Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics.

Background: Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics. Methods: OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA). Results: Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako. Conclusions: New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics.

Injection of galanin into the dorsal hippocampus impairs emotional memory independent of 5-HT1A receptor activation.

There is evidence that interaction between the neuropeptide galanin and the 5-HT1A receptor represents an integrative mechanism in the regulation of serotonergic neurotransmission. Thus, in rats intracerebroventricular (i.c.v.) galanin did not impair retention in the passive avoidance (PA) test 24 h after training, but attenuated the retention deficit caused by subcutaneous (s.c.) administration of the 5-HT1A receptor agonist 8-OH-DPAT. This impairment has been linked to postsynaptic 5-HT1A receptor activation. To confirm these results in mice, galanin was infused i.c.v. (1 nmol/mouse) in C57BL/6/Bkl mice 30 min prior to training followed by s.c. injection (0.3 mg/kg) of 8-OH-DPAT or saline 15 min before PA training. In line with previous results, i.c.v. galanin significantly attenuated the PA impairment caused by 5-HT1A receptor activation in mice. To study if the galanin 5-HT1A receptor interaction involved the dorsal hippocampus, galanin (1 nmol/mouse) was directly infused into this brain region alone or in combination with s.c. 8-OH-DPAT. However, unlike i.c.v. galanin, galanin infusion into the dorsal hippocampus alone impaired PA retention and failed to attenuate the 8-OH-DPAT-mediated PA impairment. These results indicate that the ability of i.c.v. galanin to modify 5-HT1A receptor activation is not directly mediated via receptor interactions in the dorsal hippocampus. Instead, the galanin-mediated PA impairment suggests an important inhibitory role of galanin receptors in the dorsal hippocampus for acquisition (encoding) and/or consolidation of emotional memory. In addition, the interaction between galanin and 5-HT1A receptors probably involves a wide serotonergic network that is important for the integration of emotional and cognitive behaviors.

Neuropeptide and Small Transmitter Coexistence: Fundamental Studies and Relevance to Mental Illness.

Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanin's role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression-like behavior. The translational potential of these results was tested by studying the galanin system in postmortem human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a 'brake' to prevent overexcitation, a brake that is also part of resilience to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting - a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment.

Significant association and synergistic adverse prognostic effect of podocalyxin-like protein and epidermal growth factor receptor expression in colorectal cancer.

BACKGROUND: Podocalyxin-like 1 (PODXL) is an anti-adhesive transmembrane protein that has been demonstrated to be an independent factor of poor prognosis in colorectal cancer (CRC). The gene encoding PODXL is located to chromosome 7, which also harbours the gene for the epidermal growth factor receptor (EGFR). The aim of this study was to examine the associations between PODXL and EGFR expression in CRC in vitro and in vivo. METHODS: EGFR expression was analysed in tumours from three independent patient cohorts; cohort 1 (n = 533), cohort 2 (n = 259) and cohort 3 (n = 310), previously analysed for immunohistochemical PODXL expression and KRAS and BRAF mutations (cohort 1 and 3). Levels of EGFR and PODXL were determined by western blot in six different CRC cell lines. RESULTS: High expression of PODXL was significantly associated with high EGFR expression (p < 0.001) in all three cohorts, and with BRAF mutation (p < 0.001) in cohort 1 and 3. High EGFR expression correlated with BRAF mutation (p < 0.001) in cohort 1. High EGFR expression was associated with adverse clinicopathological factors and independently predicted a reduced 5-year overall survival (OS) in cohort 1 (HR 1.77; 95 % CI 1.27-2.46), cohort 2 (HR 1.58; 95 % CI 1.05-2.38) and cohort 3 (HR 1.83; 95 % CI 1.19-2.81). The highest risk of death within 5 years was observed in patients with tumours displaying high expression of both EGFR and PODXL in cohort 1 and 3 (HR 1.97; 95 % CI 1.18-3.28 and HR 3.56; 95 % CI 1.75-7.22, respectively). Western blot analysis showed a uniform expression of PODXL and EGFR in all six examined CRC cell lines. CONCLUSIONS: The results from this study demonstrate that high expression of EGFR is an independent factor of poor prognosis in CRC. Moreover, strong links have been uncovered between expression of the recently proposed biomarker candidate PODXL with EGFR expression in CRC in vivo and in vitro, and with BRAF mutation in vivo. High expression of both PODXL and EGFR may also have a synergistic adverse effect on survival. These findings suggest a potential functional link in CRC between PODXL, EGFR and BRAF, all originating from chromosome 7, which may be highly relevant in the clinical setting and therefore merit future in-depth study.

