Upregulation of BDNF mRNA and trkB mRNA in the nigrostriatal system and in the lesion site following unilateral transection of the medial forebrain bundle

We have performed unilateral transection of the medial forebrain bundle (MFB) and studied BDNF mRNA and trkB mRNA levels at different postlesion times in the nigrostriatal system by means of in situ hybridization. BDNF mRNA levels were transiently induced in the substantia nigra pars compacta at 1 day postaxotomy. The disposition of BDNF mRNA expressing cells at this postlesion time in substantia nigra mimicked that of the dopaminergic neurons expressing the mRNA for the dopamine transporter. TrkB mRNA levels remained unaltered in the ventral mesencephalon at the different postlesion times examined-1 to 14 days. In contrast, trkB mRNA levels were significantly induced in the striatum at the longer postlesion time examined-14 days-when all neurodegenerative events are completed. It is becoming apparent that nigral BDNF mRNA levels are anterogradely transported to its target tissue in striatum. However, following axotomy, the lesion site represents a second potential target for BDNF action. Consequently, we also analyzed the pattern of mRNA expression for BDNF and trkB at the lesion site where dopaminergic axons are disconnected. There, we found notable inductions of both BDNF mRNA and trkB mRNA levels at 4 days postaxotomy. BDNF mRNA expressing cells were confined at the site of axotomy, which coincided precisely to that showing induction of trkB mRNA. Altogether, our results anticipate promising trophic roles of BNDF in the injured nigrostriatal system.     

Electro-acupuncture improves behavior and upregulates GDNF mRNA in MFB transected rats

Low and high frequency electro-acupuncture (EA) stimulation was used in rats that had been lesioned by medial forebrain bundle transection. Behavioral tests showed that both low and high frequency EA stimulation significantly reduced the amphetamine-induced rotation 2 weeks after the lesion but only high frequency EA improved the rotational behavior at 4 weeks. Analysis of the dopamine content in the striatum did not show any significant change after EA. In situ hybridization showed that high frequency EA stimulation up-regulated the glial cell line-derived neurotrophic factor (GDNF) mRNA in both sides of the globus pallidus, while low frequency EA only affected the unlesioned side. It suggests that the retrograde nourishment of GDNF to the dopaminergic neurons and the balanced activity of different nuclei in the basal ganglia circuit after EA may contribute to the behavioral improvement in these rats, which might be the factors that underlie the effectiveness of EA in the treatment of Parkinson's disease.     

Differential expression of GDNF, BDNF, and NT-3 in the aging nigrostriatal system following a neurotoxic lesion

Protein levels for brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were measured in the striatum and ventral midbrain of young and aged Brown Norway/F344 F1 (F344BNF(1)) hybrid rats following a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. At 2 weeks post-lesion, protein levels of BDNF and GDNF were higher in the denervated striatum when compared to the intact striatum for young (4-5 months old) but not old (31-33 months old) rats. Interestingly, in old rats BDNF protein in the denervated striatum was significantly lower than that measured in the intact striatum. At the same time point BDNF protein levels in the ventral midbrain were higher on the lesioned versus intact side for both young and old rats while no significant side differences were detected for GDNF protein in the ventral midbrain of young or old rats. No significant differences in NT-3 protein levels were detected between the lesioned and intact sides for striatal or ventral midbrain regions in either young or old brain. While no significant age effects were detected for BDNF or NT-3 protein, young rats showed higher GDNF protein levels in both the striatum (lesioned or intact) and ventral midbrain (lesioned or intact) than old rats. These data show that two endogenous neurotrophic factors, BDNF and GDNF, are differentially affected by a 6-OHDA lesion in the aging nigrostriatal system with young brain showing a significant compensatory increase of these two factors in the denervated striatum while no compensatory increase is observed in aged brain.     

