormone relative to diabetic heart.

Galanin and galanin-like peptide modulate vasopressin and oxytocin release in vitro: the role of galanin receptors.

Galanin (Gal) and galanin-like peptide (GALP) may be involved in the mechanisms of the hypothalamo-neurohypophysial system. The aim of the present in vitro study was to compare the influence of Gal and GALP on vasopressin (AVP) and oxytocin (OT) release from isolated rat neurohypophysis (NH) or hypothalamo-neurohypophysial explants (Hth-NH). The effect of Gal/GALP on AVP/OT secretion was also studied in the presence of galantide, the non-selective galanin receptors antagonist. Gal at concentrations of 10(-10 )M and 10(-8 )M distinctly inhibited basal and K(+)-stimulated AVP release from the NH and Hth-NH explants, whereas Gal exerted a similar action on OT release only during basal incubation. Gal added to the incubation medium in the presence of galantide did not exert any action on the secretion of either neurohormone from NH and Hth-NH explants. GALP (10(-10 )M and 10(-9 )M) induced intensified basal AVP release from the NH and Hth-NH complex as well as the release of potassium-evoked AVP from the Hth-NH. The same effect of GALP has been observed in the presence of galantide. GALP added to basal incubation medium was the reason for stimulated OT release from the NH as well as from the Hth-NH explants. However, under potassium-stimulated conditions, OT release from the NH and Hth-NH complexes has been observed to be distinctly impaired. Galantide did not block this inhibitory effect of GALP on OT secretion. It may be concluded that: (i) Gal as well as GALP modulate AVP and OT release at every level of the hypothalamo-neurohypophysial system; (ii) Gal acts in the rat central nervous system as the inhibitory neuromodulator for AVP and OT release via its galanin receptors; (iii) the stimulatory effect of GALP on AVP and OT release is likely to be mediated via an unidentified specific GALP receptor(s).

Antinociceptive effects of galanin in the nucleus accumbens of rats.

It has been demonstrated that galanin plays important roles in the modulation of nociceptive information in rats. The present study is performed to investigate the regulating role of galanin in nociception in the nucleus accumbens (NAc) of rats. Intra-NAc administration of galanin induces dose-dependent increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats. Furthermore, the galanin-induced antinociceptive effects are blocked by following intra-NAc injection of the galanin receptor antagonist galantide. The results demonstrate that galanin induces antinociceptive effects in the NAc of rats, and galanin receptors are involved in the galanin-induced antinociception effects.

The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: implications for neural control of cardiac excitability.

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 µM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p<0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n=6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n=6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 µM, n=5), and completely reversed with the NPY Y(2) receptor antagonist BIIE 0246 at all time points (1 µM, n=6). Exogenous galanin (n=6, 50-500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n=6). Galanin (500 nM) also significantly attenuated the release of (3)H-acetylcholine from isolated atria during field stimulation (5 Hz, n=5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n=5). Importantly the GalR(1) receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n=6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500 nM, n=6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR(1) receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.

Galanin regulation of LH release in male rats.

The present study examines the role of cerebroventricular administered (IIIrd ventricle) galanin on LHRH and LH release in adult and immature male rats. In both age groups, galanin stimulated LHRH synthesis and release from the hypothalamus, leading to a higher release of pituitary LH which in turn increased plasma LH levels. Galantide, a galanin receptor blocker, on the other hand, drastically reduced hypothalamic LHRH and plasma LH while increasing pituitary LH. In vitro incubation of anterior pituitary cells with galanin followed by LHRH resulted in increased release of pituitary LH but not by galanin alone. Galantide exhibited no such effect either alone or with LHRH. These results indicate that galanin is an important regulator for both hypothalamic LHRH and hypophysial LH and its role is independent of age in the case of male rats.

The combination of neurokinin-1 and galanin receptor antagonists ameliorates caerulein-induced acute pancreatitis in mice.

Both galanin and substance P have been separately implicated in the pathogenesis of acute pancreatitis. We compared the efficacy of the combination of the galanin antagonist galantide and the neurokinin-1 receptor antagonist L703,606 with that of either alone in the treatment of acute pancreatitis. Acute pancreatitis was induced in mice with 7-hourly caerulein injections. Galantide was co-administered with each caerulein injection commencing with the first injection (prophylactic) or 2h after the first injection (therapeutic). L703,606 was administered either 30 min before (prophylactic), or 2h after the first caerulein injection (therapeutic). Combination of the two agents was also administered. Control groups received galantide, L703,606, or saline, without caerulein. Pancreata were harvested for histological examination and estimation of myeloperoxidase activity. Plasma amylase activity was measured. Prophylactic and therapeutic administration of galantide reduced the hyperamylasemia by 37% and 30% respectively whereas only prophylactic L703,606 reduced hyperamylasemia (by 34%). Prophylactic administration of the combined antagonists reduced the hyperamylasemia by 44%. In contrast, therapeutic administration of the combination significantly increased plasma amylase levels by 27%. The plasma amylase activity in the control groups was similar to basal levels. Prophylactic and therapeutic administration of either antagonist or the combination significantly reduced myeloperoxidase activity. Galantide and L703,606 individually, and in combination, significantly reduced the acute pancreatitis-induced necrosis score. The administration of the combined antagonists does not offer any further benefit as compared to galantide alone. An interaction between neurokinin-1 and galanin receptors may occur to modulate amylase secretion.

