Vasopressin V1b receptor knockout reduces aggressive behavior in male mice.

Increased aggression is commonly associated with many neurological and psychiatric disorders. Current treatments are largely empirical and are often accompanied by severe side effects, underscoring the need for a better understanding of the neural bases of aggression. Vasopressin, acting through its 1a receptor subtype, is known to affect aggressive behaviors. The vasopressin 1b receptor (V1bR) is also expressed in the brain, but has received much less attention due to a lack of specific drugs. Here we report that mice without the V1bR exhibit markedly reduced aggression and modestly impaired social recognition. By contrast, they perform normally in all the other behaviors that we have examined, such as sexual behavior, suggesting that reduced aggression and social memory are not simply the result of a global deficit in sensorimotor function or motivation. Fos-mapping within chemosensory responsive regions suggests that the behavioral deficits in V1bR knockout mice are not due to defects in detection and transmission of chemosensory signals to the brain. We suggest that V1bR antagonists could prove useful for treating aggressive behavior seen, for example, in dementias and traumatic brain injuries.

Promoter sequence, expression, and fine chromosomal mapping of the human gene (MLP) encoding the MARCKS-like protein: identification of neighboring and linked polymorphic loci for MLP and MACS and use in the evaluation of human neural tube defects.

The MARCKS-like protein (MLP), also known as F52, MacMARCKS, or MARCKS-related protein, is a widely distributed substrate for protein kinase C (PKC). Recent studies using gene disruption in vivo have demonstrated the importance of both MARCKS and MLP to the development of the central nervous system; specifically, mice lacking either protein exhibit a high frequency of neural tube defects. We isolated a genomic clone for human MLP and discovered a directly linked polymorphism (MLP1) useful for genetic linkage analysis. The MLP promoter was 71% identical over 433 bp to that of the corresponding mouse gene, Mlp, with conservation of many putative transcription factor-binding sites; it was only 36% identical over 433 bp to the promoter of the human gene, MACS, which encodes the MLP homologue MARCKS. This 433-bp fragment drove expression of an MLP-beta-galactosidase transgene in a tissue-specific and developmental expression pattern that was similar to that observed for the endogenous gene, as shown by in situ hybridization histochemistry. In contrast to MACS, the MLP and Mlp promoters contain a TATA box approximately 40 bp 5' of the presumed transcription initiation site. MLP was localized to chromosome 1p34-->1pter by analysis of human-mouse somatic cell hybrid DNA and to 1p34 by fluorescence in situ hybridization. Radiation hybrid mapping of MLP placed it between genetic markers D1S511 (LOD > 3.0) and WI9232. MACS was localized to 6q21 between D6S266 (LOD > 3.0) and AFM268uh5 by the same technique. We tested the novel MLP1 polymorphism and the MACS flanking markers in a series of 43 Caucasian simplex families in which the affected child had a lumbosacral myelomeningocele. We found no evidence of linkage disequilibrium, suggesting that these loci were not major genes for spina bifida in these families. Nonetheless, the identification of linked and neighboring polymorphisms for MACS and MLP should permit similar genetic studies in other groups of patients with neural tube defects and other neurodevelopmental abnormalities.

Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution.

Programmed cell death (PCD) of mammary alveolar cells during involution commences within hours of the end of suckling. Locally, milk accumulates within alveolar lumens; systemically, levels of lactogenic hormones fall. Four experimental models were used to define the role of local factors as compared with systemic hormones during the first and second stages of involution. In three models, milk release was disrupted in the presence of systemic lactogenic hormones: (i) sealing of the teats, (ii) mammary gland transplants that cannot release milk due to the absence of a teat connection, and (iii) inactivation of the oxytocin gene. The ability of systemic hormones to preserve lobular-alveolar structure without blocking PCD was illustrated using a fourth transgenic model of lactation failure. During the first stage of involution, local signals were sufficient to induce alveolar PCD even in the presence of systemic lactogenic hormones. PCD coincided with bax induction, decreased expression of milk proteins, block of prolactin signal transduction through Stat5a and 5b, and activation of Stat3. The two stages of mammary gland involution are regulated by progressive gain of death signals and loss of survival factors. This study demonstrates that genetic events that occur during the first reversible stage are controlled by local factors. These mammary-derived death signals are dominant over protective effects related to systemic hormone stimulation.

