It is generally assumed that the inhibitory neurotransmitter GABA and the stimulatory neurotransmitter glutamate are released from different neurons in adults. However, this tenet has made it difficult to explain how the same afferent signals can cause opposite changes in GABA and glutamate release. Such reciprocal release is a central mechanism in the neural control of many physiological processes including activation of gonadotropin-releasing hormone (GnRH) neurons, the neural signal for ovulation. Activation of GnRH neurons requires simultaneous suppression of GABA and stimulation of glutamate release, each of which occurs in response to a daily photoperiodic signal, but only in the presence of estradiol (E 2 ). In rodents, E 2 and photoperiodic signals converge in the anteroventral periventricular nucleus (AVPV), but it is unclear how these signals differentially regulate GABA and glutamate secretion. We now report that nearly all neurons in the AVPV of female rats express both vesicular glutamate transporter 2 (VGLUT2), a marker of hypothalamic glutamatergic neurons, as well as glutamic acid decarboxylase and vesicular GABA transporter (VGAT), markers of GABAergic neurons. These dual-phenotype neurons are the main targets of E 2 in the region and are more than twice as numerous in females as in males. Moreover, dual-phenotype synaptic terminals contact GnRH neurons, and at the time of the surge, VGAT-containing vesicles decrease and VGLUT2-containing vesicles increase in these terminals. Thus, we propose a new model for ovulation that includes dual-phenotype GABA/glutamate neurons as central transducers of hormonal and neural signals to GnRH neurons.

Nitric oxide (NO) generated from neuronal nitric oxide synthase (NOS-1) in intrinsic cardiac ganglia has been implicated in parasympathetic-induced bradycardia. We provide direct evidence that NOS-1 acts in a site-specific manner to promote cardiac vagal neurotransmission and bradycardia. NOS-1 gene transfer to the guinea pig right atrium increased protein expression and NOS-1 immunolocalization in cholinergic ganglia. It also increased the release of acetylcholine and enhanced the heart rate (HR) response to vagal nerve stimulation (VNS) in vitro and in vivo. NOS inhibition normalized the HR response to VNS in the NOS-1-treated group compared with the control groups (enhanced green fluorescent protein and sham) in vitro. In contrast, an acetylcholine analogue reduced HR to the same extent in all groups before and during NOS inhibition. These results demonstrate that NOS-1-derived NO acts presynaptically to facilitate vagally induced bradycardia and that upregulation of NOS-1 via gene transfer may provide a novel method for increasing cardiac vagal function.T he biological messenger nitric oxide (NO) is thought to be a fundamental signaling molecule in the regulation of cardiac cholinergic function. [1][2][3][4] Neuronal nitric oxide synthase (NOS-1), the enzyme responsible for NO synthesis, colocalizes with choline acetyltransferase in the intracardiac ganglia. 5 Functionally, pharmacological evidence suggests that NO generated from NOS-1 directly enhances the negative chronotropic effect of cholinergic stimulation 6,7 by activating the guanylate cyclase/ cGMP pathway 7,8 to facilitate the release of acetylcholine (ACh), 9 and indirectly via endothelial NOS-3 M 2 receptor coupled inhibition of I Ca-L in pacemaking cells, 10 although this latter point is disputed. 11 Moreover, the vagal heart rate (HR) response to modulators of the NO-cGMP pathway is not mimicked by the stable analogue of ACh, carbachol, suggesting that the dominant functional role of this pathway is presynaptic to the neuroeffector junction. 7,8 We tested the hypothesis that NOS-1 gene transfer into the right atrium would enhance vagal-induced neurotransmission and bradycardia but would be ineffective when HR was decreased by carbachol. Materials and MethodsDetailed methods for gene transfer, 12 immunohistochemistry, 13 fluorescence microscopy, 14 confocal imaging, 5 immunoblotting, 7 measurements of ACh release, 9 and in vivo or in vitro autonomic phenotyping 7,8 can be found in the online data supplement, available at http://www.circresaha.org. Results and DiscussionQualitative examination using NADPH-diaphorase staining of tissue cryosections showed greater expression in atrial tissue after replication-deficient adenoviral vector transfection with neuronal NOS (Ad.NOS-1) ( Figure 1A). This was confirmed by Western blotting in which Ad.NOS-1-treated atria (nϭ8) showed significantly greater expression of NOS-1 protein compared with atria infected with an adenoviral vector encoding recombinant enhanced green fluorescent protein (Ad.eGFP) (nϭ8, ...