Galanin receptor antagonist m35 but not m40 or c7 ameliorates cerulein-induced acute pancreatitis in mice.

BACKGROUND/AIMS: We compared the galanin antagonists C7, M35, M40 and galantide, for their ability to ameliorate acute pancreatitis (AP). METHODS: Galanin antagonists were co-administered with 7 hourly cerulein injections used to induce AP. Plasma amylase and lipase activities were measured as indices of AP, and pancreata were harvested at 12 h for histological examination and estimation of myeloperoxidase (MPO) activity. RESULTS: Treatment with galantide, M35 and C7 ameliorated the AP-induced plasma hyperenzymemia by 40-75%. Administration of M40 did not significantly alter plasma hyperenzymemia. Galantide, M35 and M40 significantly reduced the pancreatic MPO activity by 65-80%, whereas C7 increased MPO activity. Galantide and M35 but not C7 or M40 treatment significantly reduced the AP-induced necrosis score by 30-50% compared to the AP alone group. C7 alone increased plasma lipase activity and the pancreatic necrosis score compared with saline treatment alone, whereas the other antagonists were without effect. CONCLUSION: Galantide and M35 ameliorated the severity of AP, but M40 and C7 had mixed effects. Complex galanin pathways may be involved in cerulein-induced AP. M35 and galantide are potential therapeutic peptides for the treatment of AP and further evaluation should be considered. and IAP.

Galanin modulates vagally induced contractions in the mouse oesophagus.

Nitrergic myenteric neurons co-innervating motor endplates were previously shown to inhibit vagally induced contractions of striated muscle in the rodent oesophagus. Immunohistochemical demonstration of putative co-transmitters, e.g. galanin, in enteric neurons prompted us to study a possible role of galanin in modulating vagally mediated contractions in an in vitro vagus nerve-oesophagus preparation of the mouse. Galanin (1-16) (1-100 nmol L(-1)), in the presence of the peptidase inhibitor, phenanthroline monohydrate, inhibited vagally induced contractions in a concentration-dependent manner (control: 100%; galanin 1 nmol L(-1): 95.6 +/- 1.6%; galanin 10 nmol L(-1): 57.3 +/- 6.5%; galanin 100 nmol L(-1): 31.2 +/- 8.1%, n = 5). The non-selective galanin receptor antagonist, galantide (100 nmol L(-1)), blocked the inhibitory effect of galanin (10 nmol L(-1)) while the selective non-galanin receptor 1 and galanin receptor 3 antagonists, M871 (1 micromol L(-1)) and SNAP37889 (100 nmol L(-1)), respectively, and the nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME) (200 micromol L(-1)), failed to affect this galanin-induced response. Simultaneous application of galantide (100 nmol L(-1)) and L-NAME (200 micromol L(-1)) significantly reduced the inhibitory effect of capsaicin (30 mumol L(-1)) on vagally induced contractions when compared with its effect in the presence of L-NAME alone or in combination with the selective galanin receptor 2 or 3 antagonists. An inhibitory effect of piperine on vagally induced contractions was reduced neither by galantide nor by L-NAME. Immunohistochemistry revealed galanin immunoreactive myenteric neurons and nerve fibres intermingling with cholinergic vagal terminals at motor endplates. These data suggest that galanin from co-innervating enteric neurons co-operates with nitric oxide in modulating vagally induced contractions in the mouse oesophagus.

Galanin in Alzheimer's disease: neuroinhibitory or neuroprotective?

Galanin (GAL) and GAL receptors (GALRs) are overexpressed in degenerating brain regions associated with cognitive decline in Alzheimer's disease (AD). The functional consequences of GAL plasticity in AD are unclear. GAL inhibits cholinergic transmission in the hippocampus and impairs spatial memory in rodent models, suggesting GAL overexpression exacerbates cognitive impairment in AD. By contrast, gene expression profiling of individual cholinergic basal forebrain (CBF) neurons aspirated from AD tissue revealed that GAL hyperinnervation positively regulates mRNAs that promote CBF neuronal function and survival. GAL also exerts neuroprotective effects in rodent models of neurotoxicity. These data support the growing concept that GAL overexpression preserves CBF neuron function which in turn may slow the onset of AD symptoms. Further elucidation of GAL activity in selectively vulnerable brain regions will help gauge the therapeutic potential of GALR ligands for the treatment of AD.

Galanin, galanin receptors and drug targets.

Galanin, a neuropeptide widely expressed in the central and peripheral nervous systems and in the endocrine system, has been shown to regulate numerous physiological and pathological processes through interactions with three G-protein-coupled receptors, GalR1 through GalR3. Over the past decade, some of the receptor subtype-specific effects have been elucidated through pharmacological studies using subtype selective ligands, as well as through molecular approaches involving knockout animals. In the present review, we summarize the current data which constitute the basis of targeting GalR1, GalR2 and GalR3 for the treatment of various human diseases and pathological conditions, including seizure, Alzheimer's disease, mood disorders, anxiety, alcohol intake in addiction, metabolic diseases, pain and solid tumors.