The class III POU factor Brn-4 interacts with other class III POU factors and the heterogeneous nuclear ribonucleoprotein U.

The class III POU proteins are expressed throughout the central nervous system, including the hypothalamus, where they are often co-localized. Presumably, these POU proteins (Brain-1, Brain-2, Brain-4 and SCIP) serve as transcriptional transactivators. That they are co-expressed in some neurons suggests that, if they were to form homomeric and heteromeric complexes with each other, depending on the particular combination, they might have different DNA-binding specificities and, thus, activate different genes. We used purified fusion proteins of the four class III POU proteins in far-western assays to show that the proteins can interact. We confirmed their interactions using a two-hybrid system. Both techniques indicate that the interaction occurs through the POU domain. The far-western technique also allowed us to identify a 120-kDa nuclear protein that interacts with Brain-4. Subsequent affinity purification and microsequencing identified the protein as the heterogeneous nuclear ribonucleoprotein U (hnRNP U). This result suggests another mechanism by which a POU protein can influence gene expression: by facilitating the processing of pre-mRNA whose transcription it has stimulated.

A microscopic view of helix propagation: N and C-terminal helix growth in alanine helices.

Molecular dynamics simulations with umbrella sampling are used to perform free energy simulations of C-terminal and N-terminal helix propagation in small helices of Ace-(Ala)n-NMe, with n= (4,5,10,15), in water. From the resulting free energy surfaces, computed as a function of the terminal psi dihedral angle, the roles of length and end effects in helix propagation are explored. An energetic analysis of the helices, both formed and partially formed, is used to develop a molecular rationalization for the observed trends in helix stability. We find that the microscopic helix propagation parameters vary significantly depending on the end and length of the helix in which the terminal hydrogen bond is forming. A model which considers propagation of the helices from either end as statistically independent yields Zimm-Bragg s parameters in the range of 0.5 to 1.5, depending on helical length. Analysis of the mechanism of helix propagation suggests that 3(10)-helix plays a role in helix formation but its population should be low in the helical state of these model peptides.

Estrogen increases renal oxytocin receptor gene expression.

Estrogens have been implicated in the sodium and fluid imbalances associated with the menstrual cycle and late pregnancy. An estrogen-dependent role for renal oxytocin receptors in fluid homeostasis is suggested by the present findings which demonstrate that estradiol benzoate treatment increases the expression of the oxytocin receptor messenger ribonucleic acid and 125I-OTA binding to oxytocin receptors in the renal cortex and medullary collecting ducts of ovariectomized female rats. Moreover, estradiol induced high levels of oxytocin receptor expression in outer stripe proximal tubules of ovariectomized female and adrenalectomized male rats. Proximal tubule induction was inhibited in a dose-dependent manner by the antiestrogen tamoxifen, but cortical expression of oxytocin receptors in macula densa cells was unaffected by tamoxifen. These data demonstrate cell-specific regulation of oxytocin receptor expression in macula densa and proximal tubule cells, and suggest a important role for these receptors in mediating estrogen-induced alterations in renal fluid dynamics by possibly affecting glomerular filtration and water and solute reabsorption during high estrogen states.

Topography of neurons expressing luteinizing hormone-releasing hormone gene transcripts in the human hypothalamus and basal forebrain.

The distribution of neurons expressing luteinizing hormone-releasing hormone (LHRH) gene transcripts was mapped in the human hypothalamus and basal forebrain by in situ hybridization and computer-assisted microscopy. Hypothalamic blocks were dissected from five adult males and one adult female and snap frozen in isopentane. The blocks were serially sectioned either in the coronal or in the sagittal plane at a thickness of 20 microns. Approximately every twentieth section was incubated with a 35S-labeled cDNA probe complementary to LHRH mRNA. Specificity was confirmed by hybridization of adjacent sections with a probe targeted to the gonadotropin-associated protein (GAP) region of LHRH messenger ribonucleic acids (mRNA). Maps of neurons containing LHRH mRNA were manually digitized with the aid of an image-combining computer microscope system. We report a much wider distribution and greater numbers of LHRH neurons than have been previously described in the human brain. Three morphological subtypes were observed based on cell size and labeling density: 1) small, heavily labeled, oval or fusiform neurons, located primarily in the medial basal hypothalamus, ventral preoptic area, and periventricular zone; 2) small, oval, sparsely labeled neurons located in the septum and dorsal preoptic region and scattered from the bed nucleus of the stria terminalis to the amygdala ("extended amygdala"); and 3) large round neurons (> 500 microns 2 sectional profile area), intermediate in labeling density, scattered within the magnocellular basal forebrain complex, extended amygdala, ventral pallidum, and putamen. The pronounced differences in morphology, labeling density, and location of the three subtypes suggest that distinct functional subgroups of LHRH neurons exist in the human brain.

