Suzanne Numan

Distribution of estrogen receptor α and β in the mouse central nervous system: In vivo autoradiographic and immunocytochemical analyses

Although the distribution of estrogen receptor β (ERβ) immunoreactivity in the rat central nervous has been reported, no such data are available in the mouse. The present study used in vivo autoradiography utilizing a 125I‐estrogen that has equal binding affinity for both receptors as well as immunohistochemistry for ERβ and ERα, to investigate and compare the distribution of the two ERs in the mouse CNS. The use specific antisera against ERα and ERβ allowed us to evaluate the contribution of these receptors to the binding detected with autoradiography. In addition, data were collected in ovariectomized wildtype and ERα KO (knockout) mice to examine developmental regulation of ERβ expression by ERα. These studies revealed that in the mouse CNS, combining immunoreactivity for ERα with that for ERβ accounted for all regions where binding was seen using autoradiography. Therefore, these data strongly suggest that the major contributors of estrogen binding in the mouse CNS are ERα and ERβ. Together, these data provide an anatomical foundation for future studies and advance our understanding of estrogen action in the CNS. Moreover, since the immunocytochemical images were similar in wildtype and ERα KO mice, these studies suggest that the lack of ERα does not influence the expression of ERβ in the central nervous system. J. Comp. Neurol. 473:270–291, 2004.

Alterations in BDNF and NT-3 mRNAs in rat hippocampus after experimental brain trauma

Previous studies have suggested that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are neuroprotective or neurotrophic for certain subpopulations of hippocampal neurons following various brain insults. In the present study, the expression of BDNF and NT-3 mRNAs in rat hippocampus was examined after traumatic brain injury. Following lateral fluid percussion (FP) brain injury of moderate severity (2.0-2.1 atm) or sham injury, the hippocampi from adult rats were processed for the in situ hybridization localization of BDNF and NT-3 mRNAs using 35S-labeled cRNA probes at post-injury survival times of 1, 3, 6, 24 and 72 h. Unilateral FP injury markedly increased hybridization for BDNF mRNA in the dentate gyrus bilaterally which peaked at 3 h and remained above control levels for up to 72 h post-injury. A moderate increase in BDNF mRNA expression was also observed bilaterally in the CA3 region of the hippocampus at 1, 3, and 6 h after FP injury, but expression declined to control levels by 24 h. Conversely, NT-3 mRNA was significantly decreased in the dentate gyrus following FP injury at the 6 and 24 h survival times. These results demonstrate that FP brain injury differentially modulates expression of BDNF and NT-3 mRNAs in the hippocampus, and suggest that neurotrophin plasticity is a functional response of hippocampal neurons to brain trauma.

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: A double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high‐affinity receptors TrkB (for BDNF and NT‐4/5) and TrkC (for NT‐3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double‐labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA‐containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT‐3 in the adult midbrain in vivo. Moreover, because BDNF and NT‐3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms. J. Comp. Neurol. 403:295–308, 1999.

Increased expression of 5HT2 receptor mRNA in rat striatum following 6-OHDA lesions of the adult nigrostriatal pathway

Neonatal destruction of the dopaminergic nigrostriatal system with the specific neurotoxin 6-hydroxydopamine (6-OHDA) leads to increases in several components of the adult serotonergic raphe-striatal system. Although results following similar lesions of adult ventral midbrain dopaminergic neurons are less consistent, increases in striatal serotonin (5-hydroxytryptamine; 5HT) fiber density, content, and metabolites have been reported. The effect of such lesions upon gene expression for striatal 5HT receptors, however, has not been determined. The purpose of the present study was to investigate possible changes in expression of several 5HT receptor mRNAs in rat striatum following destruction of the adult nigrostriatal pathway. In situ hybridization for 5HT1A, 5HT1C, and 5HT2 receptor subtype mRNAs was performed in rat striatum following unilateral injection of 6-OHDA into the medial forebrain bundle or directly into the ventral midbrain. Compared to the uninjected control side, a significant increase in the hybridization density for 5HT2 receptor mRNA was observed in the caudate-putamen ipsilateral to the 6-OHDA lesion (P < 0.05). In contrast, no significant changes in the hybridization densities for 5HT1A or 5HT1C receptor mRNAs were detected. The observed increase in striatal 5HT2 receptor mRNA levels after the dopamine-depleting lesion provides evidence for plasticity of the serotonergic raphe-striatal system in the adult rat at the level of striatal gene expression. Furthermore, the present data indicate that dopaminergic mechanisms differentially regulate the expression of 5HT receptor mRNAs in adult rat striatum.

