Galanin-Expressing GABA Neurons in the Lateral Hypothalamus Modulate Food Reward and Noncompulsive Locomotion.

The lateral hypothalamus (LHA) integrates reward and appetitive behavior and is composed of many overlapping neuronal populations. Recent studies associated LHA GABAergic neurons (LHA GABA ), which densely innervate the ventral tegmental area (VTA), with modulation of food reward and consumption; yet, LHA GABA projections to the VTA exclusively modulated food consumption, not reward. We identified a subpopulation of LHA GABA neurons that coexpress the neuropeptide galanin (LHA Gal ). These LHA Gal neurons also modulate food reward, but lack direct VTA innervation. We hypothesized that LHA Gal neurons may represent a subpopulation of LHA GABA neurons that mediates food reward independent of direct VTA innervation. We used chemogenetic activation of LHA Gal or LHA GABA neurons in mice to compare their role in feeding behavior. We further analyzed locomotor behavior to understand how differential VTA connectivity and transmitter release in these LHA neurons influences this behavior. LHA Gal or LHA GABA neuronal activation both increased operant food-seeking behavior, but only activation of LHA GABA neurons increased overall chow consumption. Additionally, LHA Gal or LHA GABA neuronal activation similarly induced locomotor activity, but with striking differences in modality. Activation of LHA GABA neurons induced compulsive-like locomotor behavior; while LHA Gal neurons induced locomotor activity without compulsivity. Thus, LHA Gal neurons define a subpopulation of LHA GABA neurons without direct VTA innervation that mediate noncompulsive food-seeking behavior. We speculate that the striking difference in compulsive-like locomotor behavior is also based on differential VTA innervation. The downstream neural network responsible for this behavior and a potential role for galanin as neuromodulator remains to be identified.SIGNIFICANCE STATEMENT The lateral hypothalamus (LHA) regulates motivated feeding behavior via GABAergic LHA neurons. The molecular identity of LHA GABA neurons is heterogeneous and largely undefined. Here we introduce LHA Gal neurons as a subset of LHA GABA neurons that lack direct innervation of the ventral tegmental area (VTA). LHA Gal neurons are sufficient to drive motivated feeding and locomotor activity similar to LHA GABA neurons, but without inducing compulsive-like behaviors, which we propose to require direct VTA innervation. Our study integrates galanin-expressing LHA neurons into our current understanding of the neuronal circuits and molecular mechanisms of the LHA that contribute to motivated feeding behaviors.

Dnmt3a in Sim1 neurons is necessary for normal energy homeostasis.

Obesity rates continue to rise throughout the world. Recent evidence has suggested that environmental factors contribute to altered energy balance regulation. However, the role of epigenetic modifications to the central control of energy homeostasis remains unknown. To investigate the role of DNA methylation in the regulation of energy balance, we investigated the role of the de novo DNA methyltransferase, Dnmt3a, in Single-minded 1 (Sim1) cells, including neurons in the paraventricular nucleus of the hypothalamus (PVH). Dnmt3a expression levels were decreased in the PVH of high-fat-fed mice. Mice lacking Dnmt3a specifically in the Sim1 neurons, which are expressed in the forebrain, including PVH, became obese with increased amounts of abdominal and subcutaneous fat. The mice were also found to have hyperphagia, decreased energy expenditure, and glucose intolerance with increased serum insulin and leptin. Furthermore, these mice developed hyper-LDL cholesterolemia when fed a high-fat diet. Gene expression profiling and DNA methylation analysis revealed that the expression of tyrosine hydroxylase and galanin were highly upregulated in the PVH of Sim1-specific Dnmt3a deletion mice. DNA methylation levels of the tyrosine hydroxylase promoter were decreased in the PVH of the deletion mice. These results suggest that Dnmt3a in the PVH is necessary for the normal control of body weight and energy homeostasis and that tyrosine hydroxylase is a putative target of Dnmt3a in the PVH. These results provide evidence for a role for Dnmt3a in the PVH to link environmental conditions to altered energy homeostasis.