The diurnal rhythm in vasopressin V1a receptor expression in the suprachiasmatic nucleus is not dependent on vasopressin.

The mammalian suprachiasmatic nucleus is the site of the circadian rhythm generator. The degrees of expression there of several neuropeptides, including vasopressin and vasoactive intestinal polypeptide, follow a diurnal rhythm. The vasopressin V1a receptor, whose activation results in phosphoinositol hydrolysis and mobilization of intracellular calcium, is expressed in this nucleus. This study used double simultaneous hybridization histochemistry to show that V1a receptor transcripts are present in both vasopressin and vasoactive intestinal polypeptide neurons and that their levels follow a diurnal rhythm. Furthermore, the expression of the V1a receptor is 12 h out of phase from that of vasopressin. However, the receptor's diurnal rhythm is still maintained in the vasopressin-deficient Brattleboro rat, indicating that the V1a receptor's rhythm is independent of any vasopressin feedback.

Regulation of gene expression in the hypothalamus: hybridization histochemical studies.

Hybridization histochemistry has bridged molecular biology and neuroanatomy to provide nearly dynamic views of gene expression in the brain--perhaps especially in the hypothalamus. These snapshots of transcript levels with precise anatomical localization have revealed new insights into gene regulation in the hypothalamus under specific conditions. Magnocellular neurons in the paraventricular and supraoptic nuclei produce vasopressin and oxytocin. Transcript levels for these hormones are affected by hyperosmolality, as are those for many other neuropeptides. Patterns of gene expression in the magnocellular neurons in these nuclei during development and under different physiological conditions have been studied less extensively. The parvocellular neurons of the paraventricular nucleus produce corticotropin-releasing factor and thyrotropin-releasing hormone. Expression of the corticotropin-releasing factor gene is regulated by glucocorticoids. Physiological stresses, which activate the hypothalamo-pituitary-adrenal axis, also affect gene expression in the parvocellular paraventricular nucleus. Thyrotropin-releasing hormone is synthesized in a different set of parvocellular neurons in the paraventricular nucleus and in other neurons of the hypothalamus. Expression of the thyrotropin-releasing hormone gene is regulated by thyroid hormone. The suprachiasmatic nucleus contains neurons that produce vasopressin or vasoactive intestinal polypeptide in a circadian rhythm. Future studies using combinations of classical neuroanatomical techniques, hybridization histochemistry and immunohistochemistry will further our understanding of hypothalamic responses to various stimuli.

Hypertrophy and increased gene expression of neurons containing neurokinin-B and substance-P messenger ribonucleic acids in the hypothalami of postmenopausal women.

