Fukuko Kimura

Acetylcholine release in the rat hippocampus as measured by the microdialysis method correlates with motor activity and exhibits a diurnal variation

Extracellular levels of acetylcholine were measured by the microdialysis method coupled to high performance liquid chromatography in the dorsal hippocampus of freely moving rats over a period of 24 h to examine whether the acetylcholine release in the hippocampus exhibited a diurnal variation. Spontaneous motor activity was simultaneously measured with an automatic animal activity monitor. The amount of acetylcholine collected per 20-min sample varied markedly, in a range from about 5 to 90 pmol. There appeared to be variations in the amount with a 2-4 h periodicity as well as an apparent diurnal periodicity. In all five rats studied, the overall mean value for the dark cycle (11.1-34.5, average 20.9 pmol/20 min) was significantly greater than that for the light cycle (5.1-21.3, average 12.3 pmol/20 min), showing a 70% average increase. Cross-correlation analysis performed between the amount of acetylcholine and the motor activity count for the animal during the sampling revealed a significant positive correlation coefficient in four rats studied. The present study demonstrates for the first time that the acetylcholine release shows a diurnal variation.

Naloxone Increases the Frequency of the Electrical Activity of Luteinizing Hormone-Releasing Hormone Pulse Generator in Long-Term Ovariectomized Rats

Characteristic increases in neuronal activity accompanied by the initiation of each luteinizing hormone (LH) pulse have been successfully recorded in freely moving ovariectomized rats by means of multiunit activity (MUA) recording techniques. In the present study, the effect of the opioid receptor antagonist naloxone on this neuronal activity was examined in rats ovariectomized for 6-10 weeks. Electrodes were chronically implanted into the medial basal hypothalamus (n = 78) or the medial preoptic area (n = 29). During the MUA recording, blood samples were taken through an indwelling atrial cannula at 3- or 6-min intervals to determine the serum LH concentration. Naloxone was administered intravenously for 1 h by either intermittent injection every 6 min (0.05 mg/kg, 10 times) or continuous infusion (0.1, 0.2 and 0.5 mg/kg/h). Explosive rises in the MUA (volleys) associated with the initiation of LH pulse were recorded in 7 animals in which the tips of electrodes were located in the arcuate nucleus-median eminence region. In 5 animals studied, the mean (+/- SE) duration and interval of the MUA volleys were 2.1 +/- 0.1 and 22.1 +/- 0.9 min, respectively. Naloxone given every 6 min significantly increased the duration to 2.6 +/- 0.1 min, and decreased the interval to 9.5 +/- 0.9 min. This naloxone treatment disturbed the clear pulsatility of LH secretion observed in the pretreatment control period. Analysis of the effects of naloxone infused at 3 different doses revealed that the facilitatory effects of naloxone on the duration and frequency of the MUA volleys were dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in the basolateral amygdala: the extracellular levels of serotonin and dopamine, and their responses to restraint stress in rats

The sex difference in the emotional response to stress suggests a sex‐specific stress response in the amygdala. To examine the sex difference in extracellular levels of serotonin (5HT) and dopamine (DA) in the basolateral amygdala (BLA) and their responses to restraint stress, in vivo microdialysis studies were performed in male and female rats. In experiment I, dialysates were collected from the BLA at 15‐min intervals under the freely moving condition. Mean extracellular levels of 5HT or DA in the BLA were higher in male rats than in female rats. In experiment II, rats were subjected to restraint stress for 60 min to examine the stress response of 5HT or DA levels. Although restraint stress significantly increased extracellular 5HT levels in both sexes of rats, female rats showed a greater response than male rats. Moreover, restraint stress significantly increased extracellular DA levels in female rats, but not in male rats. In experiment III, rats were subjected to restraint stress for 30 min to examine behavioral responses to restraint stress. Although no sex difference was observed in the number of audible vocalizations, male rats defecated a larger number of fecal pellets than female rats. In experiment IV, rats were tested for freezing behavior to examine contextual fear responses. Conditioned male rats showed a longer freezing time than conditioned female rats. We found sex differences in the extracellular levels of 5HT and DA in the BLA and their responses to restraint stress, which may be involved in the sex‐specific emotional response to stress in rats.

Sex Differences in the Stress-Induced Release of Acetylcholine in the Hippocampus and Corticosterone from the Adrenal Cortex in Rats

To assess sex differences in stress-induced acetylcholine (ACh) release in the hippocampus and corticosterone (CS) release from the adrenal cortex, an in vivo microdialysis study was performed in intact male (n = 6) and cycling female (diestrus 1, n = 5; proestrus, n = 6) rats. Dialysates and blood samples were taken from 11.00 to 16.00 h in freely moving rats, but restraint stress was applied from 12.00 to 13.00 h. Basal ACh release in the hippocampus was low without significant differences between groups. Although ACh release promptly increased with the onset of restraint stress in both sexes, the magnitude of the increase in males was significantly greater than in female rats during diestrus and proestrus (p < 0.01). Basal serum CS concentrations were significantly greater in proestrus than in diestrus (p < 0.01) or in male rats (p < 0.01). Serum CS concentrations significantly increased at the onset of restraint stress in both sexes (p < 0.01), but the magnitude of the increase was significantly greater in female than in male rats (p < 0.01). These results indicate that sex and/or gonadal steroid environment can affect the stress-induced ACh release in the hippocampus and CS release from the adrenal cortex.

