Meeting the constraints upon behavioral expression through neural and genomic interactive mechanisms.
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Biomedical subjects
D W Pfaff
Publications and source records attributed to D W Pfaff.
Estrogen regulation of preproenkephalin messenger RNA in the forebrain of female mice.
We studied the effects of 10 micrograms of 17-beta-estradiol-3-benzoate treatment on preproenkephalin (PPE) mRNA expression in female ovariectomized (OVX) Swiss Webster mice after 0, 1, 6, 12, 24, or 48 h, using the in situ hybridization technique. The VMH showed a 1.6- and 3.3-fold increase in PPE mRNA levels after 24 and 48 h of estrogen treatment (respectively) when compared to OVX females. No differences at 1, 6 or 12 h of estrogen treatment groups were observed compared to control groups. PPE mRNA levels were also increased at 24 and 48 h after estrogen treatment in the posterior medial nucleus of the amygdala (MeAmyg) by 3.3- and 2.5-fold, respectively, and in the arcuate nucleus (ARC) by 2- and 1.9-fold, respectively. No effects of estrogen were observed on PPE mRNA levels in the caudate-putamen (CPu) or the posterior lateral cortical nucleus of the amygdala (plCoAmyg). Furthermore, basal levels of PPE mRNA expression in the VMH and MeAmyg of female mice were lower than those observed in rats, although levels in the CPu, plCoAmyg, and ARC were similar between females of the two species. In conclusion, we have found two differences between the species. First, Swiss mice demonstrated a slower time course of estrogen induction of PPE mRNA in the VMH, ARC. and MeAmyg compared to female rats. Second, there are differences in basal levels of PPE in the MeAmyg and VMH.
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Hypothalamic cellular and molecular mechanisms helping to satisfy axiomatic requirements for reproduction.
In the absence of universal equations expressing neurobiological findings, the safest theoretical approach for the neuroendocrinologist is to start from axiomatic requirements for biologically adaptive neural mechanisms, in our case for reproduction. From this emerge two themes: the likely importance of interactions between internal (hormonal) and external signals in controlling gene expression relevant to reproductive functions; and, second, the vision of molecular interactions on DNA subserving environmental impacts on reproduction. The first theoretical notion has so far yielded data showing a role for synaptic inputs during the onset of estradiol actions for the hormone's induction of enkephalin mRNA, a finding which parallels earlier behavioral results. As well, noxious somatosensory inputs interact with estrogens and progesterone in their influence on enkephalin gene expression. The second theme led to novel investigations of thyroid influences on reproductive molecular biology and behavior, including the ability of exogenous or endogenous thyroid hormones to reduce female mating responses. Since elevated thyroid hormone levels could signal environmental cold, our experiments offer the possibility of explaining ethological facts at a molecular level. More generally, nuclear hormone receptor interactions on the surface of DNA may offer a new level of neural integration revealed first by hormone effects in neuroendocrine cells.
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Expression of EGFR-, p75NGFR-, and PSTAIR (cdc2)-like immunoreactivity by proliferating cells in the adult rat hippocampal formation and forebrain.
Immunocytochemical and autoradiographic techniques were used to examine proliferating cells in the adult rat brain along with the expression of specific growth factor receptors and cell cycle proteins. Two hours following an injection of [3H]thymidine ([3H]Thy), dividing cells were detected in the subgranular region of the dentate gyrus and in the subependymal region (SER) extending into the olfactory bulb. Many cells continued to divide over the next 24 h as demonstrated by the ability for thymidine-labeled cells to incorporate bromodeoxyuridine (BrdU); however, the results of BrdU, PSTAIR, and vimentin staining suggest that the majority of the progeny cells detectable by [3H]Thy autoradiography at 3 days and 1 week after injection are postmitotic and at least partially differentiated. Significant numbers of thymidine-labeled cells detected 2 h following thymidine injection in the subgranular region of the dentate gyrus and in the SER of the lateral ventricle stained positively for epidermal growth factor receptor-, vimentin-, and PSTAIR-like immunoreactivity. Significant numbers of thymidine-labeled cells in the SER also stained positively for the low-affinity neurotrophin receptor p75NGFR. No [3H]Thy/p75NGFR-labeled cells were detected in the dentate gyrus. In addition, very few [3H]Thy/PSTAIR- or [3H]Thy/ vimentin-labeled cells were detected in region CA4. These data suggest that proliferating cells located in different regions of the adult brain may not be homogeneous and may be subject to different growth factor regulation.