Inverse relationship between the contents of neuromelanin pigment and the vesicular monoamine transporter-2: human midbrain dopamine neurons

The dopaminergic neurons in the ventral substantia nigra (SN) are significantly more vulnerable to degeneration in Parkinson's disease (PD) than the dopaminergic neurons in the ventral tegmental area (VTA). The ventral SN neurons also contain significantly more neuromelanin pigment than the dopaminergic neurons in the VTA. In vitro data indicate that neuromelanin pigment is formed from the excess cytosolic catecholamine that is not accumulated into synaptic vesicles by the vesicular monoamine transporter-2 (VMAT2). By using quantitative immunohistochemical methods in human postmortem brain, we sought to examine the relative contents of VMAT2 within neurons that contain different amounts of neuromelanin pigment. The immunostaining intensity (ISI) was measured for VMAT2 and also for the rate-limiting enzyme for the synthesis of dopamine, tyrosine hydroxylase (TH). ISI measures were taken from the ventral SN region where neurons are most vulnerable to degeneration in PD, nigrosome-1 (N1); from the ventral SN region where cells are moderately vulnerable to degeneration in PD, the matrix (M); and from VTA neurons near the exit of the third nerve (subregion III). The data indicate that 1) subregion III neurons have significantly higher levels of VMAT2 ISI compared with N1 neurons (more than twofold) and M neurons (45%); 2) there is an inverse relationship between VMAT2 ISI and neuromelanin pigment in the N1 and III neurons; 3) there is an inverse relationship between VMAT2 ISI and the vulnerability to degeneration in PD in the N1, M, and III subregions; and 4) neurons with high VMAT2 ISI also have high TH ISI. These data support the hypothesis that midbrain dopaminergic neurons that synthesize greater amounts of dopamine have more vesicular storage capacity for action potential-induced release of transmitter and that the ventral SN neurons accumulate the most neuromelanin pigment, in part because they have the least VMAT2 protein.     

Lesion-induced increase of BDNF is greater in the striatum of young versus old rat brain

Young (4-5 month old) and old (32-34 month old) Brown Norway/F344 hybrid rats were given unilateral 6-OHDA lesions of the nigrostriatal pathway. Four weeks later tissue from the lesioned or intact striatum or ventral midbrain was dissected and analyzed for brain-derived neurotrophic factor (BDNF) protein levels using an enzyme-linked immunosorbent assay. BDNF protein content was greater in the lesioned striatum than in the intact striatum for all young rats, and the increased BDNF content in the lesioned striatum of young rats was directly correlated with severity of lesion as determined by rotational scores. BDNF content in the lesioned striatum increased in less than half of the old rats and was not significantly different than BDNF content in the intact striatum. BDNF content in the lesioned substantia nigra/ventral tegmental area (SN/VTA) was greater than BDNF content in the intact SN/VTA for both young and old rats. These data suggest that an age-related difference in activity of at least one neurotrophic factor, BDNF, occur within the denervated striatum following a neurotoxic lesion of the nigrostriatal pathway.     

Reduced BDNF mRNA expression in the Parkinson's disease substantia nigra

Brain-derived neurotrophic factor (BDNF) has potent effects on survival and morphology of dopaminergic neurons and thus its loss could contribute to death of these cells in Parkinson's disease (PD). In situ hybridization revealed that BDNF mRNA is strongly expressed by dopaminergic neurons in control substantia nigra pars compacta (SNpc). In clinically and neuropathologically typical PD, SNpc BDNF mRNA expression is reduced by 70% (P = 0.001). This reduction is due, in part, to loss of dopaminergic neurons which express BDNF. However, surviving dopaminergic neurons in the PD SNpc also expressed less BDNF mRNA (20%, P = 0.02) than their normal counterparts. Moreover, while 15% of control neurons had BDNF mRNA expression >1 SD below the control mean, twice as many (28%) of the surviving PD SNpc dopaminergic neurons had BDNF mRNA expression below this value. This 13% difference in proportions (95% CI 8-17%, P < or = 0.000001) indicates the presence of a subset of neurons in PD with particularly low BDNF mRNA expression. Moreover, both control and PD neurons displayed a direct relationship between the density of BDNF mRNA expression per square micrometer of cell surface and neuronal size (r(2) = 0.93, P 相似文献   

