Nigrostriatal denervation does not affect glutamate transporter mRNA expression but subsequent levodopa treatment selectively increases GLT1 mRNA and protein expression in the rat striatum

There is growing evidence that the loss of the nigrostriatal dopaminergic neurones induces an overactivity of the corticostriatal glutamatergic pathway which seems to be central to the physiopathology of parkinsonism. Moreover, glutamatergic mechanisms involving NMDA receptors have been shown to interfere with the therapeutical action of levodopa. Given the key role played by uptake processes in glutamate neurotransmission, this study examined the effects of nigrostriatal deafferentation and of levodopa treatment on the striatal expression of the glutamate transporters GLT1, GLAST and EAAC1 in the rat. No significant changes in striatal mRNA levels of these transporters were detected after either levodopa treatment (100 mg/kg; i.p., twice a day for 21 days) or unilateral lesion of the nigrostriatal pathway by intranigral 6-hydroxydopamine injection. In contrast, animals with the lesion subsequently treated with levodopa showed a selective increase (36%) in GLT1 mRNA levels in the denervated striatum versus controls. These animals also showed increased GLT1 protein expression, as assessed by immunostaining and western blotting. These data provide the first evidence that levodopa therapy may interfere with striatal glutamate transmission through change in expression of the primarily glial glutamate transporter GLT1. We further suggest that levodopa-induced GLT1 overexpression may represent a compensatory mechanism preventing neurotoxic accumulation of endogenous glutamate.     

Up-regulation of GLT1 reverses the deficit in cortically evoked striatal ascorbate efflux in the R6/2 mouse model of Huntington's disease

A corticostriatal-dependent deficit in the release of ascorbate (AA), an antioxidant vitamin and neuromodulator, occurs concurrently in striatum with dysfunctional GLT1-dependent uptake of glutamate in the R6/2 mouse model of Huntington's disease (HD), an autosomal dominant condition characterized by overt corticostriatal dysfunction. To determine if deficient striatal AA release into extracellular fluid is related to altered GLT1 activity in HD, symptomatic R6/2 mice between 6 and 9 weeks of age and age-matched wild-type (WT) mice received single daily injections of 200 mg/kg ceftriaxone, a β-lactam antibiotic that elevates the functional expression of GLT1, or saline vehicle for five consecutive days. On the following day, in vivo voltammetry was coupled with corticostriatal afferent stimulation to monitor evoked release of AA into striatum. In saline-treated mice, we found a marked decrease in evoked extracellular AA in striatum of R6/2 relative to WT. Ceftriaxone, in contrast, restored striatal AA in R6/2 mice to WT levels. In addition, intra-striatal infusion of either the GLT1 inhibitor dihydrokainic acid or dl-threo-beta-benzyloxyaspartate blocked evoked striatal AA release. Collectively, our results provide compelling evidence for a link between GLT1 activation and release of AA into the striatal extracellular fluid, and suggest that dysfunction of this system is a key component of HD pathophysiology.     

Long-Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6-hydroxydopamine-induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1- and 6-month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.     

Regional Deafferentiation Down-Regulates Subtypes of Glutamate Transporter Proteins

Abstract: Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d -[³H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.     

Effect of Cortico-Striate Pathway Lesion on the Activities of Enzymes Involved in Synthesis and Metabolism of Amino Acid Neurotransmitters in the Striatum

The activities of several enzymes involved in the metabolism of aspartate and glutamate were measured in striatal (nucleus caudatus and putamen) homogenates 2-3, 6-7, and 35-40 days following frontoparietal and frontal cortical ablation. The activity of glutamine synthetase (GS) was substantially increased (46-48%) on the operated side 6-7 days following the lesion whereas smaller changes were observed at 2-3 and 35-40 days after lesion. In contrast, decreased levels of glutaminase and malate dehydrogenase (MDH) were observed by 6-7 days while no significant change was found at either 2-3 or 35-40 after the lesion. The activities of glutamate dehydrogenase (GDH) and glutamate decarboxylase (GAD) were elevated after 35-40 days whereas no changes in the levels of either GDH or aspartate aminotransferase (ASAT) were found at 2-3 or 6-7 days after the fronto-parietal decortication. When only the frontal cortex was removed quantitatively similar changes were observed in striatal GS and glutaminase activity. The content of glutamate and glutamine in the denervated striatum followed qualitatively the changes in glutaminase and GS. The results indicate that the degeneration of cortico-striatal terminals causes a profound glial reaction in the striatum, and both glutaminase and MDH are present in relatively high concentrations in the corticostriatal terminals.     

Serotonin depletion produces long lasting increase in striatal glutamatergic transmission

The ability of serotonin (5-HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7-dihydroxytryptamine, producing 80% decreases in striatal tissue 5-HT levels, no changes were observed in the glutamatergic transmission. When 5-HT depletion was almost complete (21 days post-lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1 mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as L-trans-pyrrolidine-2,4-dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post-lesion. These results suggest that 5-HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.     

Effects of subthalamic nucleus lesions and stimulation upon corticostriatal afferents in the 6-hydroxydopamine-lesioned rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.     