We have previously described hypertrophy of neurons containing estrogen receptor mRNA in the infundibular nucleus of postmenopausal women. In the present investigation we identified peptide mRNAs in the hypertrophied neurons and determined whether postmenopausal neuronal hypertrophy was accompanied by changes in gene expression. In the first study in situ hybridization was performed on sections from hypothalami of postmenopausal women (n = 3) using synthetic 35S-labeled cDNA probes complementary to mRNAs encoding estrogen receptor, substance-P (SP), neurokinin-B (NKB), POMC, cholecystokinin, dynorphin, CRF, enkephalin, galanin, neuropeptide-Y, GH-releasing hormone, and tyrosine hydroxylase. Neuronal cross-sectional areas and cell densities were measured with the aid of a computer microscope system. Neurons labeled with the NKB and SP probes were comparable in size, morphology, and distribution to the hypertrophied neurons containing estrogen receptor mRNA. In contrast, neurons labeled with other cDNA probes were sparsely distributed (CRF and dynorphin), smaller in size (neuropeptide-Y, galanin, GH-releasing hormone, enkephalin, cholecystokinin, and POMC), or located anterior to the hypertrophied population (tyrosine hydroxylase). In the second study sections from hypothalami of premenopausal (n = 3) and postmenopausal (n = 3) women were incubated with cDNA probes complementary to SP or NKB mRNAs. The mean cross-sectional areas of postmenopausal infundibular neurons containing NKB and SP mRNAs increased to 194% and 176% of premenopausal values, respectively. The autoradiographic grain densities of infundibular neurons labeled with either probe were also significantly increased in the postmenopausal group. Finally, the numbers of labeled neurons/tissue increased 6-fold (SP) and 15-fold (NKB) in the postmenopausal infundibular nucleus. These data demonstrate that human menopause is associated with marked increases in hypothalamic NKB and SP gene expression. We propose that neurons containing estrogen receptor, SP, and NKB mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback in the human.

Galanin mRNA in the nucleus basalis of Meynert complex of baboons and humans.

Galanin, a 29-amino acid peptide, has been shown by immunocytochemistry to occur in most large acetylcholinergic neurons of the complex that includes the nucleus basalis of Meynert and the nucleus of the diagonal band of Broca in nonhuman primates. In contrast, several studies have reported that most large neurons of the human nucleus basalis of Meynert complex appear to lack galanin immunoreactivity. We investigated this apparent species-difference by hybridization histochemistry for galanin messenger ribonucleic acid (mRNA) in humans and baboons. The results confirm previous immunocytochemical data; very few large neurons of the nucleus basalis of Meynert complex in humans contained detectable galanin messenger RNA, whereas most such cells in baboons were labeled by the oligodeoxynucleotide probe. The few labeled neurons in humans were primarily medial or ventral to the main body of the nucleus basalis of Meynert and corresponded in location to a minor population of relatively intensely labeled cells in baboons. These findings indicate that the indetectability of immunoreactive galanin in most cells of the nucleus basalis of Meynert complex in humans is due to a paucity or an absence of galanin messenger RNA and not to differences in posttranslational processing or transport of the peptide. Inasmuch as the probe labeled neurons in several other nuclei of both species, it is unlikely that differences in galanin messenger RNA sequences underlie the species-related disparity in hybridization in the nucleus basalis of Meynert complex. The indetectability of galanin messenger RNA in most cells of the human nucleus basalis of Meynert complex indicates that the expression of the galanin gene is regulated by as yet unidentified influences that differ in human and nonhuman primates. The varying phenotypes of galanin in primates suggest potentially important species-differences in the function of galanin in neurons of the nucleus basalis of Meynert complex.

Postmenopausal hypertrophy of neurons expressing the estrogen receptor gene in the human hypothalamus.

Computer microscopy and in situ hybridization were used to investigate neuronal hypertrophy in the infundibular nucleus of postmenopausal women. In the first experiment, hypothalami from premenopausal (n = 3) and postmenopausal (n = 3) women were formalin fixed, paraffin embedded, serially sectioned, and stained with cresyl violet. Soma areas of more than 3500 neurons were digitized using an image-combining computer microscope. The mean cross-sectional area of infundibular neurons in the postmenopausal women was 30% greater than that in premenopausal women, with no change in cell density. The mean cross-sectional area of mammillary neurons was unchanged, indicating that the infundibular neuronal hypertrophy was not an artifact of tissue processing. In the second experiment, hypothalami from premenopausal (n = 3) and postmenopausal (n = 2) women were frozen, serially sectioned, and incubated with a 48-base synthetic cDNA probe complementary to estrogen receptor (ER) mRNA. Adjacent sections were incubated with a cDNA probe complementary to GnRH mRNA. Morphometric analysis revealed that the mean cross-sectional area of infundibular neurons expressing the ER gene in the postmenopausal women was twice as large as the mean area in premenopausal hypothalami. The hypertrophied neurons did not contain GnRH mRNA. Finally, analysis of the infundibular nucleus from an oophorectomized 38-yr-old woman also revealed hypertrophied neurons containing ER mRNA. These data support the hypothesis that hypertrophy of infundibular neurons in postmenopausal women is secondary to loss of the inhibitory feedback of ovarian steroids.

