Seiichiro Kawashima

Effects of a Novel Cognitive Enhancer, Spiro[imidazo-[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446), on Learning Impairments Induced by Amyloid-β1–40 in the Rat

We have previously shown that intracerebroventricular (i.c.v.) infusion of amyloid-β (Aβ)1–40 produces oxidative stress and cholinergic dysfunction, as well as learning and memory deficits, in rats. In the present study, effects of a newly synthesized azaindolizinone derivative, spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446), were assessed in rats with learning deficits induced by Aβ1–40 or scopolamine. The i.c.v. infusion of Aβ1–40 caused impairments in spontaneous alternation behavior in a Y-maze task, spatial reference and short-term memory in a water-maze task, and retention of passive-avoidance learning. Aβ1–40-infused rats also showed reduction in choline acetyltransferase (ChAT) activity in the medial septum and hippocampus, but not in the basal forebrain and cortex, and a decrease in glutathione S-transferase (GST)-like immunoreactivity in the cortex. Nicotine-stimulated acetylcholine (ACh) release in Aβ1–40-infused rats was lower than that in vehicle-infused rats. Oral administration of ZSET1446 at the dose range of 0.01 to 1 mg/kg ameliorated Aβ1–40-induced learning impairment in Y-maze, water-maze, and passive-avoidance tasks. ZSET1446 reversed the decrease of ChAT activity in the medial septum and hippocampus, GST-like immunoreactivity in the cortex, and nicotine-stimulated ACh release of Aβ1–40-treated rats to the levels of vehicle-infused control rats. Furthermore, 0.001 to 0.1 mg/kg ZSET1446 showed ameliorative effects on learning impairments caused by scopolamine in a passive-avoidance task. These results suggest that ZSET1446 may be a potential candidate for development as a therapeutic agent to manage cognitive impairment associated with conditions such as Alzheimers disease.

Mitotic activity of prolactin cells in the pituitary glands of male and female rats of different ages

SummaryThe anterior pituitary of colchicine-pretreated male and female rats from 20 days to 12 months of age was stained immunohistochemically with anti-rat prolactin serum. Immunoreactive mitotic cells were identified in all groups of rats. In adult female rats the mitotic index of prolactin cells was higher at oestrus than at other stages of the oestrus cycle and than that in male rats of comparable ages. If adult female rats were ovariectomized on the second day of dioestrus or on the day of proestrus, the mitotic indices at presumptive oestrus were less than those in sham-operated controls at oestrus. Estrogen administration to ovariectomized rats significantly elevated the mitotic index of prolactin cells at 48 h after the treatment. The mitotic indices of prolactin cells in female rats reached a peak at 60 days of age, and then decreased with age. In male rats the mitotic indices showed a steady decrease from the value at 20 days of age. A sex difference in the mitotic indices of prolactin cells was noted from 60 days to 12 months of age. The present results clearly demonstrate that differentiated prolactin cells can undergo mitosis and that a sex difference in the mitotic activity of prolactin cells is present during adult life.

Age-Related Changes in Prolactin Cell Percentage and Serum Prolactin Levels in Intact and Neonatally Gonadectomized Male and Female Rats

Electron microscopically the percentages of various pituitary cell types were calculated at 30 and 90 days of age. Prolactin cell percentage was more at 90 days of age than at 30 days in both intact male and female rats. No sexual difference was observed in the percentage of prolactin cells at 30 days of age, but at 90 days female pituitaries contained more numerous prolactin cells than males ones. Neonatal ovariectomy did not affect the prolactin cell percentage at 30 days of age, while it lowered the percentage at 90 days. Neonatal orchidectomy did not affect the prolactin cell percentage at both 30 and 90 days of age. Mitotic prolactin cells were more frequently observed in intact female rats at estrus than in intact male rats at 90 days of age. These results shown the presence of the sexual difference in the proliferation of prolactin cells. Serum prolactin levels increased with age in intact male and female rats. In neonatally gonadectomized male and female rats the serum prolactin levels failed to increase at 60 and 90 days of age. In general conclusion, the changes in serum prolactin levels are in line with the changes in prolactin cell population during postnatal development.

