Research on the neurobiology of sexual behavior at the turn of the millennium.
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Animals↗
Biomedical subjects
D W Pfaff
Publications and source records attributed to D W Pfaff.
Survival of reproductive behaviors in estrogen receptor beta gene-deficient (betaERKO) male and female mice.
Previously, it was shown that the lack of a functional estrogen receptor (ER) alpha gene (ERalpha) greatly affects reproduction-related behaviors in both female and male mice. However, widespread expression of a novel second ER gene, ERbeta, demanded that we examine the possible participation of ERbeta in regulation of these behaviors. In dramatic contrast to our results with ERalpha knockout (alphaERKO) males, betaERKO males performed at least as well as wild-type controls in sexual behavior tests. Moreover, not only did betaERKO males exhibit normal male-typical aggressive behavior, including offensive attacks, but they also showed higher levels of aggression than wild-type mice under certain conditions of social experience. These data revealed a significant interaction between genotype and social experience with respect to aggressive behavior. Finally, females lacking a functional beta isoform of the ER gene showed normal lordosis and courtship behaviors, extending in some cases beyond the day of behavioral estrus. These results highlight the importance of ERalpha for the normal expression of natural reproductive behaviors in both sexes and also provide a background for future studies evaluating ERbeta gene contributions to other, nonreproductive behaviors.
Aggression↗
Frequent colocalization of mu opioid and NMDA-type glutamate receptors at postsynaptic sites in periaqueductal gray neurons.
In the ventrolateral periaqueductal gray (PAG), endogenous pathways which dampen pain transmission can be activated by either opioids or excitatory amino acids such as N-methyl D-aspartate (NMDA). The effects of these ligands may converge, because morphine-produced analgesia in the PAG can be blocked by NMDA receptor antagonists. To determine the relationship between the subcellular sites where opioid ligands of the mu opioid receptor (MOR) and NMDA receptor ligands may act, we studied the ultrastructural distribution of immunolabeling for MOR and the R1 subunit of the NMDA receptor (NR1) in the ventrolateral PAG. MOR labeling was most commonly distributed along extrasynaptic regions of the plasma membrane of neuronal dendrites (80% or 245/306). In addition, MOR labeling was found presynaptically in axon terminals (13% or 39/306) which preferentially formed symmetric (inhibitory-type) synapses. NR1 immunoreactivity was also prevalent in dendrites (72% or 242/335), but in contrast to MOR, was usually associated with a subset of postsynaptic densities. Axon terminals (5%, 17/335) and glial processes (18%, 61/335) comprised the remainder of NR1-labeled profiles. There was a striking colocalization of MOR and NR1 labeling within dendrites. The majority of NR1-labeled dendrites contained MOR labeling (72%, 176/242) and likewise, the majority of MOR-labeled dendrites contained NR1 labeling (72%, 176/245). Thus, mu opioid and NMDA receptor ligands may act at several overlapping subcellular sites to modulate behaviors subserved by the ventrolateral PAG, such as antinociception.
Animals↗
The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus.
Estrogen (E) and progesterone (P) orchestrate many cellular responses involved in female reproductive physiology, including reproductive behaviors. E- and P-binding neurons important for lordosis behavior have been located within the ventromedial hypothalamus (VMH), and several hormone-responsive genes have been observed there as well. In attempts to identify additional E- and P-responsive genes in the VMH that may contribute to sexual behaviors, we used the differential display mRNA screening technique. One of the genes identified encodes the 73-kDa heat shock cognate protein (Hsc73). Quantitative in situ hybridization analysis of brains from naturally cycling female rats revealed a significant increase in Hsc73 mRNA in the VMH and arcuate nucleus of animals during proestrus compared with those at diestrus-1. To confirm that these increases were steroid hormone dependent, we compared vehicle-treated ovariectomized females with ovariectomized females treated with estradiol benzoate and P. Northern analysis and in situ hybridizations showed that the Hsc73 gene is enhanced by E and P in the pituitary and subregions of the VMH. Incidentally, by examining the primary amino acid sequence of rat, human, and chicken progesterone receptors, we noticed that putative Hsc73 binding sites are conserved across species with similar sites existing in the androgen and glucocorticoid receptors. Together these findings suggest a possible mechanism through which E could influence the activities of progesterone, androgen, and glucocorticoid receptors, by enhancing the expression of Hsc73 in cells where these proteins colocalize.