SATB1 is an independent prognostic factor in radically resected upper gastrointestinal tract adenocarcinoma.

Gastric cancer is the second most common cause of cancer-related death worldwide, and the incidence of esophageal adenocarcinoma is rising. While some progress has been made in treatment strategies, overall survival remains very poor for patients with adenocarcinoma in the upper gastrointestinal tract. Special AT-rich sequence binding protein 1 (SATB1) is a global genome organizer that has been demonstrated to promote aggressive tumor behavior in several different types of cancer, including gastric cancer. The prognostic value of SATB1 expression in esophageal cancer has, however, not yet been described. In this study, expression of SATB1 was examined by immunohistochemistry on tissue microarrays prepared from tissue samples from 175 patients with adenocarcinoma of the esophagus, cardia, or stomach and containing normal tissue, intestinal metaplasia, primary tumors, and metastases. A well-validated antibody was used. We found SATB1 to be an independent prognostic factor in patients with a radically resected tumor, correlating with shorter overall survival as well as with shorter recurrence-free survival. SATB1 expression was also found to be significantly lower in primary tumors associated with intestinal metaplasia than those without intestinal metaplasia. This observation is of potential biological interest as it has been proposed that intestinal metaplasia-associated tumors constitute a less aggressive phenotype.

Correlation between the effects of local and intracerebroventricular infusions of galanin on 5-HT release studied by microdialysis, and distribution of galanin and galanin receptors in prefrontal cortex, ventral hippocampus, amygdala, hypothalamus, and striatum of awake rats.

The neuropeptide galanin is implicated in regulation of affective behavior, including modulation of 5-HT signaling. Here, we investigated, by use of microdialysis in freely moving rats, the effects of intracerebral (i.c.) and intracerebroventricular (i.c.v.) infusions of galanin on basal extracellular 5-HT levels in medial prefrontal cortex (mPFC), CA1 area of ventral hippocampus (vHPC), central amygdaloid nucleus (CeA), ventromedial hypothalamic nucleus ventrolateral part (VMHvl), and ventromedial caudate putamen (CPu). These results were compared with a parallel immunohistochemical analysis of the distribution of galanin, 5-HT, and noradrenaline (NA) nerve terminals, and with data on galanin receptors. Galanin i.c.v. significantly decreased the 5-HT levels in mPFC to 79% and in vHPC to 72%. Local infusions of galanin caused a long-lasting decrease in 5-HT levels in vHPC to 88%, and a moderate decrease in CeA, whereas the 5-HT levels in mPFC significantly increased to 121%. These effects of i.c. galanin correlated well with the density of 5-HT and galanin nerve terminals and galanin receptors autoradiography in mPFC, vHPC, and CeA. No effects of i.c. or i.c.v. galanin on 5-HT levels were observed in CPu or VMHvl, in agreement with the low numbers of galanin-positive terminals and low/moderate galanin receptor density. Galanin was often found to coexist in NA, but could never be detected in 5-HT terminals. Together the results show a neuroanatomical correlation between the effects of galanin infusions on 5-HT release and distribution of galanin and its receptors, and that i.c.v. and i.c. administration can give opposite effects on 5-HT release.

Characterization of CGRP protein expression in "satellite-like" cells and dendritic arbours of the mouse olfactory bulb.