The midbrain dopaminergic cell groups in the baboon Papio ursinus

The present study evaluates the cytoarchitecture of midbrain dopaminergic regions in baboons using similar methodology to that recently applied to compare humans and rats. This information is relevant for the interpretation of nonhuman primate models of Parkinson's disease (PD). The midbrains of four alpha male baboons were serially sectioned into 10 evenly spaced series of 50 microm sections. Series were stained with either cresyl violet or immunohistochemically reacted for tyrosine hydroxylase, substance P, calbindin-D28k, or parvalbumin. The organization of dopaminergic cell groups and the distribution of proteins within these groups were found to be very similar to that previously described in humans [McRitchie et al., J. Comp. Neurol. 364:121-150; 1996]. Dorsal and ventral tiers of the A9 substantia nigra (SN) pars compacta and all divisions of the A8 and A10 cell groups were identified revealing a high degree of homology in the arrangement of chemically distinct midbrain neurons between primates. The major difference between the organization of human and baboon midbrain dopaminergic neurons is the anteroposterior extent of the dense cell clusters within the SN pars compacta. In baboons the dorsomedial cell cluster is absent at posterior levels. The ventral tier cell clusters, which are targeted by PD in humans, are restricted to the posterior and ventral regions of the SN pars compacta of the baboon. In humans these cell clusters are found throughout the rostrocaudal extent of the SN. These ventral cell clusters have been previously shown to have reciprocal connections with sensorimotor regions of the putamen.     

Establishment of a Survival and Toxic Cellular Model for Parkinson’s Disease from Chicken Mesencephalon

Cellular models for Parkinson’s disease (PD) represent a fast and efficient tool in the screening for drug candidates and factors involved in the disease pathogenesis. The objective of this study was to establish and characterize a survival and toxic cellular model for PD by culturing dopaminergic neurons from embryonic chicken ventral midbrain. We show that as in rodents, the common neurotrophic factors—brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and fibroblast growth factor 2 (FGF2)—are able to support the survival of chicken midbrain dopaminergic neurons. Furthermore, after treatment with MPP⁺ or rotenone as in vitro models for PD, the number of tyrosine hydroxylase-positive cells decreased drastically. This effect could be significantly rescued by treatment with BDNF or GDNF. Together, our results indicate that mechanisms of neuroprotection of dopaminergic neurons are conserved between chicken and mammals. This supports the use of primary culture from chicken embryonic midbrain as a suitable tool for the study of neuroprotection in vitro.     

Activation of the Subthalamic Nucleus and Pedunculopontine Tegmentum: Does it Affect Dopamine Levels in the Substantia Nigra,Nucleus Accumbens and Striatum?

Parkinson's disease is a neurodegenerative disorder, of which the most prominent morphological feature is the progressive loss of dopaminergic nigrostriatal neurons. Increased glutamatergic transmission in the basal ganglia has been implicated in the pathophysiology of Parkinson's disease (PD). This study investigated whether death of substantia nigra (SN) dopaminergic neurons could be caused by the hyperactivity of afferent pathways resulting in the release of a toxic dose of excitatory amino acids in the SN. Twice-daily unilateral stimulation of the subthalamic nucleus (STN) for 21 days, using two different pulse frequencies and current strengths, significantly increased amphetamine-induced rotation, whereas sham stimulated rats showed significantly reduced rotation. Striatal and SN dopamine (DA) levels were unaffected when compared to naı̈ve and sham stimulated rats. However, levels of the DA metabolite, homovanillic acid (HVA), were significantly higher in the ipsilateral anterior striata of rats that had been stimulated at high frequency (100 Hz) and low current (100 μA) as compared to sham treated animals. Stimulation of the pedunculopontine tegmentum (PPT), using a single kainic acid injection, did not affect DA concentration in the ipsilateral striatum and nucleus accumbens when compared to sham-treated rats. DA levels in the contralateral striatum and nucleus accumbens of lesioned rats were significantly higher than ipsilateral levels. DOPAC/DA ratios were lower in the contralateral striatum and nucleus accumbens, suggesting decreased DA turnover. Glutamic acid decarboxylase activity was significantly higher in the ipsilateral than the contralateral SN. The physical manifestations of PD require a large reduction in caudate and putamen DA levels and no such depletion was measured in this study. These results, therefore, do not support the hypothesis that Parkinson's disease may result from an overstimulation of substantia nigral DAneurons by glutamate afferents originating from the STN or PPT.     