The influences of hyperosmolality and synaptic inputs on galanin and vasopressin expression in the hypothalamus.

Galanin is a neuropeptide that is widely distributed throughout the rat central nervous system. It is co-localized with vasopressin in magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. Vasopressin biosynthesis is increased there by various hyperosmolar stimuli, including drinking 2% saline. We previously demonstrated that the chronically hyperosmolar Brattleboro rat has increased biosynthesis of galanin in the paraventricular and supraoptic nuclei. In this report we show using hybridization histochemistry that drinking 2% saline also increased galanin transcripts in these nuclei. We also demonstrate using hybridization histochemistry and immunohistochemistry that knife cuts that sever hypothalamo-hypophysial fibers transiently elevated galanin expression in the supraoptic nucleus ipsilateral to the lesion and depressed vasopressin expression ipsilaterally. Pituitary stalk transections also elevated galanin and decreased vasopressin transcripts. In addition, various knife cuts in the caudal hypothalamus were able to dissociate the expression of vasopressin and galanin, although co-localized and similarly affected by hyperosmolality in the supraoptic nucleus. Unilateral sagittal knife cuts that divided the posterior hypothalamus but avoided the hypothalamo-hypophysial pathway, as well as hemisections at the level of the premammillary area, resulted in ipsilateral elevations of galanin transcripts without significantly affecting vasopressin expression. These results indicate that independent intracellular signal transduction pathways exist for regulating expression of the two genes.

The gene encoding GnRH and its associated peptide GAP: some insights into hypogonadism.

The hypogonadal (hpg) mouse represents a unique animal model for hypogonadism. In this mutant the truncation of the gene encoding GnRH and its associated peptide GAP leads to drastically lowered gonadotropin levels and increased circulating prolactin. This deficiency in turn leads to a failure of testes and ovaries to develop normally. Using gene therapy we have restored the reproductive functions of the hpg mouse. The success of this therapy uniquely underscores the importance of the gene encoding the GnRH precursor and lends credence to the hypothesis that no other gene in mammals can replace it. As a consequence, defects in the control and/or structural properties of the human GnRH are expected to result in hypogonadism in humans.

Neurons with neurokinin B mRNA in the rat magnocellular basal nucleus: distribution, projection and colocalization studies.

Mapping and cortical projections of neurons containing neurokinin B (NKB) mRNA in the rat basal forebrain were studied using hybridization histochemistry and retrograde labeling. Neurokinin B mRNA was found in a small proportion of the neurons present in the magnocellular basal nucleus that project to the cortex. Most of these neurons projected to the frontal and parietal cortices, whereas only few projected to the occipital cortex and almost none to allocortical areas, including the olfactory bulb. NKB mRNA colocalization with galanin or substance P mRNAs, both found within neurons of the magnocellular basal nucleus, was not detected there. These results demonstrate a novel diffuse neocortical tachykinin input from the basal forebrain, in addition to the well-known cholinergic one.

Changes in cellular levels of messenger ribonucleic acid encoding gonadotropin-releasing hormone in the anterior hypothalamus of female rats during the estrous cycle.

Cellular levels of messenger RNA encoding GnRH were measured using quantitative in situ hybridization in coronal sections through the area of the organum vasculosum of the lamina terminalis of female rats examined at various times of the 4-day estrous cycle. GnRH mRNA levels were high on the morning of diestrus day 1, but declined throughout the day of diestrus day 2 to a nadir on the morning of proestrus. Although GnRH message levels were lowest on the morning of proestrus, they rose nearly two-fold by 1900h that evening and remained high during the day of estrus. These data support the hypothesis that GnRH synthesis is coupled to GnRH release, and indicate that GnRH biosynthesis is not stimulated on the morning of proestrus in preparation for the ovulatory surge release of GnRH and LH in the afternoon.

Galanin coexists with vasopressin in the normal rat hypothalamus and galanin's synthesis is increased in the Brattleboro (diabetes insipidus) rat.

