Bilateral control of brain activity by dopamine D1 receptors: evidence from induction patterns of regulator of G protein signaling 2 and c-fos mRNA in D1-challenged hemiparkinsonian rats.

Recent reports show that striatal dopamine D1-type receptors from one side of the normal rat brain can control brain activity (as measured by c-fos induction) on both sides of the brain. However, this phenomenon has not yet been studied in the presence of sensitized dopamine D1-type receptors. Here we address this issue by investigating the extent to which dopamine D1-type receptors control brain activation in rats with unilaterally sensitized dopamine D1-type receptors. Gene induction assays were used to identify activated regions from midbrain to forebrain in unilaterally 6-hydroxydopamine lesioned (hemiparkinsonian) rats challenged with the full dopamine D1-type agonist SKF82958 (3 mg/kg, 0.5 and 2 h). The genes used are c-fos, the proven neuronal activity marker, and Regulator of G protein Signaling 2, a gene we propose as a marker of signaling homeostasis. SKF82958-mediated induction of both genes is greatly enhanced in hemiparkinsonian rats compared with shams, in both the lesioned and the intact hemisphere. For example, in the denervated caudate-putamen at 2 h postinjection, this enhancement is more than 80-fold for c-fos and up to 20-fold for Regulator of G protein Signaling 2; for the intact side this is 35-fold for c-fos and 27-fold for Regulator of G protein Signaling 2. Cortical induction of c-fos and Regulator of G protein Signaling 2 was generalized to most neocortical regions and was essentially equivalent in both the denervated and intact hemispheres. Interestingly, hippocampal structures also showed strong bilateral induction of both genes. This overall pattern of brain activation can be accounted for by the basal-ganglia thalamocortical and hippocampal circuits which both contain hemisphere-crossing connections and which can be initially activated in the lesioned hemisphere. Some regions, such as the intact striatum or the CA1 region, showed relatively low c-fos induction and relatively high Regulator of G protein Signaling 2 induction, possibly indicating that these regions are engaged in unusually strong signaling regulation activities. Our results show that, besides basal ganglia-thalamocortical circuits, dopamine D1-type-mediated brain activation in hemiparkinsonian rats also involves hippocampal circuits.

Isoform specificity for oestrogen receptor and thyroid hormone receptor genes and their interactions on the NR2D gene promoter.

Oestrogens are critical for the display of lordosis behaviour and, in recent years, have also been shown to be involved in synaptic plasticity. In the brain, the regulation of ionotropic glutamate receptors has consequences for excitatory neurotransmission. Oestrogen regulation of the N-methyl-d-aspartate receptor subunit 2D (NR2D) has generated considerable interest as a possible molecular mechanism by which synaptic plasticity can be modulated. Since more than one isoform of the oestrogen receptor (ER) exists in mammals, it is possible that oestrogen regulation via the ERalpha and ERbeta isoforms on the NR2D oestrogen response element (ERE) is not equivalent. In the kidney fibroblast (CV1) cell line, we show that in response to 17beta-oestradiol, only ERalpha, not ERbeta, could upregulate transcription from the ERE which is in the 3' untranslated region of the NR2D gene. When this ERE is in the 5' position, neither ERalpha nor ERbeta showed transactivation capacity. Thyroid hormone receptor (TR) modulation of ER mediated induction has been shown for other ER target genes, such as the preproenkephalin and oxytocin receptor genes. Since the various TR isoforms exhibit distinct roles, we hypothesized that TR modulation of ER induction may also be isoform specific. This is indeed the case. The TRalpha1 isoform stimulated ERalpha mediated induction from the 3'-ERE whereas the TRbeta1 isoform inhibited this induction. This study shows that isoforms of both the ER and TR have different transactivation properties. Such flexible regulation and crosstalk by nuclear receptor isoforms leads to different transcriptional outcomes and the combinatorial logic may aid neuroendocrine integration.