Dynamics of appetite-mediated gene expression in daidzein-fed female rats in the meal-feeding method.

We previously found that daidzein decreased food intake in female rats. The present study aimed to elucidate the relationship between dynamics of appetite-mediated neuropeptides and the anorectic effect of daidzein. We examined appetite-mediated gene expression in the hypothalamus and small intestine during the 3 meals per day feeding method. Daidzein had an anorectic effect specifically at the second feeding. Neuropeptide-Y (NPY) and galanin mRNA levels in the hypothalamus were significantly higher after feeding in the control but not in the daidzein group, suggesting that daidzein attenuated the postprandial increase in NPY and galanin expression. The daidzein group had higher corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamus after feeding, and increased cholelcystokinin (CCK) mRNA levels in the small intestine, suggesting that CCK is involved in the hypothalamic regulation of this　anorectic effect. Therefore, daidzein may induce anorexia by suppressing expression of NPY and galanin and increasing expression of CRH in the hypothalamus.

Neurochemical heterogeneity of rats predicted by different measures to be high ethanol consumers.

BACKGROUND: Alcoholism is a heterogeneous disease, with subjects possibly differing both in the best measure that predicts their excess consumption and in their most effective pharmacotherapy. Two different measures, high novelty-induced activity and high-fat-induced triglycerides (TGs), are known to identify subgroups of animals prone to consuming higher amounts of ethanol (EtOH). The question investigated here is whether these subgroups are, in fact, similar in their neurochemical phenotype that may contribute to their overconsumption. METHODS: EtOH-naïve, Sprague-Dawley rats were subgrouped based on the 2 predictor measures of activity or TG levels, and then quantitative real-time polymerase chain reaction and digoxigenin-labeled in situ hybridization were used to measure their expression of hypothalamic peptides that affect EtOH intake. In additional subgroups subsequently trained to drink 9% EtOH, the opioid antagonist and alcoholism medication, naltrexone, was tested at a low dose (0.02 mg/kg, s.c.) to determine the rats' sensitivity to its effects. RESULTS: The 2 measures, while both effective in predicting amount of EtOH intake, were found to identify distinctive subgroups. Rats with high compared to low activity exhibited significantly greater expression of galanin and enkephalin in the paraventricular nucleus (PVN) and of orexin in the perifornical lateral hypothalamus (PFLH), but no difference in melanin-concentrating hormone in PFLH or neuropeptide Y in arcuate nucleus. This contrasts with rats having high TG, which exhibited greater expression only of PVN galanin, along with reduced PFLH orexin. The high-activity rats with elevated enkephalin, but not high-TG rats, were also unusually sensitive to naltrexone, which significantly reduced their alcohol intake. CONCLUSIONS: In addition to revealing differences in endogenous peptides and drug responsiveness in predicted high EtOH drinkers, this study demonstrates that these disturbances differ markedly between the 2 at-risk subgroups. This indicates that simple tests may be effective in identifying subjects most responsive to a specific pharmacotherapy.

Exogenous galanin attenuates spatial memory impairment and decreases hippocampal ß-amyloid levels in rat model of Alzheimer's disease.

One of the major pathological characteristics of Alzheimer's disease (AD) is the presence of enhanced deposits of beta-amyloid peptide (Aß). The neuropeptide galanin (GAL) and its receptors are overexpressed in degenerating brain regions in AD. The functional consequences of galaninergic systems plasticity in AD are unclear. The objective of the present study was to investigate whether exogenous galanin could attenuate spatial memory impairment and hippocampal Aß aggregation in rat model of AD. The effects of Aß, galanin, galanin receptor 1 agonist M617 and galanin receptor 2 agonist AR-M1896 on spatial memory were tested by Morris water maze. The effects of Aß, galanin, M617 and AR-M1896 on hippocampal Aß protein expression were evaluated by western blot assay. The expression of galanin, galanin receptors 1 and 2 in rats' hippocampus were detected by real time PCR and western blot assay. The results showed that (1) Galanin administration was effective in improving the spatial memory and decreasing hippocampal Aß levels after intracerebroventricular injection of Aß; (2) AR-M1896 rather than M617 could imitate these effects of galanin; (3) GAL and GALR2 mRNA and protein levels increased significantly in hippocampus after Aß administration, while GALR1 mRNA and protein levels did not change; (4) GAL, AR-M1896 and M617 administration did not show significant effect on GAL, GalR1 and GalR2 mRNA and protein levels in hippocampus after Aß administration. These results implied that galanin receptor 2, but not receptor 1 was involved in the protective effects against spatial memory impairment and hippocampal Aß aggregation.

