Quantitative expression profile of distinct functional regions in the adult mouse brain

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ～50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.     

Biochemical analysis of selenoprotein expression in brain cell lines and in distinct brain regions

Selenium is present in various biologically important selenoproteins. The preferential incorporation of selenium into the brain indicates its significance for this organ, but so far knowledge concerning the cerebral selenoproteome is scarce. We therefore investigated the expression of selenoproteins in various regions of the rat brain, various subcellular fractions and several brain cell lines by (75)Se-labelling, gel electrophoretic separation and autoradiography, with the (75)Se tracer as the selenoprotein marker. Quantitative evaluation of the labelled proteins in selenium-deficient rats revealed information regarding preferentially supplied selenoproteins and their distribution; 21 selenoproteins could be distinguished, among them a novel or modified 15-kDa selenoprotein enriched in the cerebellum cytosol. The selenoproteins differed in the degree of their expression among the brain regions and within a region among the subcellular fractions. Some cell-type-specific selenium-containing proteins were found in the cell lines. Differences in the distribution patterns between mono-cultured and co-cultured endothelial cells and astrocytes showed that mediators produced by other cells could affect the selenoprotein expression of a specific cell-type. This effect might play a role in the uptake and distribution of selenium in the brain but could also be of significance in the selenium metabolism of other tissues.     

Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats

The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of tamoxifen treatment at adolescent age on the sexual behaviour and steroid hormone receptor binding of adult female rats

Hormonal imprinting takes place perinatally, at the first encounter between the target hormone and its developing receptor. However, there is a secondary critical period of imprinting at puberty. In these periods molecules similar to the hormones (members of the same hormone family, antagonists, certain environmental pollutants, etc.) can cause faulty imprinting with lifelong consequences. In the present experiments 5+2 days of tamoxifen treatment (120 microg/day) at adolescent age dramatically (from approx. 40% to 10%) reduced the sexual activity (Meyerson index and lordosis quotient) of female rats, soon after the finishment of the treatment and between four to six weeks after treatment. Similar results were observed in animals neonatally treated with allylestrenol and tamoxifen treated at puberty. Thymic glucocorticoid receptor and uterine estrogen receptor binding capacity were not influenced.     

Effects of food deprivation and high fat diet on immunoreactive beta-endorphin levels in brain regions of Zucker rats

The levels of immunoreactive beta-endorphin (ir-beta-EP) were measured in the brain and pituitary of lean Zucker rats subjected to food deprivation for 72 h and to a high fat diet, and in fatty Zucker rats after food deprivation for 72 h. Ir-beta-EP was increased in the neurointermediate (NI-) pituitary lobe but reduced in the medulla-pons of fatty rats when compared to lean littermates fed ad libitum. Food deprivation decreased ir-beta-EP in the cortex and medulla-pons of lean rats and in the cortex, midbrain and NI-pituitary of fatty rats. In contrast, ir-beta-EP was increased in the anterior pituitary of lean rats and in the striatum of fatty rats after deprivation. The high fat diet produced a decrease in ir-beta-EP in the cortex, midbrain and NI-pituitary with an increase in the striatum and hypothalamus of lean rats. These results suggest that the ir-beta-EP concentration could be differentially affected in different brain regions of Zucker rats by changes in the energy balance.     

Effects of an anabolic steroid on reproduction in female rats

Mature female rats given food and water ad libitum and maintained under a lighting schedule of 12h light/12h dark were treated with boldenone undecylenate. Neither dosage nor duration of treatment had a significant effect on weight gain when compared to controls. All treated females were characterized by the absence of cyclic ovarian activity that continued for several weeks following termination of treatment. Long term reproductive performance was altered by a significant reduction in both conception rate and neonatal survival. Behavioral patterns were modified for several months following steroid withdrawal.     

Effect of some contraceptive steroids on pituitary growth hormone content in female rats

At the University of Missouri-Kansas City, School of Dentistry, the effect of mestranol and/or norethynodrel on growth hormone (GH) was studied with an anterior pituitary gland (AP) donor-recipient technique in rats. Experimental donor rats received, per 100 g of body weight, subcutoneous injections of 50 mcg of mestranol (16 rats), 1250 mcg of norethynodrel (18), or their combination (23) on Monday, Wednesday, and Friday for 10 weeks. 12 control donor rats received vehicle alone. P values greater than .05 were considered significant. Mean final body weight, body weight gain, and absolute AP weight of the 3 steroid-treated donor groups were significantly less than the control values. Body sizes in the 3 donor groups were markedly smaller than in the control group. The effect of donor AP given in ip injections to hypophysectomized recipient rats was studied by measurement of the tibial epiphyseal cartilage width. Judged in this indirect manner, total GH in the AP was seen to be significantly decreased by all 3 steroid treatments but still present in moderate amounts. The treatments may have, directly or indirectly through the hypothalamus, inhibited pituitary GH synthesis and release, or the decreased total GH in the AP may have been due simply to the significant decrease in gland size. The estrogenic activities of mestranol and norethynodrel may have been responsible for an inhibition of GH synthesis. On the other hand, the steroids may have stimulated GH release from the AP as is the case in humans but concurrently exerted antagonistic action on GH at the level of peripheral tissues.     

Prenatal haloperidol alters the expression of DNA polymerases in brain regions of neonate rats

1. Previous studies have reported a marked reduction in the [3H]thymidine incorporation in forebrain after administration of a dopamine antagonist such as haloperidol. 2. We have investigated the possibility that the expression levels of genes related to DNA metabolism could be altered by haloperidol treatment. 3. By Northern blot analysis, we have studied the steady-state mRNA levels for genes involved in DNA metabolism, in neonate rat mesencephalon and forebrain, after chronic prenatal blockade of dopamine receptors with haloperidol. 4. We found that the expression levels for DNA polymerases alpha and beta were clearly reduced in forebrain by haloperidol treatment. On the contrary, the expression of DNA polymerase beta was increased in mesencephalon. 5. Our results suggest that dopamine receptors occupancy may be a critical factor in controlling cell proliferation during brain development, through a mechanism(s) involving changes in the expression of DNA polymerases.     