Subthalamic nucleus lesion improves cell survival and functional recovery following dopaminergic cell transplantation in parkinsonian rats

Subthalamic nucleus (STN) modulation is currently the gold standard in the treatment of Parkinson's disease (PD) cases refractory to medication. Cell transplantation is a tissue‐restorative approach and is a promising strategy in the treatment of PD. One of the obstacles to overcome in cell therapy is the poor dopaminergic cell survival. Our experiment investigates the impact of a partial subthalamotomy prior to ventral mesencephalic (VM) embryonic cell transplantation on dopaminergic cell survival and functional outcome. Unilateral dopamine depletion was carried out in rats, via medial forebrain bundle (MFB) injection of 6‐hydroxydopamine, and half of the animals went on to receive unilateral excitotoxic lesions of the STN/Zone Incerta (ZI) causing partial lesion of these structures on the same side as the MFB lesion. All MFB‐lesioned animals, with or without the STN/ZI lesion, received striatal ipsilateral embryonic VM cell grafts. The data suggest that the STN/ZI lesion could boost the dopamine cell survival in the grafts by 2.6‐fold compared with the control grafted‐only group. Moreover, performance on the drug‐induced rotation and the spontaneous behavior tests were ameliorated on the STN/ZI‐lesioned group to a significantly greater extent than the grafted‐only group. These data suggest that the STN/ZI partial lesion optimized the striatal environment, promoting an improvement in cell survival. Further studies are needed to see whether the synergy between STN modulation via deep brain stimulation and cell therapy might have clinical applications in the management of PD.     

APP knockout attenuates microglial activation and enhances neuron survival in substantia nigra compacta after axotomy

Focal microglial activation and progressive dopaminergic neurodegeneration in substantia nigra compacta (SNc) have characterized Parkinson's disease (PD). We have hypothesized that the microglial response may be provoked by molecular signals from chronically stressed SNc neurons. To test whether amyloid precursor protein (APP) could serve as such a signal, we evaluated microglial activation in SN after unilateral transection of the medial forebrain bundle (MFB) in mice either wild-type (WT) or null (KO) for APP. WT and KO mice displayed comparable microglial response at the MFB transection site. In WT mice microglial activation was first apparent in the ipsilateral SN at 3 days postlesion (dpl), marked by morphological change and increased isolectin immunoreactivity. The microglial response intensified at 7 dpl and persisted in the medial nigra through 14 dpl. In contrast, in KO mice activated microglia appeared predominantly at 7 dpl, with little activation at 3 dpl and none at 14 dpl. Neuron number in affected WT SNc at 14 dpl was significantly reduced compared with loss in affected KO SNc. The delayed and limited local microglial activation and increased neuron survival in response to distal axotomy of SNc neurons in APP KO mice are consistent with the important role APP in neuronal stress responses in vivo.     

Opposite medial thalamic unit responses to rewarding and aversive brain stimulation

Medial thalamus receives fibers from both medial forebrain bundle (MFB) and hindbrain and midbrain reticular formation (RET). The MFB and RET stimulations are rewarding and aversive respectively. In 32 unanesthetized cerveau isolé rats, 158 units were recorded. The MFB and RET effects converge on two thirds of the units recorded in the dorsal medial and paracentral nuclei of thalamus and are opposite in the following ways: post-stimulus pattern of unit discharge during 7 Hz stimulation; slow-wave recruiting with MFB, but not RET, 7 Hz stimulation; and at a “desynchronization” stimulus frequency (20 Hz), MFB elicits decreased unit discharge and RET elicits increased unit discharge, compared to the 7 Hz rates. In the intralaminar and parafasicular nuclei, post-train (60 Hz, 0.2 sec) decreases and increases in unit firing lasting seconds are often elicited with MFB and RET trains respectively. Stimulation of hypothalamic sites outside MFB did not elicit these MFB effects; parafasicular stimulation did not elicit the RET effects. The MFB effects were not seen in habenula or ventral basal thalamus. Hippocampal, habenular, and ventral medial thalamic units did not show opposite MFB and RET effects. Threshold currents for the opposite MFB and RET effects are similar to those eliciting self-stimulation and escape respectively in several operated rats tested both behaviorally and neurophysiologically.     