The main olfactory bulb (OB) is made up of several concentric layers, forming circuitries often involving dendro-dendritic synapses. Important interactions between OB neurons occur in the external plexiform layer (EPL), where dendrites of tufted and Van Gehuchten cells form synapses with dendrites of deeper lying mitral, tufted, and granule cells. OB neurons display a variety of neurotransmitters. Here, the focus is on calcitonin gene-related peptide (CGRP), a 37-amino acid neuropeptide transmitter that is widely distributed in the central and peripheral nervous system. In the OB, CGRP-immunoreactive (ir) cell bodies were mostly observed in the mitral cell layer (MCL) of normal mice, and their number increased following colchicine treatment. Sparsely distributed CGRP-ir cell bodies were also found in the EPL and granular cell layer. Double-immunofluorescence experiments revealed a lack of co-localization between CGRP-like immunoreactivity (LI) and corticotropin-releasing factor- or galanin-LI, two markers for mitral cells, and no CGRP-LI was found in cholecystokinin-, parvalbumin-, or vasoactive intestinal polypeptide-ir tufted/Van Gehuchten cells. CGRP-ir cell bodies were not found to co-localize glutamic acid decarboxylase 67 (GAD67)-green fluorescence protein, gamma-aminobutyric acid (GABA)-, or calretinin-LI, although the possibility remains that CGRP-ir cells may contain low levels of GABA and/or GAD67 not detected by our methodology. Dendrites of CGRP-ir cells extensively ramified deep in the EPL and double-immunofluorescence revealed them to be adjacent with, often apparently contacting, dendrites of granule, mitral, tufted, and Van Gehuchten cells. We propose that these CGRP-ir cell bodies in the mouse OB are "satellite-like" cells within and, occasionally, close to the MCL.

Galanin, galanin receptor subtypes and depression-like behaviour.

The pathophysiology of depression remains unclear, but involves disturbances in brain monoaminergic transmission. Current antidepressant drugs, which act by enhancing this type of neurotransmission, have limited therapeutic efficacy in a number of patients, and also cause serious side-effects, which limits their compliance. Increasing evidence suggests that neuropeptides, including galanin, can be of relevance in mood disorders. Galanin is co-expressed with and modulates noradrenaline and serotonin transmission, both implicated in depression. Pharmacological and genetic studies suggest a role for galanin in depression-like behaviour in rodents, involving specific receptor subtypes. Thus, stimulation of GalR1 and/or GalR3 receptors results in depression-like phenotype, while activation of the GalR2 receptor reduces depression-like behaviour in the rat. These findings suggest that galanin receptor subtypes may represent novel targets for the development of antidepressant drugs.

Neuropeptides in learning and memory processes with focus on galanin.

Neuropeptides represent by far the most common signalling molecules in the central nervous system. They are involved in a wide range of physiological functions and can act as neurotransmitters, neuromodulators or hormones in the central nervous system and in the periphery. Accumulating evidence during the past 40 years has implicated a number of neuropeptides in various cognitive functions including learning and memory. A major focus has been on the possibility that neuropeptides, by coexisting with classical neurotransmitters, can modulate classical transmitter function of importance for cognition. It has become increasingly clear that most transmitter systems in the brain can release a cocktail of signalling molecules including classical transmitters and several neuropeptides. However, the neuropeptides seem to come into action mainly under conditions of severe stress or aversive events, which have linked their action also to regulation of affective components of behaviour. This paper summarises some of the results of three neuropeptides, which can impact on hippocampal cognition by intrinsic (dynorphins, nociceptin) or extrinsic (galanin) modulation. The results obtained with these neuropeptides in rodent studies indicate that they are important for various aspects of hippocampal learning and memory as well as hippocampal plasticity. Recent studies in humans have also shown that dysregulation of these neuropeptides may be of importance for both neurodegenerative and neuropsychiatric disorders associated with cognitive impairments. It is concluded that compounds acting on neuropeptide receptor subtypes will represent novel targets for a number of disorders, which involve cognitive deficiencies.