Sexually dimorphic brain nuclei underlie gender-specific neural functions and susceptibility to disease, but the developmental basis of dimorphisms is poorly understood. In these studies, we focused on the anteroventral periventricular nucleus (AVPV), a nucleus that is larger in females and critical for the female-typical cyclic surge pattern of luteinizing hormone (LH) release. Sex differences in the size and function of the AVPV result from apoptosis that occurs preferentially in the developing male. To identify upstream pathways responsible for sexual differentiation of the AVPV, we used targeted apoptosis microarrays and in vivo and in vitro follow-up studies. We found that the tumor necrosis factor ␣ (TNF␣)-TNF receptor 2 (TNFR2)؊NFB cell survival pathway is active in postnatal day 2 (PND2) female AVPV and repressed in male counterparts. Genes encoding key members of this pathway were expressed exclusively in GABAergic neurons. One gene in particular, TNF receptor-associated factor 2 (TRAF2)-inhibiting protein (trip), was higher in males and it inhibited both TNF␣-dependent NFB activation and bcl-2 gene expression. The male AVPV also had higher levels of bax and bad mRNA, but neither of these genes was regulated by either TNF␣ or TRIP. Finally, the trip gene was not expressed in the sexually dimorphic nucleus of the preoptic area (SDN-POA), a nucleus in which apoptosis is higher in females than males. These findings form the basis of a new model of sexual differentiation of the AVPV that may also apply to the development of other sexually dimorphic nuclei.NFkB ͉ TNFR2 ͉ TNF␣ ͉ sexual dimorphism ͉ apoptosis S ex differences in the morphology of brain nuclei were first observed over 30 years ago (1), but the physiological significance of these differences is known in only a few cases. The neural control of gonadotropin release in rodents is perhaps the best studied example of sex-specific physiology linked to neural dimorphisms. Females have a cyclic pattern that culminates in the preovulatory surge of luteinizing hormone (LH) release on one day of the cycle (2). This female-typical pattern of LH release is controlled by the anteroventral periventricular nucleus (AVPV) (3-6), one of the few sexually dimorphic nuclei that is larger in females. Clearly, sexual dimorphisms of the nucleus are linked to function, because developmental manipulations that alter the size of the AVPV region (7) result in inappropriate gonadotropin release patterns and infertility in adulthood (8).Sexual differentiation of the AVPV, like that of other dimorphic nuclei, is thought to be through apoptosis (9). Consistent with this idea, cells in the developing male AVPV express higher levels of the proapoptotic gene, bax, while expression of the prosurvival gene, bcl2, is more abundant in the developing female AVPV (10). The Bcl2/Bax ratio determines whether or not apoptosis will be triggered in the AVPV, because genetic manipulations of either Bcl2 or Bax expression alter the volume of the nucleus (11, 12). Unfortunately, we still have ...

Historically, much of the research on health effects of environmental pollutants focused on ascertaining whether compounds were carcinogenic. More recent findings show that environmental contaminants also exert insidious effects by disrupting hormone action. Of particular concern are findings that developmental exposure to dioxins, chemicals that act through the aryl hydrocarbon receptor pathway, permanently alters sexually differentiated neural functions in animal models. In this review, we focus on mechanisms through which dioxins disrupt neuroendocrine development as exemplified by effects on a brain region critical for ovulation in rodents. We also provide evidence that dysregulation of GABAergic neural development may be a general mechanism underlying a broad spectrum of effects seen after perinatal dioxin exposure.