Galanin is a peptide containing 29 amino acid residues, that is present in the median eminence, in the magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus and in the posterior pituitary. We report here that: (1) immunoreactivity for galanin (GAL) and vasopressin coexist in the SON of normal rats, (2) levels of mRNA encoding preprogalanin are markedly elevated in the PVN and SON of Brattleboro (diabetes insipidus) rats, as determined by in situ hybridization histochemistry but (3) levels of GAL-like immunoreactivity (GAL-LI) are significantly reduced in the posterior pituitary of these rats, as determined by radioimmunoassay. We suggest that production and possibly secretion of the peptide GAL may be increased in the Brattleboro rat.

In situ hybridization histochemistry for messenger ribonucleic acid (mRNA) encoding gonadotropin-releasing hormone (GnRH): effect of estrogen on cellular levels of GnRH mRNA in female rat brain.

Using in situ hybridization histochemistry, we have detected perikarya containing mRNA encoding GnRH and GnRH-associated peptide (GAP) in rat brains. Synthetic DNA oligomers with sequences complementary to the rat cDNA encoding the GnRH structural region and the GAP structural region were hybridized to formaldehyde-fixed coronal sections. The distribution and number of cells containing GnRH/GAP mRNA were similar to those shown by immunocytochemical studies. The areas in which GnRH mRNA perikarya were shown included the medial septal area, the diagonal band of Broca, the preoptic area, and the anterior hypothalamus. Up to 55 cells were detected in a single 12-micron section containing the diagonal band and organum vasculosum lamina terminalis (OVLT) whereas cell numbers diminished in more caudal regions. In addition, both probes labeled the same cells contained within adjacent sections. We used this technique to examine the effect of estrogen on GnRH mRNA levels in the area of the OVLT of normal and androgen-sterilized female rats, using an estrogen treatment paradigm previously characterized in studies investigating the hypothalamic regulation of negative and positive estrogen feedback. We found that 7 days after ovariectomy, 2 days of estrogen treatment resulted in a significant reduction in the average cellular level of GnRH mRNA in both normal and androgen-sterilized females. Analysis of histograms relating the intensity of labeling to the abundance of cells suggested that a small population of GnRH cells responded to the estrogen treatment. However, we found no evidence for a discrete neuroanatomical segregation of such a subpopulation of GnRH-responsive cells within the area of the OVLT.

Plasma hyperosmolality increases G protein and 3',5'-cyclic adenosine monophosphate synthesis in the paraventricular and supraoptic nuclei.

Hyperosmotic stimuli produce profound changes in cellular morphology and biosynthetic activities within the hypothalamic paraventricular and supraoptic nuclei (SON) of the rat. The mechanisms by which osmoreceptive signals are transduced within these nuclei are poorly understood. We examined several components of the cAMP-associated second messenger system after giving rats 2% saline to drink for one week, a strong hyperosmotic stimulus. We found that mRNA levels for both the stimulatory and inhibitory guanine-nucleotide binding protein alpha-subunits were increased in the paraventricular nucleus and SON. In the SON, these changes were accompanied by increased basal cAMP levels, cholera toxin-stimulated adenylate cyclase, and Gs alpha. Our results suggest that Gs alpha levels are not saturated with respect to adenylate cyclase coupling and that osmoreception activates the cAMP second messenger system.

Corticotropin-releasing factor as a transmitter in the human olivocerebellar pathway.

This study demonstrates that the neuropeptide, corticotropin-releasing factor (CRF), is present in neurons of the human inferior olivary complex (IOC). The medulla (including the inferior olive) and the anterior vermis of the cerebellum of 6 human controls obtained at autopsy were immunostained with an antibody directed against CRF. CRF receptors in cerebellum were localized with labeled CRF using in vitro receptor autoradiography. The great majority of neurons in all divisions of the IOC expressed CRF immunoreactivity, and CRF-immunoreactive fibers were demonstrated in the hilus of the olive and in the molecular layer of the cerebellum, where they closely resembled climbing fibers as visualized with other methods. CRF receptors were enriched in the cerebellum, with the highest density in inner portions of the molecular layer. These findings in human brain, consistent with studies in tissues from rat, cat, and monkey, demonstrate that CRF may be a peptidergic transmitter in the IOC climbing fiber system and that CRF receptors are expressed by cellular targets in the cerebellum.