Differential role of galanin receptors in the regulation of depression-like behavior and monoamine/stress-related genes at the cell body level.

The present study on rat examined the role of galanin receptor subtypes in regulation of depression-like behavior as well as potential molecular mechanisms involved in the locus coeruleus (LC) and dorsal raphe (DR). The effect of intracerebroventricular (i.c.v.) infusion of galanin or galanin receptor GalR1- and GalR2-selective ligands was studied in the forced swim test, followed by quantitative in situ hybridization studies. Naive control, non-treated (swim control), saline- and fluoxetine-treated rats were used as controls in the behavioral and in situ hybridization studies. Subchronic treatment with fluoxetine reduced immobility and climbing time. Intracerebroventricular infusion of galanin, the GalR1 agonist M617 or the GalR2 antagonist M871 increased, while the GalR2(R3) agonist AR-M1896 decreased, immobility time compared to the aCSF-treated animals. Galanin also decreased the time of climbing. Galanin mRNA levels were upregulated by the combination of injection+swim stress in the saline- and the fluoxetine-treated groups in the LC, but not in the DR. Also tyrosine hydroxylase levels in the LC were increased following injection+swim stress in the saline- and fluoxetine-treated rats. Tryptophan hydroxylase 2 and serotonin transporter mRNAs were not significantly affected by any treatment. 5-HT(1A) mRNA levels were downregulated following i.c.v. galanin, M617 or AR-M1896 infusion. These results indicate a differential role of galanin receptor subtypes in depression-like behavior in rodents: GalR1 subtype may mediate 'prodepressive' and GalR2 'antidepressant' effects of galanin. Galanin has a role in behavioral adaptation to stressful events involving changes of molecules important for noradrenaline and/or serotonin transmission.

Galanin enhances and a galanin antagonist attenuates depression-like behaviour in the rat.

The effect of intracerebroventricular infusion of galanin and/or the galanin antagonist M35 was studied in the forced swim test. Animals were pre-exposed to water for 15 min 24 h prior to test. Immobility and climbing were assessed during the second, 5 min exposure to water. Rats receiving a single infusion of galanin (3 nmol) displayed a significant increase of immobility. This effect was blocked by co-administration of M35 (1 nmol). M35 alone (1 nmol) produced a significant decrease of immobility. The results further support the hypothesis that galanin may play a role in mood disorders, and that galanin antagonists may represent new candidates for antidepressant treatment.

Galanin receptor antagonists : a potential novel pharmacological treatment for mood disorders.