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Estradiol regulation of nitric oxide synthase mRNAs in rat hypothalamus.
Expression and estrogen regulation of the genes for nitric-oxide (NO)-synthesizing enzymes (NO synthase, NOS) were investigated by in situ hybridization. This study focused on regions of the hypothalamus that contain estrogen receptors and regulate specific neuroendocrine functions related to female sexual behavior and food intake, among others. Ovariectomized (OVX) rats were treated with vehicle or 3 micrograms/100 g estradiol benzoate (EB) for 7 days. Brains were sectioned and hybridized with antisense riboprobes for neuronal NOS, macrophage NOS and endothelial NOS. In the hypothalamus, mRNA was clearly detectable only for the neuronal NOS with the probes used. A strong hybridization signal was observed in the supraoptic paraventricular and ventromedial nuclei (SON, PVN and VMN, respectively). Quantitative analysis showed an increase in neuronal NOS mRNA in the VMN of the OVX rats treated with EB. The increase was mainly in the ventrolateral aspect of the VMN. No significant changes were observed in the hypothalamic SON and PVN. The data suggest that the expression of neuronal NOS mRNA in VMN can be regulated by estrogen.
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Reversal of sex roles in genetic female mice by disruption of estrogen receptor gene.
Deficiency of normal estrogen receptor(ER) gene function led to behavioral change in female mice (ERKO females). Maternal behavior as measured by retrieving of pups was reduced. In some cases, pups were killed by the ERKO females, which was not seen in wild-type animals. Aggression toward other females was increased. Female-typical lordosis behavior was reduced for at least two reasons: less response to somatosensory stimuli on the hindquarters, as well as the fact that ERKO females were immediately treated as intruder males by resident 'stud' males and were thus attacked. In sum, disruption of the ER gene led to a pattern of hormonal and neural changes which caused the females to lose their normal female-typical behavior and to behave and be treated more like males.
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Embryonic development of gonadotropin-releasing hormone neurons in the sockeye salmon.
Immunocytochemistry and in situ hybridization were used to test the hypothesis that gonadotropin-releasing hormone (GnRH) neurons are formed in the olfactory placode during embryonic development in a salmonid, Oncorhynchus nerka. The development of GnRH neurons and the pituitary cell types was examined from 19 through 910 days after fertilization. Immunoreactive GnRH was first detected at 19 days in the cells of the olfactory placode. GnRH immunoreactivity was not detected in any other structure of the central nervous system at this age. By day 24, GnRH-immunoreactive neurons were seen in the apical, intermediate, and basal layers of the olfactory placode. From days 30 through 51, GnRH neurons were seen emerging from the epithelium, along the olfactory nerve, and at the rostral olfactory bulb. By day 41, GnRH immunoreactivity was lost in the nasal epithelium. In the 72-day-old fish, most of the GnRH neuronal population was found in ganglia of the nervus terminalis, at the cribriform bone (gCB), and at the rostral olfactory bulb (gROB). On day 293, a decrease in GnRH-immunoreactive neurons in the gCB and gROB was concomitant with an initial appearance of GnRH-immunoreactive neurons and fibers along the caudoventral olfactory bulb. By day 462, the distribution of GnRH neurons and fibers was almost similar to adults. In maturing adults (910 days), GnRH-immunoreactive neurons were rarely seen in the nasal regions, but were primarily found in the basal forebrain. GnRH fibers were widespread in the brain, proximal para distalis, and in the pars intermedia of the pituitary. Our study supports the notion that neurons expressing salmon-GnRH mRNA and peptide originate in the medial olfactory placode and migrate into the basal forebrain during development. The midbrain neurons did not express salmon-GnRH mRNA or peptide in the larval and juvenile fish.
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Cellular uptake of intracerebrally administered oligodeoxynucleotides in mouse brain.
Intracerebral diffusion, cellular uptake and intracellular localization of oligodeoxynucleotides (ODN) after their microinjection in mouse brains were examined. Using either tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC)- or gamma-33P ATP-labeled ODNs, it was found that both phosphodiester ODNs (D-ODN) and phosphorothioate ODNs (S-ODN) quickly diffused (up to about 500 microns) and were taken up by many cells around the injection site as early as 15 min after administration. Fluorescence labeling intensity and silver grain accumulation of D-ODNs were greatly reduced by 4 h after injection, whereas those of S-ODNs were stable beyond at least 8 h after injection. Most of labeled ODNs were found in neuronal cells as identified by immunocytochemistry for neurofilament, NF 200, and to a much lesser extent in astrocytic cells as identified by immunocytochemistry for glial fibrillary acidic protein.
