BM88 is an early marker of proliferating precursor cells that will differentiate into the neuronal lineage

Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.     

Circulating ghrelin levels and central ghrelin receptor expression are elevated in response to food deprivation in a seasonal mammal (Phodopus sungorus)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). However, the functional interaction of ligand and receptor is not very well understood. We demonstrate that GHSR mRNA is up-regulated after food deprivation (48 h) in the hypothalamic arcuate nucleus and ventromedial nucleus of the seasonal Siberian hamster, Phodopus sungorus. This increase is accompanied by a two-fold elevation of circulating ghrelin concentration. Chronic changes in feeding state imposed by food restriction over a period of 12 weeks during long day-length induced increased GHSR gene expression, whereas food restriction for 6 weeks had no effect. Phodopus sungorus reveals remarkable seasonal changes in body weight, fat mass and circulating leptin levels. Ghrelin is generally regarded as having opposing effects on appetite and body weight with respect to those exhibited by leptin. However, our study revealed that seasonal adaptations were not accompanied by changes in either GHSR gene expression or circulating ghrelin concentration. Therefore, we suggest that ghrelin only plays a minor role in modulating long-term seasonal body weight cycles. Our findings imply that ghrelin predominantly acts as a short-term regulator of feeding.     

Vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2 and angiopoietin-2 expression in the mouse choroid plexuses

In this study, for the first time, we investigated about the localization of VEGF-A, VEGFR-2 and Ang-2 in the choroid plexuses of the adult mouse by Western blot and immunohistochemistry. Results showed that VEGF-A stained epithelial cells, while anti-VEGFR-2 and -Ang-2 antibodies stained endothelial cells. These data suggest that Ang-2, converting blood vessels into a more plastic and immature phenotype, would provide more accessibility of VEGF-A to endothelial cells.     

Leptin inhibits norepinephrine efflux from the hypothalamus in vitro: role of gamma aminobutyric acid

Leptin, a hormone secreted by adipocytes, produces a number of central and neuroendocrine effects, the mechanisms behind which are not completely understood. Hypothalamic norepinephrine (NE) is involved in many of the neuroendocrine effects that are associated with leptin. Therefore, we hypothesized that leptin could affect hypothalamic NE activity to bring about its central and neuroendocrine effects. Because gamma aminobutyric acid (GABA) is known to affect the release of NE, we also tested the possibility that leptin-induced changes in NE could be mediated through GABA. The mediobasal hypothalami from adult male rats were incubated in an in vitro incubation system for four consecutive incubation periods of 60 min each at 37 degrees C in Krebs Ringers Henseleit (KRH) solution in an atmosphere of 95% O(2) and 5% CO(2.) After determining the basal release, the hypothalami were challenged with 0, 0.1, 1 or 10 nm of leptin, bicuculline (a GABA-A receptor antagonist; 10 microM) and bicuculline (10 microM) +10 nM of leptin during the second incubation period. Residual effects of leptin were measured in the third incubation where tissues were incubated with KRH alone, and the viability of tissues was determined in the fourth incubation when tissues were exposed to high K(+) KRH. NE levels in the incubation medium were measured using high-performance liquid chromatography with electrochemical detection (HPLC-EC). Leptin inhibited NE efflux from the hypothalamus in a dose-dependent manner. Moreover, incubation of hypothalami with 10 nM of leptin and bicuculline, a completely blocked the leptin-induced decrease in NE efflux. These results demonstrate for the first time that leptin could act directly on the hypothalamus to inhibit NE efflux through GABA. It was concluded that leptin could probably produce its central and neuroendocrine effects by modulating NE and GABA levels in the hypothalamus.     

Brain Na+,K+-ATPase isozyme activity and protein expression in ouabain-induced hypertension

In normotensive rats, chronic infusion of exogenous ouabain causes hypertension involving central mechanisms. To determine whether ouabain-induced hypertension is associated with specific changes in brain Na+,K+-ATPase activity and expression, we assessed brain Na+,K+-ATPase isozyme activity and protein expression in rats treated with ouabain (50 microg/day s.c. or 10 microg/day i.c.v. for 14 days). Resting mean arterial pressure (MAP) was higher in s.c.- and i.c.v.-ouabain-treated animals vs. control (124+/-2 vs. 105+/-2 and 130+/-2 vs. 109+/-2, respectively, p<0.01). Ouabain infused s.c. or i.c.v. for 14 days had no effect on Na+,K+-ATPase isozyme activity in hypothalamic, pontine/medullary or cortical microsomes. However, the percent increase in total Na+,K+-ATPase activity produced in vitro by antibody Fab fragments that bind ouabain with high affinity (Digibind) was two-fold greater in s.c.- and i.c.v.-ouabain-treated rats vs. control, but only in hypothalamic microsomes. Thus, ouabain infused s.c. or i.c.v. does appear to directly inhibit Na+,K+-ATPase activity in the hypothalamus. On the other hand, in the hypothalamus, s.c.- and i.c.v.-ouabain infusions tended to increase alpha3 (by 30-44%), but had no effect on alpha1 or alpha2 Na+,K+-ATPase isozyme protein expression. In addition, ouabain was found to partially dissociate from the Na+,K+-ATPase enzyme following sample processing. Thus, the inability to detect a decrease in enzyme activity in the hypothalamus in response to ouabain may be due, in part, to an increase in enzyme expression and the dissociation of ouabain during sample processing.     

