Search

Genes induced by estrogens in the mammalian forebrain influence a variety of neural functions. Among them, reproductive behavior mechanisms are very well understood. Their functional genomics provide a theoretical paradigm for linking genes to neural circuits to behavior. We propose that estrogen-induced genes are organized in modules: Growth of hypothalamic neurons; Amplification of the estrogen effect by progesterone; Preparative behaviors; Permissive actions on sex behavior circuitry; and Synchronization of mating behavior with ovulation. These modules may represent mechanistic routes for CNS management of successful reproduction. Moreover, new microarray results add estrogen-dependent genes, including some expressed in glia, suggesting possible hormone-dependent neuronal/glial coordination.

In female rodents, sleep and activity levels fluctuate over the estrous cycle. When estradiol (E2) levels are highest, sleep is reduced whereas locomotion is increased. The preoptic area (POA) is a key site for estrogenic regulation of these functions. However, molecular mechanisms by which E2 acts to reduce sleep and increase activity are unclear. Recently, we demonstrated a twofold reduction in lipocalin-type prostaglandin D synthase (L-PGDS) transcript levels, after E2 treatment, in the ventrolateral POA (VLPO), a putative sleep-active nucleus. Catalytic activity of L-PGDS produces PGD2, an endogenous somnogen. Thus, we hypothesized that decreases in PGD2 in the VLPO may contribute to the generalized arousal mediated by estrogens. To test this, we infused (i) antisense oligonucleotides (oligos), containing locked nucleic acid moieties (an improved technology), targeted to L-PGDS mRNA, (ii) scrambled sequence control oligos, or (iii) saline vehicle into the VLPO of ovariectomized female mice treated with E2 or oil. Arousal states and activity levels were assessed in response to a series of sensory stimuli (vestibular, olfactory, and somatosensory). The vestibular stimulus, which was administered first, resulted in the strongest responses and elicited significantly different responses among the groups: all groups in the E2 cohort demonstrated increases in overall home cage activity and duration of that activity compared with the oil-treated control groups. As predicted from E2 suppression of L-PGDS transcript levels, the responses of the locked nucleic acid antisense oligo-treated animals from the oil cohort did not differ from the E2-treated groups, such that they also demonstrated increases in activity and duration of activity compared with their controls. Thus, reducing L-PGDS in the VLPO of oil-treated females mimicked the effect of E2 on activity and arousal and represents a unique molecular pathway through which E2 may modulate these functions.

Estrogen treatment induces transcription and increases excitability and reproductive behavior. Estrogens provide the structural basis for increased synaptic activity and greater behavior-facilitating output. Administration of progesterone amplifies the effect of estrogens on mating behavior. The role of GnRH is to synchronize reproductive behavior with the ovulatory surge of LH. A causal connection can be charted from one individual gene to human social behavior, but only via six causal links. Glia, meninges and neurons may participate, under the influence of sex hormones, in the direction of sex behavior. Neural and genetic mechanisms for motivation may lead to biological understanding of functions that apply to the most primitive aspects of human mental functioning. With respect to aggression, besides testosterone and its metabolites, serotonergic projections to the forebrain play an important role.

Microarrays comprise an efficient approach to discovering large numbers of differentially expressed mRNA transcripts in the CNS resulting from changes in hormonal milieu. We used high-density oligonucleotide microarrays to examine the short- and long-term actions of estradiol (E(2)) on the transcriptomes from the medial basal hypothalamus and other brain regions of E(2)-treated (10 microg) adult female mice. Our results have revealed several unanticipated gene regulations. Most striking is lipocalin prostaglandin D(2) synthase (L-PGDS), which catalyzes the conversion of prostaglandin (PG) H(2) to PGD(2), a neuromodulator involved in a variety of functions, including sleep, pain, and odor responses. In situ hybridization revealed significant increases in L-PGDS expression in the arcuate and ventromedial nucleus of the medial basal hypothalamus compared with vehicle controls. The magnitude of these changes is approximately 2-fold and suggests a modulatory role for PGD(2) in E(2)-controlled neuroendocrine secretions and behaviors. Surprisingly, L-PGDS gene expression is reduced 2-fold after E(2) treatment in the ventrolateral preoptic area (VLPO), the suspected site of action for the sleep-promoting effects of PGD(2). Finally, whereas L-PGDS has been reported to be expressed primarily in oligodendrocytes of the adult rodent brain, we demonstrate, immunocytochemically, that L-PGDS is also expressed in a population of VLPO neurons. Thus, our data suggest the intriguing possibility that E(2) modulation of L-PGDS plays a role in the regulation of sleep-wake states through hitherto unknown mechanisms in VLPO neurons and through hormone-dependent neuronal-glial cooperation.

