Role of the gonads in sex differentiation of growth hormone-releasing hormone and somatostatin neurons in the mouse hypothalamus during postnatal development

We clarify the mechanism of sexual dimorphism of growth hormone releasing hormone (GHRH) neurons in the arcuate nucleus (ARC) and somatostatin (SS) neurons in periventricular nucleus (PeN), by studying the role of the gonads during the neonatal period and after puberty using immunohistochemical and morphometric methods. As in our previous works the numbers of ARC GHRH-ir and PeN SS-ir neurons were significantly greater in adult normal male (NM) mice than in adult normal female (NF) mice. Adult female mice that were ovariectomized neonatally (NOF) increased the expression of GHRH-ir neurons to the male pattern, but adult female mice ovariectomized after puberty (APO) did not change. Adult male mice castrated neonatally and after puberty (NCM and APC, respectively) were not significantly different from NM mice. However, NCT male mice, which were castrated neonatally and transplanted with ovary just before puberty, showed a significantly reduced number of GHRH-ir neurons compared with NCM mice, but no significant difference from NM and NF mice. On the other hand, the PeN SS-ir neuron expression in NCM mice and APC mice showed a significant reduction compared with NM mice, but no significant difference from NF mice. The number of PeN SS-ir neurons in NOF increased to match that of NM mice. Our results suggest that the presence of the ovary during postnatal life inhibits the development of ARC GHRH-ir neurons. The presence of the testis during postnatal life may stimulate the development of PeN SS-ir neurons, while the presence of the ovary during neonatal period may inhibit the development of PeN SS-ir neurons; the presence of ovary after puberty does not inhibit.     

Functional maturation of periodontal mechanoreceptors during development in rats

The influence of development on periodontal mechanoreceptors (PMRs) was investigated in four groups of male Wistar albino rats aged 1, 3, 5 weeks and 6 months using an in vitro jaw-nerve preparation. The mean values of conduction velocities of the nerve innervating PMRs in 5-week and 6-month groups were significantly higher than those in the other two groups. All fiber types obtained in the 5-week and 6-month groups were Abeta. The mechanical thresholds of 5-week and 6-month groups were significantly higher than those of 1- and 3-week groups. These data suggest that the response properties of rat's PMRs are matured by 5-week after birth, when functional molar occlusion and transition of dietary contents from liquid to hard-diet can be achieved.     

Transient expression of somatostatin receptors in the rat cerebellum during development

Somatostatin and somatostatin receptors have not been identified in adult rat cerebellum. In contrast, during the development, somatostatin-containing neurons have been visualized in the deep layers of the cerebellum. The present study shows that during ontogenesis, somatostatin receptors are present in close association with the external granule cell layer of the cerebellum. No correlation was found between the location of immunoreactive somatostatin and the distribution of somatostatin receptors. The disappearance of somatostatin receptors, from postnatal day 13 to 23, was concomitant with the involution of the external germinal layer.     

Sex differentiation of growth hormone-releasing hormone and somatostatin neurons in the mouse hypothalamus: an immunohistochemical and morphological study

We examine sexual dimorphism in growth hormone-releasing hormone (GHRH) in the arcuate nucleus (ARC), and somatostatin (SS) in the periventricular nucleus (PeN) of the hypothalamus, and investigate when it becomes evident. Using immunohistochemical staining and morphometry, we observed ARC GHRH-immunoreactive (ir) neurons, ARC SS-ir neurons and PeN SS-ir neurons in male and female mice at 5, 20, 30, 40 and 60 days old. The number of ARC GHRH-ir neurons was significantly higher in males than females, after 20 days old. ARC SS-ir neurons showed no significant differences between sexes. On the other hand, PeN SS-ir neurons were significantly more numerous in males at 30, 40 and 60 days than in females. During postnatal development, these GHRH- and SS-ir neurons changed in different patterns from ages 20 to 60 days. The number of ARC GHRH-ir neurons in both sexes decreased from 5 to 20 days, increased until day 40, and then decreased at day 60, while ARC SS-ir neurons in both sexes increased from day 5 to day 60. PeN SS-ir neurons in both sexes increased from days 5 to 20 to 116% in males and 189% in females. Furthermore, in male mice, the increase continued until 40 days of age, while in females, there was no significant difference from days 20 to 60. There were no apoptotic cells; a few proliferating cell nuclear antigen (PCNA) stained cells were found in the ARC and PeN. Our results suggest that the sex difference of ARC GHRH neurons and PeN SS neurons appears by stimulation with testosterone during the development life. The developmental fluctuation in the number of ARC GHRH-ir neurons may not be modulated by testosterone, but by ARC SS neurons.     

Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm-generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0-P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several-fold during the next days. Hypoxia increased breathing frequency from E18-P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0-P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40-50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0-P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16-E18, in agreement with the concept of multiple rhythmogenic mechanisms.     

In vitro electrophysiological study of spontaneous activity in neonatal mouse vestibular ganglion neurons during development

Spontaneous discharges from vestibular ganglion neurons have been studied in mice between 0 and 10 days in freshly isolated in vitro preparations. The vestibular ganglion neurons were electrophysiologically active at birth with irregular activities. From the 3rd day. the activities could be divided into irregular and regular discharging units based on the coefficient of variation of their interspike intervals. This study provides two criteria for maturation of vestibular activities: a decreasing number of irregular units with age and an increase of the spontaneous discharge frequencies.     

Identification of multiple somatostatin receptors in the rat somatosensory cortex during development

The present study was aimed at identifying somatostatin receptor subtypes on the basis of their ligand-binding properties in the rat somatosensory cortex during fetal and postnatal development. Characterization of somatostatin-binding sites was performed in individual cortical layers by using three radioligands and eight competitors with known selectivities for the five somatostatin receptor subtypes. Binding sites sensitive to sst2-selective ligands were detected with high densities in the intermediate zone of the fetal cortex. From embryonic day 21 to 21 days postnatal (P21), mixed populations of receptors were detected in the cortical plate and emerging layers I-VI. Putative sst2 receptors were detected throughout the entire period but displayed different affinities for somatostatin and analogs, and a different sensitivity to GTP, depending on the developmental stage and the cortical layer considered. High densities of binding sites exhibiting characteristics of the sst1, sst3/5, and sst4 receptor subtypes were observed from P4 to P7, P7 to P14, and P7 to P21, respectively. In addition, each type of site exhibited a particular distribution pattern across the cortical layers that varied during the development. In the adult cortex, binding sites with sst1 and sst2 receptor characteristics were predominant. This study provides evidences of developmental expression windows of four sst receptor subtypes in selected areas of the rat cerebral cortex.     

Delayed expression of somatostatin mRNA in GDNFs-dependent rat sensory neurons during postnatal development

Gene expression of somatostatin (SST) and preprotachykinin A (PPTA) in lumbar DRG neurons of postnatal developing rats was examined by in situ hybridization. SST mRNA signals were not seen in DRG neurons until postnatal day 1 to 7, and were detected in about 10% of DRG neurons of 2- and 8-week-old rats. The positive neurons expressed c-ret mRNA in 8-week-old rats. On the other hand, PPTA mRNA signals were constantly seen in about 30% of DRG neurons. This study demonstrates the differential expression patterns of SST and PPTA mRNAs in DRG neurons of developing rats.     

Spatiotemporal distribution of dying neurons during early mouse development

Apoptosis is a critical cellular event during several stages of neuronal development. Recently, we have shown that biotinylated annexin V detects apoptosis in vivo in various cell lineages of a wide range of species by binding to phosphatidylserines that are exposed at the outer leaflet of the plasma membrane. In the present study, we tested the specificity by which annexin V binds apoptotic neurons, and subsequently investigated developmental cell death in the central and peripheral nervous system of early mouse embryos at both the cellular and histological level, and compared the phagocytic clearance of apoptotic neurons with that of apoptotic mesodermal cells. Our data indicate: (i) that biotinylated annexin V can be used as a sensitive marker that detects apoptotic neurons, including their extensions at an early stage during development; (ii) that apoptosis plays an important part during early morphogenesis of the central nervous system, and during early quantitative matching of brain-derived neurotrophic factor and neurotrophic factor 3 responsive postmitotic large clear neurons in the peripheral ganglia with their projection areas; and (iii) that apoptotic neurons are removed by a process that differs from classical phagocytosis of non-neuronal tissues.     

AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitro

Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca²⁺-activated non-specific cation current (I_CAN). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca²⁺ channels to directly evoke I_CAN. Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca²⁺ release also evoked I_CAN. Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca²⁺ influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic I_CAN, which underlies inspiratory drive potentials. The coupling of AMPA receptors with I_CAN suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network.     

Neuronal differentiation and maturation in the mouse trigeminal sensory system, in vivo and in vitro.

We have isolated and characterized four monoclonal antibodies (mAbs B33, E1.9, B30, and B10) that recognize mouse trigeminal sensory neurons at specific times during development. These antibodies permit the study of neuronal differentiation, axon outgrowth, and neuronal maturation in the trigeminal sensory system. With B33, we can follow migrating neural crest and placode cells into the anlagen of the trigeminal ganglion. E1.9 immunoreactivity marks neuronal differentiation and appears in the central nervous system at embryonic day 8.5 (E8.5) and in the peripheral nervous system at E9, E1.9 and B30 show the axonal outgrowth of trigeminal sensory neurons and reveal the pioneering of the peripheral tracts by an early population of ganglionic neurons. At this stage, in the central nervous system, mesencephalic trigeminal neurons are also E1.9 and B30 positive as they migrate to their final location in the rostral metencephalon. B30 and B10 allow us to follow the maturation of these neurons. Also, in about 1% of the embryos, we identified mispositioned or misrouted trigeminal neurons. Furthermore, these biochemical markers facilitate the study of neuronal development in vitro. We find that, based on morphological and biochemical criteria, the maturation of trigeminal neurons in culture is target independent.     

Dynamic loss of surface-expressed AMPA receptors in mouse cortical and striatal neurons during anesthesia

Ionotropic glutamate receptors, especially the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype, undergo dynamic trafficking between the surface membrane and intracellular organelles. This trafficking activity determines the efficacy and strength of excitatory synapses and is subject to modulation by changing synaptic inputs. Given the possibility that glutamate receptors in the central nervous system might be a sensitive target of anesthetic agents, this study investigated the possible impact of anesthesia on trafficking and subcellular expression of AMPA receptors in adult mouse brain neurons in vivo. We found that anesthesia induced by a systemic injection of pentobarbital did not alter total protein levels of three AMPA receptor subunits (GluR1-3) in cortical neurons. However, an anesthetic dose of pentobarbital reduced GluR1 and GluR3 proteins in the surface pool and elevated these proteins in the intracellular pool of cortical neurons. The similar redistribution of GluR1/3 was observed in mouse striatal neurons. Pentobarbital did not significantly alter GluR2 expression in the two pools. Chloral hydrate at an anesthetic dose also reduced surface GluR1/3 expression and increased intracellular levels of these proteins. The effect of pentobarbital on subcellular distribution of AMPA receptors was reversible. Altered subcellular distribution of GluR1/3 returned to normal levels after the anesthesia subsided. These data indicate that anesthesia induced by pentobarbital and chloral hydrate can alter AMPA receptor trafficking in both cortical and striatal neurons. This alteration is characterized by the concurrent loss and addition of GluR1/3 subunits in the respective surface and intracellular pools.     

Retrograde axonal transport of asialoglycoproteins in mouse trigeminal neurons in vivo and in rat dorsal root ganglia neurons in vitro

Different terminal sugars of the glycoprotein orosomucoid were exposed by sequential glycosidase digestions. The orosomucoid and its different derivatives were conjugated to horseradish peroxidase by a two-step glutaraldehyde coupling procedure, injected into the snout of 12-day-old mice or exposed to dorsal root ganglia neurons from embryonic rats, cultivated in a two chamber system. A marked increase in transport of the conjugates in the trigeminal and dorsal root ganglia neurons was observed histochemically after removal of sialic acid, exposing galactose as the terminal sugar. Quantitative hydrolysis of galactose residues resulted in reduced uptake. The data suggest the presence of a galactose-recognition molecule in the axon-terminal membrane, involved in retrograde axonal transport.     