[Establishment of an N-2a cell line stably expressing mouse galanin and the effect of over-expressed galanin on the proliferation and apoptosis of N-2a cell].

OBJECTIVE: To construct an N-2a cell line stably expressing PcDNA 3.1-platelet derived growth factor-galanin (GAL) and explore the effect of over-expressed GAL on proliferation and apoptosis of N-2a cell in vitro. METHODS: The vector containing the target gene was transfected into N-2a cells by liposome, and cell clones stably over-expressing GAL was obtained via G418 screening. GAL mRNA and protein levels were determined by reverse transcriotion-polymerase chain reaction (RT-PCR) and Western blot. The proliferation of N-2a cells was detected by MTT.The cell cycle and apoptosis were detected by flow cytometry. RESULTS: RT-PCT and Western blot indicated that GAL genes were highly expressed in the transfected N-2a cells (i.e.GAL-N-2a). As shown by MTT, the proliferation of the N-2a cells transfected with PcDNA 3.1-PDGF-GAL was significantly slower than the control group(P<0.05). Compared with the non-transfected cells in the control group, the N-2a cells with endogenously overexpressed GAL were arrested at the G0/G1 phases, and the over-expressed GAL protein significantly induced the N-2a cell apoptosis in a concentration-dependent fashion. CONCLUSION: Eukaryotic expression vector PcDNA 3.1-PDGF-GAL can encode the expression of GAL in N-2a cells. Aslo, it can inhibit cell proliferation and promote the cell apoptosis.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Nerve growth factor regulates galanin and neuropeptide Y expression in primary cultured superior cervical ganglion neurons.

Both galanin and neuropeptide Y (NPY) are expressed in superior cervical ganglion (SCG) neurons. Following nerve transection or axotomy galanin is strongly upregulated and NPY is downregulated in SCG neurons because target-derived nerve growth factor (NGF) content decreased. It is not known whether or to what extent NGF affects both galanin and NPY expression in primary cultured SCG neurons. In the present study we examine whether exogenous NGF affects expression of neuropeptides for galanin and NPY in primary cultured SCG neurons. In addition, we explore whether mRNAs for galanin and NPY are affected by administration of exogenous NGF in SCG cultures. The significance of expression of galanin and NPY and their mRNAs was revealed by performing experiments without and with administration of exogenous NGF. Galanin and its mRNA expression was attenuated by administration of exogenous NGF in SCG cultures. The enhancement of NPY and its mRNA expression by administration of exogenous NGF in SCG cultures was dose-dependent. The physiological or pathophysiological mechanisms of the alterations of galanin and NPY expression affected by NGF in primary cultured SCG neurons are still unknown. The present data provide basic knowledge about the expression of galanin and NPY in primary cultured SCG neurons of rats, which may further improve our understanding of the functional significance of galanin and NPY expression affected by NGF.

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo.

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.

Galanin: presence and distribution in the brain and pituitary of Rhinella arenarum (Amphibia: Anura) during development.

