Expression and estrogen regulation of progesterone receptor mRNA in neurons of the mediobasal hypothalamus: an in situ hybridization study.

Diverse effects of steroid hormones on different tissues result from the tissue-specific regulation of target gene expression by steroid hormone receptors. These receptors belong to a family of transacting factors that regulate transcriptional activation of target genes by binding to DNA recognition sequences located in the 5'-flanking region of the target gene. In the brain, receptors for the gonadal steroid hormones estrogen (E) and progesterone (P) are present in discrete neuronal populations. These steroid hormone receptor-containing neurons mediate the effects of the gonadal steroids on a number of neural processes, including reproductive behavior. Using in situ hybridization we have found progesterone receptor (PR) mRNA-containing neurons present in specific hypothalamic nuclei and in the amygdala. E regulates PR mRNA levels in specific neuronal cell groups which express both ER and PR (in basomedial hypothalamus), but not in others (medial amygdala). The E-induced increase in P-responsive neurons in ventromedial hypothalamus can account for the permissive influence of E on P-facilitated reproductive behavior. This is the first demonstration that synthesis of a transcription factor (PR) can be related to a mammalian behavior.

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat.

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.

Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization.

To determine the localization in rat brain and spinal cord of individual neurons that contain the messenger RNA coding for the opioid peptide precursor preproenkephalin, we performed in situ hybridization with a tritiated cDNA probe complementary to a protion of preproenkephalin mRNA. We observed autoradiographic signal over the cytoplasm of neurons of many regions of the central nervous system. Several types of controls indicated specificity of the labeling. Neurons containing preproenkephalin mRNA were found in the piriform cortex, ventral tenia tecta, several regions of the neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (especially central nucleus, with fewer labeled neurons in all other nuclei), hippocampal formation, anterior hypothalamic nucleus, perifornical region, lateral hypothalamus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, dorsal and ventral premamillary nuclei, medial mamillary nucleus, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral parabrachial nuclei, pontine and medullary reticular formation, several portions of the raphe nuclei, nucleus of the solitary tract, nucleus of the spinal trigeminal tract (especially substantia gelatinosa), ventral and dorsal cochlear nuclei, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, gracile nucleus, superior olive, nucleus of the trapezoid body, some deep cerebellar nuclei, Golgi neurons in the cerebellum, and most laminae of the spinal cord. In most of these brain regions, the present results indicate that many more neurons contain preproenkephalin mRNA than have been appreciated previously on the basis of immunocytochemistry.

Estrogen regulation of proenkephalin gene expression in the ventromedial hypothalamus of the rat: temporal qualities and synergism with progesterone.

Estrogen has been shown to increase proenkephalin (PE) mRNA levels in neurons of the ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM). In this series of experiments, we examined the temporal qualities of this induction by determining both the latency of the estrogen-induced elevation in PE mRNA levels and the rate at which the message levels decline following removal of estrogen. In addition we have examined the effects of progesterone on PE gene expression in the VL-VM of estrogen-primed rats. The latency of the estrogen-induced elevation in PE mRNA levels was found to be relatively short: PE mRNA levels were increased 2-fold within 1 h of estrogen replacement. Following estrogen removal the levels of PE mRNA declined rapidly. Progesterone treatment attenuated this decline, prolonging the estrogen-induced elevation of PE mRNA levels. These results suggest that estrogen rapidly increases PE mRNA levels through a mechanism that probably involves alterations in both the rate of appearance and the rate of degradation of the message. Together, the short latency of the estrogen-induced elevation and the rapid rate of decay following estrogen removal indicate that PE gene expression is highly sensitive to fluctuating estrogen levels. The effect of progesterone suggests that this enkephalinergic system may be regulated by both estrogen and progesterone during the estrous cycle.

Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat.

Hormone effects on proenkephalin (PE) mRNA allow an opportunity to compare a brain region-specific molecular change with a quantifiable behavior. Slot blots were used to measure PE mRNA levels in the ventromedial hypothalamus (VMN) and preoptic area (POA) as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PE mRNA level in VMN, while only a small effect was observed in POA at the higher estradiol doses. Lordosis behavior, assessed manually and in mating tests, also increased monotonically with estradiol dose. The data indicate that an apparent 'threshold' level of PE mRNA in VMN coincided wit display of behavior, and suggest further that high levels of PE mRNA, alone, are not sufficient for lordosis. While the exact relationship of the eventual product, Met-enkephalin, to female reproductive behavior remains to be determined, the parallel changes in PE mRNA level and behavior encourage further analysis.

Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in situ Hybridization Study.

Abstract We have used in situ hybridization to investigate estradiol regulation of estrogen receptor (ER) mRNA in regions of the mediobasal hypothalamus which contain ER and are related to specific neuroendocrine functions. Ovariectomized rats were treated with oil or 10 mug estradiol benzoate for 2, 4, 18 or 24 h. Brains were sectioned and hybridized with a [(3) H]single-stranded DNA probe prepared from the pORF cDNA of the human ER gene and exposed to autoradiographic emulsion for 4 months. Specificity of labeling was determined by counting the number of grains over cells in hypothalamic regions known to bind estradiol, compared to cells in the thalamus and cortex, and by comparing with sections pretreated with ribonuclease or hybridized with a [(3) H]single-stranded message-sense (control) probe. Labeling for ER mRNA was distributed differently than glucocorticoid and thyroid hormone receptor mRNAs, and was regulated by estrogen differently than progestin receptor mRNA. These differences indicated specific hybridization for ER mRNA. ER-expressing cells constituted 11.5% of the cells in the dorsomedial nucleus, 30% of the cells in the arcuate nucleus and 43% in the ventromedial nucleus, in close accordance with previous studies of ER autoradiography and binding. Quantitative analysis showed that the highest level of ER mRNA was present in the ovariectomized controls. ER mRNA levels fell 42% (ventromedial), 64% (arcuate), or remained unchanged (dorsomedial) 18 h following estradiol benzoate treatment. The pattern of decrease was similar for cells in the ventromedial nucleus and arcuate nucleus. These data show that estrogen regulation of ER mRNA in brain parallels that reported for MCF-7 cells and rat uterus. These results also demonstrate that in situ hybridization can be used to detect and measure the relative level of a low abundance mRNA in a heterogeneous tissue in which only 12% to 40% of the cells in limited regions express the message.

Gene expression for estrogen and progesterone receptor mRNAs in rat brain and possible relations to sexually dimorphic functions.

A clear neuroendocrine sex difference lies in the ability of the female rat to produce an ovulatory surge of luteinizing hormone. Preoptic neurons, as they respond to estrogen and progesterone, have been proven to be involved in this mechanism, with an emphasis on the possible participation of neurons in the anteroventral periventricular nucleus and the suprachiasmatic portion of the preoptic area (POA). Further, prominent morphological sex differences have been reported in the rat medial POA. To examine expression of the estrogen receptor (ER) and the progesterone receptor (PR) messenger RNAs (mRNAs) in these critical preoptic neurons, we have used in situ hybridization with tritiated single-stranded DNA probes complimentary for ER and PR mRNA. ER mRNA containing cells were found in the periventricular, suprachiasmatic and medical preoptic cell groups, in a manner which agrees with steroid hormone autoradiography. In the female rat, preoptic neurons expressing PR mRNA were distributed very similarly to those for ER mRNA. Moreover, in the male rat brain, all subsets of preoptic neurons which express the PR gene in the female were also detected in the male. Thus, the distribution of PR expressing cells was very similar between females and males. We conclude that the insensitivity to the male to progesterone, as regards the hormonal control of ovulation, cannot be due to a total failure of PR gene expression in a specific subset of POA neurons. Instead, male preoptic neurons must be less sensitive to neural or hormonal inducers in the physiological range or perhaps lack sufficient levels of a transcription factor linking progesterone responsive elements to the start sites of hormone-controlled genes.

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior.

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization.

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.

Bipap improves survival and rate of pulmonary function decline in patients with ALS.

