Distribution of galanin receptor 2b neurons and interaction with galanin in the zebrafish central nervous system.

Galanin is a multifunctional neuropeptide that is implicated in the modulation of physiological processes, including nociception, cognition, feeding behavior, neuronal growth, and reproduction. The physiological effects of galanin are mediated through its interaction with three different G protein-coupled receptors, i.e., GALR1, GALR2, and GALR3. Unlike mammals, zebrafish have four different receptors for galanin, diversified from GALR1 (GAL1a and GALR1b) and GALR2 (GALR2a and GALR2b). Despite the importance of galanin in the central nervous system (CNS), no information has been reported regarding GalR2 in zebrafish CNS. In this study, we found that galr2a is expressed at low levels in restricted areas of the brain; however, galr2b was widely expressed in CNS including olfactory bulb, midbrain tegmentum, preoptic region, dorsal thalamus, posterior tuberculum, postoptic commissure, hindbrain, and spinal cord. To further analyze the distribution of GALR2b neurons and their interaction with GAL, we generated Tg(galr2b:egfp) zebrafish, which express enhanced green fluorescent protein (EGFP) under the control of a galr2b promoter. Investigation of the CNS of transgenic reporter zebrafish revealed that galr2b:EGFP(+) neurons are distributed and interact with galanin-immunoreactive (galanin-IR) cells in various regions of the brain and spinal cord. We found that in some regions of the brain and spinal cord, galanin-IR nerve cells were not observed near galr2b:EGFP neurons, suggesting that GALR2b may have the potential to interact with other ligands instead of galanin in these regions.

Distribution of Galanin and Galanin Receptor 2 in the Pre-optic Area of the Female Guinea Pig.

This study describes the distribution of galanin (Gal) and galanin receptor 2 (GalR2) in the pre-optic area (POA) of the female guinea pig. Frozen sections were undergone for a routine immunofluorescence labelling. Gal and GalR2 display immunoreactivity in all parts of the pre-optic area. Gal shows reactivity both in perikarya and fibres, whereas GalR2 was observed only in perikarya. Gal- and GalR2-immunoreactive (-ir) perikarya were the most numerous in the medial pre-optic area (MPA) with the highest reactivity in its dorsal part. In the median pre-optic nucleus (MPN) and periventricular pre-optic nucleus (PPN), only single Gal- and GalR2-ir neurons were observed. The highest density of Gal-ir fibres was revealed in the PPN and the lowest in the lateral pre-optic area (LPA). The results of this study indicate that the distribution pattern of Gal containing neurons overlaps well with the distribution pattern of GalR2-positive neurons, especially in the MPA. This may suggest GalR2-dependent activity in this brain region.

Analyzing the validity of GalR1 and GalR2 antibodies using knockout mice.

G-protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors and are the major drug targets for the treatment of various human diseases. The lack of sensitive and selective antibodies capable of recognizing endogenous GPCRs, however, hampers the progress of research on this class of receptors. GalR1 through GalR3, GPCRs for the neuropeptide galanin, are potential drug targets for seizure, Alzheimer's disease, depression and anxiety, as well as pain and metabolic syndrome; therefore, determining the cellular and subcellular localization of galanin receptors is of high interest. Several Antibodies raised against galanin receptors are currently available from commercial or academic sources. We have tested several antibodies to GalR1 and GalR2 on tissues from respective knockout mice. Unexpectedly, the immunoreactivity patterns are the same in wild-type and in knockout mice, suggesting that current GalR1 and GalR2 antibodies, under standard immunodetection conditions, might not be suitable for mapping the receptors. These findings argue for taking precaution when using antibodies to galanin receptors.

Immunohistochemical localization of galanin receptors (GAL-R1, GAL-R2, and GAL-R3) on myenteric neurons from the sheep and dog stomach.