Galanin in the regulation of pancreatic vascular perfusion.

OBJECTIVES: Acute pancreatitis is associated with compromised pancreatic microcirculation. Galanin is a vasoactive neuropeptide, but its role in the regulation of pancreatic vascular perfusion (PVP) is unclear. METHODS: Localization of galanin immunoreactivity was investigated by immunohistochemistry, and the effects of bolus doses of galanin or the antagonist galantide on blood pressure (BP) and PVP (by laser Doppler fluxmetry) were determined in anesthetized possums. RESULTS: Galanin immunoreactivity was abundant in the possum pancreas particularly around blood vessels. Galanin (0.001-10 nmol) produced a dose-dependent increase in BP (to 177% of baseline) and a complex PVP response consisting of a transient increase, then a fall below baseline with recovery to above baseline. Galantide (0.003-30 nmol) caused a dose-dependent biphasic response in BP, with a reduction, recovery, then a further fall, followed by recovery, whereas PVP increased (178%) then fell (to 56%) of baseline. Similar effects were produced by continuous intravenous infusion of galanin (1 and 10 nmol) or galantide (3 and 30 nmol). The second-phase response of these agents is probably a passive response of the pancreatic vasculature to systemic cardiovascular effects. CONCLUSIONS: These data suggest that galanin acutely reduces PVP, whereas galantide increases it, implying galanin may be important in the regulation of PVP.

The role of the neuropeptide galanin in forming type-specific behavioral characteristics.

Intranasal administration of a galanin receptor blocker to rats was found to change their behavioral type on being placed in an unfamiliar environment, with decreases in movement and investigative activity and increases in the level of anxiety in the open field test. The basal level of expression of the galanin precursor mRNA in the anterior hypothalamus was significantly higher in rats with the active type of behavior in the open field test. In conditions of galanin receptor blockade, there was also a faster increase in the serum corticosterone level in response to a stress situation (forced swimming test), which was accompanied by a reduction in the immobilization time. These data support the involvement of galanin in the formation of individual-typological behavioral characteristics and demonstrate its important role in adaptation to stress.

The role of galanin receptors in anticonvulsant effects of low-frequency stimulation in perforant path-kindled rats.

Low-frequency stimulation (LFS) has antiepileptogenic effects on kindled seizures. In the present study, the role of galanin receptors in the inhibitory effect of LFS on perforant path kindling acquisition was investigated in rats. Animals were kindled by perforant path stimulation in a rapid kindling manner (six stimulations per day). LFS (0.1 ms pulses at 1 Hz, 600 pulses, and 80-150 microA) was applied immediately after termination of each kindling stimulation. M35 (0.5 and 1.0 nM per site), a nonselective galanin receptor antagonist and M871 (1.0 microM per site), a selective galanin receptor type 2 (GalR2) antagonist, were daily microinjected into the dentate gyrus before starting the stimulation protocol. The expression of GalR2 in the dentate gyrus was also investigated using semi-quantitative RT-PCR. Application of LFS significantly retarded the kindling acquisition and delayed the expression of different kindled seizure stages. In addition, LFS significantly reduced the increment of daily afterdischarge duration during kindling development. Intra-dentate gyrus microinjection of both M35 and M871 significantly prevented the inhibitory effects of LFS on kindling parameters. During the focal kindled seizure stages (1-3) M871 had no significant effect. However, during generalized seizure stages (4 and 5), M871 had the same effect as M35. Semi-quantitative RT-PCR also showed that after kindling acquisition, the GalR2 mRNA level decreased in the dentate gyrus but application of LFS prevented this decrease. Obtained results show that activation of galanin receptors by endogenous galanin has a role in mediating the inhibitory effect of LFS on perforant path-kindled seizures. This role is exerted through GalR1 during focal- and through GalR2 during generalized-kindled seizures.

Differential effect of galanin on proliferation of PC12 and B104 cells.

The effect of galanin (GAL) on neural cell proliferation was studied using PC12 and B104 cells. Reverse transcription-PCR was used to determine the expression of GAL and GAL receptors and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay of cell viability was employed to detect the effects of GAL on cell proliferation. These studies revealed firstly that PC12 cells express mRNAs encoding all three GAL receptors (GalR1-3) but not GAL mRNA, whereas B104 cells express GAL, GalR2 and GalR3 mRNAs, but not GalR1 mRNA; and secondly that GAL inhibited the proliferation of PC12 cells, but in contrast significantly activated the proliferation of B104 cells. Moreover, these effects of GAL were blocked by M35, a nonselective, chimera peptide antagonist of GAL receptors. These data suggest that GAL can alter neural cell proliferation via GAL receptor activation, and that different GAL receptors and/or cellular complements of receptors produce different net effects via activation of different signaling pathways.