Jun Arita

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.

Age-related disturbance of memory and CREB phosphorylation in CA1 area of hippocampus of rats.

In the early process of long-term memory formation, cyclic AMP response element-binding protein (CREB), a transcription factor on which multiple signal transduction pathways converge, has been implicated. We examined whether the age difference in the performance of contextual fear conditioning (CFC) is associated with a change in activation of CREB in the hippocampus which is an important neural structure for long-term memory. The activation of CREB in the hippocampus in young (15 weeks old) and old (120 weeks old) male rats was determined immunohistochemically with an antibody that specifically recognizes the phosphorylated form of CREB (pCREB). Young rats exhibited better performance than old rats with respect to the freezing time in CFC. Phosphorylation of CREB as revealed by the ratio of the pCREB-immunoreactive cell number to the CREB-immunoreactive cell number was increased in the CA1 region, but not in other hippocampal regions following training for CFC. The close relationship between behavioral performance and CREB phosphorylation in the CA1 region suggests that hippocampal CREB is involved in age-related decline of learning and memory.

Effects of preoptic injections of gastrin, cholecystokinin, secretin, vasoactive intestinal peptide and PHI on the secretion of luteinizing hormone and prolactin in ovariectomized estrogen-primed rats

The effects of intracerebral injections of brain-gut peptides in the preoptic region on the secretion of luteinizing hormone (LH) and prolactin (PRL) were examined in ovariectomized, estrogen-primed rats. Gastrin or cholecystokinin-octapeptide injection in the preoptic region induced a moderate increase of circadian rise of LH secretion in some animals but no appreciable change in PRL secretion. Secretin injection was strikingly effective in increasing circadian rise of LH and PRL secretion. Vasoactive intestinal peptide injection completely abolished the occurrence of circadian rise of LH secretion whereas PHI injection facilitated LH secretion undergoing the circadian rhythm. The results suggest that those peptides act as neurotransmitters or neuromodulators in the preoptic region and are implicated in the regulation of LH and PRL secretion.

Distribution of prolactin-releasing peptide-immunoreactive neurons in the rat hypothalamus

A newly identified hypothalamic peptide whose specific receptors are present in the anterior pituitary gland is a selective and potent stimulator of prolactin secretion and is therefore termed prolactin-releasing peptide (PRP). We investigated the distribution of PRP-containing neurons in the hypothalamus of female rats by immunocytochemical techniques. Immunocytochemistry using a specific antibody raised to PRP revealed that PRP-immunoreactive perikarya were located in the posteroventral part of the dorsomedial nucleus of the hypothalamus. PRP-immunoreactive nerve terminals were present in high concentrations in a region ventral to the bed nucleus of the stria terminalis, but scarcely observed in the external layer of the median eminence in which well known hypothalamic hormones such as growth hormone-releasing hormone and somatostatin were abundantly detected. This specific distribution in the hypothalamus suggests a novel route of the hypophysiotropic action of PRP.

Effects of intracerebroventricular administration of growth hormone-releasing factor and corticotropin-releasing factor on somatostatin secretion into rat hypophysial portal blood.

To clarify the regulatory mechanisms for the secretion of somatostatin (SRIF) from the hypothalamus, the effects of intracerebroventricular (i.c.v.) administration of growth hormone-releasing factor (GRF) and corticotropin-releasing factor (CRF) on SRIF secretion into hypophysial portal blood were examined in pentobarbital-anesthetized male rats. Neither the concentration of SRIF in portal plasma nor the secretion rate of SRIF was changed after i.c.v. administration of 0.9% saline. Administration of 10 ng or 5 micrograms human GRF i.c.v. produced a significant increase in the portal plasma concentration and secretion rate of SRIF. Likewise, 5 micrograms CRF significantly increased the portal plasma concentration and secretion rate of SRIF. These results suggest that the neuropeptides GRF and CRF centrally influence SRIF secretion into hypophysial portal blood.

Differential Regulation of Prolactin Release and Lactotrope Proliferation during Pregnancy, Lactation and the Estrous Cycle

The proestrous surge of prolactin (PRL) secretion and subsequent proliferation of lactotropes at estrus have been suggested to be induced by a common hypothalamic hormone. We investigated changes in lactotrope proliferation at other reproductive stages of female rats when PRL secretion was stimulated. To assess proliferative activity of lactotropes, incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA was measured by double immunostaining for PRL and BrdU. BrdU-labeling indices, determined by BrdU injections at 10.00 h, revealed low levels of proliferative activity of lactotropes at the reproductive stages including diestrus, days 6 and 13 of pregnancy, and day 6 of lactation while high levels were detected on estrus and the day of parturition. When BrdU-labeling indices were determined at 3-hour intervals through day 6 of pregnancy to find an increase in lactotrope proliferation which might occur at times other than 10.00 h, proliferative activity of lactotropes remained at low levels with a slight increase in the afternoon. Such a diurnal change as observed in early pregnancy was not detected on day 13 of pregnancy. In contrast, short-interval determinations of BrdU-labeling indices during a period from day 20 of pregnancy to day 2 of lactation revealed a marked increase in proliferative activity on the day of parturition with a peak at 18.00 h, which was comparable to that observed at estrus. To investigate involvement of ovarian steroids in suppression of lactotrope proliferation as observed during early pregnancy and lactation, ovariectomized and pup-removed lactating rats were given one of treatment combinations of estradiol and suckling. In pup-removed lactating rats, estradiol treatment alone induced neither a PRL surge nor an increase in BrdU-labeling indices. Suckling stimuli, which were effective in increasing serum PRL concentrations irrespective of estradiol treatment, elicited a marked increase in BrdU-labeling indices in the presence of estradiol but not in its absence. These results suggest that proliferative activity of rat lactotropes does not necessarily correlate with PRL secretion during pregnancy and lactation. In contrast to PRL release, lactotrope proliferation requires both a hypophysiotropic stimulatory input from the hypothalamus and a sensitizing action of estradiol, an observation which may account for the fact that proliferation does not occur during pregnancy and lactation in spite of elevated PRL release.