Co-expression of estrogen and thyroid hormone receptors in individual hypothalamic neurons.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are members of the nuclear receptor family of transcription factors that induce or repress the expression of target genes. Previous behavioral studies in female rodents have demonstrated that thyroid hormones can antagonize the effects of estrogen in the central nervous system (CNS), particularly by attenuating estrogen's ability to facilitate reproductive behaviors. Additional molecular studies have suggested a mechanism for this antagonism by showing that ligand-activated ER alpha and TRs have the potential to interact in their transcriptional controls. Although the expression patterns of ER alpha and TRs in the rodent brain appear to overlap in behaviorally relevant areas, it remained to be determined whether these two classes of proteins coexist in vivo at the level of single neurons. To address this possibility, we employed a highly sensitive double-label in situ hybridization technique using digoxigenin and (35)S-labeled cRNA probes to analyze, in detail, the expression of ER alpha mRNA with TR alpha 1 and TR alpha 2 mRNAs in the same neurons of the ovariectomized (OVX) adult mouse brain. Our results demonstrate that a large majority of the ER alpha-positive neurons also expresses TR alpha 1 and TR alpha 2 mRNAs. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 81.5% and 80.5% of the neurons endowed with ER alpha mRNA also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. In the amygdala, more than 60.5% and 67% of ER alpha-positive cells also contain TR alpha 1 and TR alpha 2 mRNAs, respectively. These findings provide the first anatomical evidence that ER and TR can be found in the same neurons, including hypothalamic neurons. This coexpression of ER alpha and TR provides the cellular basis for a new level of neuronal integration in a brain region where estrogens control female reproductive behaviors.

Estrogens, brain and behavior: studies in fundamental neurobiology and observations related to women's health.

Mechanisms and consequences of the effects of estrogen on the brain have been studied both at the fundamental level and with therapeutic applications in mind. Estrogenic hormones binding in particular neurons in a limbic-hypothalamic system and their effects on the electrophysiology and molecular biology of medial hypothalamic neurons were central in establishing the first circuit for a mammalian behavior, the female-typical mating behavior, lordosis. Notably, the ability of estradiol to facilitate transcription from six genes whose products are important for lordosis behavior proved that hormones can turn on genes in specific neurons at specific times, with sensible behavioral consequences. The use of a gene knockout for estrogen receptor alpha (ERalpha) revealed that homozygous mutant females simply would not do lordosis behavior and instead were extremely aggressive, thus identifying a specific gene as essential for a mammalian social behavior. In dramatic contrast, ERbeta knockout females can exhibit normal lordosis behavior. With the understanding, in considerable mechanistic detail, of how the behavior is produced, now we are also studying brain mechanisms for the biologically adaptive influences which constrain reproductive behavior. With respect to cold temperatures and other environmental or metabolic circumstances which are not consistent with successful reproduction, we are interested in thyroid hormone effects in the brain. Competitive relations between two types of transcription factors - thyroid hormone receptors and estrogen receptors have the potential of subserving the blocking effects of inappropriate environmental circumstances on female reproductive behaviors. TRs can compete with ERalpha both for DNA binding to consensus and physiological EREs and for nuclear coactivators. In the presence of both TRs and ERs, in transfection studies, thyroid hormone coadministration can reduce estrogen-stimulated transcription. These competitive relations apparently have behavioral consequences, as thyroid hormones will reduce lordosis, and a TRbeta gene knockout will increase it. In sum, we not only know several genes that participate in the selective control of this sex behavior, but also, for two genes, we know the causal routes. Estrogenic hormones are also the foci of widespread attention for their potential therapeutic effects improving, for example, certain aspects of mood and cognition. The former has an efficient animal analog, demonstrated by the positive effects of estrogen in the Porsolt forced swim test. The latter almost certainly depends upon trophic actions of estrogen on several fundamental features of nerve cell survival and growth. The hypothesis is raised that the synaptic effects of estrogens are secondary to the trophic actions of this type of hormone in the nucleus and nerve cell body.

Thyroid hormone coadministration inhibits the estrogen-stimulated elevation of preproenkephalin mRNA in female rat hypothalamic neurons.

