Mitsuhiro Kawata

The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus

The localization of estrogen receptor-immunoreactive (ER-IR) cells in the medial preoptic nucleus (MPN) and the effect of estrogen upon these cells were quantitatively analyzed by immunohistochemistry using a monoclonal antibody to estrogen receptor (ER) protein in rat. ER was distributed in the nucleus of MPN neurons and was not detected in either cytoplasm or glial cells. There were more ER-IR cells with higher immunoreactivities in ovariectomized (OVX) rats than in estradiol (E2)-treated rats. The number of ER-IR cells in E2-treated compared with OVX rats was reduced by 43%. In particular, the number of ER-IR cells in the central part of the MPN was largely decreased. Our data indicate that the central part of the MPN is most sensitive to estrogen.

Estrogen as a growth factor to central nervous cells. Estrogen treatment promotes development of acetylcholinesterase-positive basal forebrain neurons transplanted in the anterior eye chamber.

In a previous report, we demonstrated in vivo ameliorating effects of conjugated estrogen in women suffering from senile dementia-Alzheimers type. To investigate the effects of estrogen on the growth of cholinergic neurons, the present study was performed using rat cholinergic tissue implanted into the anterior chamber of the eye. Fetal diagonal band tissue containing cholinergic neurons was grafted into the anterior eye chamber of adult female rats that had either been treated or not with 2 mg estradiol valerate injected every 3 days after oophorectomy. Two and four weeks after transplantation, the axonal and/or dendritic growth of cholinergic neurons in the graft was studied using acetylcholinesterase histochemistry. At both times, acetylcholinesterase positive processes were densely distributed in the grafts of estradiol valerate treated rats, while in rats without estradiol valerate treatment acetylcholinesterase positive reaction was essentially localized only on the cell bodies. These findings were more obvious at 2 weeks after transplantation than at 4 weeks. These results suggest that estrogen acts on cholinergic neurons as a growth factor.

Rapid changes in the content of proenkephalin A and corticotrophin releasing hormone mRNAs in the paraventricular nucleus during morphine withdrawal in urethane-anaesthetized rats

Quantitative in situ hybridization was used to measure corticotrophin-releasing hormone (CRH) and proenkephalin A mRNA in the medial parvocellular paraventricular nucleus (PVN) 4 h after test procedures. Urethane anaesthesia alone resulted in a significant increase in both CRH and proenkephalin transcripts. The additional stimulus of i.p. hypertonic saline, however, resulted in a further significant increase in both mRNA species. Female rats were given intracerebroventricular (i.c.v.) infusion for 5 days of either morphine sulphate to induce tolerance and dependence, or vehicle, via a subcutaneous osmotic minipump implanted under ether anaesthesia. The rats were then anaesthetized with urethane, fitted with an intravenous cannula for injections and 65 min later either naloxone (5 mg/kg) or vehicle was injected. Naloxone alone in the i.c.v. vehicle rats had no effect on CRH or proenkephalin A mRNA. In i.c.v. morphine-infused rats proenkephalin a mRNA in the PVN was significantly less than in controls. Naloxone given to i.c.v. morphine-infused rats resulted in a doubling of hybridization to proenkephalin mRNA in the PVN which was significantly greater than that seen in the i.c.v. vehicle group. CRH mRNA in the PVN was not altered either by naloxone in control rats, or by chronic i.c.v. morphine infusion. By contrast, naloxone did increase CRH mRNA by ca. 40% in morphine-infused rats. The results show that stress-induced increases in CRH and enkephalin mRNAs in the PVN do not require conscious appreciation of the stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative in situ hybridization to measure single-cell changes in vasopressin and oxytocin mRNA levels after osmotic stimulation.

Summary1.The measurement of cellular mRNA content by quantitativein situ hybridization is a valuable approach to the study of gene expression in brain since this tissue exhibits a high degree of phenotypic heterogeneity.2.The cellular content of vasopressin and oxytocin mRNA in hypothalamo-neurohypophysial system neurons was altered by maintaining rats for 24 hr on 2% sodium chloride water.3.Statistical and graphical techniques were then used to analyze cell by cell how mRNA levels were altered as a result of osmotic stimulation. We propose that the negative binomial probability distribution is a suitable model to describe how mRNA content varies across a defined cell population. For both measures of oxytocin and vasopressin mRNA levels, maximum-likelihood estimation indicated that this model adequately described empirical findings obtained from rats drinking tap water or salt water.4.Both graphical and statistical analyses suggested how the defined neural system responds to osmotic stimulation: mRNA content was altered as a multiplicative function of “initial state.” The utility and limitations of the quantitative approach are discussed.

Time-related changes in the labeling pattern of motor and sensory neurons innervating the gastrocnemius muscle, as revealed by the retrograde transport of the cholera toxin B subunit

SummaryMorphological changes in the motor and sensory neurons in the lumbar spinal cord and the dorsal root ganglia were investigated at different survival times following the injection of the B subunit of cholera toxin (CTB) into the medial gastrocnemius muscle. Unconjugated CTB, visualized immunohistochemically, was found to be retrogradely transported through ventral and dorsal roots to motor neurons in the anterior horn, each lamina in the posterior horn, and ganglion cells in the dorsal root ganglia at L3–L6. The largest numbers of labeled motor neurons and ganglion cells were observed 72 h after the injection of CTB. Thereafter, labeled ganglion cells were significantly decreased in number, whereas the amount of labeled motor neurons showed a slight reduction. Motor neurons had extensive dendritic trees filled with CTB, reaching lamina VII and even the pia mater of the lateral funiculus. Labeling was also seen in the posterior horn, but the central and medial parts of laminae II and III had the most extensively labeled varicose fibers, the origin of which was the dorsal root ganglion cells. The results indicate that CTB is taken up by nerve terminals and can serve as a sensitive retrogradely transported marker for identifying neurons that innervate a specific muscle.

