Makoto Yokosuka

Postnatal development and sex difference in neurons containing estrogen receptor-alpha immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat.

Estrogen has been considered as a key substance that induces sexual differentiation of the brain during fetal and neonatal life in the rat. Thus, to define the brain regions involved in the brain sexual differentiation, we examined the regions where the estrogen receptor (ER) is located in the developing rat brain. We examined immunohistochemical distribution of the cells containing estrogen receptor‐α (ER‐α) in the preoptic region, the diencephalon, and the amygdala in male and female rats on postnatal days 1–35 (PD1–PD35). The antibody used recognizes ER‐α equally well for both occupied and unoccupied forms. ER‐α immunostaining was restricted to the cell nuclei of specific cell groups. In PD1 rats, ER‐α‐immunoreactive (ER‐IR) signals were detected in the lateral septum, the organum vasculosum lamina terminalis, the medial preoptic nucleus (MPN), the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, the lateral habenula, the posterodorsal part of the medial amygdala nucleus, the posterior part of the cortical amygdala nucleus, the hypothalamic ventromedial nucleus (VMH), the hypothalamic arcuate nucleus, and the posterior hypothalamic periventricular nucleus. The distribution pattern of ER‐IR cells in the newborn rat was much the same as that in the adult in the preoptic‐hypothalamic and amygdala regions. Moreover, the signals in the MPN and the VMH were stronger in the female than in the male, perhaps reflecting the ability of estrogen generated by aromatization of testosterone in the male to down‐regulate the ER signal. Thus, the brain regions showing sex differences may be sites of sexual differentiation of the brain by aromatizable androgen during the neonatal period. J. Comp. Neurol. 389:81–93, 1997.

Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats.

In an attempt to define the role of the pineal secretory melatonin and an analogue, 6‐hydroxymelatonin (6‐OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)‐induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6‐OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague–Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co‐treatment of CP‐treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the GSH/GSSG ratio was prevented. Treatment of CP‐enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH‐Px), decreased as a result of CP administration, which was restored to control levels with melatonin co‐treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP‐treated rats; these changes were prevented by melatonin treatment. 6‐OHM has been shown to have some antioxidative capacity, however, the protective effects of 6‐OHM against CP‐induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co‐treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH‐Px activating property, and/or to its regulatory activity for renal function.

Neural substrates for leptin and neuropeptide Y (NPY) interaction: hypothalamic sites associated with inhibition of NPY-induced food intake

Intracerebroventricular (i.c.v.) injection of leptin, the adipocyte hormone, inhibits neuropeptide Y (NPY)-induced feeding in the rat. To identify the neural substrate for leptin and NPY interaction in the hypothalamus, we evaluated the expression of c-fos-like immunoreactivity (FLI), an early marker of neuronal activation, in response to icv administration of leptin, NPY and leptin plus NPY. As expected, leptin significantly decreased NPY-induced feeding in leptin plus NPY-treated rats. A comparative evaluation of the number of FLI-positive neurons in hypothalamic sites showed that both leptin and NPY activated FLI in the parvocellular subdivision of the paraventricular nucleus (pPVN), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). NPY also augmented the FLI response in the magnocellular PVN (mPVN) and supraoptic nucleus (SON), two sites where leptin alone was ineffective. Combined leptin and NPY treatment significantly decreased the number of FLI-positive neurons in the magnocellular PVN but increased their number in the dorsomedial nucleus as compared to the number of FLI-expressing neurons in these sites after NPY and leptin alone. Because there is morphologic evidence of a link between magnocellular PVN and dorsomedial nucleus, these results suggest the functional involvement of leptin plus NPY responsive elements in these sites in reduction of NPY-induced feeding by leptin.

Inhibition of neuropeptide Y (NPY)-induced feeding and c-Fos response in magnocellular paraventricular nucleus by a NPY receptor antagonist: a site of NPY action.

Neuropeptide Y (NPY) is one of the important endogenous orexigenic peptides. In these studies we employed c-Fos immunostaining and a selective NPY Y1 receptor antagonist to identify the site of action of NPY in the hypothalamus. The results showed that intracerebroventricular administration of NPY stimulated feeding and increased immunostaining of c-Fos, a product of the immediate early gene c-fos, in several hypothalamic sites, including the dorsomedial nucleus, the supraoptic nucleus, and the two subdivisions of the paraventricular nucleus (PVN), the parvocellular PVN, and magnocellular PVN (mPVN). Intracerebroventricular administration of 1229U91, a selective NPY Y1 receptor antagonist, affected neither food intake nor c-Fos-like immunoreactivity (FLI) in these hypothalamic sites. Coadministration of NPY and NPY Y1 receptor antagonist inhibited NPY-induced food intake by 48%, but failed to affect NPY-induced FLI in the supraoptic nucleus, dorsomedial nucleus, and parvocellular PVN. However, this combin...