Expression of the enkephalin gene in ventromedial hypothalamus (VMH) of the female rat has been correlated with the performance of lordosis behavior. By antisense DNA evidence, it has been drawn into a causal role as well. Here, we explored whether, parallel to earlier molecular and behavioral results, thyroid hormone coadministration could disrupt the estrogenic induction of preproenkephalin (PPE) mRNA. As expected, estradiol benzoate treatment to ovariectomized rats led to a large and significant increase in PPE gene expression in the VMH. This increase was inhibited by coadministration of thyroid hormone. The thyroid hormone interference in PPE gene expression was specific to the VMH, as there were no significant effects in the central nucleus of the amygdala or in the caudate/putamen. These in situ hybridization histochemical results form a direct parallel both to previous transcriptional measurements and to reproductive behavior assays in which thyroid hormones were able to oppose estrogenic facilitation. Previous evidence supports the notion of competitive DNA binding and protein/protein interactions providing mechanisms for nuclear thyroid hormone receptors to affect estrogen receptor function, but other, additional mechanisms cannot be ruled out. To date, both oxytocin and PPE gene expression represent potential hypothalamic systems by which thyroid hormones could interfere with estrogen-stimulated female rat reproductive behavior.

Ultrastructural localization of 5-hydroxytryptamine1A receptors in the rat brain.

5-Hydroxytryptamine1A (5-HT1A) receptors have been visualized at the electron microscopic level in selected areas (dorsal raphe nucleus, hippocampus, septum) of the rat brain using specific anti-peptide antibodies. 5-HT1A receptor immunoreactivity was found almost exclusively in the somatodendritic compartment of neurons and was very rarely observed within processes possibly belonging to glial cells. The immunoenzymatic reaction product was associated exclusively with dendritic spines in the dorsal hippocampus, whereas in the dorsal raphe nucleus and the septal complex, immunoreactivity was found in both dendritic processes and somata. Although some immunolabeling was observed within the cytoplasm of cell bodies, 5-HT1A receptor immunoreactivity was essentially confined to the plasma membrane where it was unevenly distributed. It was frequently associated with synapses (except in the dorsal raphe nucleus), but was also found extrasynaptically in both somata and dendrites. These data suggest that the action of serotonin via 5-HT1A receptor could occur through junctional as well as nonjunctional transmission.

Serotonin1A receptors are expressed by a subpopulation of cholinergic neurons in the rat medial septum and diagonal band of Broca--a double immunocytochemical study.

The possible colocalization of 5-hydroxytryptamine1A receptors and choline acetyltransferase in the same neurons of the medial septum and diagonal band of Broca was investigated using double immunocytochemical techniques, either on the same section or on adjacent thin sections of the rat brain. The presence of both antigens in the same neurons was demonstrated at the light and electron microscopic levels. The proportion of cholinergic neurons that express 5-hydroxytryptamine1A receptors was similar in the different parts of the septal complex (around 25%). By contrast, the proportion of 5-hydroxytryptamine1A receptor-positive neurons also exhibiting choline acetyltransferase immunoreactivity was much higher (40-44%) in the dorsal and ventral groups of cholinergic cells, than in the intermediate group (18%). In line with the topographical distribution of cholinergic projections, this result points out the potential involvement of 5-hydroxytryptamine1A receptors in the control of the septohippocampal cholinergic projection by serotonin. This connection might be relevant to learning and memory, and in the appearance of age-dependent or neurodegenerative cognitive deficits, which have been shown to involve alterations in both the serotoninergic and the cholinergic systems.

Immunocytochemical localization of serotonin1A receptors in the rat central nervous system.