The arcuate nucleus of the hypothalamus is one of several sexually dimorphic hypothalamic nuclei. We have previously demonstrated that astrocytes in the neonatal arcuate nucleus exhibit a marked sexually dimorphic morphology as a result of differential exposure to gonadal steroids by postnatal day (PN) 2, with males having complex stellate cells compared to the simple bipolar ones found in females. Here, we present data demonstrating that arcuate astrocytes are sexually dimorphic by the day of birth and continue as such throughout postnatal development (PN0-PN15), and persist into adulthood. These data suggest that early gonadal steroid exposure permanently organizes arcuate astrocyte morphology. The male versus female difference in astrocyte morphology may contribute to the sexually dimorphic regulation of neuroendrocrine secretions from the pituitary in the adult.

There is a growing appreciation for the importance of astrocytes, a type of nonneuronal glial cell, to overall brain functioning. The ability of astrocytes to respond to gonadal steroid hormones with changes in morphology has been well documented in the adult brain. It is also apparent that astrocytes of the developing brain are permanently differentiated by the neonatal hormonal milieu, in particular by estradiol, resulting in sexually dimorphic cell morphology, synaptic patterning, and density in males and females. The mechanisms of hormonally mediated astrocyte differentiation are likely to be region specific. In the arcuate nucleus of the hypothalamus, neuron-to-astrocyte signaling appears to play a critical role in estradiol-induced astrocyte differentiation during the first few days of life. Gamma aminobutyric acid (GABA) is an amino acid neurotransmitter that is synthesized and released exclusively by neurons. The levels of GABA are increased in the arcuate nucleus of neonatal males versus females. Preventing the increase in males or mimicking GABA action in females modulates astrocytes accordingly. Speculation about and evidence in support of the functional significance of this dimorphism to adult reproductive functioning is the topic of this review.

A central goal of neuroendocrinology is the understanding of how hormones modulate a variety of neurobiological functions including releasing factors for anterior pituitary secretions and behavior. We know that mechanisms of hormone actions clearly include the activation and repression of genes either directly through nuclear hormone receptors or indirectly, through a series of transduced signals originating from membrane receptors. Until recently, identification of the differentially expressed genes has been a "gene-at-a-time" proposition. With the advent of the completion of sequencing of several genomes including those of the human and mouse, new methods for the simultaneous assessment of many genes' expression are proving especially timely. Two such methods, differential display PCR and gene microarrays, are based on the well-established principles of DNA amplification and nucleic acid hybridization, respectively. With properly designed and well-executed experiments, these methods are powerful tools in the assessment of differentially expressed genes yielding results both expected and unanticipated.

Our previous work has demonstrated that astrocytes in the developing arcuate nucleus of the rat hypothalamus are sexually dimorphic as a result of differential exposure to oestradiol. Moreover, our experiments in neonatal rats suggest an absence of oestrogen receptors (ER) in arcuate nucleus astrocytes in vivo. This, along with the conspicuous lack of evidence in the literature confirming the presence of ER in arcuate nucleus astrocytes of the neonatal rat brain, led us to question the mechanism by which oestrogen induces changes in arcuate nucleus astrocyte morphology. Based on our previous findings that oestradiol increases gamma-aminobutyric acid (GABA) levels in the neonatal rat arcuate, we hypothesize that GABA is released from neighbouring oestrogen-sensitive neurones and alters arcuate nucleus astrocyte morphology. Here, we report that in vivo reduction of GABA synthesis in the neonatal rat brain by antisense oligodeoxynucleotides to glutamic acid decarboxylase prevented gonadal steroid-induced astrocyte differentiation in males and testosterone-treated females. Conversely, activation of GABAA receptors with the agonist muscimol increased astrocyte differentiation in females in the absence of gonadal steroids. Given that GABA is made only in neurones and that its synthesis is increased by oestradiol, we conclude that it acts as a diffusible factor inducing the differentiation of neighbouring astrocytes.

