Estradiol receptor-alpha expression in hypothalamic and limbic regions of ewes is influenced by physiological state and maternal experience

The influence of estrus, pregnancy, parturition, and maternal experience on the expression of estrogen receptor-alpha (ERalpha) was investigated in hypothalamic and limbic regions of the sheep brain, using immunocytochemistry. Four days before parturition, previous maternal experience was associated with a higher density of ERalpha-labeled neurons in the paraventricular and supraoptic nuclei, the medial preoptic area, and the medial amygdala, but not in the mediobasal hypothalamus. Furthermore, an interaction was found between physiological state and experience in the peripartum period as the effect of experience existing 4 days prepartum was not found at parturition, when densities were lowest both in primiparous and in multiparous ewes. An additional effect of physiological state was also observed between parturition and estrus, densities being significantly lower at parturition than at estrus in the SON, PVN, and MPOA, but not in the medial amygdala. These results indicate that in sheep ERalpha expression is influenced by previous physiological and/or maternal experience at specific times of the reproductive cycle. They are also congruent with the higher ability of multiparous than nulliparous ewes to show maternal behavior several days prepartum.     

Serotonin-mediated increases in the extracellular levels of beta-endorphin in the arcuate nucleus and nucleus accumbens: a microdialysis study

Although the involvement of both endogenous opioid and serotonergic systems in modulation of pain and emotion was suggested, the neurochemical interaction between these systems in the brain has not previously been studied directly. Herein, the effects of the local application of serotonin (5-HT) and fluoxetine (a 5-HT reuptake inhibitor) on extracellular levels of beta-endorphin in the arcuate nucleus and nucleus accumbens were assessed in freely moving rats using in vivo microdialysis. The mean basal concentrations of beta-endorphin in dialysates obtained from the arcuate nucleus and nucleus accumbens were 259.9 and 143.3 pM, respectively. Specific lesion of the serotonergic system by 5,7-dihydroxytryptamine (5,7-DHT) caused a significant decrease in these dialysate beta-endorphin levels. When 5-HT (0.25-5 microM) was added to the perfusion solution, the levels of beta-endorphin in the dialysate from the arcuate nucleus increased (186-296% of baseline), in a concentration-dependent manner. In the nucleus accumbens, 0.5 and 2 microM 5-HT in the perfusion fluid did not affect the levels of beta-endorphin in the dialysate, whereas 5 and 10 microM 5-HT caused an increase of approximately 190% of baseline. When fluoxetine (250 microM) was present in the perfusing solution, the levels of beta-endorphin in the dialysates from the arcuate nucleus and nucleus accumbens increased two- to threefold. This effect was not obtained in the 5,7-DHT-lesioned rats. Thus, 5-HT, either endogenously or exogenously delivered, appears to facilitate the release of beta-endorphin in the arcuate nucleus and nucleus accumbens. This indication of an interaction between serotonergic and endorphinic systems may be relevant for assessing pain and mood disorder circuits and the mode of action of antidepressant drugs.     

Different effects of five dopamine receptor subtypes on nuclear factor-kappaB activity in NG108-15 cells and mouse brain

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-kappaB activity. To investigate whether other dopamine receptor subtypes regulate NF-kappaB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-kappaB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-kappaB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element-luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-kappaB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-kappaB activity. By contrast, risperidone treatment enhanced NF-kappaB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-kappaB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-kappaB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.     

Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

Protein kinase B (also known as Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase 3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of three daily injections of 15 mg/kg cocaine or 1 mL/kg saline spaced 1 h apart for 1, 3, or 14 days. Amygdala, nucleus accumbens, caudate putamen, and hippocampus tissues were dissected 30 min following the last injection and analyzed for phosphorylated and total Akt and GSK3(alpha and beta) protein levels using western blot analysis. Phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.     