Specific anti-rat 5-hydroxytryptamine1A (serotonin1A) receptor antibodies raised in a rabbit injected with a synthetic peptide corresponding to a highly selective portion of the third intracellular loop of the receptor protein (El Mestikawy et al. [1990] Neurosci. Lett. 118:189-192) were used for immunohistochemical mapping of serotonin1A receptors in the brain and spinal cord of adult rats. The highest density of immunostaining was found in limbic areas (lateral septum, CA1 area of Ammon's horn and dentate gyrus in the hippocampus, and frontal and entorhinal cortices), in the anterior raphe nuclei, and in the interpeduncular nucleus, in agreement with previous autoradiographic studies with selective radioligands showing the enrichment of these regions in serotonin1A receptor binding sites. Serotonin1A receptor-like immunoreactivity was also present, but at a moderate level, in the neocortex, in some thalamic and hypothalamic nuclei, in the nucleus of the solitary tract, in the dorsal tegmentum, in the nucleus of the spinal tract of the trigeminal nerve, and in the superficial layers of the dorsal horn in the spinal cord. In contrast, extrapyramidal areas, including the caudate putamen, the globus pallidus, and the substantia nigra as well as the cerebellum, exhibited very low to no immunostaining by antiserotonin1A receptor antibodies. At the cellular level, both the plasma membrane of neuronal perikarya and fine neuronal processes probably corresponding to dendritic fields were found to bind antiserotonin1A receptor antibodies. Regional differences were noted regarding these two types of immunostaining, because only dendrites bound antibodies within the hippocampus and the lateral septum, whereas both dendrites and neuronal cell bodies were immunoreactive in the medial septum, in the diagonal band of Broca, and in the dorsal and median raphe nuclei. Therefore, differential addressing of serotonin1A receptors could occur from one neuron to another. In general, the distribution and density of serotonin1A receptor-like immunoreactivity in the whole brain and in spinal cord were consistent with the mapping of serotonin1A receptor binding sites and serotonin1A receptor mRNA previously established by immunoautoradiographic and in situ hybridization procedures.

Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization.

Specific antibodies raised against a fusion protein containing the amino acid sequence of the putative second intracellular loop of the cloned 5-HT3-A receptor subunit were used for the immunohistochemical visualization of 5-HT3 receptors in the rat spinal cord. A dense 5-HT3-like immunoreactivity was found in the superficial layers of the dorsal horn, which closely matched the labelling of 5-HT3 binding sites by [125I]iodo-zacopride. This immunostaining was markedly decreased following unilateral rhizotomy, consistently with a preferential location of 5-HT3 receptors on terminals of primary afferent fibres, and with the presence of 5-HT3 mRNA in dorsal root ganglia. However, a significant proportion of 5-HT3 receptors persisted after rhizotomy, and the corresponding mRNA was found in the dorsal horn of the spinal cord. 5-HT3 receptors are therefore also located on intrinsic neurones of the spinal cord.

Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor.

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [3H]8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

Postnatal development and localization of 5-HT1A receptor mRNA in rat forebrain and cerebellum.

The localization of the rat brain 5-HT1A receptor mRNA was analyzed by RNAse mapping and in situ hybridization during postnatal development, particularly in the cerebellum. The regional distribution of 5-HT1A mRNA during the first 2 postnatal weeks was different from that found in adults. In some areas of the immature brain (hippocampus, cerebral cortex), 5-HT1A mRNA was found in lower density than in the adult brain. In contrast, high concentrations of the transcript were present in other brain structures only during the first days after birth. Thus, in the cerebellum, the density of 5-HT1A mRNA decreased markedly from day 2 to day 9 after birth and could hardly be detected in the adult animal. The localization of the mRNA in the molecular/Purkinje cell layer of the immature cerebellum agreed with that of the 5-HT1A receptor protein visualized by immunocytochemistry and was consistent with the hypothesis that Purkinje cells express this receptor.

Immunocytochemical localization of 5-HT1A receptors in the rat immature cerebellum.

The serotonin 5-HT1A receptors were visualized in the cerebellar vermis of 8-day-old and adult rats by immunocytochemistry using anti-5-HT1A receptor antibodies raised against a synthetic peptide corresponding to a highly selective portion of the receptor amino acid sequence (El Mestikawy et al, Neurosci Lett 118, 189-192, 1990). The 5-HT1A receptor-like immunoreactivity was particularly abundant in the posterior lobules (IXB-X) of the immature vermis where it was found in the molecular-Purkinje cell layers. Immunostaining was confined to the plasmic membrane of the Purkinje cell somas, dendrites and perhaps axons (at their somatic emergence) suggesting that 5-HT receptors might participate in non-junctional 5-HT neurotransmission in the immature cerebellum.