Amino Acid Sequence↗
In the ventromedial nucleus of the rat hypothalamus, GABA-immunolabeled neurons are abundant and are innervated by both enkephalin- and GABA-immunolabeled axon terminals.
Immunohistochemical-labeling for the neurochemicals gamma-aminobutyric acid (GABA) and enkephalin are abundant in the ventromedial nucleus of the hypothalamus (VMN). In VMN, both GABA and enkephalin may function to regulate feeding behavior, as well as other hormone-controlled behaviors. Importantly, in several brain areas, enkephalin is often thought to modulate GABAergic neurotransmission. Therefore, we used dual-labeling immunohistochemistry with electron microscopic analysis to study the circuitry of neurons containing GABA- and/or enkephalin-labeling within the VMN. Somato-dendritic profiles containing GABA-labeling were three fold more abundant than GABA-labeled axon terminals (117 soma or dendrites vs. 34 axons). In addition, axon terminals containing GABA-labeling sometimes synapsed onto GABA-labeled somata or dendrites (25% or 9/34). In contrast, under these conditions labeling for enkephalin was primarily restricted to axon terminals, which were very abundant throughout VMN. Enkephalin-containing terminals accounted for a large fraction (25% 23/92) of the axons in contact with GABA-labeled dendrites, although they also contacted unlabeled dendrites. These observations suggest that a population of VMN neurons are GABAergic. These may be either local circuit 'interneurons' or projection neurons. In addition, GABA-labeled VMN neurons may be regulated by either enkephalin or GABA. These morphologic observations provide the basis for disinhibitory mechanisms to function within the VMN.
Animals↗
Neural oxytocinergic systems as genomic targets for hormones and as modulators of hormone-dependent behaviors.
At the molecular level, estradiol turns on the gene for oxytocin in a subset of paraventricular hypothalamic neurons and turns on the gene for the oxytocin receptor in other limbic and hypothalamic cell groups. As a result, oxytocin deposition, whose signal is transduced both through G alpha (q/11) and Gi to stimulate phosphatidylinositol turnover, facilitates electrical activity in certain hypothalamic neurons. Consequently, affiliative behaviors including those closely associated with reproduction--mating behaviors and parental behaviors--are promoted. One important aspect of this effect is the preservation of instinctive behaviors associated with reproduction, in the face of disturbances due to mild stress.
Animals↗
Thyroid hormones and estrogen affect oxytocin gene expression in hypothalamic neurons.
The oxytocin (OT) gene promoter has a composite hormone response element, such that several members of the steroid/thyroid hormone superfamily of nuclear receptors can interact at this response element in vitro. To investigate this in brain tissue, parallel to foregoing behavioural experiments, we used in situ hybridization histochemistry to seek interactions between estrogen and thyroid hormones on OT mRNA in the hypothalamus. In ovariectomized (OVX) rats, high doses of triiodothyronine (T3) elevated OT mRNA levels in the paraventricular (PVN) nucleus, while treatment with estradiol benzoate (EB) alone had no significant effect. In contrast, animals that were thyroidectomized (TX) in addition to OVX had dramatically elevated levels of OT gene expression in the PVN following EB treatment. That is, endogenous thyroid hormones interfered with EB-induction of gene expression. Moreover, in both OVX and TX/OVX animals, OT gene expression was reduced to values equivalent to controls when T3 was given together with EB. Particular subdivisions of the PVN responded differentially to T3 and EB treatment, demonstrating marked heterogeneity of OT-containing neurons in this nucleus. Thus, parallel to and perhaps related to the manner in which thyroid hormones reduced estrogen-stimulated behaviour, endogenous or exogenous thyroid hormones interfered with estrogen stimulation of OT mRNA. These data demonstrate competition between nuclear proteins, transcription factors, in hypothalamic neurons.
Animals↗
Thyroid hormone coadministration inhibits the estrogen-stimulated elevation of preproenkephalin mRNA in female rat hypothalamic neurons.