The developing brain is exquisitely sensitive to gonadal steroid hormones, which permanently differentiate the neural substrate during a critical developmental period. One of the more striking sexual dimorphisms in the adult rat brain is synaptic patterning in the arcuate nucleus (ARC); females have twice the number of axospinous synapses as males (Matsumoto and Arai [1980] Brain Res. 190:238-242). Previously, we have demonstrated that a similar dimorphism in spine densities on ARC dendrites is present as early as early as postnatal day 2 (PN2) in Golgi-impregnated rat brains (Mong et al. [1999] J. Neurosci. 19:1464-1472). Males have 37% fewer dendritic spines than females. Moreover, these spine densities are sensitive to changes in the hormonal milieu such that males castrated on the day of birth have a significant increase in spine density, whereas females masculinized at birth by gonadal steroid exposure have a decreased dendritic spine density. One of the limitations of the Golgi technique is the inability to confirm the presence of synapses. The current study used quantitative electron microscopy and demonstrated that testosterone exposure dramatically reduced axospinous synapses in the ARC by PN 2. Males had 54% fewer and masculinized females had 77% fewer axospinous synapses than females (P < 0.05 and P < 0.01, respectively). We previously reported that gonadal steroids induce coincident changes in neuronal and astrocyte morphology in the neonatal ARC (Mong et al., 1999), and here confirm that these changes include an altered synaptic pattern that is strikingly similar to that observed in the adult (Matsumoto and Arai, 1980)., (Copyright 2001 Wiley-Liss, Inc.)

Antisense oligodeoxynucleotides have been highly successful agents at modulating gene expression in the adult brain and widely exploited in the field of neuroendocrinology. We have also used this technique in the developing brain to explore the role of select proteins during sensitive periods of development, particularly those influenced by steroid hormones. Presented here are the technical details of using antisense oligodeoxynucleotides in the neonatal brain, as well as a review of some of our successes and failures. Our goal is to illustrate the relative ease of use of this technique in neonates and demonstrate the power such an approach offers so that other investigators will also begin to take advantage of this useful tool., (Copyright 2000 Academic Press.)

We have previously demonstrated that astrocytes in the developing arcuate nucleus of the rat hypothalamus exhibit a sexually dimorphic morphology as a result of differential exposure to gonadal steroids. Testosterone via its aromatized byproduct, estrogen, induces arcuate astrocytes to undergo differentiation during the first few days of life. These differentiated astrocytes exhibit a stellate morphology. Coincident with the steroid-induced increase in astrocyte differentiation is a reduction of dendritic spines on arcuate neurons. As a result, the arcuate nucleus of males has fewer axodendritic spine synapses than females and this dimorphism is retained throughout life. In the immediately adjacent ventromedial nucleus, neonatal astrocytes are immature and unresponsive to steroids. Neurons in this region show no change in dendritic spines in the first few days of life but do exhibit increased dendritic branching as a result of testosterone exposure. These findings illustrate the importance of distinct populations of astrocytes in restricted brain regions and their potential importance to the establishment of regionally specific synaptic patterning. Conflicting reports leave the site of steroid-mediated astrocyte responsiveness in the arcuate nucleus unresolved: Are gonadal steroids acting directly on astrocytes or are steroid-concentrating neurons mediating astrocytic responsiveness? In this review, we discuss the current understanding of astrocyte-neuron interactions and the possible mechanisms for steroid-mediated, astrocyte-directed synaptic patterning in the developing hypothalamus., (Copyright 1999 John Wiley & Sons, Inc.)

One of the more striking sexual dimorphisms in the adult brain is the synaptic patterning in some hypothalamic nuclei. In the arcuate nucleus (ARC) males have twice the number of axosomatic and one-half the number of axodendritic spine synapses as females. The opposite pattern is observed in the immediately adjacent ventromedial nucleus (VMN). In both cases, early exposure to testosterone dictates adult dimorphism, but the exact timing, mechanism, and site of steroid action remain unknown. Astrocytes also exhibit sexual dimorphisms, and their role in mediating neuronal morphology is becoming increasingly evident. Using Golgi-Cox impregnation to examine neuronal morphology and glial fibrillary acidic protein immunoreactivity (GFAP-IR) to characterize astrocytic morphology, we compared structural differences in dendrites and astrocytes from the ARC and VMN in postnatal day 2 rat pups from four hormonally different groups. Consistent with previous observations, testosterone exposure induced a rapid and dramatic stellation response in ARC astrocytes. Coincident with this change in astrocytic morphology was a 37% reduction in the density of dendritic spines on ARC neurons. In contrast, astrocytes in the VMN were poorly differentiated and did not respond to testosterone exposure, nor were there any changes in neuronal dendrite spine density. However, VMN neurons exposed to testosterone had almost double the number of branches compared with that in controls. These data suggest that the degree of maturation and the differentiation of hypothalamic astrocytes in vivo are correlated with the ability of neurons to sprout branches or spines in response to steroid hormones and may underlie regionally specific differences in synaptic patterning.