Galanin exerts its biological activities (inhibitory or excitatory) via three different G protein-coupled receptors. In the present study, double immunocytochemical labeling was used to localize GAL-R1, GAL-R2 and GAL-R3 on PGP 9.5-positive myenteric neurons from the dog and sheep stomach/forestomachs. In both species, the occurrence of galanin in neurons and nerve fibers of gastric ganglia was also studied. Myenteric ganglia of the dog stomach were supplied with numerous, mainly varicose, galanin-immunoreactive (IR) nerve terminals whereas the frequency of galanin-positive nerve fibers in myenteric ganglia of the ovine stomach and forestomachs was moderate. The number of PGP 9.5-IR/galanin-IR myenteric neurons was significantly lower in the dog stomach (12.3+/-1.3%) as compared to the sheep rumen (20.1+/-0.7%), omasum (19.5+/-2.9%), abomasum (23.8+/-1.2%) but not reticulum (8.1+/-0.8%). In the canine stomach the frequencies of GAL-R1, GAL-R2 and GAL-R3 expressing myenteric neurons were statistically equivalent (4.4+/-0.9%, 3.5+/-0.7% and 3.1+/-0.5%, respectively). Immunoreactivity to GAL-R1 was absent in myenteric ganglia from the ovine rumen, reticulum as well as omasum. GAL-R1 was localized on 0.5+/-0.3% of myenteric perikarya from the abomasum. GAL-R2 bearing myenteric neurons were localized in the ovine rumen (0.6+/-0.3%), reticulum (0.5+/-0.3%), omasum (1.0+/-0.2%) and abomasum (1.1+/-0.3%). The percentages of PGP 9.5-IR/GAL-R3-IR neurons were 0.8+/-0.2% in the rumen, 0.6+/-0.3% in the reticulum, 0.7+/-0.2% in the omasum and 0.9+/-0.3% in the abomasum. In all compartments of the sheep stomach, the proportions of GAL-R1, GAL-R2 and GAL-R3 expressing neurons were significantly lower when compared to analogous neuronal subpopulations present in the dog. It is suggested that, although endogenous galanin may potentially inhibit or stimulate the activity of sparse gastric enteric neurons, its general role in indirect mediation of gastric motility and/or secretion seems to be of minor importance.

Prenatal exposure to undernutrition and programming of responses to high-fat feeding in the rat.

Fetal undernutrition programmes risk of later metabolic disorders. Postnatal factors modify the programmed phenotype. This study aimed to assess the effects of a postnatal high-fat (HF) challenge on body weight gain, adiposity and gene expression following prenatal undernutrition. Pregnant rats were fed either a control diet or a low-protein (LP) diet, targeted at days 0-7 (LPE), days 8-14 (LPM), or days 15-22 (LPL) gestation. At 12 weeks of age offspring were either fed standard laboratory chow diet (4.13 % fat), or a 39.5 % fat diet, for 10 weeks. LP exposure had no effect on weight gain or abdominal fat in males. Females exposed to LP diet in utero exhibited a similar weight gain on HF diet as on the chow diet. Programming of fat deposition was noted in LPE females and males of the LPM and LPL groups (P = 0.019). Hypothalamic expression of galanin mRNA was similar in all groups, but expression of the galanin-2 receptor was modified by LP exposure in female offspring. Hepatic expression of sterol response element binding protein (SREBP-1c) was decreased by LP at both the mRNA (P = 0.008) and protein (P < 0.001) level. HF feeding increased expression of SREBP-1c mRNA three-fold in controls, with little response noted in the LP groups. Interactions of factors such as postnatal diet, age and sex act together with prenatal factors to determine metabolic function and responsiveness at any stage of postnatal life. This study further establishes a role for prenatal nutrition in programming the genes involved in lipid metabolism and appetite regulation.

Galanin receptor subtype GalR2 mediates apoptosis in SH-SY5Y neuroblastoma cells.

Recently we have shown that galanin binding significantly correlates with survival in neuroblastoma patients, indicating a possible modulatory role of galanin receptors in neuroblastic tumor biology. However, the molecular mechanisms beyond this correlation have not been elucidated. Here, the cellular effects on activation of specific galanin receptor subtypes in human SH-SY5Y neuroblastoma cells were analyzed using a tetracycline-controlled expression system. Pharmacological studies confirmed the inducible expression of high affinity binding sites for galanin in SH-SY5Y cells transfected with the galanin receptors GalR1 (SY5Y/GalR1) and GalR2 (SY5Y/GalR2). Microphysiometry revealed that both receptor subtypes were able to mediate an intracellular signal upon galanin application. Interestingly, induction of receptor expression and treatment with 100 nm galanin resulted in a dramatic decrease in cell viability in SY5Y/GalR2 cells (93 +/- 3%) compared with a less pronounced effect in SY5Y/GalR1 cells (19 +/- 10%). The antiproliferative potency of galanin was 100-fold higher in SY5Y/GalR2 (50% effective concentration, 1.1 nm) than in SY5Y/GalR1 cells (50% effective concentration, 190 nm). Furthermore, activation of receptor expression and exposure to galanin resulted in apparent morphological changes indicative of apoptosis in SY5Y/GalR2 cells only. Induction of cell death by the apoptotic process was confirmed by poly-(ADP-ribose)-polymerase cleavage, caspase-3 activation, and the typical laddering of DNA. This study indicates that a high level of GalR2 expression is able to inhibit cell proliferation and induce apoptosis in neuroblastoma cells and therefore identifies GalR2 as a possible target for pharmacological intervention in neuroblastoma.