Precision in mouse behavior genetics.
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Animals↗
Biomedical subjects
D Pfaff
Publications and source records attributed to D Pfaff.
Statistical analysis of measures of arousal in ovariectomized female mice.
In preparation for evaluating hormone effects and hormone receptor gene influences on fundamental processes of behavioral arousal, we subjected 48 ovariectomized female mice to a rigid protocol of several tests bearing on arousal concepts. The central hypothesis was that results would organize themselves according to capacities for sensory alertness, motor activity, and emotional reactivity. The large table of across-mouse correlation was subjected to factor analysis and cluster analysis. Results provided evidence for a general arousal (one-factor solution) which, however, accounted for only 29.72% of the variance. Four-factor (72.03%) matched four-cluster and six-factor (84.38%) matched six-cluster lineups of behavior components quite well, but did not conform to the main hypothesis; e.g., home cage exploration frequency and duration variables did not covary, and locomotor wheel activity grouped with fear indices. Besides providing a baseline for ongoing estrogen/thyroid and related genetic experiments, this statistical approach should be useful for a variety of hormonal studies of complex behaviors in mice.
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Crosstalk between oestrogen receptors and thyroid hormone receptor isoforms results in differential regulation of the preproenkephalin gene.
Nuclear receptors are ligand-activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)alpha-mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand-binding TR isoforms on the ER-mediated induction of the PPE gene in transient transfection assays in CV-1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ER alpha and beta isoforms mediate a four to five-fold induction by oestrogen. Cotransfection of TR alpha 1 along with ER alpha inhibited the ER alpha transactivation of PPE by approximately 50%. However, cotransfection with either TR beta 1 or TR beta 2 expression plasmids produced no effect on the ER alpha or ER beta mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TR alpha 1 DNA-binding mutant could also inhibit ER alpha transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC-1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ER beta is cotransfected, TR alpha 1 stimulated ER beta-mediated transactivation of PPE by approximately eight-fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.
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Differential interaction of estrogen receptor and thyroid hormone receptor isoforms on the rat oxytocin receptor promoter leads to differences in transcriptional regulation.
Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, alpha and beta, exist. The TRalpha and beta isoforms are products of two distinct genes that are further differentially spliced to give TRalpha1 and alpha2, TRbeta1 and beta2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17beta-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERalpha could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERbeta was incapable of inducing the gene in either cell line. ERalpha is therefore not equivalent to ERbeta on this physiological promoter. Indeed, in the neural cell line, ERbeta can inhibit ERalpha-mediated induction from the OTR promoter. While the TRalpha1 isoform inhibited ERalpha-mediated induction in the neural cell line, the TRbeta1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERalpha-mediated induction of the rat OTR gene promoter by the TRalpha1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRalpha1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRalpha-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.
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Hormones, genes and the structure of sexual arousal.
Despite the inherent difficulty of connecting individual genes with integrated mammalian behaviors, it has been determined that a series of genes are turned on by estrogenic hormones acting in forebrain. Their products are, in turn, facilitatory for female reproductive behaviors such as lordosis. The causal routes by which two genes contribute to the control of lordosis behavior, the classical estrogen receptor gene (ER-alpha) and a thyroid hormone (TH) receptor gene (TR-beta), have been delineated. Beyond the mechanisms underlying the expression of concrete, specific natural behaviors, lies the question of sexual motivation. Required as an intervening variable to explain fluctuations in natural behaviors in the face of constant stimuli, motivational states have both general and specific features. Most theoretical and experimental approaches toward the general aspects of motivation have depended heavily on concepts of 'arousal.' Sexual arousal is likely to depend both on very general, broadly distributed neuronal influences and on specific affiliative and sexual tendencies. Is 'general arousal' a monolithic, undifferentiated process? In no way can a review at this time settle such issues, but the reasons behind six new experimental approaches to these questions are described.
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Catechol-O-methyltransferase-deficient mice exhibit sexually dimorphic changes in catecholamine levels and behavior.