Expression of the enkephalin gene in ventromedial hypothalamus (VMH) of the female rat has been correlated with the performance of lordosis behavior. By antisense DNA evidence, it has been drawn into a causal role as well. Here, we explored whether, parallel to earlier molecular and behavioral results, thyroid hormone coadministration could disrupt the estrogenic induction of preproenkephalin (PPE) mRNA. As expected, estradiol benzoate treatment to ovariectomized rats led to a large and significant increase in PPE gene expression in the VMH. This increase was inhibited by coadministration of thyroid hormone. The thyroid hormone interference in PPE gene expression was specific to the VMH, as there were no significant effects in the central nucleus of the amygdala or in the caudate/putamen. These in situ hybridization histochemical results form a direct parallel both to previous transcriptional measurements and to reproductive behavior assays in which thyroid hormones were able to oppose estrogenic facilitation. Previous evidence supports the notion of competitive DNA binding and protein/protein interactions providing mechanisms for nuclear thyroid hormone receptors to affect estrogen receptor function, but other, additional mechanisms cannot be ruled out. To date, both oxytocin and PPE gene expression represent potential hypothalamic systems by which thyroid hormones could interfere with estrogen-stimulated female rat reproductive behavior.
Amygdala↗
Olfactory bulb development is altered in small-eye (Sey) mice.
Small-eye (Sey) is a spontaneous, semidominant murine mutation that results from a point mutation in the Pax-6 gene. Both the eyes and the olfactory system fail to develop in homozygotes and these animals die neonatally. Heterozygotes (Sey/+) have different degrees of eye abnormalities including decreased lens size and cataracts. In the present study, we examined whether one mutated allele of Pax-6 also affects olfactory system development. By 42 days of age, main olfactory bulb volume was significantly decreased in Sey/+ animals compared with wild-type littermates, and this effect was even more dramatic in 70-day-old animals. In contrast, there was no effect on accessory olfactory bulb, olfactory epithelial, or vomeronasal organ development at any age in Sey/+ animals, demonstrating the specificity of the effect. In the main olfactory bulb, the largest differences in laminar volume were found in the glomerular and granule cell layers. These layers contain the olfactory bulb interneurons, and a subpopulation of these cells were found to be Pax-6 immunoreactive. Examination of the neurochemical consequences of this mutation showed that the number of both tyrosine hydroxylase (TH)- and gamma-aminobutyric acid (GABA)-immunoreactive profiles were dramatically decreased in Sey/+ animals as compared with controls. In contrast, neither calretinin nor calbindin immunoreactivity was affected by this mutation. Dual-labeling immunohistochemistry showed that nearly all TH-immunoreactive cells and a subpopulation of GABA-immunoreactive cells coexpressed Pax-6. However, calretinin- and calbindin-immunoreactive cells were not Pax-6 immunopositive. These data indicate that two normal alleles of Pax-6 are required for normal olfactory bulb development and, as part of this effect, this gene may be involved in the development of specific neurotransmitter systems.
Aging↗
Estrogen alters behavior and forebrain c-fos expression in ovariectomized rats subjected to the forced swim test.
Estrogen has been implicated in brain functions related to affective state, including hormone-related affective disorders in women. Although some reports suggest that estrogen appears to decrease vulnerability to affective disorders in certain cases, the mechanisms involved are unknown. We used the forced swim test (FST), a paradigm used to test the efficacy of antidepressants, and addressed the hypotheses that estrogen alters behavior of ovariectomized rats in the FST and the FST-induced expression of c-fos, a marker for neuronal activity, in the rat forebrain. The behaviors displayed included struggling, swimming, and immobility. One hour after the beginning of the test on day 2, the animals were perfused, and the brains were processed for c-fos immunocytochemistry. On day 1, the estradiol benzoate-treated animals spent significantly less time struggling and virtually no time in immobility and spent most of the time swimming. Control rats spent significantly more time struggling or being immobile during a comparable period. On day 2, similar behavioral patterns with still more pronounced differences were observed between estradiol benzoate and ovariectomized control groups in struggling, immobility, and swimming. Analysis of the mean number of c-fos immunoreactive cell nuclei showed a significant reduction in the estradiol benzoate versus control groups in areas of the forebrain relating to sensory, contextual, and integrative processing. Our results suggest that estrogen-induced neurochemical changes in forebrain neurons may translate into an altered behavioral output in the affective domain.
Animals↗
Regulation of neuronal nitric oxide synthase mRNA in lordosis-relevant neurons of the ventromedial hypothalamus following short-term estrogen treatment.
Short-term estrogenic regulation of neuronal nitric oxide synthase (nNOS) mRNA in the ventrolateral subdivision of the ventromedial nucleus (VLVMN), an area central to lordosis, was demonstrated using in situ hybridization. Estrogen-treated animals showed a significantly greater signal in the VLVMN, but not the arcuate or supraoptic nuclei, compared to ovariectomized controls. Neuronal NOS may be involved in early actions of estrogen in the VLVMN.