The immunohistochemical distribution of galanin (Gal) in the brain and pituitary of Rhinella arenarum was studied during development. Gal-immunoreactivity was first observed in the brain just after hatching in anterior preoptic area, infundibular area, median eminence and pars distalis of the pituitary as well as in the olfactory epithelium. At the beginning of prometamorphosis new Gal-immunoreactive (ir) cells were observed in the olfactory nerve and bulb. Later in prometamorphosis new Gal-ir cells were observed in the telencephalon, suprachiasmatic nucleus, rostral rhombencephalon and in the pars nervosa of the pituitary. The most numerous accumulations of Gal-ir neurons throughout the larval development were observed in the ventral hyphothalamus where numerous Gal-ir cells of cerebrospinal fluid-contacting type were found. During metamorphic climax and soon after we did not detect Gal-ir neurons in the pallium, medial or pretectal dorsal thalamus. In the median eminence and pars distalis of the pituitary many Gal-ir fibers were found during development indicating that Gal may play a role in the modulation of hypophyseal secretion. Furthermore, the distribution of Gal-ir elements observed throughout larvae development indicates that galaninergic system maturation continues until sexual maturity.

Changes in the expression and localization of signaling molecules in mouse facial motor neurons during regeneration of facial nerves.

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.

Galanin is up-regulated in colon adenocarcinoma.

The early diagnosis of colorectal cancer and the early detection of recurrence are central to effective treatment, as prognosis is directly related to the stage of the disease. When colorectal cancer is diagnosed at an early, localized stage, 5-year survival is 90%. There is substantial interest in the identification of circulating human tumor-derived proteins in serum for the purposes of early cancer diagnosis. We have implemented an approach based on the analysis of microarray data for the identification of tumor proteins that may have utility as biomarkers in colon cancer. Expression analysis of microarray data obtained from a variety of 290 tumors and normal tissues revealed that galanin was maximally expressed in colon cancer. These findings were corroborated by real-time quantitative PCR, in which the colon cancer cell lines LOVO, HCT15, SW480, and SW620 cell showed significantly higher levels of galanin expression than did noncolon cancer cell lines. To evaluate galanin as a potential biomarker of colon cancer, a preliminary "training" set of serum from 40 healthy donors and 55 colon cancer patients was analyzed by ELISA. The data pattern was confirmed by an independent set of 90 masked serum samples: 24 from healthy donors and 66 from colon cancer patients. This result yielded a sensitivity of 69.7% [95% confidence interval (95% CI), 57.1-80.4], specificity of 75.0% (95% CI, 53.3-90.2), and positive predictive value of 88.5% (95% CI, 76.6-95.7). The galanin expression level was significantly increased with tumor size and tumor stage. These findings justify a prospective assessment of serum galanin protein as a screening tool for colon cancer.

Galanin gene transfer curtails generalized seizures in kindled rats without altering hippocampal synaptic plasticity.

Gene therapy-based overexpression of endogenous seizure-suppressing molecules represents a promising treatment strategy for epilepsy. Viral vector-based overexpression of the neuropeptide galanin has been shown to effectively suppress generalized seizures in various animal models of epilepsy. However, it has not been explored whether such treatment can also prevent the epileptogenesis. Using a recombinant adeno-associated viral (rAAV) vector, we induced hippocampal galanin overexpression under the neuron specific enolase promoter in rats. Here we report that in animals with galanin overexpression, the duration of electrographic afterdischarges was shortened and initiation of convulsions was delayed at generalized seizure stages. However, the hippocampal kindling development was unchanged. Short-term plasticity of mossy fiber-cornu ammonis (CA) 3 synapses was unaltered, as assessed by paired-pulse and frequency facilitation of field excitatory postsynaptic potentials (fEPSPs) in hippocampal slices, suggesting that despite high transgene galanin expression, overall release probability of glutamate in these synapses was unaffected. These data indicate that hippocampal rAAV-based galanin overexpression is capable of mediating anticonvulsant effects by lowering the seizure susceptibility once generalized seizures are induced, but does not seem to affect kindling development or presynaptic short-term plasticity in mossy fibers.

Differentiation of human embryonal carcinomas in vitro and in vivo reveals expression profiles relevant to normal development.

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Increase of galanin in trigeminal ganglion during tooth movement.