Deep brain stimulation of the accumbens increases dopamine,serotonin, and noradrenaline in the prefrontal cortex

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is effective in treatment‐refractory obsessive‐compulsive disorder and major depressive disorder. However, little is known about the neurobiological mechanisms underlying the rapid and effective changes of DBS. One of the hypotheses is that DBS modulates activity of monoamine neurotransmitters. In this study, we evaluated the effects of DBS in the NAc core on the extracellular concentration of monoaminergic neurotransmitters in the medial (mPFC) and orbital prefrontal cortex (OFC). Freely moving rats were bilaterally stimulated in the NAc core for 2 h while dopamine, serotonin, and noradrenaline were measured using in vivo microdialysis in the mPFC and the OFC. We report rapid increases in the release of dopamine and serotonin to a maximum of 177% and 127% in the mPFC and an increase up to 171% and 166% for dopamine and noradrenaline in the OFC after onset of stimulation in the NAc core. These results provide further evidence for the distal effects of DBS and corroborate previous clinical and pre‐clinical findings of altered neuronal activity in prefrontal areas.     

Sex differences in oxytocin receptor binding in forebrain regions: Correlations with social interest in brain region- and sex- specific ways

Social interest reflects the motivation to approach a conspecific for the assessment of social cues and is measured in rats by the amount of time spent investigating conspecifics. Virgin female rats show lower social interest towards unfamiliar juvenile conspecifics than virgin male rats. We hypothesized that the neuropeptide oxytocin (OT) may modulate sex differences in social interest because of the involvement of OT in pro-social behaviors. We determined whether there are sex differences in OT system parameters in the brain and whether these parameters would correlate with social interest. We also determined whether estrus phase or maternal experience would alter low social interest and whether this would correlate with changes in OT system parameters. Our results show that regardless of estrus phase, females have significantly lower OT receptor (OTR) binding densities than males in the majority of forebrain regions analyzed, including the nucleus accumbens, caudate putamen, lateral septum, bed nucleus of the stria terminalis, medial amygdala, and ventromedial hypothalamus. Interestingly, male social interest correlated positively with OTR binding densities in the medial amygdala, while female social interest correlated negatively with OTR binding densities in the central amygdala. Proestrus/estrus females showed similar social interest to non-estrus females despite increased OTR binding densities in several forebrain areas. Maternal experience had no immediate or long-lasting effects on social interest or OT brain parameters except for higher OTR binding in the medial amygdala in primiparous females. Together, these findings demonstrate that there are robust sex differences in OTR binding densities in multiple forebrain regions of rats and that OTR binding densities correlate with social interest in brain region- and sex-specific ways.     

Heterosexual housing increases the retention of courtship behavior following castration and elevates metabolic capacity in limbic brain nuclei in male whiptail lizards,Cnemidophorus inornatus

In male vertebrates the display of courtship behavior depends on the presence of testicular androgens. However, social experiences in adulthood can alter the hormonal dependence of courtship behavior in a variety of species, and we have previously proposed that these behavioral changes are linked to changes in neural metabolic capacity (cytochrome oxidase activity). Here we investigated the effects of prior social experience (housing with females vs housing in isolation) on the retention of courtship behavior following gonadectomy and on cytochrome oxidase (CO) activity in male little striped whiptail lizards, Cnemidophorus inornatus. In Experiment 1, we found that males that were previously housed with females (HWF males) continued to display courtship behavior longer after castration than males previously housed in isolation (ISOLATE males). This is similar to the behavioral plasticity found in rodents and cats. On the other hand, courtship behavior while gonadally intact was indistinguishable between HWF and ISOLATE males. Because all males were housed individually following castration, the difference is due to different social experiences prior to castration. In Experiment 2, we found that gonadally intact HWF males had significantly elevated CO activity in the preoptic area, amygdala, and anterior and ventromedial hypothalamic areas relative to intact ISOLATE males. No significant differences in metabolism were found in the lateral septum, lateral hypothalamus, and habenula or in hindlimb muscle, suggesting that the increase in metabolism is specific to brain nuclei involved in courtship behavior. Altogether, this demonstrates that elevations in metabolic capacity correlate with experience-dependent increases in robustness to castration.     