The neonatal ventral hippocampal lesion model of schizophrenia: effects on dopamine and GABA mRNA markers in the rat midbrain

The neonatal ventral hippocampal lesion in the rat has been used as a model of schizophrenia, a human disorder associated with changes in markers of dopamine and gamma-aminobutyric acid (GABA) circuits in various regions of the brain. We investigated whether alterations in mRNA markers related to the activity of midbrain dopaminergic and GABAergic neurons are associated with this model. We used in situ hybridization histochemistry to assess expression of mRNAs for dopamine transporter (DAT), tyrosine hydroxylase (TH) and glutamate decarboxylase-67 (GAD67) in the midbrain of adult rats with neonatal and adult ibotenic acid lesions of the ventral hippocampus. Neonatally lesioned rats showed in adulthood significantly reduced expression of DAT mRNA in the substantia nigra and the ventral tegmental area but no changes in the expression of TH and GAD67 mRNAs in these midbrain regions. Adult lesioned rats showed no changes in the expression of any of these genes. As the neonatal ventral hippocampal lesion reproduces many aspects of schizophrenia and is used as an animal model of this disorder, these results suggest that the reduction in DAT mRNA could result from developmental neuropathology in the ventral hippocampus and may thus represent a molecular substrate of the disease process.     

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 Parkinson's 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.     

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. © 1999 Wiley-Liss, Inc.     

帕金森病大鼠黑质NurrlmRNA表达的动态变化

目的　探讨帕金森病大鼠黑质核内受体相关因子 1(Nurr1)mRNA表达的动态变化。方法　通过脑立体定位注射 6 羟基多巴胺 (6 OHDA)的方法建立大鼠帕金森病 (Parkinson’sdisease,PD)模型 ,采用HE染色 ,酪氨酸羟化酶 (TH)免疫组织化学染色、原位杂交技术 ,选择 6 OHDA注射术后 1d、3d、5d、7d、2 1d为研究时点 ,观察大鼠PD模型形成过程中黑质TH 多巴胺细胞数量及Nurr1mRNA表达的改变。结果　与健侧比较 ,注射 6 OHDA 5d组损毁侧黑质TH 细胞显著减少 (P <0 .0 1) ,2 1d时仅为健侧的 15 %且出现明显的旋转行为 ;同时 ,注射 6 OHDA 1d组损毁侧黑质Nurr1mRNA表达即开始下降 ,且以 3d组最为显著 (P <0 .0 1) ,7d以后各组完全消失。结论　本实验研究结果表明 ,6 OHDA能下调大鼠黑质Nurr1mRNA的表达早于诱导多巴胺细胞的死亡     

Mitochondria mass is low in mouse substantia nigra dopamine neurons: implications for Parkinson's disease

In Parkinson's disease (PD) there is a selective loss of certain midbrain dopaminergic (DA) neurons. The most vulnerable neurons reside in the substantia nigra zona compacta (SNC), whereas the DA neurons in the ventral tegmental area (VTA) and interfascicular (IF) nucleus are less vulnerable to degeneration. Many sporadic PD patients have a defect in mitochondria respiration, and some of the genes that cause PD are mitochondrial-related (e.g., PINK1, Parkin, DJ1). The present study sought to determine whether mitochondria mass is different in SNC neurons compared to other midbrain DA neurons and to non-DA neurons in the mouse. At the electron microscopic level, mitochondria in the SN DA neurons occupy 40% less of the soma and dendritic area than in the SN non-DA neurons. The area occupied by mitochondria in the SN DA neurons is also lower than in the VTA neurons, although not different from the IF neurons. The red nucleus somata have the largest percentage of the somata occupied by mitochondria (12%). Mitochondria size is related to somata size; the largest mitochondria are found in the red nucleus neurons and the smallest mitochondria are found in the IF neurons. At the light microscopic level, SNC, VTA and IF DA neurons have <50% of the cytoplasm immunostained with the mitochondrial antibody 1D6, whereas non-DA neurons in the same midbrain regions contain mitochondria areas up to >65% of the cytoplasm area. These data indicate that mitochondria size and mass are not the same for all neurons, and the SNC DA neurons have relatively low mitochondria mass. The low mitochondria mass in SNC DA neurons may contribute to the selective vulnerability of these neurons in certain rodent models of PD.