Impairment of maze learning in rats following long-term glucocorticoid treatments

The present study examined the influence of long-term glucocorticoid treatment on a maze learning task on a radial 8-arm maze in rats. Either 100 mg cholesterol (as a control), or corticosterone, bead was implanted in rats for a period of 3 months, beginning at 12 weeks of age. The effect of this treatment on the maze learning task was evaluated during or 4 weeks after the treatments. In both experiments, corticosterone-implanted rats showed an increase in number of trials to attain at least seven correct choices in the first eight choices in five consecutive trials (P < 0.05). We concluded that long-term glucocorticoid exposure resulted in an impairment of the hippocampal functions, i.e. learning and memory, similar to that found in aged hippocampus.

Age-related changes in diurnal acetylcholine release in the prefrontal cortex of male rats as measured by microdialysis

Extracellular levels of acetylcholine in the prefrontal cortex were measured using the micro-dialysis method in freely moving young (three to four months old) and old (23 to 24 months old) male rats over a period of 24 h to examine the effect of aging on prefrontal acetylcholine release. Prefrontal acetylcholine release during a 24 h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in young rats but not in old rats. In addition, prefrontal acetylcholine release was closely associated with spontaneous activity in young rats but not in old rats. The present study suggests that aging reduces diurnal changes in the prefrontal acetylcholine release and that there is a cross-correlation between the prefrontal acetylcholine release and spontaneous locomotor activity in male rats.

Gonadotropin-Releasing Hormone Exhibits Circadian Rhythm in Phase with Arginine-Vasopressin in Co-Cultures of the Female Rat Preoptic Area and Suprachiasmatic Nucleus

To determine whether the suprachiasmatic nucleus can drive a circadian release of gonadotropin‐releasing hormone (GnRH) in the preoptic area, we measured the release of GnRH, arginine‐vasopressin and vasoactive intestinal polypeptide (VIP) in cocultures of the preoptic area and the suprachiasmatic nucleus at 2‐h intervals over a period of 120 h. The release of GnRH in cocultures exhibited a significant circadian rhythm in the presence of oestrogen but not in the absence of oestrogen. The period of the GnRH circadian rhythm was the same as that of the arginine‐vasopressin circadian rhythm, and different from the VIP circadian rhythm in each coculture. Furthermore, the peak phase of the GnRH rhythm occurred at the time same as that of the arginine‐vasopressin rhythm in each coculture. However, the peak phase of the GnRH rhythm was not always the same as that of the VIP rhythm. Administration of arginine‐vasopressin significantly increased GnRH release in single preoptic area cultures in the presence of oestrogen, but VIP did not. The result suggests that, in cocultures of the suprachiasmatic nucleus and the preoptic area, arginine‐vasopressin neurones drive the circadian release of GnRH in the presence of oestrogen. We suggest that arginine‐vasopressin neurones in the suprachiasmatic nucleus mediate the clock information to GnRH neurones in vivo as well.

Changes in estrogenic regulation of estrogen receptor α mRNA and progesterone receptor mRNA in the female rat hypothalamus during aging: an in situ hybridization study

We examined two molecular responses to estrogen, reduction in estrogen receptor alpha (ER alpha) mRNA and increase in progesterone receptor (PR) mRNA, in the hypothalamus of 3- (young) and 10-month-old (middle-aged) cycling, and 15-month-old (old) acyclic, Fischer 344 female rats. The rats were ovariectomized and then given silastic capsules containing 5% 17beta-estradiol. or empty implants, and killed 4 days after implantation. By means of in situ hybridization, we found that, in young rats, estrogen reduced ER alpha mRNA in both the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) but not in the preoptic area (POA). In contrast, the effect of estrogen on ER alpha mRNA in the VMH and ARC of middle-aged and old rats was not statistically significant. On the other hand in all regions the induction of PR mRNA by estrogen was at least as strong in middle-aged and old as in young rats. The present study revealed that the induction of PR mRNA by estrogen in the hypothalamus was not impaired with age but ER alpha mRNA in the VMH and ARC was significantly impaired with age, but not in the POA.

GABAergic modulation of gap junction communication in slice cultures of the rat suprachiasmatic nucleus.

We employed morphological and electrophysiological methods in order to elucidate mechanisms which are responsible for communication between cellular oscillators in the cultured rat suprachiasmatic nucleus, the site of the endogenous biological clock that regulates circadian rhythms in mammals. When a gap junction-permeable dye, Lucifer Yellow, was injected into single neurons in the suprachiasmatic nucleus culture, the dye was transferred to neighboring cells in a gap junction blocker-sensitive manner. Optical imaging of neural activity evoked by electrical stimulation in the culture revealed that the spread of depolarization was inhibited by gap junction blockers but not by a blocker of voltage-dependent Na(+) channels. Depolarization propagation was inhibited by muscimol, a GABA(A) receptor agonist, in a dose-dependent manner and the inhibition was reversed by bicuculline, a GABA(A) receptor antagonist. Furthermore, muscimol inhibited dye-transfer between neurons in the suprachiasmatic nucleus culture in a dose-dependent fashion.These independent lines of evidence suggest that the gap junction communication is involved in interneuronal communication in the suprachiasmatic nucleus slice culture and that the coupling state between neurons is not static but dynamically regulated via GABA(A) receptor systems.

Intraventricular injection of antiserum to nerve growth factor delays the development of amygdaloid kindling

To investigate the possibility that nerve growth factor (NGF) may play some role in the development of kindling, rabbit anti-NGF serum was injected into the third ventricle on the first 3 days of daily electrical stimulation of the basolateral amygdala. The number of stimulations required to reach full amygdaloid kindling increased significantly in the rat injected with anti-NGF serum compared to that in the rat injected with normal rabbit serum. It was confirmed that anti-NGF serum did not act as an anticonvulsant. The results demonstrate that NGF may be important for the long-lasting neuronal changes induced by daily electrical stimulation of the amygdala.