During normal development there is a perinatal sensitive period during which the male brain is exposed to high levels of gonadal steroids, resulting in permanent differentiation of neural substrates. The cellular mechanisms mediating hormonally induced sexual differentiation remain largely unknown. In the adult brain, steroids exert profound influences on the amino acid transmitters, GABA, and glutamate. We have found steroid regulation of amino acid neurotransmission during the perinatal sensitive period and propose this may be functionally related to sexual differentiation of the brain. Specifically, the mRNA coding for the rate-limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD), is up to twice as high in some steroid-concentrating regions of the neonatal male brain compared to females, including the arcuate nucleus, dorsomedial nucleus, and the CA1 region of hippocampus. Sex differences in GABA tissue concentrations positively correlate with GAD mRNA differences in several brain regions. There are also sex differences in protein levels of GABA(A) receptor subunits. In parallel with these findings are significantly higher levels of binding to the non-NMDA glutamate receptor in steroid-concentrating regions of male brain. Given that GABA is an inhibitory transmitter and glutamate is an excitatory amino acid, these results initially appear paradoxical. However, in contrast to its inhibitory action in the adult brain, early in development GABA is actually excitatory and acts in a manner analogous to glutamate. Therefore, the combination of increased excitatory GABAergic and glutamatergic activity should result in substantially higher levels of neuronal excitation in the male brain. We speculate that an increased level of neuronal excitation is a potential mechanism mediating the permanent masculinization of the brain.

It is well established that gonadal steroids mediate sexual differentiation of the brain via direct effects on neurons during a restricted critical period. In addition, estrogen can influence glial morphology in the adult brain, and in vitro studies suggest estrogen induces glial differentiation. However, there is a lack of in vivo evidence for steroid effects on glia during the critical period. We report here a hormone-mediated sexual differentiation of arcuate glia as early as Postnatal Day 1. Using glial fibrillary acidic protein immunoreactivity (GFAP-ir), we compared the responsiveness of astroglia in the rat arcuate nucleus among five hormonally different groups. The results indicate increased GFAP-ir cell surface area 24 hr after hormonal manipulation in castrate males compared to intact males, intact females (ANOVA; P < 0.01), and females injected with testosterone propionate (50 microg; ANOVA; P < 0.05). However, astroglia in intact males extended their processes significantly greater distances from the cell body compared to all other treatment groups (ANOVA; P < 0.01). The GFAP-ir cells were categorized into four distinct classes ranging from a simple bipolar to a fully stellate morphology. The frequency distribution of classes varied between groups with more stellate cells found in intact males. Finally, these sex differences in arcuate glia persisted into adulthood. We hypothesize that during the critical period, testosterone, or its metabolite estrogen, induce sexual differentiation of glia. We further hypothesize that in females glial cells remain partially undifferentiated and this may be important to glial plasticity seen in adult female arcuate.

Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

Nitric oxide appears to mediate the immune functions of macrophages, the influence of endothelial cells on blood vessel relaxation, and also to serve as a neurotransmitter in the central and peripheral nervous system. Macrophage nitric oxide synthase is inducible with massive increases in new nitric oxide synthase protein synthesis following immune stimulation of macrophages. By contrast, endothelial nitric oxide synthase and neuronal nitric oxide synthase are thought to be constitutive with activation induced by calcium entry into cells in the absence of new protein synthesis. Developmental studies showing the transient expression of neuronal nitric oxide synthase in embryonic and early postnatal life in rodent spinal motoneurons and cerebral cortical plate neurons (Bredt and Snyder, unpublished observations) implies inducibility of neuronal nitric oxide synthase. Moreover, neuronal nitric oxide synthase expression is greatly enhanced in sensory ganglia following peripheral axotomy. Staining for NADPH diaphorase in spinal motoneurons is greatly increased following ventral root avulsion. In many parts of the Central Nervous System NADPH diaphorase staining reflects nitric oxide synthase. In the present study, we have combined in situ hybridization for neuronal nitric oxide synthase, immunohistochemical staining of neuronal nitric oxide synthase, and NADPH diaphorase staining to establish that neuronal nitric oxide synthase expression is markedly augmented in spinal motoneurons following avulsion. The generality of this effect is evident from augmented staining in nucleus dorsalis following spinal cord transection.