Organizational effects of estrogens on brain vasotocin and sexual behavior in quail

Reproductive behavior is sexually differentiated in quail: The male-typical copulatory behavior is never observed in females even after treatment with high doses of testosterone (T). This sex difference in behavioral responsiveness to T is organized during the embryonic period by the exposure of female embryo to estrogens. We showed recently that the sexually dimorphic medial preoptic nucleus (POM), a structure that plays a key role in the activation of male copulatory behavior, is innervated by a dense steroid-sensitive network of vasotocin-immunoreactive (VT-ir) fibers in male quail. This innervation is almost completely absent in the female POM and is not induced by a chronic treatment with T, suggesting that this neurochemical difference could be organizational in nature. This idea was tested by injecting fertilized quail eggs of both sexes on day 9 of incubation with either estradiol benzoate (EB) (25 μg, a treatment that suppresses the capacity to show copulatory behavior in adulthood) or the aromatase inhibitor R76713 (10 μg, a treatment that makes adult females behaviorally responsive to T), or with the solvents as a control (C). At 3 weeks posthatch, all subjects were gonadectomized and later implanted with Silastic capsules filled with T. Two weeks later, all birds were perfused and brain sections were processed for VT immunocytochemistry. Despite the similarity of the adult endocrine conditions of the subjects (all were gonadectomized and treated with T Silastic implants providing the same plasma level of steroid to all subjects), major qualitative differences were observed in the density of VT-ir structures in the POM of the different groups. Dense immunoreactive structures (fibers and a few cells) were observed in the POM of C males but not females; EB males had completely lost this immunoreactivity (and lost the capacity to display copulatory behavior); and, conversely, R76713 females displayed a male-typical VT-ir system in the nucleus (and also high levels of copulatory behavior). Similar changes in immunoreactivity were seen in the nucleus of the stria terminalis and in the lateral septum (VT-ir fibers only in this case) but not in the magnocellular vasotocinergic system. These neurochemical changes closely parallel the effects of the embryonic treatments on male copulatory behavior. The vasotocinergic system of the POM can therefore be considered an accurate marker of the sexual differentiation of brain circuits mediating this behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 684–699, 1998     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

3,4-Methylenedioxymethamphetamine increases interleukin-1beta levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5-HT depletion

3,4-Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5-HT release. Interleukin-1beta (IL-1beta) is a pro-inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL-1beta concentration and glial activation and their relationship with acute hyperthermia and 5-HT depletion. MDMA, given to rats housed at 22 degrees C, increased IL-1beta levels in hypothalamus and cortex from 1 to 6 h and [(3)H]-(1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)3-isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX-42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL-1 receptor antagonist did not modify MDMA-induced hyperthermia indicating that IL-1beta release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4 degrees C, hyperthermia was abolished and the IL-1beta increase significantly reduced. The MDMA-induced acute 5-HT depletion was prevented by fluoxetine coadministration but the IL-1beta increase and hyperthermia were unaffected. Therefore, the rise in IL-1beta is not related to the acute 5-HT release but is linked to the hyperthermia. Contrary to IL-1beta levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.     

Plasminogen activator inhibitor-2 (PAI-2) mRNA is localized in the accumbens nucleus of the mouse brain and is induced in specific brain sites after kainate excitation

Plasminogen activator inhibitor-2 (PAI-2) specifically inhibits plasminogen activators, extracellular fibrinolytic serine proteases that are also implicated in brain plasticity and toxicity. Primarily localized intracellularly, PAI-2 is thought to also counteract apoptosis mediated by a currently undefined intracellular protease. Here we localized PAI-2 mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate excitation. We found that in the normal brain PAI-2 mRNA was confined to an area within the accumbens nucleus shell. After kainate was injected (i.p.), PAI-2 mRNA was substantially and rapidly (within 2 h) induced in neuron-like cells primarily in layers II–III of the neocortex; the cingulate, piriform, entorhinal and perirhinal cortices; the olfactory bulb, nucleus and tubercle; in the accumbens nucleus, shell and core; throughout the caudate putamen and the amygdaloid complex; in the CA1 and CA3 areas of the hippocampus, and in the parasubiculum. These findings suggest that PAI-2 could play a role in the accumbens nucleus as well as in activity-related events associated with olfactory, striatal, and limbic structures.