Naoto Sensui

Acetylcholine in the hippocampus during the discrimination learning performance in a rat model of chronic cerebral ischaemia

Acetylcholine (ACh) release in the hippocampus of Wistar strain rats with permanent bilateral occlusion of the common carotid arteries was examined during a discrimination learning task using a microdialysis method. Such occlusion resulted in obvious impairment of the discrimination performance. The state, the basal value and released patterns, of ACh in the hippocampus differed in the sham-operated control and the experimental group, while ACh release was elevated during the dialysis experiment in both groups. These findings suggested that the bilateral occlusion produced persistent learning deficits from an early stage after the operation and that the impaired discrimination learning performance might be related to the diffusion of ACh in the hippocampus.

Exogenous Oxytocin Attenuates Suckling-Induced Prolactin Release but Not Maternal or Infant Behavior in Lactating Rats

The influence of exogenous oxytocin (OT) on maternal and infant behavior over 60 min of suckling, which followed 6 h of isolation, was investigated on Day 12 of lactation in rats. Mothers administered 1 IU of OT or saline through an indwelling atrial catheter and their litters indicated a similar nursing and suckling pattern, which was estimated by the crouching time of the mothers and the number of stretch reactions performed by the litters during a suckling period. To assess the alteration of the suckling intensity by OT administration, the plasma prolactin (PRL) level was determined by an Nb2 lymphoma cell bioassay. In the control group, the plasma PRL level increased and reached a peak at 45 min after the onset of suckling in 60% of the animals. The suckling-induced PRL release was completely inhibited and/or markedly delayed by OT administration. The difference in body weight of the litters before and after a suckling period was estimated as an index of the amount of milk suckled by the litters. There was no difference in the amount of milk between the control and OT-treated groups during a 60-min suckling period. However, it was significantly greater in the OT-treated group during the first 20 min of the suckling period. These results indicate that a dose of OT is a factor in the attenuation of the intensity of suckling done by the pups, whereas the nursing and suckling behavior is not influenced by OT administration.

Effect of estradiol and FBS on PRL cells, GH cells, and PRL/GH cells in primary cultures of pituitary cells from prenatal rats.

The effects of estradiol (E2) treatment on prolactin (PRL) cells, GH cells, and PRL/GH cells in immature pituitary cells were determined using primary cultures from prenatal rats and immunocytochemistry with fluorescent antibodies. Anterior pituitaries obtained from fetuses on day 22 of pregnancy were monodispersed and cultured in chemically defined medium or medium containing 10% fetal bovine serum (FBS). After preincubation for 24 hr, E2 (final concentrations were 0 M, 10-8 M, 10-7 M, and 10-6 M) was added into each medium. After 72 hr of incubation, cells were subjected to Immunocytochemistry. E2 stimulated the increase of PRL cells in a dose-dependent manner, and the PRL cell percentage cultured with FBS in all groups was significantly higher than that cultured in chemically-defined medium. PRL/GH cells also responded to E2 in the same manner as PRL cells. E2 was not effective in proliferating GH cells, and GH cell percentage significantly decreased with the addition of FBS into the medium. These results suggest that E2 is dose-dependently capable of increasing immature PRL cells and/or PRL/GH cells in vitro. Moreover, there is a factor(s) in FBS that regulates the increase of these cells.

Induction of cholinergic differentiation by 5-azacytidine in NG108-15 neuronal cells.

The DNA-demethylating agent 5-azacytidine (5-azaC) causes extensive genomic demethylation of 5-methyl-cytosine residues and reduces DNA methyltransferase activity in cells. This study evaluated the effect of 5-azaC on neuronal differentiation in proliferating NG108-15 neuronal cells, which exhibit cholinergic traits. The expression of choline acetyltransferase, an enzyme responsible for acetylcholine synthesis, was increased at both the mRNA and protein level, and neurite outgrowth was markedly induced with an increase of neurofilament-heavy chain protein, in the 5-azaC-treated cells. These findings show that global DNA demethylation markedly induces the expression of the neurotransmitter phenotype and morphological differentiation in NG108-15 neuronal cells as a model for cholinergic neuron.

Suppression of Lactation by Pregnancy-Dependent Mammary Tumors in GR/A Mice

Abstract Pregnancy-dependent mammary tumors (PDMT) in GR/A mice appear during pregnancy, disappear soon after parturition, and appear again during subsequent pregnancies. The retardation of pup growth, an indication of the level of milk production, was also observed with the advance of lactation numbers in this strain. This study was performed to elucidate the relationship between PDMT and lactational performance. At the end of the second pregnancy, mice were divided into two groups according to the presence of PDMT [PDMT(-) and PDMT(+) groups]. Although all PDMT disappeared within a day after parturition, the weight and growth of pups on Day 12 of lactation were significantly less in the PDMT(+) group than in the PDMT(-) group. Associated with this, the DNA and RNA contents of the mammary glands were apparently lower in the former than in the latter, although the differences were not statistically significant. There was little difference in mammary RNA/DNA ratio between groups. No difference was also observed between groups in endocrine organ weights, mother body weights, morphology of the mammary glands, adrenals and ovaries and plasma prolactin and progesterone levels. These results suggest that PDMT suppression of lactation is principally due to the retardation of mammary gland growth. Furthermore, no significant correlations were obtained between the size of PDMT and the parameters for mammary gland function. The data suggest that the development of PDMT per se is important for the retarded mammary gland growth.