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Estrogen modulation of mRNA levels for the two forms of glutamic acid decarboxylase (GAD) in female rat brain.
Two separate forms of glutamic acid decarboxylase, now termed GAD65 and GAD67, are the rate limiting enzymes for synthesis of gamma-aminobutyric acid (GABA). Because of the significance of GABA to neuroendocrine processes, numerous attempts have been made to determine the impact of gonadal steroids on enzyme functioning with inconclusive results. Therefore, we attempted to determine the impact of estradiol on mRNA levels for each form of GAD by quantitative in situ hybridization histochemistry in various brain regions. Ovariectomized rats were treated with estradiol benzoate or oil vehicle on 2 consecutive days and the brains collected on the third day. DNA probes specific for GAD65 and GAD67 were radiolabeled with CTP32 using asymmetric polymerase chain reaction. Results of in situ hybridizations for each probe on alternate sections from the same animals were analyzed for magnocellular preoptic area (McPOA), dorsal medial nucleus of the hypothalamus (DMN), zona incerta (ZI), and midbrain central gray (MCG). In the McPOA, estradiol exerted opposite effects on the frequency distribution of pixels per cell for two GAD mRNA probes, significantly increasing GAD65 (P < .05) and decreasing GAD67 (P < .01; Kolmogorov-Smirnov). In the DMN, estradiol treatment significantly increased GAD67 by 60% (P < .05; two-way ANOVA) but decreased GAD65 mRNA by 73% (P < .01). Note the direction of effects are opposite between McPOA and DMN. In MCG, analysis showed no estradiol effect on GAD mRNA levels/cells, but the proportion of cells expressing detectable levels of GAD65 or GAD67 increased by 33-40% in estradiol-treated rats (chi 2, P < .001).
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In vitro electro-pharmacological and autoradiographic analyses of muscarinic receptor subtypes in rat hypothalamic ventromedial nucleus: implications for cholinergic regulation of lordosis.
Muscarinic agonists can act through the hypothalamic ventromedial nucleus (VMN) to facilitate lordosis. To elucidate the neuronal mechanism(s) underlying this muscarinic facilitation, effects of muscarinic agents on the single-unit activity of VMN neurons recorded in brain tissue slices of estrogen-primed female rats were analyzed. All the agonists tested, including acetylcholine (ACh), oxotremorine-M (OM), carbachol (CCh) and McN-A-343 (McN), evoked primarily excitation (80-100%), some inhibition (0-20%) and occasional biphasic responses (0-8%). By comparing the response magnitude and the effectiveness in evoking a response, the rank order for evoking excitation, the primary response, was found to be: OM > CCh > ACh approximately McN, which is consistent with that (OM > CCh > McN) for facilitating lordosis reported by others. This consistency and the frequency of its occurrence suggest that the excitatory electric action of the muscarinic agonists is related to their facilitatory behavioral effect. Experiments with antagonists selective for M1 (pirenzepine), M2 (AF-DX 116) and M3 (4-DAMP and p-F-HHSiD) indicate that muscarinic excitations are mediated by M1 and/or M3, but not M2. Since M1 receptors have been shown to be neither sufficient nor necessary to mediate the muscarinic facilitation, M3 receptor may be crucially involved in this behavioral effect. Autoradiographic assays of binding to [3H]4-DAMP with or without pirenzepine and AF-DX 116, also indicate the presence of M3 receptors in the VMN. Quantitative analyses show that the M3 binding was not affected by the in vivo estrogen priming required to permit muscarinic agonists to facilitate lordosis. Thus, while the excitation mediated by M3 is likely to be involved in muscarinic facilitation of lordosis, the regulation of M3 receptor density does not seem to be involved in the permissive
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Functional analyses of alpha 1-adrenoceptor subtypes in rat hypothalamic ventromedial nucleus neurons.
