Evaluation of WGA-Cre-dependent topological transgene expression in the rodent brain.

Novel neuromodulation techniques in the field of brain research, such as optogenetics, prompt to target specific cell populations. However, not every subpopulation can be distinguished based on brain area or activity of specific promoters, but rather on topology and connectivity. A fascinating tool to detect neuronal circuitry is based on the transsynaptic tracer, wheat germ agglutinin (WGA). When expressed in neurons, it is transported throughout the neuron, secreted, and taken up by synaptically connected neurons. Expression of a WGA and Cre recombinase fusion protein using a viral vector technology in Cre-dependent transgenic animals allows to trace neuronal network connections and to induce topological transgene expression. In this study, we applied and evaluated this technology in specific areas throughout the whole rodent brain, including the hippocampus, striatum, substantia nigra, and the motor cortex. Adeno-associated viral vectors (rAAV) encoding the WGA-Cre fusion protein under control of a CMV promoter were stereotactically injected in Rosa26-STOP-EYFP transgenic mice. After 6 weeks, both the number of transneuronally labeled YFP+/mCherry- cells and the transduced YFP+/mCherry+ cells were quantified in the connected regions. We were able to trace several connections using WGA-Cre transneuronal labeling; however, the labeling efficacy was region-dependent. The observed transneuronal labeling mostly occurred in the anterograde direction without the occurrence of multi-synaptic labeling. Furthermore, we were able to visualize a specific subset of newborn neurons derived from the subventricular zone based on their connectivity.

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons.

Gamma-aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin-releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools.

Effects of the blood components on the AMPA and NMDA synaptic responses in brain slices in the onset of hemorrhagic stroke.

Blood-borne events play a major role in post bleeding disturbances of the neuronal network. However, very little is known about the early effects of blood plasma, leucocytes, and the red blood cells on the AMPA and NMDA-mediated synaptic responses in the onset of experimental intracranial hemorrhage (ICH). In this study, we used the technique of on-line monitoring of electrophysiological parameters referred to synaptic activity in piriform cortex of SHR rat slice. We exposed the olfactory cortex slices to diluted autologous blood or its components and compared with effects of ferric chloride. Whole blood exerted a total inhibition of synaptic activity in piriform cortex within first 5 min. Dilution of blood induced prolonged epileptic synaptic activation of NMDA receptors. Blood plasma and fraction of leucocytes induced hyperactivation of neurons transforming to epileptiform discharges. Fraction of red blood cells acted biphasic, an initial sharp activity of AMPA- and NMDA-mediated receptors replaced by a following total depression. Our slice-based models of experimental stroke revealed the mechanism of the earliest pathophysiologic events occur in brain tissue during bleeding that may be relevant to the human ICH.

[Detection of c-fos expression in animal brain in a pilocarpine model of temporal lobe epilepsy].

The dynamics of the involvement of different brain structures in a pathological process is very important for decoding the mechanisms of temporal lobe epilepsy. In this work, the experimental model of temporal lobe epilepsy induced by lithium chloride and pilocarpine was used. The method of immunochemical detection of the immediate early gene c-fos was used as an indicator of functioning neurons in the brain. The c-fos expression was determined at different time points (30, 60 and 90 min) after the pilocarpine injection. An increase in the c-fos expression was observed in neuronal populations during the development of the status epilepticus, the time and degree of involvement of different brain structures being different. The expression of c-fos was first observed in the piriform cortex, the olfactory tubercle, thalamic nuclei, lateral habenular nuclei, and the caudate putamen. Then the hippocampus, the septal formation, the amygdala, and basal ganglia were involved in the activation process. In the hypothalamic areas, c-fos expression was observed latest. These data contribute to understanding the mechanisms of temporal lobe epilepsy and searching for the ways of its therapy.

Uranium-induced sensory alterations in the zebrafish Danio rerio.