Animals↗
The gonadotropin-releasing hormone system does not develop in Small-Eye (Sey) mouse phenotype.
This study examined the development of the gonadotropin releasing-hormone (GnRH) system in a spontaneous mouse mutation, Small-Eye (Sey). This phenotype is due to a point mutation in the developmental control gene Pax-6 and results in failed development of the eye and olfactory placodes in homozygous (Sey/Sey) embryos and a variety of eye abnormalities in heterozygotes (Sey/+). Therefore, Sey/Sey embryos provided a naturally occurring olfactory placode ablation to ask whether all of the GnRH neurons found in the adult mouse forebrain arise from the olfactory epithelium. In Sey/Sey embryos, GnRH-immunoreactive neurons were not present in either the presumptive nasal regions or in any area of the brain at any embryonic age. In contrast, in Sey/+ embryos, there was no apparent effect on either GnRH cell proliferation or migration. These data support and extend the hypothesis that GnRH neurons in mice originate in the olfactory placodes and also demonstrate that two normal alleles of Pax-6 are not required for GnRH system development.
Alleles↗
Differential regulation of AP-1 DNA binding activity in rat hypothalamus and pituitary by estrogen.
Ap-1 proteins such as Fos and Jun are nuclear transcription factors that have been postulated to function as third messengers in signal transduction pathways to regulate target gene expression. Using electrophoretic mobility shift assays (EMSA), we have studied estrogen (E) effects on regulation of AP-1 DNA binding activity in the rat hypothalamus and pituitary. AP-1 binding is defined herein as the specific association with a consensus AP-1 site during EMSA. Specific AP-1 binding activity was observed in nuclear extracts from the hypothalamus and pituitary of ovariectomized (OVX) female and castrated (CAS) male rats. Treatment with E increased the levels of AP-1 binding activity in the pituitary and uterus, whereas E decreased the levels of AP-1 binding in the hypothalamus, of OVX females. These effects were observed within 60 min and maintained for at least 72 h after a single dose of estrogen. Estrogen-induced changes in AP-1 binding were much more prominent in OVX females than in CAS males. Treatment with progesterone in OVX females had no significant effects on AP-1 binding activity in either pituitary or hypothalamus. Analysis of AP-1 binding activity in both hypothalamus and pituitary by supershift, immunodepletion and shift-Western blot indicated that part of the AP-1 binding was due to the presence of Fos and Jun proteins. However, Western blot analysis shows that the levels of Fos and Jun proteins in the hypothalamic nuclear extracts were not altered by E treatment. We conclude that E produced tissue and sex-differentiated alterations in AP-1 DNA binding activity in the hypothalamus and pituitary of female rats, which may be related to differential estrogenic actions on gene regulation.
Animals↗
In situ PCR for in vivo detection of foreign genes transferred into rat brain.
Here we describe the use of in situ PCR to detect a viral transgene in rat brain. Previously, we have reported in vivo gene transfer by using a defective herpes simplex viral vector in mammalian brain (Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S.P., Hanlon, B.A., Rabkin, S.D., Pfaff, D.W., Mol. Cell. Neurosci. 2 (1991) 320-330). For detection of the LacZ transgene, we have used histochemical staining for the protein product, beta-galactosidase, and in situ hybridization for its mRNA, but the DNA itself cannot be reliably detected with conventional methods. Therefore we have adapted the technique of in situ PCR, so that we may detect minute quantities of transgenic vector DNA following in vivo gene. The brain sections, prefixed, were treated with PBS-detergent before PCR amplification to increase permeability for peptides and oligonucleotides across cellular barriers in brain tissue. Pretreatment with detergent retained better brain morphology than the more widely used proteinase treatment. The PCR mixture containing dNTPs, primers, digoxigenin-dUTP (Dig-dUTP) and buffer was loaded onto each brain section. Slides containing brain sections were placed in an aluminum boat and then on the block of the thermal cycler. Temperature was brought to 82 degrees C before adding Taq polymerase ('hot start' method). Dig-labeled PCR amplified fragments were then detected by alkaline-phosphatase-linked anti-digoxigenin-antibody. Positive signals were seen within the nucleus of transduced neurons, indicating presence of viral DNA. Enhanced specificity was observed with the use of Dig-labeled primers which eliminates the possibility of non-specific viral DNA detection through primer-independent reactions. Overall, this technique can serve not only as an internal control for transgene presence during comparisons of experimental groups of animals, but may also have clinical applications including the detection of viral infection in human brain such as HIV in pathology specimens.