Brain natriuretic peptide in the porcine spinal cord: an immunohistochemical investigation of its localization and the comparison with atrial natriuretic peptide, substance p, calcitonin gene-related peptide, and enkephalin

Immunohistochemistry was used to localize brain natriuretic peptide in the porcine spinal cord and to compare it with that of atrial natriuretic peptide, substance P, calcitonin gene-related peptide and [Met]enkephalin. Brain natriuretic peptide-immunoreactive varicose fibers were observed in lamina I and the inner portion of lamina II of the dorsal horn. Semiquantitative analysis showed that the highest density of brain natriuretic peptide-immunoreactive varicosities was in the lumbosacral and coccygeal segments. The distributional pattern of brain natriuretic peptide-immunoreactive nerve fibers in the spinal cord was unique and quite distinct from that of the other neuropeptides studied. These neuroanatomical findings suggest that brain natriuretic peptide may play a role in the regulation of nociceptive processing in the spinal cord, either alone or with bioactive substances.

Effects of hyperosmotic stimulation and adrenalectomy on vasopressin mRNA levels in the paraventricular and supraoptic nuclei of the hypothalamus: in situ hybridization histochemical analysis using a synthetic oligonucleotide probe.

The effects of salt loading and adrenalectomy on arginine vasopressin (AVP) mRNA levels in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus were studied by semiquantitative in situ hybridization histochemistry, using a synthetic oligonucleotide probe and a computer-assisted image analysis system. Salt loading (2% NaCl) for 7 days produced marked increases in AVP mRNA levels in the magnocellular neurons of the PVN, SON, and accessory nuclei. Adrenalectomy caused an increase in AVP mRNA expression in the magnocellular part of the PVN and the expansion of hybridization signals into its medial parvocellular region, where the cell bodies of corticotropin-releasing hormone (CRH) neurons are located. No apparent alteration of AVP mRNA levels was observed in the SON following adrenalectomy. These results indicate that hyperosmotic stimulation and the loss of circulating glucocorticoids had differential effects on AVP gene expression in the PVN and SON, and that the magnocellular PVN and SON neurons responded in different manners to the loss of feedback signals.

Semi-quantitative analysis of the effects of estrogen on CGRP- and methionine-enkephalin-immunoreactivity in the periventricular preoptic nucleus and the medial preoptic area of female rats

The effects of 17 beta-estradiol (E2) on calcitonin gene-related peptide (CGRP)- and methionine-enkephalin (Met-Enk)-immunoreactive (IR) nerve fibers in the periventricular preoptic nucleus (Pe) and the medial preoptic area, including the medial preoptic nucleus (MPN), of the female rat were studied semi-quantitatively by using a computer-based image analysis system. The area occupied by CGRP- and Met-Enk-IR fibers was significantly increased in the Pe and the MPN after 28-day exposure to E2. Computer-based analysis of CGRP- and Met-Enk-IR fibers in an area of 50-microns intervals from the wall of the third ventricle showed a low flat histogram pattern in ovariectomized rats, but E2 treatment caused a diphasic pattern, corresponding to the Pe and the MPN, respectively. Since the Pe and the MPN contain a high population of estrogen receptors, it is suggested that E2 may have an influence on the neuronal configuration of afferent fibers to these areas.

Does acute, intense stimulation of oxytocin neurones in the supraoptic nucleus increase their content of oxytocin mRNA?

We investigated whether a sustained increase in oxytocin secretion, with or without enhanced electrical activity of the cell-bodies of oxytocin neurones, leads to a rapid increase in oxytocin mRNA content in these neurones. To stimulate oxytocin release, naloxone (2.5 mg/kg i.v. twice, 30 min apart) was given to urethane-anaesthetized female rats after intracerebroventricular (i.c.v.) morphine or vehicle infusion for 5 days; in the latter, naloxone acts on the neurohypophysis to increase oxytocin release without affecting the electrical activity of oxytocin neurone cell-bodies, but in the former, naloxone acts both on the neucohypophysis and on the cell-bodies to excite them electrically. Oxytocin content in peripheral plasma was measured intermittently by radioimmunoassay for 4 h after i.v. naloxone or vehicle, then the brain was removed and cryostat sections were cut through the supraoptic nucleus (SON). Oxytocin mRNA content in individual neurones (25-50 per rat) was measured semiquantitatively by in situ hybridisation histochemistry, using a tritiated synthetic cDNA 25-mer oligonucleotide probe, autoradiographical visualisation, and computer-assisted image-analysis to measure silver grain density. Nalaxone increased oxytocin content in plasma 7-fold for at least 40 min in i.c.v. vehicle-infused rats, and 40-fold for at least 40 min in i.c.v. morphine-infused rats. Naloxone had no significant effect on the oxytocin mRNA content in labelled cells in the SON, and no effect on the proportion of labelled cells, in either the i.c.v. morphine- or i.c.v. vehicle-infused rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Collateral sprouting of serotonergic fibers in the cingulate cortex and the septum following cortical-hippocampal lesions

The parietal cortex and dorsal hippocampus of adult rats were unilaterally ablated. One and 3 months after this operation, changes in serotonergic fiber distribution in the forebrain were studied immunohistochemically. At 1 month, increased numbers of serotonergic fibers were seen in the cingulate cortex and the medial and lateral septal nuclei of the lesioned side. This increase continued to 3 months in the ipsilateral cingulate cortex. In the present study, two different processes of serotonergic fiber collateral sprouting were noted. Plastic changes in serotonergic fibers in the ipsilateral septal nuclei and cingulate cortex may be attributable to the collateral sprouting underlying reactive synaptogenesis and the pruning effect, respectively.