Catechol-O-methyltransferase (COMT) is one of the major mammalian enzymes involved in the metabolic degradation of catecholamines and is considered a candidate for several psychiatric disorders and symptoms, including the psychopathology associated with the 22q11 microdeletion syndrome. By means of homologous recombination in embryonic stem cells, a strain of mice in which the gene encoding the COMT enzyme has been disrupted was produced. The basal concentrations of brain catecholamines were measured in the striatum, frontal cortex, and hypothalamus of adult male and female mutants. Locomotor activity, anxiety-like behaviors, sensorimotor gating, and aggressive behavior also were analyzed. Mutant mice demonstrated sexually dimorphic and region-specific changes of dopamine levels, notably in the frontal cortex. In addition, homozygous COMT-deficient female (but not male) mice displayed impairment in emotional reactivity in the dark/light exploratory model of anxiety. Furthermore, heterozygous COMT-deficient male mice exhibited increased aggressive behavior. Our results provide conclusive evidence for an important sex- and region-specific contribution of COMT in the maintenance of steady-state levels of catecholamines in the brain and suggest a role for COMT in some aspects of emotional and social behavior in mice.
Amino Acid Sequence↗
Aspects of GnRH neurobiology conserved across vertebrate forms.
The decapeptide gonadotropin-releasing hormone (GnRH) came into prominence because of its roles in releasing luteinizing hormone and follicle-stimulating hormone and promoting reproductive behavior. At least three aspects of GnRH neurobiology have features which may be universal among vertebrates. First, the GnRH neuronal migration from the olfactory placode into the basal forebrain appears to hold true for forms ranging from fish to humans. Second, for proper agonist activity in the anterior pituitary, GnRH must be released in a pulsatile fashion. Since GT-1 neuronal cell cultures can demonstrate pulsatile release, it must be concluded that GnRH neuronal networks themselves can manage this type of pulsatility. Using a neuronal mathematical model with "minimalist" assumptions, we demonstrated that a network of identical neurons can achieve this self-organizing property without the use of, or spontaneous appearance of, "pacemaker cells." Indeed, since many parameter combinations worked, and since no information about species identity or chemical cell type is provided to the model, this conclusion could apply across many vertebrate forms and, perhaps, even for other neuroendocrine cell types. Third, Fernald and colleagues (this issue of General and Comparative Endocrinology) have demonstrated remarkable effects of social context on GnRH expression in fish. Reviewed here are some data in musk shrews suggesting that behavioral and social stimuli can also modify GnRH neurons in mammals. Therefore, although GnRH neuronal mechanisms are adapted to meet species-typical variations in environment and physiology, some of the important features of this system appear to be widely conserved.
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Differential LHRH secretion, dye coupling, and protein expression in two morphologically distinct cell types identified in GT1-7 cultures.
The immortalized neuronal cell line, GT1-7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1-7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two-fold shorter than that for G cells and N cells displayed 'piling up' whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye-coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron-specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV-40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1-7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.
Adenylyl Cyclases↗
Genes and behavior as studied through gonadotropin-releasing hormone (GnRH) neurons: comparative and functional aspects.
1. GnRH neurons migrate from olfactory placode into the developing basal forebrain in a manner which appears remarkably constant across all vertebrates studied, from fish to human beings. 2. Interruption of this migration can result in Kallmann's Syndrome. Absence of libido by individuals suffering from Kallmann's has allowed us to chart a causal route from a specific gene to a human social behavior.
Aging↗
Vasoactive mediators and the progression from oedematous to necrotising experimental acute pancreatitis.
Little is known about the pathophysiological factors that determine the clinical severity of acute pancreatitis. Because impairment of pancreatic circulation and oxygenation is associated with greater disease severity and morphological damage in experimental pancreatitis it has been suggested that various vasoactive mediators might participate in the progression from the oedematous to the necrotising variety of the disease. This study used an animal model of acute pancreatitis induced by intravenous caeruleint (10 micrograms/kg/h for up to six hours), which does not entail either haemorrhage or significant necrosis of the pancreas. This study considered whether the administration or the inhibition of either nitric oxide, bradykinin, or adrenergic mediators can convert this mild variety into haemorrhagic and necrotising pancreatitis. Neither nitric oxide nor catecholamines were involved in the progression from oedematous to haemorrhagic pancreatitis. Their substitution, activation, and inhibition all failed to change the severity of the disease process. Bradykinin alone seemed to be critically involved in the pathogenesis of pancreatic haemorrhage and necrosis. However, the inhibition of bradykinin and not its activation or substitution increased the severity of the disease.
