Anne M. Etgen

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.

Estradiol rescues neurons from global ischemia-induced cell death: Multiple cellular pathways of neuroprotection

The potential neuroprotective role of sex hormones in chronic neurodegenerative disorders and acute brain ischemia following cardiac arrest and stroke is of a great therapeutic interest. Long-term pretreatment with estradiol and other estrogens affords robust neuroprotection in male and female rodents subjected to focal and global ischemia. However, the receptors (e.g., cell surface or nuclear), intracellular signaling pathways and networks of estrogen-regulated genes that intervene in neuronal apoptosis are as yet unclear. We have shown that estradiol administered at physiological levels for two weeks before ischemia rescues neurons destined to die in the hippocampal CA1 and ameliorates ischemia-induced cognitive deficits in ovariectomized female rats. This regimen of estradiol treatment involves classical intracellular estrogen receptors, transactivation of IGF-1 receptors and stimulation of the ERK/MAPK signaling pathway, which in turn maintains CREB activity in the ischemic CA1. We also find that a single, acute injection of estradiol administrated into the brain ventricle immediately after an ischemic event reduces both neuronal death and cognitive deficits. Because these findings suggest that hormones could be used to treat patients when given after brain ischemia, it is critical to determine whether the same or different pathways mediate this form of neuroprotection. We find that an agonist of the membrane estrogen receptor GPR30 mimics short latency estradiol facilitation of synaptic transmission in the hippocampus. Therefore, we are testing the hypothesis that GPR30 may act together with intracellular estrogen receptors to activate cell signaling pathways to promote neuron survival after global ischemia.

The effects of Δ9-tetrahydrocannabinol on potassium-evoked release of dopamine in the rat caudate nucleus: an in vivo electrochemical and in vivo microdialysis study

The effect of systemically administered delta 9-tetrahydrocannabinol (THC), the psychoactive ingredient in marijuana, on the potassium-evoked release of dopamine (DA) was examined in the neostriatum of the chloral hydrate anesthetized rat. Both in vivo electrochemical and in vivo microdialysis techniques were employed. A low dose of THC (0.5 mg/kg, i.p.) increased the time course of potassium-evoked in vivo electrochemical signals corresponding to released extracellular DA. In vivo microdialysis showed an increase in potassium-evoked DA release following 0.5 and 2.0 mg/kg doses of THC. Potassium-evoked electrochemical signals corresponding to released extracellular DA were augmented in time course following i.p. administration (5.0 mg/kg) of nomifensine, a recognized and potent catecholaminergic reuptake blocker. In addition, in vivo brain microdialysis studies of nomifensine (5.0 mg/kg i.p.) on neostriatal potassium-evoked DA release showed that DA levels were augmented in magnitude over the time course of the microdialysis. Taken together, these studies indicate that THC has a potent presynaptic augmenting effect on at least the neostriatal portions of the mesotelencephalic DA system in the rat, although the possibility that this effect could be mediated transsynaptically cannot be ruled out. Given the previous extensive evidence for an involvement of portions of the mesotelencephalic DA system in mediating the reinforcing and euphorigenic properties of many classes of abused drugs, and in mediating direct electrical brain stimulation reward, we suggest that the presently demonstrated effects of THC on forebrain dopamine function may be related to marijuanas euphorigenic properties and, thus, to its abuse potential.

Effects of prenatal exposure to morphine on the development of sexual behavior in rats

Females exposed to morphine sulfate in utero (5-10 mg/kg twice a day on days 11-18 of gestation) displayed precocious vaginal opening and had increased body weight from the 8th week after weaning. In addition, there was a substantial inhibition in adult feminine sexual behavior. Male rats that received either morphine or saline prenatally did not show any body weight differences, and most of the measures of masculine sexual behavior did not differ between the two groups. However, the male rats exposed to morphine had a significantly shorter post-ejaculatory intromission latency than the saline controls. Examination of cytosol estrogen receptor levels in the hypothalamus-preoptic area (HPOA) of both saline and morphine sulfate-treated female rats revealed essentially identical patterns of depletion and replenishment. Additionally, estrogen treatment was equally effective at inducing HPOA progestin receptor synthesis in both groups. These results show that prenatal morphine treatment at the times and dose level administered disrupts the development of reproductive function in females but has only minor effects on male reproductive function.