Changes in prolactin bioactivity of milk following isolation from litter and oxytocin administration in rats

Changes in prolactin (PRL)-like bioactivity in rat milk after a longer isolation of litters and the effects of oxytocin (OT) were determined by Nb2 lymphoma cell bioassay and compared with the immunoreactivity using an enzyme immunoassay. First, we confirmed the appropriate dilution rates of rat milk which could neutralize the influence of an antimitogenic factor(s) in milk to Nb2 lymphoma cell proliferation. To prolong the litter removal, i.e. 6 h to 20 h, resulted in the increase in an antimitogenic factor(s) for Nb2 lymphoma cells in milk and in the significant decrease in milk PRL concentration measured by both assays, and plasma bioactive PRL level increased. The bioactivity/immunoreactivity ratio of PRL in milk accumulated in the mammary gland was 0.65 in rats after a 20-h isolation, but 1.18 after a 6-h isolation. The milk PRL concentration in rats isolated for 6 h decreased significantly within 45 min after the administration of OT and the plasma PRL concentration increased only slightly, but OT had no effect in rats after isolation for 20 h. The present findings indicate that milk PRL might transfer to plasma with the excessive engorgement of milk in the mammary gland and with OT administration. Moreover, PRL trapped in milk for a long period loses its biological activity more rapidly than its immunological activity, since bioactivity/immunoreactivity ratio in the 20-h isolation group was significantly lower than in the 6-h isolation group.

Inhibition of Suckling-Induced Prolactin Release by Estrogen in Ovariectomized Lactating Rats: Bioassay versus Radioimmunoassay

Abstract Plasma levels of prolactin (PRL) were determined before and during suckling in intact and ovariectomized (OVX) lactating rats treated 5 days earlier with a single sc injection of 50 μg polyestradiol phosphate (PEP) or saline. Radioimmunoassay (RIA) and the Nb2 lymphoma cell bioassay (BA) were used to measure plasma PRL. Pituitary PRL concentrations were also determined by the two assays. In addition, lactational performance was estimated by measuring litter weight gain before and after estrogen treatment. Lactation was inhibited by PEP in intact rats; however, PEP did not alter plasma or pituitary levels of PRL in this group. Lactation was not inhibited by estrogen in OVX rats, but plasma PRL levels were significantly reduced at 10, 30, and 45 min of suckling. In addition, there was no evidence of an estrogen-induced afternoon prolactin surge in either PEP-treated group. The lactational inhibition in the intact, PEP-treated rats was probably due to combined effects of estrogen and progesterone. Plasma progesterone concentration was 64 ± 8 ng/ml in the intact PEP-treated group compared to 3 ± 1 ng/ml in the OVX, PEP-treated rats. The exact mechanism for the supression or delay in suckling-induced plasma PRL in the OVX, PEP-treated females remains unresolved but it was not due to an increase in suckling-induced corticosterone levels. The qualitative changes in plasma PRL during suckling in the various groups detected by RIA were also detected by the BA. However, there were slight quantitative differences between the two assays. When plasma PRL was low (nonsuckled states) the BA/RIA ratio was less than that observed when the plasma PRL was high (during suckling), i.e., more bioactive PRL was apparently released by suckling. However, these differences between RIA and BA may have been due in part to the fact that very low or very high levels of prolactin approached the minimal and maximal assay limits of the RIA but not the BA indicating that care be exercised when making such assay comparisons.

Early milk availability modulates the activity of choline acetyltransferase in the cerebral cortex of rats.

The purpose of the present study was to investigate the effect of milk in the early stage of lactation on the maturation of cholinergic neurons in the cerebral cortex of rats. Pups were removed from their mothers immediately following parturition and placed with foster dams at days 5-7 of lactation. At days 18 and 56 after birth, the activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine synthesis, in different areas of the cerebral cortex was examined by high-performance liquid chromatography electrochemical detection. In the frontal and hindlimb/parietal regions of the cerebral cortex, the lack of early milk significantly decreased ChAT activity at days 18 and 56. There was no effect on gains in the body or brain weight of infants. ChAT activity in the occipital area tended to be lower in the early milk-deprived rats. The intake of early milk potentially contributes not only to nutrients for the growth of newborn infants, but also to the functional maturation of the cholinergic neurotransmission system in a region-specific manner.

Changes with age of mammary glands in male and female soft-furred rat, Millardia meltada, in relation to prolactin and testosterone

The soft-furred rat, millardia (Millardia meltada), is characterized by the development of androgen-dependent mammary tumours only in males. The age-related changes of the activities of thymidylate synthetase (TS) and thymidine kinase (TK), which contribute to DNA synthesis through de novo and salvage pathways, respectively, and structure in the mammary glands were studied in both males and females of this species between 5-28 months of age. While TK activity had no relation to age, TS activity decreased with age in males. In the females, TK activity increased with age, but not TS activity. These enzyme activities were generally higher in females than in males. The mammary glands of both sexes consisted of fine ducts with small end-buds and the glands of males contained mostly black pigments at any age examined. In either males or females, serum levels of prolactin and testosterone related little with age, DNA synthesizing enzyme activities or structure of the mammary glands. Furthermore, elevation by pituitary grafting of circulating prolactin affected neither DNA synthesizing enzyme activities nor structure of mammary glands in both sexes. The histological structures of adrenal, testis, ovary, ventral prostate and uterus of millardia were essentially similar to those of mice or rats.