The effect of chronic exposure to uranium ions (UO(2)(2+)) on sensory tissues including the olfactory and lateral line systems was investigated in zebrafish (Danio rerio) using scanning electron microscopy. The aim of this study was to determine whether exposure to uranium damaged sensory tissues in fish. The fish were exposed to uranium at the concentration of 250 µg l(-1) for 10 days followed by a depuration period of 23 days. Measurements of uranium uptake in different fish organs: olfactory rosettes and bulbs, brain, skin, and muscles, were also determined by ICP-AES and ICP-MS during the entire experimental period. The results showed that uranium displayed a strong affinity for sensory structures in direct contact with the surrounding medium, such as the olfactory and lateral line systems distributed on the skin. A decreasing gradient of uranium concentration was found: olfactory rosettes>olfactory bulbs>skin>muscles>brain. At the end of the experiment, uranium was present in non-negligible quantities in sensory tissues. In parallel, fish exposed to uranium showed severe sensory tissue alterations at the level of the olfactory and lateral line systems. In both sensory systems, the gross morphology was altered and the sensory hair cells were significantly damaged very early after the initiation of exposure (from the 3rd day). At the end of the experiment, after 23 days of depuration, the lateral line system still displayed slight tissue alterations, but approximately 80% of the neuromasts in this system had regenerated. In contrast, the olfactory system took more time to recover, as more than half of the olfactory rosettes observed remained destroyed at the end of the experiment. This study showed, for the first time, that uranium is able to damage fish sensory tissues to such an extent that tissue regeneration is delayed.

Brain processing of the mammary pheromone in newborn rabbits.

Chemosignals strongly contribute to social interactions in mammals, including mother-young relationships. In the European rabbit, a volatile compound emitted by lactating females in milk, the 2-methylbut-2-enal, has been isolated. Carrying the properties of a pheromone, in particular the spontaneous ability to release critical sucking-related movements in newborns, it has been called the mammary pheromone (MP). Lesion of the vomeronasal organ and preliminary 2-deoxyglucose data suggested that the MP could be processed by the main olfactory system. However, the neuronal substrate that sustains the MP-induced response of neonates remained unknown. Here, we evaluated Fos expression in 4-day-old-rabbits exposed to the MP (in comparison with control neonates exposed to non-relevant odorant, no odorant or unmanipulated pups) both at the level of the olfactory bulb and central brain regions. Evidence of high and widespread Fos immunoreactivity in the main olfactory bulb appear in MP pups while the accessory olfactory bulb exhibits a negligible staining. However, no obvious bulbar pattern of Fos expression is observed, when in contrast a certain pattern emerges with the neutral odorant. Compared to this latter, the MP exposure increases Fos expression in the anterior piriform cortex, the organum vasculosum of the lamina terminalis and the habenula, with a tendency in the lateral preoptic region. For the first time, a pheromone essential for mother-young interaction is thus highlighted for its processing by the main olfactory system, the whole olfactory bulb, and by brain regions involved in osmoregulation, thirst and motivation-guided motor responses.

Development of a MR-visible compound for tracing neuroanatomical connections in vivo.

Traditional studies of neuroanatomical connections require injection of tracer compounds into living brains, then histology of the postmortem tissue. Here, we describe and validate a compound that reveals neuronal connections in vivo, using MRI. The classic anatomical tracer CTB (cholera-toxin subunit-B) was conjugated with a gadolinium-chelate to form GdDOTA-CTB. GdDOTA-CTB was injected into the primary somatosensory cortex (S1) or the olfactory pathway of rats. High-resolution MR images were collected at a range of time points at 11.7T and 7T. The transported GdDOTA-CTB was visible for at least 1 month post-injection, clearing within 2 months. Control injections of non-conjugated GdDOTA into S1 were not transported and cleared within 1-2 days. Control injections of Gd-Albumin were not transported either, clearing within 7 days. These MR results were verified by classic immunohistochemical staining for CTB, in the same animals. The GdDOTA-CTB neuronal transport was target specific, monosynaptic, stable for several weeks, and reproducible.

Ghrelin, leptin and adiponectin as possible predictors of the hedonic value of odors.

Several lines of evidence point to a close relationship between the hormones of energy homeostasis and the olfactory system. Examples are the localization of leptin and adiponectin receptors in the olfactory system or increased activation of brain regions related to the palatability and the hedonic value of food in response to food pictures after application of ghrelin. In this preliminary study, we tested in 31 subjects (17 male and 14 female) if and to what extent the peripheral blood concentrations of "satiety" hormones, such as leptin, adiponectin, and ghrelin (acyl and total), are correlated with the self-ratings of odor pleasantness and with the objective olfactory and gustatory ability. The hedonic values of some odors were found to be differently rated between donors depending on gender and body weight. The concentrations of leptin, adiponectin and total ghrelin were significantly associated with the hedonic value of pepper black oil, but failed to show significant correlations for 5 other odors tested. Except for a significant association between leptin and odor identification, hormone concentrations were not linked to the abilities of smell and taste. Peripheral adipokines and gut hormones may alter the perception and pleasantness of specific odors, presumably either directly through their receptors in the olfactory system or indirectly through central interfaces between the regulation systems of olfaction, appetite control, memory and motivation.