Acute Administration of Non-Classical Estrogen Receptor Agonists Attenuates Ischemia-Induced Hippocampal Neuron Loss in Middle-Aged Female Rats

Background Pretreatment with 17β-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown. Methodology/Principal Findings We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 µg), G1 (50 µg) or STX (50 µg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 µg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55–60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices. Conclusions/Significance These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.

Estradiol and progesterone modulation of norepinephrine neurotransmission : implications for the regulation of female reproductive behavior

A long range objective of research in behavioral neuroendocrinology is to elucidate the mechanisms by which hormones modify behavior. Significant progress in understanding hormone-brainbehavior relationships is most likely to be achieved if one studies a behavior: 1) which can be measured with a high degree of validity and reliability, 2) whose occurrence can be manipulated in a predictable fashion by hormone administration, and 3) whose neural circuitry, including the necessary and sufficient sensory inputs and motor outputs and the sites of hormone action, has been established. One of the few mammalian behaviors which fits these criteria is the lordosis response of female rodents (for detailed descriptions of the lordosis posture and underlying neural circuitry, see 1). In rodents, the expression of this component of female reproductive (sexual, estrous, mating) behavior is strictly dependent on sequential exposure of neurons in specific hypothalamic sites to the ovarian steroids, estradiol (E,) and progesterone (P) (1-3). It should be emphasized that hormones do not ‘elicit’ or ‘activate’ lordosis; rather, the hormonal milieu determines the probability that animals display the behavior in response to appropriate sensory stimulation (in this case, flank and perineal stimulation). The lordosis response is also of great physiological importance; unless female rodents assume the lordosis posture when mounted by males, penile insertion and hence fertilization cannot take place. In addition, the hormonal control of reproduction in female vertebrates is an elegant example of neuroendocrine integration. Ovarian E2 and P act in extensively interconnected neuronal populations (4) to ensure that the release of pituitary gonadotropins which trigger ovulation (especially luteinizing hormone; LH) coincides with the expression of behavioral receptivity (5) . This neuroendocrine coordination of physiology and behavior maximizes the probability that a female will contact and be inseminated by a conspecific male at the optimal time for achieving fertilization. If one accepts the view that behavior is the product of neuronal activity, then it is reasonable to examine the influence of hormones on brain cells in the neural circuits that mediate hormoneregulated behaviors. Indeed, there is compelling evidence that E2 facilitation of lordosis behavior in rats requires increased excitability of neurons in the ventromedial hypothalamus (VMH) which project to the midbrain central gray, a critical site of sensory and motor integration of lordosis (1, 4, 6, 7). Similarly, it is rational to propose that hormone-dependent changes in chemical neurotransmission in specific neural circuits are likely to produce behavioral changes. In keeping with this perspective, numerous reports of alterations in neurotransmitter metabolism, release, and receptor binding as a function of estrous cycle stage and/or experimental manipulation of circulating E, and P have appeared (5, 8). Likewise, pharmacological manipulation of a variety of neurotransmitter systems can either facilitate or inhibit hormonedependent lordosis (810). Nevertheless, we still have only a rudimentary knowledge of which molecular components of specific neurotransmitter systems are regulated by E, and/or P in brain regions that control reproductive behavior, and of how the regulated molecules alter neuronal function such that stimuli which have a low probability of eliciting lordosis responses in hormone-deprived rodents have a high probability of doing so in hormone-exposed animals. Moreover, the development over the past decade of sensitive new methods for monitoring transmitter release in vivo and for mapping the qualitative and quantitative distribution of putative neurotransmitters, neuromodulators and their receptors in discrete neuronal populations, has produced an explosive increase in the number of potential molecular targets for hormonal regulation. In an attempt to begin developing a coherent picture of the neural mechanisms that mediate ovarian steroid regulation of lordosis, we have elected to focus on the monoamine neurotransmitter norepinephrine (NE). As summarized below, consideration of a variety of neuroendocrine, behavioral, neuroanatomical and neurophysiological observations which had accumulated by the mid 1980s led us to this choice.

Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology.

In many mammalian species, the ovarian steroid hormones estradiol (E(2)) and progesterone (P) act in the hypothalamus and preoptic area to coordinate the timing of female sexual receptivity with ovulation. We study lordosis behavior, an important component of sexual receptivity in rats, and its regulation by E(2) and P as a model system for understanding how hormonal modulation of synaptic neurotransmission influences reproductive physiology and behavior. Our findings suggest that E(2) and P extensively regulate synaptic communication involving the catecholamine norepinephrine (NE) in the hypothalamus. Estrogen priming shifts the balance of postsynaptic NE receptor signaling in the hypothalamus and preoptic area away from beta-adrenergic activation of cAMP synthesis toward alpha(1)-adrenergic signaling pathways. Attenuation of beta-adrenergic signal transduction is achieved by receptor-G-protein uncoupling, apparently due to stable receptor phosphorylation. E(2) modification of alpha(1)-adrenergic signaling includes both increased expression of the alpha(1B)-adrenoceptor subtype and a dramatic, P-induced reconfiguration of the biochemical responses initiated by agonist activation of alpha(1)-adrenoceptors. Among these is the emergence of alpha(1)-adrenergic receptor coupling to cGMP synthesis. We also present evidence that estrogen promotes novel, functional interactions between insulin-like growth factor-1 (IGF-1) and alpha(1)-adrenergic receptor signaling in the hypothalamus and preoptic area. Thus, estrogen amplification of signaling mediated by alpha(1)-adrenoceptors is multifaceted, involving changes in gene expression (of the alpha(1B)-adrenoceptor), switching of receptor linkage to previously inactive intracellular pathways, and the promotion of cross talk between IGF-1 and NE receptors. We propose that this hormone-dependent remodeling of hypothalamic responses to NE maximizes reproductive success by coordinating the timing of the preovulatory release of gonadotropins with the period of behavioral receptivity in female rodents.

Acute and chronic estradiol treatments reduce memory deficits induced by transient global ischemia in female rats

Transient global ischemia induces selective, delayed neuronal death in the hippocampal CA1 and delayed cognitive deficits. Estrogen treatment ameliorates hippocampal injury associated with global ischemia. Although much is known about the impact of estrogen on neuronal survival, relatively little is known about its impact on functional outcome assessed behaviorally. We investigated whether long-term estradiol (21-day pellets implanted 14 days prior to ischemia) or acute estradiol (50 microg infused into the lateral ventricles immediately after ischemia) attenuates ischemia-induced cell loss and improves visual and spatial working memory in ovariectomized female rats. Global ischemia significantly impaired visual and spatial memory, assessed by object recognition and object placement tests at 6-9 days. Global ischemia did not affect locomotion, exploration, or anxiety-related behaviors, assessed by an open-field test at 6 days. Long-term estradiol prevented the ischemia-induced deficit in visual working memory, maintaining normal performance in tests with retention intervals of up to 1 h. Long-term estradiol also prevented ischemia-induced deficits in spatial memory tests with short (1 and 7 min), but not longer (15 min), retention intervals. Acute estradiol significantly improved visual memory assessed with short retention intervals, but did not prevent deficits in spatial memory. Acute estradiol significantly increased the number of surviving CA1 neurons, assessed either at 7 days after ischemia or after the completion of behavioral testing 9 days after ischemia. In contrast, chronic estradiol did not reduce CA1 cell death 9 days after ischemia. Thus, long-term estradiol at near physiological levels and acute estradiol administered after ischemic insult improve functional recovery after global ischemia. These findings have important implications for intervention in the neurological sequellae associated with global ischemia.