The pathophysiology of mood disorders involves several genetic and social predisposing factors, as well as a dysregulated response to chronic stress. Accumulated evidence during the last two decades has implicated disturbances in brain serotonin and/or noradrenaline (norepinephrine) neurotransmission in the aetiology of depression. In fact, current pharmacological treatment for mood disorders is based on the use of drugs that act mainly by enhancing brain serotonin and noradrenaline neurotransmission by blockade of the active reuptake mechanism for these neurotransmitters. However, current antidepressant drugs have a delayed onset of therapeutic action, and a substantial number of patients do not respond adequately to them. In addition, these drugs have a number of adverse effects that limit patient compliance. In view of this, there is an intense search to identify novel (receptor) targets for antidepressant therapy. Recent studies have indicated that several neuropeptides and their receptors are potential candidates for the development of novel antidepressant treatment. In this context, galanin is of particular interest, since it is co-localised with serotonin in the dorsal raphe nucleus and with noradrenaline in the locus coeruleus, nuclei known to play a major role in affective disorders and in the action of antidepressant drugs. The actions of galanin are mediated by three receptor subtypes (GAL1, GAL2 and GAL3), which are coupled to different intracellular effector systems. Studies in rats have shown that galanin administered intracerebroventricularly is a potent inhibitor of mesencephalic serotonergic neurotransmission, as indicated by a long-lasting reduction in the release of serotonin in the hippocampus. This inhibitory effect is related to activation of the galanin receptors located on the dorsal raphe neurons. Moreover, intracerebroventricular galanin alters the gene expression of serotonin 5-HT1A autoreceptors in the dorsal raphe and also changes their functional activity. In addition, galanin produces a functional blockade of postsynaptic 5-HT1A receptor-mediated responses. Both pharmacological and genetic studies suggest a role for galanin in depression-like behaviour in rodent models. Transgenic mice overexpressing galanin under the control of the platelet-derived growth factor-beta promoter display increased immobility in the forced swim test. Intracerebroventricular administration of galanin in the rat increases depression-like behaviour, and this is fully blocked by the nonselective peptide galanin receptor antagonist M35. Importantly, M35 alone administered intracerebroventricularly produces an antidepressant-like effect. Recently, newly developed receptor-specific nonpeptidergic galanin GAL3 receptor antagonists (SNAP-37889 and SNAP-398299), which cross the blood-brain barrier after systemic administration, have shown antidepressant-like activity in several animal models. On the other hand, stimulation of the GAL2 receptor at the raphe level by local application of the GAL2 receptor agonist galanin (2-11) has been shown to increase serotonin levels in the hippocampus and dorsal raphe. These results indicate an important (mainly inhibitory) role of galanin as a regulator of brain serotonin and 5-HT1A receptor-mediated transmission, which may be of potential importance for understanding mood disorders and for the development of antidepressant drugs. Taken together, the present evidence suggests that antidepressant efficacy may be associated with compounds acting as antagonists at the GAL3 and/or possibly GAL1 receptors, and/or agonists at the GAL2 receptor.

Galanin expressed in the excitatory fibers attenuates synaptic strength and generalized seizures in the piriform cortex of mice.

The neuropeptide galanin is considered to be an endogenous antiepileptic agent, presumably acting via inhibition of glutamate release. Previously, we have demonstrated that in mice ectopically overexpressing galanin in cortical and hippocampal neurons, particularly in granule cells and their axons, the mossy fibers, hippocampal kindling epileptogenesis is suppressed and is associated with attenuated frequency facilitation in mossy fiber-CA3 cell synapses. We hypothesized that changes in synaptic transmission might occur also in other excitatory synapses of the galanin overexpressing (GalOE) mouse, contributing to seizure suppression. Lateral olfactory tract (LOT) synapses, formed by axons of olfactory bulb (OB) mitral cells and targeting piriform cortex (PC) pyramidal cells, ectopically express galanin in GalOE mice. Using whole-cell patch-clamp recordings, we found that excitatory synaptic responses recorded in PC pyramidal cells during high frequency stimulation of the LOT were attenuated in GalOE mice as compared to wild-type controls. This effect was mimicked by bath application of galanin or its agonist galnon to wild-type slices, supporting the notion of ectopic galanin action. Since the high frequency activation induced in vitro resembles epileptic seizures in vivo, we asked whether the observed synaptic inhibition would result in altered epileptogenesis when animals were kindled via the same synapses. In male GalOE mice, we found that the latency to convulsions was prolonged, and once animals had experienced the first stage 5 seizure, generalized seizures were less sustainable. These data indicate that the PC is a possible target for epilepsy treatment by ectopically overexpressing galanin to modulate seizure activity.

Age-related impairments of synaptic plasticity in the lateral perforant path input to the dentate gyrus of galanin overexpressing mice.