Effects of sex steroids on the growth of neuronal processes in neonatal rat hypothalamus-preoptic area and cerebral cortex in primary culture

Dissociated cells of neonatal rat hypothalamus‐preoptic area and cerebral cortex were grown in primary culture, and the effects of sex steroids on neurons were morphometrically studied. In cultures of the hypothalamus‐preoptic area estradiol‐17β or testosterone propionate significantly increased the total neuronal process length as compared with control cultures given no steroids in the medium. Significant stimulatory effects were detected on 1, 2 and 3 days after plating, while, 5a‐dihydrotestosterone failed to show any stimulatory influence. In contrast, none of these steroids was effective in cultures of cerebral cortical cells. Based on the number of processes arising from the soma, neurons were divided into unipolar, bipolar and multipolar neurons. Percentage of multipolar neurons of the hypothalamus‐preoptic area was more in cultures treated with estradiol‐17β or testosterone propionate than the controls. Significant effect was not noted in cultures with 5α‐dihydrotestosterone, while, in cultures of the cerebral cortical cells, effects on sprouting of these steroids were far less marked than in cultures of the hypothalamus‐preoptic area. The present findings substantiate the importance of aromatization of androgen to exert its influence on neuronal process growth in cell culture.

Human type II GnRH receptor mediates effects of GnRH on cell proliferation.

Abstract GnRH (gonadotropin-releasing hormone) is well-known as the central regulator of the reproductive system through its stimulation of gonadotropin release from the pituitary. Progress in studies on GnRH demonstrated that GnRH has both inhibitory and stimulatory effects on cell proliferation depending on the cell type, and the mechanisms of these effects have been intensively studied. However, even human GnRH receptors which mediate GnRH stimulation have not been completely identified. In the present study, we showed that the inhibitory and stimulatory effects of GnRH on colony-formation using four cell lines and have demonstrated that the inhibitory and stimulatory effects of GnRH exhibit distinctly different patterns of ligand sensitivity. This result strongly suggests that the two opposite effects of GnRH occur via different types of GnRH receptors, however expressional analyses of human GnRH receptors did not exhibit the significantly different pattern between negatively and positively responding cell lines. Then, in order to identify the GnRH receptors involved in the two opposite effects, effects of GnRH were analysed under the conditions that human GnRH receptors were knocked down by the technique of RNA interference. Consequently, it was found that human type II GnRH receptor mediates GnRH stimulation and its splice variant determines the direction of the response to GnRH. These results are the first clear evidence for the functionality of human type II GnRH receptor. Therefore our novel findings are quite noticeable and will greatly contribute to the studies on the mechanisms of the effects of GnRH on cell proliferation in the future.

Ultrasonic vocalizations and aggressive behavior in male rats

The aim of the present study was to know whether 22-28-kHz vocalizations have any communicatory role in the regulation of aggressive behavior in male rats of the Wistar strain. In pairs of intact rats 22-28-kHz vocalizations showed a positive correlation with the extent of aggressive behavior. The pattern of aggressive behavior during ultrasonic vocalizations was different from that just before and just after the vocalizations. However, surgically deafened rats were less active in aggressive behavior and more active in ambulatory activity in the open field than the controls. Muted rats were not different from the controls in both aggressive behavior and ambulatory activity. The present result that the deprivation of ultrasonic signals failed to increase aggressive behavior does not support the classical hypothesis that ultrasonic vocalizations inhibit the initiation of aggressive behavior. It is concluded that ultrasounds emitted during aggressive encounters may have little communicative value in male rats.