Age-related changes of elements in the anterior commissures and the relationships among their elements.

To elucidate compositional changes of the anterior commissure with aging, the authors investigated age-related changes of elements in the anterior commissures and the relationships among their elements. After ordinary dissection at Nara Medical University was finished, the anterior commissures were resected from 45 subjects, ranging in age from 70 to 101 years. The subjects consisted of 22 men and 23 women. After ashing with nitric acid and perchloric acid, the element content of the anterior commissures was determined by inductively coupled plasma-atomic emission spectrometry. The seven element contents of Ca, P, S, Mg, Zn, Fe, and Na did not change significantly in the anterior commissures with aging. Regarding the relationships among their element contents, significant correlations were found among the contents of Ca, Mg, Zn, and Na in the anterior commissures. The gender difference that the Zn content was significantly higher in men than in women was found in the anterior commissure.

Trpc2 gene impacts on maternal aggression, accessory olfactory bulb anatomy and brain activity.

The Trpc2 gene codes for an ion channel found in the vomeronasal organ (VNO). Studies using the Trpc2(-/-) (KO) mouse have exploited the gene's role in signal transduction to explore the VNO's role in pheromonally mediated behaviors. To date, no study has evaluated the impact of the Trpc2 gene on activity within the brain. In this study, we examine the gene's effect on brain regions governing maternal aggression. We intruder-tested lactating dams and then quantified Fos immunoreactivity (Fos-IR) in the vomeronasal amygdala, hypothalamus, olfactory regions and accessory olfactory bulb (AOB). Our data confirm previous reports that loss of the Trpc2 gene severely diminishes maternal aggression. We also show that deletion of the gene results in differential hypotrophy of the glomerular layer (GlA) of the AOB, with the anterior portion the GlA resembling that of wild-type mice, and the posterior portion reduced or absent. This anatomy is suggestive of residual functioning in the apical VNO of these animals. Our Fos study describes an impact of the deletion on a network of 21 brain regions involved in emotion, aggression and olfaction, suggesting that signals from the VNO mediate activity throughout the brain. Home-cage observations of KO dams show specific deficits in nest-building, suggesting a role for pup pheromones in inducing and maintaining pup-directed maternal behaviors as well as maternal aggression.

[Comparison of the distribution of breviscapine in the brain by different administration routes].

By comparing the drug distribution of breviscapine administered intranasally, orally and intrgvenous injected in rats' brain. After 0.4 mg x kg(-1) breviscapine was given by tail vein, intranasal and gastric perfusion administration to SD rats, cerebrospinal fluid was obtained by erebellomedllery cisternal puncture at different times. 125I labeling was used to determine the drug content of cerebrospinal fluid, cerebrum, cerebellum, medulla oblongata, olfactory region, olfactory bulb and blood in rats. AUCs were calculated by trapezoidal rule. The results showed that AUCs(0-240 min) (microg x min x g(-1)) of brain tissues were 11.686 +/- 1.919, 5.676 +/- 1.025, 7.989 +/- 0.925, 7.956 +/- 1.159, 17.465 +/- 2.136, 24.2 +/- 2.906 and 78.51 +/- 12.05, respectively, in the intranasal administration group; while those in the tail vein administration groups were 6.79 +/- 0.661, 6.251 +/- 0.40, 10.805 +/- 1.161, 9.146 +/- 1.04, 9.892 +/- 1.532, 7.871 +/- 0.842 and 173.91 +/- 10.02; and oral administration group were 0.868 +/- 0.167, 1.708 +/- 0.266, 2.867 +/- 0.725, 2.067 +/- 0.313, 1.361 +/- 0.308, 1.206 +/- 0.255 and 45.2 +/- 7.52, respectively. AUCs(0-240 min) of the brain tissues after oral, tail vein and intranasal administration were 22.29%, 29.18%, 95.49% of that of blood, respectively, it means that the absorption rate and drug distribution in the brain tissues after intranasal administration were higher than those of oral and tail vein administration. It is worth to investigate further the pharmacodynamic relationship.

Role of the olfactory receptor neurons in the direct transport of inhaled uranium to the rat brain.