It is known that nerve fibers containing neuropeptides such as galanin increase in the periodontal ligament during experimental tooth movement. However, the origin of galanin-containing nerve fibers in the periodontal ligament remains unclear. This study was conducted to examine our hypothesis that the increased galanin nerve fibers have a sensory neuronal origin, and that the peptide is associated with pain transmission and/or periodontal ligament remodeling during experimental tooth movement. In control rats, galanin-immunoreactive trigeminal ganglion cells were very rare and were observed predominantly in small ganglion cells. After 3 days of experimental tooth movement, galanin-immunoreactive trigeminal ganglion cells significantly increased, and the most marked increase was observed at 5 days after experimental tooth movement. Furthermore, their cell size spectrum also significantly changed after 3 and 5 days of movement: Medium-sized and large trigeminal ganglion cells began expressing, and continued to express, galanin until 14 days after experimental tooth movement. These findings suggest that the increase of galanin in the periodontal ligament during experimental tooth movement at least partially originates from trigeminal ganglion neurons and may play a role in pain transmission and/or periodontal remodeling.

POMC and orexin mRNA expressions induced by anticipation of a corn-oil emulsion feeding are maintained at the high levels until oil ingestion.

We investigated the gene expression dynamics of several hypothalamic neuropeptides associated with appetite regulation when rats are anticipating being fed a corn-oil emulsion. For 5 days at the same hour each day, rats were fed 5% corn oil emulsified with 0.3% xanthan gum or the vehicle for 20 min. On Day 6, the 5% corn oil emulsion or the vehicle (Vehicle) was presented to the rats, some of which (Oil-intake) were allowed to eat it and some of which (Oil-anticipation) were kept from eating it. Despite waiting a corn-oil, the mRNA levels of proopiomelanocortin (POMC), a beta-endorphin precursor, and orexin showed increases, and high levels of mRNAs of POMC and orexin were maintained for 30 min after the corn-oil was placed before the rats, and only gradually decreased through 150 min. However, the mRNA levels of POMC and orexin in the hypothalamus were decreased within 30 min after starting to ingest the corn-oil emulsion. These results suggest that POMC and orexin mRNA expression was induced by the anticipation in rats after learning the palatability of 5% corn oil emulsion, and the induced mRNA expression based on the anticipation was maintained for at least for 30 min as the rats eagerly waited for ingestion.

Amplification, expression, and steroid regulation of the preprogalanin gene in human breast cancer.

The GALN gene encodes the preprogalanin protein that is cleaved to liberate the galanin peptide, a neuropeptide and tumor cell mitogen, and the galanin message-associated peptide, which is of unknown function. GALN is located at chromosome 11q13, a frequently amplified locus in diverse tumor types including breast cancer. To determine whether GALN may contribute to the tumor phenotype resulting from 11q13 amplification, we examined GALN amplification and preprogalanin mRNA levels in breast tumors and cell lines. GALN was amplified in a subset of breast tumors and cell lines that carried 11q13 amplifications. Preprogalanin mRNA was expressed in the majority of breast cancer cell lines, but Northern analysis failed to demonstrate a relationship between GALN amplification and preprogalanin mRNA levels. Eight of eight estrogen receptor-positive cell lines expressed detectable preprogalanin mRNA, and further investigation showed that preprogalanin mRNA was increased by treatment with estradiol and progestin and decreased by the removal of serum or treatment with antiestrogens. Thus, GALN amplification is unlikely to contribute to the phenotype conferred by 11q13 amplification in breast cancer, but preprogalanin mRNA is expressed by breast cancer cells and is under steroid hormone control in estrogen receptor-positive cells, opening the wider question of the role of this steroid-regulated neuropeptide in the normal and cancerous breast.

Expression of galanin and its receptors are perturbed in a rodent model of mild, blast-induced traumatic brain injury.