Acute Disease↗
Injection of cobalt protoporphyrin into the medial nuclei of the hypothalamus elicits weight loss.
Intracerebroventricular administration of small amounts (0.1 mumol/kg body wt) of cobalt protoporphyrin (CoPP), a synthetic analogue of heme, results in transient hypophagia and prolonged reduction in body weight of rats. Statistically significant hypophagia is detectable within 3 h of CoPP infusion. These changes are accompanied by prompt and sustained reductions in running wheel revolutions, a measure of spontaneous locomotor activity. Bilateral intrahypothalamic injections of CoPP at far lower doses (4 nmol/rat) resulted in similar findings following infusion into the paraventricular, dorsomedial, and ventromedial nuclei, but the compound had no such effect when injected into the thalamus or the lateral hypothalamic area. These effects were also observed following microinjection of the natural metalloporphyrin, heme, into the medial hypothalamic nuclei. Inorganic cobalt, iron, protoporphyrin, and magnesium protoporphyrin injected similarly were without such effect. These findings provide further evidence that the site of the previously described actions of CoPP in reducing food intake and body weight in rats resides, at least in part, in the medial hypothalamus. Furthermore, this study expands the spectrum of metalloporphyrins that act in the central nervous system to elicit these changes from synthetic compounds such as CoPP to heme, the natural, physiological metalloporphyrin.
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Comprehensive polypeptide analysis of microdissected rat brain areas: combining 2-dimensional gel electrophoresis with 2-dimensional HPLC and immunoanalysis and sequencing procedures.
An improved methodology has been developed which allows resolution, identification, and quantitation of hundreds of proteins and neuropeptides from a single rat brain nucleus (5 mg wet wt.). After metabolic labelling, proteins (greater than about 15 kDa) are separated from peptides (less than about 10 kDa) by sonicating the tissue in an acidic peptide extraction buffer; after centrifugation, proteins are in the pellet, peptides in the supernatant. To quantitate peptide synthesis, peptides are resolved to purity by reverse-phase HPLC followed by ion exchange HPLC. Proteins are resolved with a two-dimensional (2-D) gel protocol optimized for neural tissue. To identify specific proteins by immunoanalysis, proteins are transferred to polyvinyl difluoride (Immobilon) and immunostained in the presence of Tween blocking buffer. After visualization with an avidin-biotin alkaline phosphatase procedure, the blot is post-stained with India ink to visualize the protein pattern context. To sequence spots, proteins are transferred to Immobilon, stained with Coomassie, and directly subjected to automated gas phase sequencing. The immunoblot procedure can detect less than 0.1 pmol protein, and the sequencing procedure can detect less than 10 pmol protein. After transfer enough material remains on the gels to allow subsequent autoradiography or silver stain. Quantitative analysis of 2-D gels is examined in a companion paper. These procedures should enhance the utility of 2-D gels in neurochemical studies.
Amino Acid Sequence↗
Similarity of an estrogen-induced protein and a luteinizing hormone releasing hormone-induced protein.
Estradiol induces a 70 kDa protein ('EI70') which is synthesized in vivo in the female rat ventromedial hypothalamus (VMH) and transported to the midbrain central gray, suggesting a role for EI70 in the female mating behavior, lordosis. Luteinizing hormone releasing hormone (LHRH), in addition to stimulating gonadotropin release, potentiates pituitary responsiveness to subsequent exposure to LHRH (the 'priming' effect), facilitates lordosis and induces the synthesis of a 70 kDa protein ('LHRH70') in pituitary in vitro. We now report that EI70 precisely co-migrates on two-dimensional (2-D) gels with the pituitary protein induced by LHRH both in vitro and in vivo. Furthermore, both proteins migrate on 2-D gels in the vicinity of a protein recognized after immunoblotting by antibodies to the heat-shock-70 kDa protein family. The induction of a common protein by estrogen or LHRH could represent a common mechanism by which these hormones facilitate secretion, and by which these hormones interact.