Uranium presents numerous industrial and military uses and one of the most important risks of contamination is dust inhalation. In contrast to the other modes of contamination, the inhaled uranium has been proposed to enter the brain not only by the common route of all modes of exposure, the blood pathway, but also by a specific inhalation exposure route, the olfactory pathway. To test whether the inhaled uranium enter the brain directly from the nasal cavity, male Sprague-Dawley rats were exposed to both inhaled and intraperitoneally injected uranium using the (236)U and (233)U, respectively, as tracers. The results showed a specific frontal brain accumulation of the inhaled uranium which is not observed with the injected uranium. Furthermore, the inhaled uranium is higher than the injected uranium in the olfactory bulbs (OB) and tubercles, in the frontal cortex and in the hypothalamus. In contrast, the other cerebral areas (cortex, hippocampus, cerebellum and brain residue) did not show any preferential accumulation of inhaled or injected uranium. These results mean that inhaled uranium enters the brain via a direct transfer from the nasal turbinates to the OB in addition to the systemic pathway. The uranium transfer from the nasal turbinates to the OB is lower in animals showing a reduced level of olfactory receptor neurons (ORN) induced by an olfactory epithelium lesion prior to the uranium inhalation exposure. These results give prominence to a role of the ORN in the direct transfer of the uranium from the nasal cavity to the brain.

Involvement of G protein-coupled receptor 30 (GPR30) in rapid action of estrogen in primate LHRH neurons.

Previously, we have reported that 17beta-estradiol (E(2)) induces an increase in firing activity of primate LH-releasing hormone (LHRH) neurons. The present study investigates whether E(2) alters LHRH release as well as the pattern of intracellular calcium ([Ca(2+)](i)) oscillations and whether G protein-coupled receptor 30 (GPR30) plays a role in mediating the rapid E(2) action in primate LHRH neurons. Results are summarized: 1) E(2), the nuclear membrane-impermeable estrogen, estrogen-dendrimer conjugate, and the plasma membrane-impermeable estrogen, E(2)-BSA conjugate, all stimulated LHRH release within 10 min of exposure; 2) whereas the estrogen receptor antagonist, ICI 182,780, did not block the E(2)-induced LHRH release, E(2) application to cells treated with pertussis toxin failed to induce LHRH release; 3) GPR30 mRNA was expressed in olfactory placode cultures, and GPR30 protein was expressed in a subset of LHRH neurons; 4) pertussis toxin treatment blocked the E(2)-induced increase in [Ca(2+)](i) oscillations; 5) knockdown of GPR30 in primate LHRH neurons by transfection with small interfering RNA (siRNA) for GPR30 completely abrogated the E(2)-induced changes in [Ca(2+)](i) oscillations, whereas transfection with control siRNA did not; 6) the estrogen-dendrimer conjugate-induced increase in [Ca(2+)](i) oscillations also did not occur in LHRH neurons transfected with GPR30 siRNA; and 7) G1, a GPR30 agonist, resulted in changes in [Ca(2+)](i) oscillations, similar to those observed with E(2). Collectively, E(2) induces a rapid excitatory effect on primate LHRH neurons, and this rapid action of E(2) appears to be mediated, in part, through GPR30.

Expression pattern of Anosmin-1 during pre- and postnatal rat brain development.

Anosmin-1 participates in the development of the olfactory and GnRH systems. Defects in this protein are responsible for both the anosmia and the hypogonadotrophic hypogonadism found in Kallmann's syndrome patients. Sporadically, these patients also manifest some neurological symptoms that are not explained in terms of the developmental defects in the olfactory system. We describe the pattern of Anosmin-1 expression in the central nervous system during rat development using a novel antibody raised against Anosmin-1 (Anos1). The areas with Anos1-stained neurons and glial cells were classified into three groups: (1) areas with immunoreactivity from embryonic day 16 to postnatal day (P) 15; (2) areas with Anosmin-1 expression only at postnatal development; (3) nuclei with immunoreactivity only at P15. Our data show that Anos1 immunoreactivity is detected in projecting neurons and interneurons within areas of the brain that may be affected in patients with Kallmann's syndrome that develop both the principal as well as sporadic symptoms.

Nitric oxide in the crustacean brain: regulation of neurogenesis and morphogenesis in the developing olfactory pathway.

Nitric oxide (NO) plays major roles during development and in adult organisms. We examined the temporal and spatial patterns of nitric oxide synthase (NOS) appearance in the embryonic lobster brain to localize sources of NO activity; potential NO targets were identified by defining the distribution of NO-induced cGMP. Staining patterns are compared with NOS and cyclic 3,5 guanosine monophosphate (cGMP) distribution in adult lobster brains. Manipulation of NO levels influences olfactory glomerular formation and stabilization, as well as levels of neurogenesis among the olfactory projection neurons. In the first 2 days following ablation of the lateral antennular flagella in juvenile lobsters, a wave of increased NOS immunoreactivity and a reduction in neurogenesis occur. These studies implicate nitric oxide as a developmental architect and also support a role for this molecule in the neural response to injury in the olfactory pathway.