In the present study, electrophysiological recordings were made from hippocampal slices obtained from mice overexpressing galanin under the promoter for the platelet-derived growth factor-B (GalOE mice). In these mice, a particularly strong galanin expression is seen in the granule cell layer/mossy fibers. Paired-pulse facilitation (PPF) of excitatory postsynaptic field potentials (fEPSPs) at the lateral perforant path (LPP)-dentate gyrus synapses was elicited in the dentate gyrus after stimulation with different interpulse intervals. Slices from young adult wild-type (WT) animals showed significant PPF of the 2nd EPSP evoked with paired-pulse stimuli, while PPF was reduced in slices from young adult GalOE mice, as well as aged WT mice, but were not observed at all in slices from aged GalOE animals. Application of the putative galanin antagonist M35 increased PPF in slices from aged WT mice as well as from adult and aged GalOE mice, but had no effect in slices taken from young adult WT mice. These data indicate that galanin is involved in hippocampal synaptic plasticity, in particular in age-related reduction of synaptic plasticity in the LPP input to the dentate gyrus. Galaninergic mechanisms may therefore represent therapeutic targets for treatment of age-related memory deficits and Alzheimer's disease.

Distribution of galanin in the brain of a galanin-overexpressing transgenic mouse.

The distribution of galanin mRNA-expressing cells and galanin-immunoreactive (IR) cell bodies and processes was studied in the brain of mice overexpressing galanin under the PDGF-B promoter (GalOE mice) and of wild type (WT) mice, both in colchicine-treated and non-treated animals. A widespread ectopic expression of galanin (both mRNA and peptide) was found, that is when neither transcript nor peptide could be seen in WT mice, not even after colchicine treatment. However, in some regions, such as claustrum, basolateral amygdala, thalamus, CA1 pyramidal cells, and Purkinje cells only galanin mRNA could be detected. The highest levels of galanin expression were observed in the forebrain structures (the mitral cells of the olfactory bulb, throughout the cortex, granular and pyramidal cell layers of the hippocampus), in the mesencephalon (nucleus ruber), in the cerebellum (lateral cerebellar nucleus), in the pons (sensory and motor nuclei of the trigeminal nerve), within the medulla oblongata (facial, prepositus and spinal trigeminal nuclei). High densities of galanin-IR fibers were found in the axonal terminals of the lateral olfactory tract, hippocampal and presumably cerebellar mossy fiber system, in several thalamic and hypothalamic regions and the lower brain stem.

Behavioural characterisation of transgenic mice overexpressing galanin under the PDGF-B promoter.

The behavioural phenotype of transgenic mice (3-5-months old) overexpressing galanin (GalOE mice) under the platelet-derived growth factor B (PDGF-B) promoter was evaluated in a battery of tests, including locomotor cages, light-dark exploration test, elevated plus-maze and the Porsolt forced swim test. Learning and memory were assessed in the Morris water maze task. GalOE mice showed a slight increase in spontaneous locomotor activity assessed in the locomotor cages, but the amphetamine-induced increase in locomotor activity was somewhat lower in GalOE mice. Anxiety-like behaviour in light-dark exploration and elevated plus-maze tests did not differ between genotypes. In the Porsolt forced swim test, GalOE mice displayed an increased time of immobility, indicative of increased learned helplessness possibly reflecting increased stress-susceptibility and/or depression-like behaviour. GalOE mice showed normal learning and memory retention in the Morris water maze tasks. These data support the hypothesis that galanin may have a role in functions related to mood states, including affective disorders.

Behavioural characterisation of young adult transgenic mice overexpressing galanin under the PDGF-B promoter.

The behavioural phenotype of transgenic mice (3- to 5-months old) overexpressing galanin (GalOE) under the platelet-derived growth factor B (PDGF-B) promoter was evaluated in a battery of tests, including open field, locomotor cages, light-dark exploration test, elevated plus-maze and the Porsolt forced swim test. Learning and memory were assessed in the passive avoidance and the Morris water maze tasks. No difference between genotypes was found in exploratory activity in the open field. GalOE mice showed a slight increase in spontaneous locomotor activity assessed in the locomotor cages, but the amphetamine-induced increase in locomotor activity was somewhat lower in GalOE mice. Anxiety-like behaviour in the three different tests including open field, light-dark exploration and elevated plus-maze did not differ between genotypes. In the Porsolt forced swim test, GalOE mice displayed an increased time of immobility, indicative of increased learned helplessness possibly reflecting increased stress-susceptibility and/or depression-like behaviour. GalOE mice showed normal learning and memory retention in the passive avoidance and the Morris water maze tasks. These data support the hypothesis that galanin may have a role in functions related to mood states including affective disorders.