The mPVN mediates blockade of NPY-induced feeding by a Y5 receptor antagonist: a c-FOS analysis.

To identify the site(s) of NPY Y5 receptor (Y5R) mediation of NPY-induced feeding, we employed c-Fos immunostaining and a selective Y5R antagonist (Y5R-A), CGP71683A, in adult male rats. Intracerebroventricular (icv) administration of NPY stimulated feeding and c-Fos-like immunoreactivity (FLI) in the dorsomedial hypothalamus, supraoptic nucleus and the two subdivision of the hypothalamic paraventricular nucleus (pPVN), the parvocellular (pPVN), and magnocellular (mPVN). Y5R-A on its own, injected either intraperitoneally or icv, neither affected feeding nor FLI in hypothalamic sites. However, Y5R-A pretreatment suppressed NPY-induced food intake and FLI selectively in the mPVN. Taken together with our previous similar finding of Y1R involvement, these results suggest that NPY receptor sites concerned with feeding behavior reside selectively in the mPVN and Y1 and Y5 receptors are either coexpressed or expressed separately in those target neurons that promote appetitive drive.

Circadian Rhythm of Melatonin Release From the Photoreceptive Pineal Organ of a Teleost, Ayu (Plecoglossus altivelis) in Flow-Thorough Culture

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow‐through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free‐running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six‐hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.

Specific expression pattern of Fos in the accessory olfactory bulb of male mice after exposure to soiled bedding of females.

The heterogeneous structure of the accessory olfactory bulb (AOB) has been demonstrated immunocytochemically. In this study, we analyzed the expression of an immediate-early gene protein, c-Fos, as a marker of neuronal activity in response to chemosensory cues was analyzed. The number of c-Fos-immunoreactive (Fos-ir) cells was measured in the rostral and caudal zones of the AOB in male ICR mice after exposure to the soiled bedding of female mice. The results revealed no significant difference in the number of Fos-ir cells in the caudal zone of the AOB between exposure to the soiled bedding of female ICR mice (ICR group) and exposure to that of female Balb mice (Balb group). In the rostral zone, however, the number of Fos-ir cells in the glomerular layer and granule cell layer was larger in the ICR group than in the Balb group. The difference in the expression of c-Fos in response to different pheromonal stimuli between the rostral and caudal zones in the mouse AOB has been shown for the first time in this study. These results strongly suggest that the heterogeneous structure of the AOB has an important role in the perception and processing of pheromones.

Transient expression of estrogen-receptor-like immunoreactivity (ER-LI) in the facial nucleus of the neonatal rat

Estrogen receptor-like immunoreactivity (ER-LI) was detected in the medial subnucleus of the facial nucleus by immunocytochemistry in both male and female neonatal rats, but not in the adult rat. Identity of the motoneurons in this subnucleus projecting to the corresponding facial muscles and the cells with ER-LI positive signals was examined by retrograde tracing. The majority of the cells marked with the tracer did not carry ER-LI signals.

Colocalization of neuronal nitric oxide synthase and androgen receptor immunoreactivity in the premammillary nucleus in rats.

To analyze the importance of premammillary region in male behavior such as aggression and mating, distribution of androgen- and estrogen-receptors (AR and ER), aromatase (ARO) and neuronal nitric oxide synthase (nNOS) was studied in the dorsal and ventral premammillary nuclei (PMd and PMv) of the rat by immunohistochemistry. The nNOS-immunoreactivities (-IR) were present both in the PMd and the PMv, while AR-IR were detected only in the PMv. AR-IR became undetectable after orchidectomy but they recovered by an injection of 5 alpha-dihydrotestosterone (DHT). In both nuclei, no clear signals of ER-IR were encountered. On the other hand, ARO-IR were found in the PMv but only very few. In the PMv, although DHT did not increase nNOS-IR significantly in castrated males, all the nNOS-IR cells contained AR-IR at least in intact and castrated-DHT injected males. Thus, involvement of nNOS-nitric oxide system in the PMv in the androgenic action on male behaviors was suggested.

Glial and neuronal localization of neuronal nitric oxide synthase immunoreactivity in the median eminence of female rats

Localization of neuronal nitric oxide synthase-immunoreactivity (nNOS-IR) in the median eminence of female rats (n=4) was examined by electron microscopy to explore the possibility that nitric oxide is involved in the terminal regulation of neurosecretory peptides such as GnRH. Under light microscopy, a dense distribution of nNOS-IR was observed in this region. Electronmicroscopically, nNOS-IR was found in glial elements and nerve terminals containing dense-core vesicles. We also found a few nNOS-immunopositive synapses, in which intense immunoreactivity was found on the postsynaptic density and mitochondrial membrane. The localization of nNOS-IR in nerve terminals and glial elements in the median eminence might indicate that nNOS plays a role in regulating the release of neurosecretory peptide.