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals.

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5-6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10-25 days pf), and by 25-30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.

Gene expression and specificity in the mature zone of the lobster olfactory organ.

The lobster olfactory organ is an important model for investigating many aspects of the olfactory system. To facilitate study of the molecular basis of olfaction in lobsters, we made a subtracted cDNA library from the mature zone of the olfactory organ of Homarus americanus, the American lobster. Sequencing of the 5'-end of 5,184 cDNA clones produced 2,389 distinct high-quality sequences consisting of 1,944 singlets and 445 contigs. Matches to known sequences corresponded with the types of cells present in the olfactory organ, including specific markers of olfactory sensory neurons, auxiliary cells, secretory cells of the aesthetasc tegumental gland, and epithelial cells. The wealth of neuronal mRNAs represented among the sequences reflected the preponderance of neurons in the tissue. The sequences identified candidate genes responsible for known functions and suggested new functions not previously recognized in the olfactory organ. A cDNA microarray was designed and tested by assessing mRNA abundance differences between two of the lobster's major chemosensory structures: the mature zone of the olfactory organ and the dactyl of the walking legs, a taste organ. The 115 differences detected again emphasized the abundance of neurons in the olfactory organ, especially a cluster of mRNAs encoding cytoskeletal-associated proteins and cell adhesion molecules such as 14-3-3zeta, actins, tubulins, trophinin, Fax, Yel077cp, suppressor of profilin 2, and gelsolin.

Distribution of hypothalamic, hippocampal and other limbic peptidergic neuronal cell bodies giving rise to retinopetal fibers: anterograde and retrograde tracing and neuropeptide immunohistochemical studies.

In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the vitreous body resulted in nerve cell body labeling in several structures: the supraoptic and paraventricular nuclei, the hippocampus (CA1, CA3), the dentate gyrus, the indusium griseum, the olfactory tubercle, and the medial habenula, all of them belong to the limbic system. We estimated that the total number of retrogradely labeled cells is 1495+/-516. We have seen fiber labeling in the retinorecipient suprachiasmatic nucleus and in the primary visual center, the lateral geniculate body, but labeled nerve cell bodies in these structures were never seen. Iontophoretic application of Fluorogold into the hippocampal formation, where the major part of the biotinylated dextran amine-labeled cell bodies was observed, resulted in labeled fibers in the optic nerve and in the retina indicating that the retrogradely labeled cells in the hippocampus and the dentate gyrus among others are the cells of origin of the centrifugal visual fibers. Sections showing biotinylated dextran amine labeling were stained for vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity using immunohistochemistry. Some biotinylated dextran amine-labeled cells also showed vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactivity. We conclude that the limboretinal pathway exists and that the cells of origin are partially vasoactive intestinal polypeptide, pituitary adenylate cyclase activating polypeptide or luteinizing hormone-releasing hormone immunoreactive.

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.

G protein-coupled galanin receptor distribution in the rat central nervous system.

The action of galanin in the central nervous system is mediated by at least three galanin receptor subtypes (GalR1, GalR2 and GalR3) which belong to the family of G protein-coupled receptors. GalR1 and GalR2 are coupled to G(i/o) proteins, although the latter may also be coupled to G(q/11) proteins. The aim of the present study was to identify the anatomical distribution and quantify the density of GalRs coupled to G proteins. The galanin (10(-6) M) stimulated guanosine 5'-(gamma-[35S] thio)triphosphate binding assay was used in tissue sections from the rat brain. Maximal percentages of stimulation over basal levels were found in the anterior olfactory nucleus and in the lateral olfactory tract nucleus ( approximately 54%). High levels of stimulation were recorded in diverse hypothalamic nuclei (16-28%), in the amygdala (central amygdaloid nucleus, 40%), in the spinal trigeminal tract (23%) and in layers 1-2 of the spinal cord (26%). Moderate binding stimulation (5-13%) was observed in thalamus, substantia nigra pars compacta, parabrachial nucleus, locus coeruleus and dorsal raphe nucleus. The lowest stimulation induced by galanin was recorded in diverse areas of the cortex, striatum, hippocampus and substantia nigra pars reticulata. The results show an anatomical distribution similar to that described for GalR1. However, in diverse brain areas, in which a high density of these receptors has previously been reported, only a moderate coupling to G proteins was found. These findings would suggest that the efficacy of galanin to induce an effective coupling of its receptors to G proteins could be different depending on the brain area.