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.

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.

Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons.

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

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.

Development of the amygdalohypothalamic projection in the mouse embryonic forebrain.

The amygdalohypothalamic projection, a major component of the stria terminalis, is involved in the conduction of emotional and olfactory information integrated in the amygdala to the hypothalamus to elicit emotional reactions. Despite the extensive studies on functional aspects of the amygdaloid complex, developmental mechanisms of the amygdala and related structures are still poorly understood. To investigate the development of the amygdalohypothalamic projection in the mouse embryonic brain, carbocyanine dye was applied to the amygdala to label the growing axons anterogradely and to the hypothalamus to label the amygdaloid neurons retrogradely. The initial outgrowth of the stria terminalis was found to be as early as E11.5. The pathway crossed in a saddle over the internal capsule, another prominent connection in the developing forebrain of the mammalian embryo. Bipolar immature neurons were distributed along the stria terminalis at the telencephalo-diencephalic boundary, and the internal capsule was also surrounded by these cells. These cells expressed immunoreactivities to calretinin and the lot-1 antigen which has been shown to be involved in guidance of the developing lateral olfactory tract. Ultrastructural analysis revealed an adherens-like junction between the stria terminalis and the apposed cells, implying contact-mediated guidance. These results suggest that, in the development of the stria terminalis, the axonal outgrowth is guided by a mechanism similar to that of the developing lateral olfactory tract, a major amygdalopetal connection.

Gonadotropin-releasing hormone (GnRH) receptor expression and membrane signaling in early embryonic GnRH neurons: role in pulsatile neurosecretion.

The characteristic pulsatile secretion of GnRH from hypothalamic neurons is dependent on an autocrine interaction between GnRH and its receptors expressed in GnRH-producing neurons. The ontogeny and function of this autoregulatory process were investigated in studies on the properties of GnRH neurons derived from the olfactory placode of the fetal rat. An analysis of immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy, demonstrated coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived and immortalized GnRH neurons (GT1-7 cells) exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are characteristics of early fetal GnRH neurons and could provide a mechanism for gene expression and regulated GnRH secretion during embryonic migration.

Patterns of neurogenesis in the midbrain of embryonic lobsters differ from proliferation in the insect and the crustacean ventral nerve cord.

Neurogenesis persists throughout life in the olfactory pathway of many decapod crustaceans. However, the relationships between precursor cells and the temporal characteristics of mitotic events in these midbrain regions have not been examined. We have conducted studies aimed at characterizing the sequence of proliferative events that leads to the production of new deutocerebral projection neurons in embryos of the American lobster, Homarus americanus. In vivo bromodeoxyuridine (BrdU) labeling patterns show that three distinct cell types are involved in neurogenesis in this region. Quantitative and temporal analyses suggest that the clearing time for BrdU is 2-3 days in lobster embryos, and that the sequence of proliferative events in the midbrain is significantly different from the stereotypical pattern for the generation of neurons in the ventral nerve cord ganglia of insects and crustaceans. The unusual pattern of proliferation in the crustacean midbrain may be related to the persistence of neurogenesis throughout life in these regions.

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters.

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.

Differing, spatially restricted roles of ionotropic glutamate receptors in regulating the migration of gnrh neurons during embryogenesis.

We have examined here the role of glutamate in regulating the process of tangential neuronal migration during embryogenesis by investigating the roles of AMPA and NMDA receptors in the migration of the gonadotropin-releasing hormone (GnRH) neurons from the nose to the hypothalamus. We first determined that GluR1-4 subunit mRNAs were present from embryonic day (E) 12.5 along the complete nose-brain migratory pathway of the GnRH neurons, whereas that of the obligatory NMDAR1 transcript was present only in brain regions of GnRH migration. In vivo studies revealed that AMPA receptor antagonism between E12.5 and E16.5 resulted in a significant (p < 0.05) accumulation of GnRH neurons in the nose adjacent to the cribiform plate. In contrast, NMDA receptor antagonism over E12.5-E16.5 or E13.5-E16.5 caused a selective increase (p < 0.05) in the number of GnRH neurons located in their final resting place within the diagonal band of Broca and preoptic area. Dual-labeling studies using GnRH promoter-LacZ transgenic mice, which facilitate the identification of receptors in GnRH neurons, identified the presence of NMDAR1 receptors in approximately 6% of embryonic GnRH neurons located throughout the migratory pathway. Postnatally, the percentage of GnRH neurons expressing NMDAR1 increased to 50%. These results indicate that tonic AMPA receptor activation enhances the migration of GnRH neurons from the nose into the brain, whereas that of NMDA receptor activation slows the final phase of GnRH migration within the forebrain. These in vivo observations demonstrate differing, spatially restricted roles for AMPA and NMDA receptor activation in the process of tangential neuronal migration.

RALDH3, a retinaldehyde dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesicle and olfactory pit during mouse development.

The enzymes that generate retinoic acid during development have been identified as members of the aldehyde dehydrogenase (ALDH) family. The developmental expression patterns of two ALDHs that function as retinaldehyde dehydrogenases, RALDH1 and RALDH2, have been described. Here we report the cloning and expression of a third retinaldehyde dehydrogenase from the mouse called RALDH3 that shares 94% amino acid sequence identity to a human retinaldehyde dehydrogenase previously named ALDH6. In mouse embryos, RALDH3 expression is first noticed in the ventral optic eminence at E8.75, then in the optic vesicle/cup, otic vesicle, and olfactory placode/pit from E9.5 to E11.5. Expression in the developing eye is primarily localized in the ventral retina, thus indicating that RALDH3 represents the V1 dehydrogenase activity described there earlier. From E8.5 to E10.5 RALDH3 expression is distinct from that of RALDH1 or RALDH2, thus indicating a unique role in sensory organ development.