Antiamnesic effects of azaindolizinone derivative ZSET845 on impaired learning and decreased ChAT activity induced by amyloid-β 25–35 in the rat

Antiamnesic effects of a newly synthesized azaindolizinone derivative ZSET845 were assessed in rats made learning ability deficient by amyloid-beta (Abeta)25-35 treatment. Intracerebroventricular injection of Abeta25-35 induced a marked decrease in step-through latency in passive avoidance task and reduction in choline acetyltransferase (ChAT) activity in the medial septum and hippocampus, but not in the basal forebrain and cortex. The number of ChAT-immunoreactive cells was decreased in the medial septum. Oral administration of ZSET845 at a dose of 1 or 10 mg/kg ameliorated learning impairment in passive avoidance task and enhanced ChAT activity in the basal forebrain, medial septum and hippocampus, and increased in the number of ChAT-immunoreactive cells in the medial septum in Abeta-treated rats to the levels of vehicle-injected control rats. These results suggest that ZSET845 is worth testing for further preclinical study aimed for the treatment of senile dementia such as Alzheimers disease.

Effect of vitamin E on the accumulation of fluorescent material in cultured cerebral cortical cells of mice.

The effect of vitamin E on the accumulation of lipofuscin-containing fluorescent material in the mouse cerebral cortical cells in primary culture was studied. Fluorescent material was extracted in ethanol:diethylether (3:1) and autofluorescence intensity of the extracts was measured by a spectrofluorophotometer. Although vitamin E at the concentration of 0.005 IU/ml was not effective, 0.01 IU/ml vitamin E inhibited the accumulation of fluorescent material. Fluorescent material accumulation was reduced to 76.3-86.4% of the control level in 6-, 12-, or 18-day treatment of 0.01 IU/ml vitamin E. High doses of vitamin E (0.05 or 0.1 IU/ml) were toxic for cultured cells. Ethanol, the vehicle of vitamin E, at the final concentration of 0.005% was also effective on the reduction of fluorescent material accumulation (81.0% of the control level at 18 days). The inhibitory effects of vitamin E as well as ethanol on the accumulation of fluorescent material in cultured cells are explained by their nature as free radical scavengers.

Differential localization of vasopressin- and oxytocin-immunoreactive cells and conventional neurosecretory cells in the ganglia of the earthworm Pheretima hilgendorfi

Cells containing arginine vasopressin (AVP)‐ and oxytocin (OXT)‐like substances were immunohistochemically visualized in the cerebral, subesophageal, and ventral nerve cord ganglia of the earthworm Pheretima hilgendorfi. Whether these anti‐AVP– and anti‐OXT–reactive cells are identical with classical aldehyde fuchsin (AF)‐positive neurosecretory cells was tested in serial sections. In all ganglia, groups of scattered neuronal cell bodies and axons strongly reactive to AVP and OXT antisera were observed, but AF‐positive cells consisting of type a (dark blue) and type b (purple) cells were predominantly present in the cerebral and subesophageal ganglia. In the cerebral and subesophageal ganglia anti‐AVP– and anti‐OXT–reactive cells were generally larger than AF‐positive cells. Some AF‐positive cells were reactive either to anti‐AVP or anti‐OXT serum, but some failed to react to either serum. Anti‐AVP– and anti‐OXT–reactive cells were not immunoreactive to OXT and AVP antisera, respectively. Electron microscopic observations showed that the granules of type a cells were larger and less electron dense than those of type b cells and anti‐AVP–reactive cells. The present cytological observations clearly showed that AVP‐ and OXT‐like substances were widely present in the ganglionic cells of the earthworm

Plasticity of vasopressin- and oxytocin-containing fibers in the median eminence in hypophysectomized young and old mice

Rearrangements of vasopressin- and oxytocin-containing fibers in the external layer of the median eminence after hypophysectomy were compared between young and old mice. In 3-month-old hypophysectomized mice, the increase in the number of fibers containing vasopressin was greater than that observed in 19-month-old hypophysectomized ones, suggesting a decrease in axonal plasticity in old mice. No difference with age was detected for the plasticity of fibers containing oxytocin.