The symptomatology, mood and cognitive disturbances seen in post-traumatic stress disorder (PTSD) and mild blast-induced traumatic brain injury (mbTBI) overlap considerably. However the pathological mechanisms underlying the two conditions are currently unknown. The neuropeptide galanin has been suggested to play a role in the development of stress and mood disorders. Here we applied bio- and histochemical methods with the aim to elucidate the nature of any changes in the expression of galanin and its receptors in a rodent model of mbTBI. In situ hybridization and quantitative polymerase chain reaction studies revealed significant, injury-induced changes, in some cases lasting at least for one week, in the mRNA levels of galanin and/or its three receptors, galanin receptor 1-3 (GalR1-3). Such changes were seen in several forebrain regions, and the locus coeruleus. In the ventral periaqueductal gray GalR1 mRNA levels were increased, while GalR2 were decreased. Analysis of galanin peptide levels using radioimmunoassay demonstrated an increase in several brain regions including the locus coeruleus, dorsal hippocampal formation and amygdala. These findings suggest a role for the galanin system in the endogenous response to mbTBI, and that pharmacological studies of the effects of activation or inhibition of different galanin receptors in combination with functional assays of behavioral recovery may reveal promising targets for new therapeutic strategies in mbTBI.

Selective activation of the extended ventrolateral preoptic nucleus during rapid eye movement sleep.

We found previously that damage to a cluster of sleep-active neurons (Fos-positive during sleep) in the ventrolateral preoptic nucleus (VLPO) decreases non-rapid eye movement (NREM) sleep in rats, whereas injury to the sleep-active cells extending dorsally and medially from the VLPO cluster (the extended VLPO) diminishes REM sleep. These results led us to examine whether neurons in the extended VLPO are activated during REM sleep and the connectivity of these neurons with pontine sites implicated in producing REM sleep: the laterodorsal tegmental nucleus (LDT), dorsal raphe nucleus (DRN), and locus ceruleus (LC). After periods of dark exposure that triggered enrichment of REM sleep, the number of Fos-positive cells in the extended VLPO was highly correlated with REM but not NREM sleep. In contrast, the number of Fos-positive cells in the VLPO cluster was correlated with NREM but not REM sleep. Sixty percent of sleep-active cells in the extended VLPO and 90% of sleep-active cells in the VLPO cluster in dark-treated animals contained galanin mRNA. Retrograde tracing from the LDT, DRN, and LC demonstrated more labeled cells in the extended VLPO than the VLPO cluster, and 50% of these in the extended VLPO were sleep-active. Anterograde tracing showed that projections from the extended VLPO and VLPO cluster targeted the cell bodies and dendrites of DRN serotoninergic neurons and LC noradrenergic neurons but were not apposed to cholinergic neurons in the LDT. The connections and physiological activity of the extended VLPO suggest a specialized role in the regulation of REM sleep.

Damage-induced neuronal endopeptidase (DINE/ECEL) expression is regulated by leukemia inhibitory factor and deprivation of nerve growth factor in rat sensory ganglia after nerve injury.

Damage-induced neuronal endopeptidase (DINE) is a novel metallopeptidase and is expressed in response to various neuronal injuries. The expression regulation of DINE mRNA in the dorsal root ganglia (DRGs) after sciatic nerve injury is examined. A substantial increase of DINE mRNA expression was observed in relatively small-sized DRG neurons after nerve injury. The expression was observed in isolectin B4-negative and partly TrkA-positive neurons, and the expression profile was fairly similar to that of the neuropeptide galanin. More than 80% of DINE mRNA-positive neurons simultaneously demonstrated galanin immunoreactivity after nerve injury. In cultured DRG, DINE mRNA expression was enhanced by leukemia inhibitory factor (LIF) but not by other growth factors and cytokines. LIF treatment to rat sciatic nerve induced DINE mRNA expression in DRG without nerve injury, and, conversely, the intranerve injection of anti-gp130 antibody after sciatic nerve injury significantly inhibited the upregulation of DINE mRNA in DRG. Furthermore, nerve growth factor (NGF) deprivation, which can induce galanin expression, also enhanced DINE mRNA expression in vitro and in vivo. Both LIF application and NGF deprivation additively enhanced DINE expression in vitro. These results suggest that DINE gene expression is regulated separately by both LIF and NGF deprivation, and this regulation pattern is similar to that of galanin gene expression. Because both DINE and galanin have a neuroprotective function, their simultaneous induction may provide more successful protection for injured sensory neurons.