Effect of short-term food deprivation on reproduction in female mice

CF-1 female mice were subjected to 24 or 48 h of food deprivation beginning when they were in estrus or diestrus, or when they were 2 or 12 days pregnant, or on Days 2 or 12 of lactation. Ovulation was delayed by a week or more when 48 h of food deprivation was initiated when the female was in diestrus; lesser delays occurred when food deprivation began in estrus. There was little effect of acute food deprivation on pregnancy. Most females deprived of food beginning on Day 2 of lactation ate their young, but females deprived on Day 12 of lactation rarely did so. These results are discussed in terms of the complexity of interacting factors that determine the degree to which each stage of the female's reproductive cycle is susceptible to disruption by acute food deprivation.     

Effect of environmental deprivation on anxiety in rats

Environmental deprivation (ED) induced a significant increase in open-field ambulation, rears, self-groomings, faecal pellets and decrease in activity in centre in Charles Foster albino rats of 30, 45 and 60 days age groups. In elevated plus maze, significant attenuation of open arm time/entries and augmentation of enclosed arm time/entries were noted in ED rats of all the three age groups. Similarly ED rats also showed significant decrease in time spent on open arms, entries, head dips and stretched attend postures in comparison to age matched rats reared under normal environmental conditions. The results indicate that imposition of environmental deprivation in rats' life consistently resulted in significant anxiogenic behaviour on all the tests. However, the anxiogenic effect of ED was less marked when it was imposed at 60th day of life in rats.     

Effect of CRF injected into the median eminence on GH secretion in female rats under different steroid status

To evaluate whether the median eminence (ME) is a site of action of CRF (corticotropin releasing factor) on GH secretion and to determine the possible role of estradiol and progesterone in modifying theses secretion, we injected CRF (0.25, 0.75, 1, and 1.5 nmol of peptide dissolved in 1 μl of water) directly into the ME in three experimental groups of rats: Long-term ovariectomized (OVX); OVX primed by estradiol (OVX±E) and OVX primed by estradiol plus progesterone (OVX±EP). Blood was collected to determine GH (30, 60, 90, and 120 min postinjection) Serum T₃, T₄, and glucose levels were measured in OVX±E rats 30 min postinjection. CRF at all doses studied significantly decreased serum GH levels in the three experimental groups. Serum T₃, T₄, and glucose levels were unchanged after CRF administration. The results suggest that: CRF inhibits “per se” GH secretion, at least in part, by a central action in the ME. The inhibitory effect of CRF on GH is independent of the estrogen/progesterone status of the animal. CRF at ME levels may participate in a variety of stress-related responses, including growth inhibition, through GH suppression.     

Modulation of luteinizing hormone subunit gene expression by intracerebroventricular microinjection of gonadotropin-releasing hormone or beta-endorphin in female rats

The effects of gonadotropin-releasing hormone (GnRH), beta-endorphin and its antagonist naloxone on the expression of luteinizing hormone (LH) subunit genes and LH secretion were examined in ovariectomized and/or cycling female rats through their direct microinjection into the third cerebral ventricle, in the proximity of the hypothalamus-pituitary complex. GnRH (1 nM) induced a significant augmentation of the pituitary content of alpha mRNA when administered 15, 30 or 60 min intervals over 5 h to ovariectomized rats whereas only the 30 and 60 min intervals were effective in increasing LHbeta mRNA, and the 60 min intervals for LH release. This was in agreement with the established concept of a pulse-dependent regulation of gonadotropin synthesis and release. Hourly pulses of GnRH also increased alpha and LHbeta mRNA levels when microinjected in female cycling rats during proestrus or diestrus II. Using this model we observed a marked negative influence of hourly intracerebral microinjections of beta-endorphin on LH mRNA content and LH release in ovariectomized rats while naloxone had no effect. This suggests that endogenous beta-endorphin was unable to exert its negative action on beta-endorphin receptors that were present and responded to the ligand. The present approach would be valuable for the exploration of the mechanisms of action of beta-endorphin or other substances on the functions of the gonadotrophs.     

The effects of paradoxical sleep deprivation on estrous cycles of the female rats

The present purpose was to examine how sleep deprivation affects the estrous cycle of the female rat. Paradoxical sleep-deprived (PSD) adult female Wistar rats were compared to home-cage control (CTRL) on their estrous cyclicity. Forty-four PSD and forty-four CTRL female rats were distributed into 4 subgroups of 11 animals each according to the phase of estrous cycle and were subjected to sleep deprivation for 96 h by the multiple platform technique. After PSD period, vaginal estrous cycles were taken for an additional 9 days. Animals that were submitted to PSD in diestrus phase (PSD-diestrus) had their estrous cycles disrupted during the recovery period by showing a constant diestrus during the first week. As for hormone alterations, progesterone concentrations were statistically higher in PSD-diestrus compared to respective phase control and to PSD-proestrus and PSD-estrus rats while CTRL-metestrus had higher levels than CTRL-proestrus and estrus groups. Testosterone was significantly decreased in PSD-estrus in relation to PSD-proestrus and PSD-diestrus groups and was lower in CTRL-diestrus rats than in home-cage rats in proestrus. In addition, PSD-diestrus phase exhibited higher concentrations of corticosterone and lower estrogen than the respective control rats. These data indicate that PSD may modulate the ovarian hormone release through alterations in hormonal-neurochemical mechanisms.