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Sex differences in response to discrete estradiol injections.
Developmental effects of perinatal androgens render adult male rats refractory to the activation of feminine sexual behavior by estradiol (E2) and progesterone (P). Recent evidence suggested that fluctuating levels of systemic E2, which are thought to approximate the ovarian secretion under physiological conditions, may reverse this insensitivity to E2 and, particularly, to the synergistic effects of P in male rats of the Wistar strain. We examined whether this hormonal regimen would reverse this insensitivity in Sprague-Dawley rats. Gonadectomized animals received two injections of E2 (1 microgram per injection) 12 hr apart at 0900 and 2100 hr followed by P (0.5 mg) or oil, at 35 hr, and a mating behavior test, at 38 hr, subsequent to the initial E2 administration. This treatment was repeated four times at 4-day intervals. The inability of Sprague-Dawley male rats to respond to E2 and P was unaffected by this pattern of exposure to exogenous E2. Receptivity scores, lordosis quotients, and proceptivity were negligible in males, and significantly less than that displayed by females. In addition, the levels of sexual receptivity and proceptivity were facilitated by the availability of P following E2 in females, but not in males. The present findings fail to support a general hypothesis that "discontinuous" E2 stimulation, achieved by two spaced injections of this hormone, reverses developmental determinants of sex differences in responsiveness to hormones mediating female sexual behaviors.
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Proteins synthesized in medial hypothalamus and transported to midbrain in estrogen-treated female rats.
Results on the anatomy of ventromedial hypothalamic outputs, together with the temporal aspects of electrophysiological studies, predict that for reproductive behavior control sex steroids alter hypothalamic protein synthesis and transport of proteins to the dorsal midbrain. We have studied labeled proteins arriving in the dorsal midbrain after local microinjection of tritiated amino acids to the ventromedial hypothalamus. Estrogen treated and control ovariectomized female rats are significantly different in this respect, and in particular some proteins appear to be synthesized or transported in greater amounts in the estrogen treated animals. Physical characterization of these proteins and comparisons under a variety of endocrine conditions will suggest whether their synthesis could be part of the mechanism by which ovarian steroids affect behavior.
Amino Acids↗
Electrophysiologic determination of projections from ventromedial hypothalamus to midbrain central gray: differences between female and male rats.
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Animals↗
Medullary reticular formation lesions and lordosis reflex in female rats.
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Animals↗
Somatosensory determinants of lordosis in female rats: behavioral definition of the estrogen effect.
By coating the ventral surface of male rats with a dye, regions of contact between male and female during male mounting were recorded precisely on the female's hair and skin. The male rat touches the female's flanks, rump, tailbase, perineum, and perivaginal surfaces during the female's initiation and maintenance of lordosis. Film analyses showed that the male's paws and pelvic thrusting stimulate the female's skin with dominant frequencies between 10 and 20 per second. Somatosensory stimuli were applied by the experimenter to the female skin locations contacted by the male. Deflection of hair on the flanks or perineum alone did not cause lordosis. Light stimulation simultaneously on flanks and perineum caused lordosis only in some females given high estrogen dosages supplemented by progesterone. When flank stimuli were followed by pressure on the rump, tailbase, and perineum, lordosis was triggered reliably in hormone-treated females. Here the estrogen-dependence of the reflex was shown, and progesterone synergized with the estrogen effect. Among lordosis components, rump and head elevations in response to pressure stimuli on the rump, tailbase, and perineum appear to be hormone-sensitive. These results help to define the minimal cutaneous sensory requirement for lordosis. In turn, the estrogen effect on lordosis may be defined behaviorally as increased responsiveness to pressure on rump, tailbase, and perineal skin, after flank stimulation. These results illustrate how estrogen, progesterone, and somatosensory stimuli interact in causing lordosis, increases in the strength of one factor compensating for decreases in another.
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