Amyotrophic Lateral Sclerosis (ALS) is a progressive motor neuron disease that frequently causes death within five years of diagnosis. The majority of deaths are due to pulmonary complications resulting from respiratory muscle weakness and bulbar involvement. A promising respiratory intervention is the recently introduced bi-level intermittent positive pressure (Bipap), which is a noninvasive ventilator modality shown to reduce the work of breathing and improve not only gas exchange, but also exercise tolerance and sleep quality. The aim of this study was to assess the utility of Bipap in prolonging survival in ALS. We retrospectively analyzed the results of Bipap use in 122 patients followed at Hahnemann University. All patients in this study were offered Bipap when their forced vital capacity (FVC) dropped below 50% of predicted value. Group 1 (n=38) accepted Bipap and used it more than 4 h/day. Group 2 (n=32) did not tolerate Bipap well and used it less than 4 h/day. Group 3 (n=52) refused to try Bipap. There was a statistically significant improvement in survival from initiation of Bipap in Group 1 (14.2 months) compared to Group 2 (7.0 months, P=0.002) or 3 (4.6 months, P<0.001) respectively. Furthermore, when the slope of vital capacity decline was examined, the group that used Bipap more than 4 h/day had slower decline in vital capacity (-3.5% change/month) compared to Group 2 (-5.9% change/month, P=0.02) and Group 3 (-8.3% change/month, P<0.001). We conclude that Bipap can significantly prolong survival and slow the decline of FVC in ALS. Our results suggest that all patients with ALS be offered Bipap when their FVC drops below 50%, at the onset of dyspnea, or when a rapid drop in %FVC is noted.

Sex difference in estradiol regulation of progestin receptor mRNA in rat mediobasal hypothalamus as demonstrated by in situ hybridization.

Previous studies have shown that estrogen increases the level of progestin receptors (PR) to a greater extent in female than in male rat hypothalamus. In order to determine if sex-specific regulation of the PR protein might be attributable to estrogenic effects on the PR message, in situ hybridization was used to assess sex differences in levels of estrogen-inducible PR mRNA in specific brain nuclei. Here, we report a sexually differentiated pattern of estrogen-regulated PR gene expression. In female hypothalamus, estrogen administered to gonadectomized rats induced a 3.6- and a 3.3-fold increase in PR mRNA in the ventrolateral aspect of the ventromedial nucleus and arcuate nucleus, respectively, but failed to alter the level of PR mRNA in the same neuronal groups of the male. Hormone treatment did not affect the levels of PR mRNA in the dorsomedial or medial amygdaloid nuclei of either sex. These results lead towards a molecular explanation of sex differences in female reproductive behavior by revealing an estrogen-dependent up-regulation of the message for PR, a transcription factor, in a region- and sex-specific fashion.

Preprotachykinin gene expression in the mediobasal hypothalamus of estrogen-treated and ovariectomized control rats.

We have determined the localization of preprotachykinin (PPT) mRNA-containing neurons in the mediobasal hypothalamus of the rat. PPT mRNA-containing neurons are present in the ventromedial nucleus (with a concentration in the ventrolateral aspect (VL-VM], the dorsomedial nucleus, the lateral hypothalamus and the arcuate nucleus. This distribution is consistent with the findings of immunocytochemical studies of substance P-immunoreactive neurons in the hypothalamus. We have also examined whether PPT gene expression is regulated by estrogen in the VL-VM by comparing the levels of PPT mRNA in the VL-VM of ovariectomized rats and ovariectomized, estrogen-replaced rats. Both in situ hybridization and slot blot hybridization analysis revealed no changes in PPT mRNA content in the VL-VM following estrogen treatment. These results suggest that estrogen does not regulate lordosis behavior by affecting PPT gene expression in VL-VM neurons.

Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex.

The cellular locations of proenkephalin mRNA have been determined for the caudate-putamen and cerebellar cortex of the rat brain by in situ hybridization. In the caudate-putamen, more than half of the neurons express the proenkephalin gene. Morphologically, they are medium-sized cells that may represent projection neurons. In the cerebellar cortex, proenkephalin mRNA is present in a subpopulation of neurons in the granule layer that appear to be Golgi cells--i.e., inhibitory interneurons. The presence of [Met]enkephalin, a pentapeptide derived from proenkephalin, in these two brain areas is consistent with a synthetic role for this mRNA and implicates proenkephalin gene expression in the control of motor function.