Activation of alpha 1-adrenoceptors in rat hypothalamic ventromedial nucleus can excite neurons and facilitate female sexual behavior. To identify the alpha 1-adrenoceptor subtype(s) involved, the alpha 1B-adrenoceptor-specific antagonist chloroethylclonidine (100 microM) and/or the alpha 1A-adrenoceptor-selective antagonist 5-methyl urapidil (1 or 2.5 microM) or WB-4101 (0.1-10 microM) were applied to a recording chamber bathing the hypothalamic slice containing the ventromedial nucleus. In all the neurons tested, both types of antagonists blocked, often completely, excitatory responses to nonselective alpha 1-adrenoceptor agonists. Since the doses used were unlikely to make these antagonists nonselective, the results suggest that activation of both alpha 1A- and alpha 1B-adrenoceptor subtypes was necessary for alpha 1-adrenoceptor agonists to evoke an excitation, or that with the present application method--injection into the continuously perfused chamber--chloroethylclonidine did not act specifically. In preincubation (at 37 degrees C for 90 min) where it was reported to act by specific alkylation, chloroethylclonidine (100 microM) but not the vehicle abolished the excitation evoked by an alpha 1-adrenoceptor agonist, but not that by carbachol or other excitants. Also, either in bath application or incubation, chloroethylclonidine worked equally efficiently on slices from ovariectomized rats, that reportedly contain few alpha 1B-adrenoceptors, and from those treated with estrogen which induces alpha 1B-adrenoceptors selectively, suggesting that alpha 1B-adrenoceptor was necessary even when in low abundance.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic alpha-Antagonists↗
Actions of sex steroids on behaviours beyond reproductive reflexes.
The actions of sex steroids in the brain have been shown, from molecular to systems levels, to control reproductive behaviour in a wide range of vertebrates. It has become increasingly clear that gonadal steroid hormones have regulatory functions which extend far beyond the direct coordination of an animal's physiological state and its display of sexual behaviour. While some of these actions may include changes in mood or other behavioural measures, such as exploration or excitability, sex steroid hormones also influence neural plasticity, neuronal activity and, possibly, learning and memory, as reflected by long-term potentiation or age-related deficits. Here we describe two systems that have been used to explore the non-reproductive roles of gonadal steroid hormones. The first of these is to examine the oestrogen-sensitive opioid peptide gene expression in the hypothalamus. Currently, we are attempting to identify the types of behaviour which may be altered consequent to the oestrogenic induction of the preproenkephalin gene. The second approach involves studying the effects of progesterone at the neuronal cell membrane and characterizing the metabolites of progesterone which have benzodiazepine-like actions in the brain. A number of studies suggest that this may provide an alternative mechanism through which progesterone can influence mood or behaviour.
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Infusions of diazepam and allopregnanolone into the midbrain central gray facilitate open-field behavior and sexual receptivity in female rats.
We have previously shown that blocking GABAA receptors with bicuculline in the dorsal and lateral quadrants of the rostral two-thirds of the midbrain central gray (MCG) in rats reduces the lordosis quotient (LQ) and proceptive behaviors without increasing the defensive index (McCarthy, Pfaff, and Schwartz-Giblin, 1991b), indicating that tonic release of endogenous GABA in the MCG has a primary facilitatory effect on reproductive behaviors. The present study tests whether two modulators of the GABAA-benzodiazepine-receptor complex, diazepam and the progesterone metabolite, allopregnanolone, facilitate female receptivity as well as open-field behavior. Ovariectomized estradiol benzoate (EB)-treated rats were bilaterally infused in the MCG with vehicle, diazepam, or allopregnanolone and tested 20 min after diazepam and 12 min after allopregnanolone. Animals were tested once each for both receptive or open-field behavior with testing sessions being at least 1 week apart in a crossover design between vehicle and one of the two drugs. Both diazepam and allopregnanolone significantly increased the lordosis quotient in a dose-dependent manner, the lowest effective dose tested being 100 and 250 ng, respectively. In the open-field test, infusion of diazepam (100 ng) in EB-treated rats significantly increased the ratio of central squares entered to total squares entered (C/T) without a change in total activity. Rats without EB elicited comparable C/T ratios when tested with five times as much diazepam. Diazepam infusions at more dorsal sites in the midbrain and in overlying cortex were without effect on C/T ratios in EB-treated rats. Allopregnanolone (500 ng) infusions in MCG of EB-treated rats did not alter C/T ratios compared to animals without EB. Our results are consistent with diazepam and allopregnanolone acting through the GABAA receptors of the MCG to enhance female sexual receptivity and with estradiol potentiating the effect of diazepam in the MCG to increase open-field behavior.
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Changes in preproenkephalin messenger RNA level in the rat ventromedial hypothalamus during the estrous cycle.