Functional Interactions between Estrogen and Insulin-Like Growth Factor-I in the Regulation of α1B-Adrenoceptors and Female Reproductive Function

The ovarian hormone estradiol (E2) and insulin-like growth factor-I (IGF-I) interact in the CNS to regulate neuroendocrine function and synaptic remodeling. Previously, our laboratory showed that 2 d E2 treatment induces α1B-adrenoceptor expression and promotes IGF-I enhancement of α1-adrenoceptor potentiation of cAMP accumulation in the preoptic area (POA) and hypothalamus (HYP). This study examined the hypothesis that E2-dependent aspects of female reproductive function, including α1B-adrenoceptor expression and function in the POA and HYP, are mediated by brain IGF-I receptors (IGF-IRs) in female rats. Ovariohysterectomized rats were implanted with a guide cannula aimed at the third ventricle and treatedin vivo with vehicle or E2 daily for 2 d before experimentation. Intracerebroventricular infusions of JB-1, a selective IGF-IR antagonist, were administered every 12 hr beginning 1 hr before the first E2 injection. Administration of JB-1 during E2 priming completely blocks hormone-induced luteinizing hormone release and partially inhibits hormone-dependent reproductive behavior. Reproductive behavior is restored by intracerebroventricular infusion of 8-bromo-cGMP, the second messenger implicated in α1-adrenergic facilitation of lordosis. In addition, blockade of IGF-IRs during E2priming prevents E2-induced increases in α1B-adenoceptor binding density and abolishes acute IGF-I enhancement of NE-stimulated cAMP accumulation in HYP and POA slices. These data document the existence of a novel mechanism by which IGF-I participates in the remodeling of noradrenergic receptor signaling in the HYP and POA after E2 treatment. These events may help coordinate the timing of ovulation with the expression of sexual receptivity.

ESTRADIOL REGULATION OF ALPHA 1B-ADRENOCEPTOR MRNA IN FEMALE RAT HYPOTHALAMUS-PREOPTIC AREA

Estradiol treatment for 48 h increases the density of α1B‐adrenoceptors in the hypothalamus‐preoptic area of ovariectomized female rats by five‐ to six‐fold. Present studies tested the hypothesis that estradiol elevation of hypothalamus‐preoptic area α1B‐adrenoceptor density is correlated with increased levels of mRNA for this receptor. We developed a semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) protocol for measuring brain α1b‐adrenoceptor mRNA. The primers chosen yielded the predicted 409 base pair PCR product when used to amplify authentic α1b‐adrenoceptor cDNA. The identity of the RT‐PCR products from rat brain was confirmed by restriction digest analysis and sequencing. Moreover, there was a good correlation between the levels of α1b‐adrenoceptor mRNA measured by RT‐PCR in liver, whole brain and cerebellum with previous measurements using Northern blots and RNAse protection assays. We then performed RT‐PCR on total RNA from hypothalamic‐preoptic area tissue taken from ovariectomized control rats and from ovariectomized rats injected once or twice with 2 μg of estradiol benzoate at 24 or 24 and 48 h before sacrifice. Exposure to estradiol for either 24 or 48 h significantly increased levels of α1b‐adrenoceptor mRNA by 86–110% in the hypothalamus‐preoptic area of ovariectomized female rats when compared to oil‐treated controls. We also examined whether estradiol regulates α1b‐adrenoceptor mRNA in the cortex. Cortical α1b‐adrenoceptor mRNA levels were reduced to approximately 20% of control levels when measured 24 h after hormone injection. A similar decrease in cortical α1b‐adrenoceptor mRNA was observed 48 h after estrogen administration. In summary, estradiol treatment significantly increases the level of α1b‐adrenoceptor mRNA in the hypothalamus‐preoptic area, a brain region involved in the control of reproductive function. In the cortex, a brain region with relatively few estrogen receptors, the same estrogen treatment reducesα1b‐adrenoceptor mRNA levels.