Increased Expression of trkB mRNA in Rat Caudate-Putamen Following 6–OHDA Lesions of the Nigrostriatal Pathway

The tyrosine kinase receptors trkB and trkC are essential components of the high‐affinity receptors for members of the neurotrophin family, including brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3). Both neurotrophin receptor mRNAs are broadly distributed throughout the caudate‐putamen. In animal models of Parkinsons disease, loss of the ventral mesencephalic dopamine projection to the striatum has been shown to alter the expression of several striatal peptides, neurotransmitter‐synthesizing enzymes and receptors. To determine if expression of trkB and/or trkC striatal mRNAs is also regulated by the integrity of the dopaminergic afferents, adult rats were given unilateral injections of 6–hydroxydopamine (6–OHDA), selective catecholamine neurotoxin, or vehicle into the right ascending medial forebrain bundle. Following 2 week survival period, in situ hybridization with 35S‐labelled cRNA probes for the kinase‐specific, full‐length form of trkB mRNA and all forms of trkC mRNA was performed in striatal sections. A significant increase in the hybridization density for trkB mRNA was observed in the caudate‐putamen ipsilateral to the 6–OHDA injection, compared with the uninjected control side (P < 0.001). In contrast, no alteration in the hybridization density for trkC mRNA was observed in the striatum of 6–OHDA‐treated rats. No alterations in trkB or trkC mRNA levels were observed in the striata of vehicle‐treated animals. These data suggest that midbrain dopaminergic afferents regulate the expression of trkB mRNA in the caudate‐putamen. Alternatively, since dopaminergic neurons of the ventral mesencephalon express BDNF mRNA, the up‐regulation of striatal trkB mRNA may reflect compensatory response by striatal neurons due to loss of anterogradely and/or retrogradely derived trophic support from the ventral midbrain.

Regulation of Phospholipase Cγ in the Mesolimbic Dopamine System by Chronic Morphine Administration

Abstract : Neurotrophic signaling pathways have been implicated in the maintenance of the mesolimbic dopamine system, as well as in changes in this system induced by chronic morphine exposure. We found that many of these signaling pathway proteins are expressed at appreciable levels within the ventral tegmental area (VTA) and related regions, although with substantial regional variation. Moreover, phospholipase Cγ1 (PLCγ1) was significantly and specifically up‐regulated within the VTA by 30% following chronic exposure to morphine. PLCγ1 mRNA expression is enriched in dopaminergic neurons within the VTA ; however, the up‐regulation of PLCγ1 in this region was not seen at the mRNA level. In contrast to PLCγ1, insulin receptor substrate (IRS)‐2, a protein involved in phosphatidylinositol 3‐kinase signaling, and another putative IRS‐like protein were significantly down‐regulated within the VTA by 49 and 45%, respectively. Levels of several proteins within the Ras‐ERK pathway were not altered. Regulation of neurotrophic factor signaling proteins may play a role in morphine‐induced plasticity within the mesolimbic dopamine system.

Expression of EGF receptor mRNA in rat nigrostriatal system.

IN SITU hybridization with a cRNA probe was used to determine the cellular expression of mRNA for the epidermal growth factor receptor (EGF-R) within the rat dopaminergic nigrostriatal system. Hybridization for EGF-R mRNA was distributed throughout the ventral mesencephalon within cells of the substantia nigra pars compacta, and ventral tegmental area (VTA). Unilateral lesion of the nigrostriatal pathway with the neurotoxin 6-hydroxydopamine resulted in substantial elimination of EGF-R cRNA hybridization in the ipsilateral substantia nigra, and VTA. EGF-R mRNA was also demonstrated within a population of striatal somata, with highest levels of expression detected neonatally. These results indicate that a subpopulation of ventral midbrain dopaminergic neurons, as well as striatal perikarya, express the EGF-R and, thus, may be directly responsive to endogenous EGF, transforming growth factor-a, or another EGF-R ligand in vivo.

Developmental expression of neurotrophins and their receptors in postnatal rat ventral midbrain

Neurotrophins are a group of structurally related polypeptides that support the survival, differentiation, and maintenance of neuronal populations that express the appropriate high-affinity neurotrophin receptors. Two members of the neurotrophin family, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to increase the survival of dopaminergic neurons from the ventral midbrain in vitro. Evidence suggests that ventral midbrain neurons might be able to derive support from these trophic factors in vivo through paracrine or autocrine interactions. Both BDNF and NT-3 mRNAs and their receptor mRNAs, trkB and trkC mRNAs, respectively, have been localized to the ventral mesencephalon. However, the relative expression levels of the neurotrophins and their receptor mRNAs throughout ontogeny and in adulthood have not been elucidated. In the present study, the postnatal developmental expression of BDNF, NT-3, trkB, and trkC mRNAs was analyzed via in situ hybridization to gain insight into the possible role of these factors in vivo. We found that there was a developmental decline in the expression of BDNF and NT-3 mRNAs in the ventral mesencephalon. In contrast, no alterations in the expression of midbrain trkB or trkC mRNAs could be discerned. The present results suggest a role for BDNF and NT-3 in the earlier postnatal developmental events of responsive populations. The continued, albeit lower, expression of the neurotrophins in the ventral mesencephalon in adulthood also suggests a role for these factors in mature neuronal systems.