Leptin receptors in estrogen receptor-containing neurons of the female rat hypothalamus

This study was undertaken to reveal whether integration of the peripheral signals, leptin and estradiol, that convey information on the metabolic state and gonadal function, respectively, might occur in the same hypothalamic neuronal perikarya. Light and electron microscopic immunolabeling for leptin receptors (LRs) and estrogen receptors (ERs) was carried out on hypothalamic sections of female rats. In the medial preoptic area, periventricular regions, including the parvicellular paraventricular nucleus, the arcuate nucleus and the ventromedial hypothalamic nucleus, all of the cells that expressed immunoreactivity for ERs were also immunopositive for LR. On the other hand, only a subpopulation of LR-containing cells was found to express ERs. The extensive colocalization of receptors for leptin and estrogen in neuronal perikarya of all parts of the hypothalamus suggests a closely coupled interaction between these peripheral signals in the regulation of a variety of behavioral and neuroendocrine mechanisms.     

Functional expression of TrkA receptors in hippocampal neurons

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types. J. Neurosci. Res. 54:424–431, 1998. © 1998 Wiley-Liss, Inc.     

Alteration of veratridine neurotoxicity in sympathetic neurons during development in vitro

Neurotoxic effects of veratridine, the activator of voltage-dependent Na+ channels, were examined at various stages of in vitro development of superior cervical ganglion cells dissociated from newborn rats. Veratridine neurotoxicity did not occur in 1DIV (days in vitro) neurons, but occurred in 7DIV neurons, both of which depend on NGF for survival, but elevated K+ supports only the latter. TUNEL and electron microscopic analyses revealed that 7DIV neurons underwent both apoptotic and necrotic cell death. Veratridine was also toxic to 21DIV neurons which are independent of NGF for survival. Nuclear features of apoptosis, however, were greatly reduced in these neurons undergoing cell death, suggesting that nuclear vulnerability is also subject to developmental regulation in vitro.     

Release of neurotransmitters and neurosecretory substances during in vitro maturation of mouse hypothalamic cultures.

The maturation of the neurosecretory activity of a hypothalamic nerve cell population grown in vitro, prepared from 10-day-old mice and cultured for 6 days, has been demonstrated in the present report. A low-molecular weight polypeptide of 30-kD was found to be released into the culture media during the 6-day period of incubation, as analyzed by SDS-polyacrylamide gel electrophoresis. Comparative electrophoresis of the in situ hypothalamic, neurohypophysis and cerebral cortex homogenates revealed the presence of a 30-kD protein component in both the hypothalamus and neurohypophysis but not in the cerebral cortex. The release of the 30-kD polypeptide into the incubation media indicates an expression of the neurosecretory activity of the peptidergic neurons of the hypothalamus during in vitro maturation. On the other hand, high pressure liquid chromatography with electrochemical detection showed appreciable quantities of released dopamine (DA), epinephrine and serotonin (5-HT) in the incubation media in which the neurons were allowed to differentiate. There was a steady release of DA during the 6-day incubation period, varying from 0.21 +/- 0.02 to 0.49 +/- 0.05 ng/mg protein. The epinephrine level increased progressively from day 1 to 6 of culture, from 3.73 +/- 0.57 to 12.08 +/- 1.81 ng/mg protein, respectively. The measured 5-HT level was 0.07 +/- 0.001 on day 2 and increased to 0.38 +/- 0.05 ng/mg protein on day 6 of culture. These data demonstrate the functional maturation of catecholaminergic, serotoninergic and peptidergic neurons in these rotary histotypic cultures of the mouse hypothalamus.     

Orexin neurons of the hypothalamus express adenosine A1 receptors

Adenosine is a putative sleep factor with effects mainly mediated by the A1 receptor. Recent studies have implicated the hypothalamic orexin/hypocretin-containing neurons in the control of sleep-wakefulness. To help determine if adenosine might play a role in the control of orexin neurons, immunohistochemistry was used to characterize the distribution of adenosine A1 receptor protein on the orexinergic neurons. About 30% of orexin-containing neurons were labeled. The data supports the presence of adenosine A1 receptors on orexinergic neurons and suggests a possible substrate for a functional role of adenosine in the regulation of orexinergic activity.