Distribution of galanin and galanin transcript in the brain of a galanin-overexpressing transgenic mouse.

The distribution of galanin mRNA-expressing cells and galanin-immunoreactive (IR) cell bodies and processes was studied in the brain of mice overexpressing galanin under the PDGF-B promoter (GalOE mice) and of wild type (WT) mice, both in colchicine-treated and non-treated animals. In this abstract, we only describe the results in GalOE mouse. A widespread ectopic expression of galanin (both mRNA and peptide) was found, that is a situation when neither transcript nor peptide could be seen in WT mice, not even after colchicine treatment. However, in some regions, such as claustrum, basolateral amygdala, thalamus, CA1 pyramidal cells, and Purkinje cells only galanin mRNA could be detected. In the forebrain galanin was seen in the mitral cells of the olfactory bulb, throughout the cortex, in the basolateral amygdaloid nucleus, claustrum, granular and pyramidal cell layers of the hippocampus, subiculum and presubiculum. In the thalamus, the anterodorsal, mediodorsal, intermediodorsal and mediodorsal lateral nuclei, the reuniens and reticular nuclei showed ectopic expression of galanin. Within the hypothalamus, neurons of the suprachiasmatic nucleus contained galanin. In the mesencephalon, the geniculate nucleus, nucleus ruber, the mesencephalic trigeminal and reticulotegmental nuclei ectopically expressed galanin. In the cerebellum, galanin was observed in the Purkinje cells and in the lateral and interposed cerebellar nuclei. In the pons, sensory and motor nuclei of the trigeminal nerve, the laterodorsal and dorsal tegmental nuclei, the pontine, reticulotegmental and gigantocellular reticular nuclei expressed galanin. Within the medulla oblongata, labeled cells were detected in the facial, ambiguus, prepositus, lateral paragigantocellular and lateral reticular nuclei, and spinal trigeminal nucleus. High densities of galanin-IR fibers were found in the axonal terminals of the lateral olfactory tract, the hippocampal and presumably the cerebellar mossy fibers system, in several thalamic and hypothalamic regions and the lower brain stem. Possible functional consequences of galanin overexpression are discussed.

Enhanced hippocampal noradrenaline and serotonin release in galanin-overexpressing mice after repeated forced swimming test.

Basal and forced swimming (FS) stress-induced release of noradrenaline (NA) and serotonin (5-HT) were determined by in vivo microdialysis in the ventral hippocampus of mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE/P) or under the dopamine beta-hydroxylase promoter (GalOE/D) (only NA). WT mice served as controls. Intraventricular infusion of galanin significantly reduced basal extracellular NA in WT mice and in GalOE/P mice (albeit less so). Microdialysis sampling during a 10-min FS showed that NA and 5-HT release were elevated to 213% and 156%, respectively, in the GalOE/P group, whereas in the WT group the increases were only 127% and 119%, respectively. The second (repeated) 10-min FS (RFS) caused a marked enhancement of NA and 5-HT release in the GalOE/P mice to 344% and 275%, respectively. However, the RFS caused only a 192% increase of extracellular NA levels in the GalOE/D mice. Pretreatment with the putative peptidergic galanin receptor antagonist M35 almost completely blocked the elevation of NA and 5-HT levels in the GalOE/P after RFS. These results suggest that the NA and 5-HT hippocampal afferents in GalOE/P mice are hypersensitive to both conditioned and unconditioned stressful stimuli, such as FS, and that this effect is mediated by galanin receptors. The present findings support a role of galanin in the regulation of release of NA and 5-HT, two neurotransmitters involved in mood control.