Animals↗
Stimulation of expression of the oxytocin gene in rat supraoptic neurons at parturition.
We measured expression of the oxytocin gene in the supraoptic nucleus (SON) during pregnancy, parturition and lactation to examine its relationship to states of accumulation or depletion of oxytocin stores and to conditions of strong excitation of oxytocin neurons. The primary transcript (heterogeneous nuclear RNA, hnRNA) of the oxytocin gene was measured using a 3H-cDNA probe against intron 1 for in situ hybridisation. Autoradiographs of the SON showed the hnRNA as discrete clumps of silver grains within the nucleus of each neuron. The number of cells expressing oxytocin hnRNA did not change during pregnancy but increased during parturition; 10-day lactating animals showed similar increases. Oxytocin mRNA was also measured by in situ hybridisation using a 3H- or 35S-labelled oligonucleotide probe against exon C: hybridisation was seen over the cytoplasm of supraoptic neurons, but no differences were measured between virgin, mid-pregnant, preparturient, parturient or 2-day lactating rats. The data suggest that enhanced oxytocin gene transcription is not necessary to increase oxytocin stores in pregnancy. However, acute stimulation of magnocellular oxytocin neurons at parturition, which strongly increases neuron activity and secretion, results in a rapid increase in the number of cells expressing oxytocin hnRNA, and increased expression is sustained in lactation.
Animals↗
Mapping of neural and signal transduction pathways for lordosis in the search for estrogen actions on the central nervous system.
Estrogen can act on the brain to regulate various biological functions and behavior. In attempts to elucidate the estrogen action, the rodent female reproductive behavior, lordosis, was used as a model. Lordosis is an estrogen-dependent reflexive behavior and, hence, is mediated by discrete neural pathways that are modulated by estrogen. Therefore, a strategy of mapping the pathways, both neural and biochemical, and examining them for estrogen effect was used to localize and subsequently analyze the central action of estrogen. Using various experimental approaches, an 'inverted Y-shaped' neural pathway both sufficient and essential for mediating lordosis was defined. The top portion is a descending pathway conveying the permissive estrogen influence which originated from hypothalamic ventromedial nucleus relayed via midbrain periaqueductal grey down to medullary reticular formation, the top of the spino-bulbo-spinal reflex arc at the bottom. This estrogen influence alters the input-output relationship, shifting the output toward more excitation. With this shift in output, estrogen can enable the otherwise ineffective lordosis-triggering sensory stimuli to elicit lordosis. In the ventromedial nucleus, the origin of the estrogen influence, a multidisciplinary approach was used to map intracellular signaling pathways. A phosphoinositide pathway involving a specific G protein and the activation of protein kinase C was found to be involved in the mediation of lordosis as well as a probable target of the permissive estrogen action. The action of estrogen on this signal transduction pathway, a potentiation, is consistent with and, hence, may be an underlying mechanism for the estrogen influenced shift toward excitation. Thus, further investigation on this specific signal transduction pathway should be helpful in elucidating the action of estrogen on the brain.
Animals↗
Current status of antisense DNA methods in behavioral studies.
The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.
Animals↗
Mathematical exploration of pulsatility in cultured gonadotropin-releasing hormone neurons.
Pulsatile gonadotropin-releasing hormone (GnRH) release has been demonstrated in cultures of an immortalized line of GnRH expressing neurons (GT1 cells) in experiments by four different research groups. Pulsatile release is known to play a crucial role in GnRH-mediated signaling in vivo, and thus deserves theoretical and quantitative consideration, especially as GT1 cells are presumably genetically homogeneous. Here we have modeled idealized GT1 cells with a differential equation/logic based modeling program, Stella II. We have created a network of 'neurons', with randomized (within the same preset limits for each neuron) thresholds, number and weight of connections to other neurons, and build-up of signal; as well as continuous decay of stored signal. Surprisingly, we found that with this minimal set of assumptions, without any sort of predefined pacemaking cells, it is possible to create pulsatility similar to that observed in the laboratory. A variety of different parameter sets was found to produce these pulses. Network behaviors similar to those of GT1 cells depended on the degree of interconnection between neurons and their functioning within a critical range of network excitability. These findings allow for a clearer consideration of the critical elements of such networks as well as experimental predictions regarding the production of pulsatile behavior.
Action Potentials↗