To gain a better understanding of the relationship between the female rat reproductive system and preproenkephalin (PPE) expressing neurons under physiological conditions, we examined changes in PPE mRNA levels in the mediobasal hypothalamus during the rat estrous cycle by means of northern blotting and in situ hybridization histochemistry (ISHH). In the Northern blot studies, we found that PPE mRNA levels in the mediobasal hypothalamus were significantly increased by noon of proestrus compared to those in the morning and stayed high until diestrus day 1, and returned toward low levels on diestrous day 2. In contrast, measured as controls, glyceraldehyde-3-phosphate-dehydrogenase mRNA levels were significantly higher on proestrus regardless of time of day compared to diestrus day 2, and levels of calcineurin mRNA on proestrous and estrous were significantly lower than diestrous day 1 and day 2. ISHH studies revealed that these changes in PPE mRNA levels were specific in the ventromedial hypothalamic nucleus pars ventrolateralis (VMHVL), since we could not see any significant changes in signal in other parts including ventromedial hypothalamic nucleus pars dorsomedialis and arcuate hypothalamic nucleus. In the VMHVL, PPE mRNA levels in the afternoon of proestrous were significantly higher than those in the afternoon of diestrous day 2 whereas no significant change in PPE mRNA was observed in the caudate-putamen. The present study provides additional information relevant to possible implications of PPE gene expression in female reproductive systems, since changes in PPE mRNA levels may be associated with estrogen as well as progesterone or other hormonal concentrations during the estrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
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Potential interactions between estrogen receptor and thyroid receptors relevant for neuroendocrine systems.
Environmental signals can profoundly affect reproductive behavior, physiology and responses to steroids. One consequence of nutritional or temperature stress is altered plasma concentrations of thyroid hormone. Recent in vivo and in vitro data indicate that manipulations of estrogen and thyroid hormone levels can alter each other's functions. One possible mechanism for interaction may be that thyroid and estrogen receptors bind to parts of the same hormone response elements of target genes and compete with each other, thus serving to integrate environmental signals with neuroendocrine responses.
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DNA binding of hypothalamic nuclear proteins on estrogen response element and preproenkephalin promoter: modification by estrogen.
Preproenkephalin (PPE) gene expression is specifically induced by estrogen in hypothalamus of ovariectomized (OVX) females, better than in male rats. To study estrogen actions on gene regulation, we have presently characterized protein-DNA interactions by use of a consensus estrogen response element (ERE) and a putative ERE from PPE gene, with nuclear extracts from hypothalamus. By use of the electrophoretic mobility shift assay (EMSA), ERE binding activity was detected in nuclear extracts from neuronal tissues including hypothalamus, hippocampus, striatum, cerebellum and frontal cortex, and non-neuronal tissues such as pituitary and uterus, but not lung of OVX female rats with a consensus ERE, as well as a 129-bp PCR fragment from PPE promoter and a hairpin oligonucleotide that contains a putative ERE of the rat PPE gene. The ERE binding was eliminated by the addition of specific ERE-containing oligonucleotide, but not control oligonucleotides. Protein and DNA associated and dissociated very rapidly. By use of supershift assay, interactions of estrogen receptor with ERE were demonstrated in hypothalamic nuclear extracts. The initial levels of specific ERE binding in the hypothalamic nuclear extracts were comparable between castrated male and OVX female rats. However, estrogen treatment, either estradiol or estradiol benzoate, produced a rapid and tissue-specific induction of a slow mobility complex of ERE binding in hypothalamic nuclear extracts from females, better than in male rats, presumably from other associated factors, or a conformational change or other posttranslational modifications. This estrogen-induced slow mobility complex of ERE binding in hypothalamus was not observed after treatment with progesterone or tamoxifen. These results suggest that specific ERE binding is present in rat hypothalamic nuclear proteins, which may contribute to the upregulation of PPE gene expression by estrogen, and that the sexually differentiated action of estrogen may be related to an estrogen-induced conformational change, but not to the initial level of ERE-binding activity.
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Rapid stimulation of oxytocin gene expression in supraoptic nucleus neurones during morphine withdrawal in rats.
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Nucleic acid sequence and DNase hypersensitive sites of the 5' region of the mouse progesterone receptor gene.
To allow study of the control of the promoter of the mouse progesterone receptor gene and its specific elements, we have isolated nine individual clones and selectively sequenced the 5' region, which includes 5' promoter sequence and the non-coding region. Sequence comparison was performed versus the progesterone receptor genes of other species. Moreover, potential estrogen response elements, progesterone response elements, and other transcription factor recognition sites were determined by sequence analysis. In addition, since it has been confirmed that chromatin structures are important for regulation of gene expression, we also examined them in different estrogen target organs using the DNase hypersensitivity assay. A different pattern of the DNase hypersensitivity sites was detected between uterus and mediobasal hypothalamus and these sites were close to specific recognition elements.
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