A selective increase in phosphorylation of cyclic AMP response element-binding protein in hippocampal CA1 region of male, but not female, rats following contextual fear and passive avoidance conditioning

Cyclic AMP response element-binding protein (CREB), a transcription factor on which multiple signal transduction pathways converge, has been implicated in long-term memory. We examined whether the sex difference in the performance of contextual fear or passive avoidance conditioning is associated with a change in the activation of CREB in the hippocampus, a neural structure important for long-term memory. The activation of CREB in different subregions within the hippocampus in male and female rats was determined immunohistochemically with an antibody that specifically recognizes the phosphorylated form of CREB (pCREB). With respect to the freezing time in contextual fear conditioning and the step-through latency in passive avoidance conditioning, male rats exhibited better performance than female rats. Phosphorylation of CREB (% pCREB) as revealed by the ratio of the pCREB-immunoreactive (pCREB-ir) cell number to the CREB-immunoreactive cell number was increased in the CA1 region, but not in CA3, CA4, or in the dentate gyrus following training for both types of conditioning in males. In females, such an increase in % pCREB was not found in any hippocampal subregion at any time after conditioning or by increasing the intensity of foot shock. Orchidectomy in males did not alter either the performance of contextual conditioning or conditioning-induced CREB phosphorylation in CA1. The close relationship between behavioral performance and CREB phosphorylation in the CA1 region suggests that hippocampal CREB is involved in the sex difference in some forms of learning and memory.

Procaine microinjection into the lower midbrain increases brown fat and body temperatures in anesthetized rats

A tonic inhibitory mechanism on heat production was studied by microinjecting procaine into various regions of the brain while recording temperature changes of the interscapular brown adipose tissue (IBAT) and rectum in urethane-anesthetized rats at room temperature of 23-25 degrees C. Procaine microinjected bilaterally (10%, 1.0 mu l/site, 1.5 mm to midline) into the midbrain and the upper- to mid-pontine area of the reticular formation increased temperatures of the IBAT and rectum. The highest temperature rise (1.02 +/- 0.11 degrees C for IBAT, 0.64 +/- 0.06 degrees C for rectum) with the shortest onset latency (1.5 +/- 0.3 min for IBAT, 4.6 +/- 1.1 min for rectum) was observed when procaine was injected into the lower midbrain (the area between 6 and 7 mm posterior to the bregma, and 6.5 to 8.5 mm deep from the cortical surface). These regions include the retrorubral field, pedunculopontine tegmental nucleus, and rubrospinal tract. Procaine-induced IBAT and rectal temperature increases were dose-dependent, and reproduced reliably from the same injection site of the same animal. Intravenous indomethacin, a prostaglandin H synthase inhibitor, did not affect procaine-induced temperature rise, and propranolol, a beta-blocker, completely blocked it. These results suggest that microinjected procaine exerts its local anesthetic effect and release a tonic inhibition resulting in a disinhibition-induced temperature increase through the enhanced central sympathetic outflow. They support the hypothesis that a bilateral tonic inhibitory mechanism on heat production exists in the lower midbrain.

Temporal changes in the expression of brain-derived neurotrophic factor mRNA in the ventromedial nucleus of the hypothalamus of the developing rat brain

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which is important for the growth, differentiation, and survival of neurons during development. We have performed a detailed mapping of BDNF mRNA in the neonatal rat brain using a quantitative in situ hybridization technique. At postnatal day (PND) 4, hypothalamic structures showed only modest expression of BDNF mRNA, with the exception of the ventromedial nucleus (VMN), where expression was higher than that detected in the hippocampus. Abundant BDNF mRNA was also found in the bed nucleus of the anterior commissure, retrosplenial granular cortex, and the posteroventral part of the medial amygdaloid nucleus. Messenger RNAs encoding other neurotrophins, including nerve growth factor (NGF) and neurotrophin-3 (NT-3) and the BDNF receptor trkB, were not selectively localized in neonatal VMN. During subsequent developmental stages, BDNF mRNA expression in the VMN changed dynamically, peaking at PND 4 and falling to minimal levels in the adult brain. In contrast, the low levels of BDNF mRNA observed in the CA3 region of the hippocampus increased to adult levels following PND 10. As the VMN undergoes sexual differentiation, we compared BDNF, NGF, NT-3, and trkB mRNA expression in the VMN in males and females at embryonic day 20 and PND 4, but found no differences between them. These results suggest that localized and high level expression of BDNF mRNA in the neonatal VMN plays an important role in its neural organization and functional development.

Spontaneous acetylcholine release in the hippocampus exhibits a diurnal variation in both young and old rats

Extracellular levels of acetylcholine (ACh) in the hippocampus were measured by the microdialysis method in freely moving young (3-4 months old) and old (18-24 months old) female rats over a period of 24 h to examine the effect of aging on hippocampal ACh release. Hippocampal ACh release during a 24-h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in old rats as well as young rats. The present study suggests that a diurnal variation in ACh release is maintained fairly well until the rats are at least 24 months old.