Upregulation of corticotropin-releasing hormone gene expression in the paraventricular nucleus of cyclophosphamide-induced cystitis in male rats

We examined the effects of cyclophosphamide (CP)-induced cystitis on the expression of corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus (PVN) and the serum levels of adrenocorticotropic hormone (ACTH) using in situ hybridization histochemistry and radioimmunoassay. In addition, the expression of AVP heteronuclear (hn) RNA and neuronal nitric oxide synthase (nNOS) mRNA was also examined in the PVN of a CP-induced cystitis model. We found that the levels of CRH mRNA were significantly increased in the PVN at 2 h after intraperitoneal administration of CP compared to those in saline-treated rats. The CRH mRNA levels in the PVN peaked at 12 h after CP administration and the levels were still significantly higher than those in saline-treated group at 24 h after CP administration. The serum ACTH levels in CP-treated group were also significantly higher compared to those in saline-treated group at any of the time points examined. Unlike previous findings showing upregulation of nNOS mRNA and AVP hnRNA under somatic nociceptive states, the levels of nNOS mRNA and AVP hnRNA were unchanged in the PVN following CP-induced cystitis, visceral nociceptive stimulation. These results suggest that visceral nociceptive stimulation as well as somatic nociceptive stimulation may activate the hypothalamo-pituitary axis but the hypothalamic neuroendocrine responses produced by visceral nociceptive stimulation may be different from those produced by somatic nociceptive stimulation.     

Electrophysiological evidences for the contribution of NMDA receptors to the inhibition of clonidine on the RVLM presympathetic neurons

The main objective of this study is to test the hypothesis that N-methyl-D-aspartate (NMDA) receptors within the rostral ventrolateral medulla (RVLM) are involved in the inhibition of clonidine on the RVLM presympathetic neurons. Totally, 22 presympathetic neurons were recorded in anesthetized and paralyzed rats. The majority of these neurons (n=16 of 22) were significantly inhibited by iontophoretic (30 nA) clonidine, the other 6 neurons were insensitive to clonidine. In seven clonidine-sensitive neurons, iontophoretic clonidine (30 nA) antagonized the neuronal excitation of iontophoretic NMDA receptor agonist NMDA (20 nA). In remaining nine clonidine-sensitive neurons, iontophoretic NMDA receptor antagonist MK801 (60 nA) significantly attenuated the neuronal inhibition of iontophoretic (30 nA) clonidine. In conclusion, these results suggest that NMDA receptors contribute to the inhibition of clonidine on the RVLM presympathetic neurons.     

Evidence for thermoregulation by dopamine D1 and D2 receptors in the anteroventral preoptic region during normoxia and hypoxia

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been called anapyrexia. Stimulation of dopamine receptors in the central nervous system (CNS) reduces Tb in rats, and dopamine D1 and D2 receptors seem to be involved in this response. Thus, we predicted that injection of SCH 23390 and haloperidol, D1 and partly D2 receptor antagonists, respectively, into the anteroventral preoptic region (AVPO, the thermointegrative region of the CNS) would lessen the hypoxia-induced anapyrexia. We measured Tb of conscious Wistar rats before and after injection of SCH 23390 (50 and 100 ng/100 nl) or haloperidol (50 e 500 ng/100 nl) or their respective vehicles (saline and DMSO 5%) into the AVPO followed by 30 min of hypoxia (7% O2). Vehicles and the lower doses of SCH 23390 and haloperidol had no effect on Tb during normoxia or hypoxia. The higher doses of SCH 23390 and haloperidol attenuated (P<0.05) the drop in Tb elicited by hypoxia. However, this higher haloperidol dose also increased Tb during normoxia. The present data is consistent with the notion that dopamine is an important thermoregulatory neurotransmitter in a way that D2 receptors are mainly involved with maintenance of Tb in euthermia, while D1 receptors are activated to induce hypoxic anapyrexia in the AVPO.