Expression of BDNF mRNA in substantia nigra is dependent on target integrity and independent of neuronal activation

We have analyzed the regulation of brain-derived neurotrophic factor (BDNF) mRNA expression in the nigrostriatal system following neurotoxin ablation of striatal targets by means of kainate (KA) or quinolinic acid (QA) injections. Loss of nigral target cells in the striatum was accompanied by significant induction of BDNF mRNA levels in the ipsilateral substantia nigra (SN) at 12 and 24 h post lesion. Dual tyrosine hydroxylase (TH) and BDNF mRNA in situ hybridization (ISH) confirmed the dopaminergic nature of the BDNF mRNA expressing cells. Analysis of neuronal activity in terms of cFos mRNA expression demonstrated intense induction of this marker in the ipsilateral SN pars reticulata (SNPR), but not in SN pars compacta. Dual glutamic acid decarboxylase (GAD) and cFos mRNA ISH confirmed this view. Colchicine injections into the medial forebrain bundle to specifically disrupt neuronal trafficking between SN and striatum induced BDNF mRNA levels in the ipsilateral SNPC, thus demonstrating that nigral expression of BDNF mRNA is dependent of striatal target tissue. In addition, we found significant elevations of BDNF in the subthalamic nucleus following striatal excitotoxic lesion, which may bring novel roles of BDNF in the basal ganglia complex.     

Increase in Glutamatergic Terminals in the Striatum Following Dopamine Depletion in a Rat Model of Parkinson’s Disease

Dopaminergic neuron degeneration is known to give rise to dendrite injury and spine loss of striatal neurons, however, changes of intrastriatal glutamatergic terminals and their synapses after 6-hydroxydopamine (6OHDA)-induced dopamine (DA)-depletion remains controversial. To confirm the effect of striatal DA-depletion on the morphology and protein levels of corticostriatal and thalamostriatal glutamatergic terminals and synapses, immunohistochemistry, immuno-electron microscope (EM), western blotting techniques were performed on Parkinson’s disease rat models in this study. The experimental results of this study showed that: (1) 6OHDA-induced DA-depletion resulted in a remarkable increase of Vesicular glutamate transporter 1 (VGlut1) + and Vesicular glutamate transporter 2 (VGlut2)+ terminal densities at both the light microscope (LM) and EM levels, and VGlut1+ and VGlut2+ terminal sizes were shown to be enlarged by immuno-EM; (2) Striatal DA-depletion resulted in a decrease in both the total and axospinous terminal fractions of VGlut1+ terminals, but the axodendritic terminal fraction was not significantly different from the control group. However, total, axospinous and axodendritic terminal fractions for VGlut2+ terminals declined significantly after striatal DA-depletion. (3) Western blotting data showed that striatal DA-depletion up-regulated the expression levels of the VGlut1 and VGlut2 proteins. These results suggest that 6OHDA-induced DA-depletion affects corticostriatal and thalamostriatal glutamatergic synaptic inputs, which are involved in the pathological process of striatal neuron injury induced by DA-depletion.

     

Structural basis of developmental plasticity in the corticostriatal system

Previous studies have shown that in developing monkey corticostriatal fibres terminate around striatal cytoarchitectonic compartments--cell islands, showing transfiguration around 105th embryonic day (E105) of gestation. In the present study we have analyzed these striatal cytoarchitectonic islands and acetylcholinesterase (AChE) rich patches in the developing human brain considering them as structural indicators of the development of the corticostriatal pathways. Postmortal brain tissue of 27 fetuses and prematurely born infants, ranging from 11-34 postovulatory weeks (POW) whose deaths were attributed to non neurological causes, were processed by Nissl method, AChE histochemistry and imunocytochemical technique (synaptophysin). All specimens are part of the Zagreb Neuroembryological Collection. Initial AChE patches, presumably corresponding to the dopaminergic islands, were seen as early as 10 POW whereas cytoarchitectonical cell islands were not observed until 14 POW The main developmental change occurs between 20-24 POW when AChE negative cell poor zones develop around cell islands. This transient AChE pattern of striatal organization reaches its peak around 28 POW being most prominent along lateral border of putamen. In one case of periventricular hemorrhagic lesion with premortem survival period we have found reorganization of AChE patches in the putamen which indicates structural plasticity of corticostriatal pathways. In conclusion we propose that cell poor zones serve as waiting compartments for growing corticostriatal fibers which approach striatum through subcallosal bundle and external capsule. The period of the existence of striatal compartments (14-30 POW) is a sensitive period for structural plasticity and vulnerability after periventricular lesions.     

Quinolinic acid lesion induces changes in rat striatal glutathione metabolism

Although the involvement of oxidative mechanisms in the cytotoxicity of excitatory amino acids has been well documented, it is not known whether the intrastriatal injection of quinolinic acid (QA) induces changes in glutathione (GSH) metabolism. In this work, the activities of the enzymes GSH reductase (GRD), GSH peroxidase (GPX), and GSH S-transferase (GST), as well as the GSH content, were studied in the striatum, hippocampus, and frontal cortex of rats 1 and 6 weeks following the intrastriatal injection of QA (225 nmol). One group of animals remained untreated. This lesion resulted in a 20% decrease in striatal GRD activity at both the 1- and 6-week postlesion times, whereas GST exhibited a 30% activity increase in the lesioned striatum observable only 6 weeks after the lesion. GPX activity remained unchanged. In addition, the QA injection elicited a 30% fall in GSH level at the 1-week postlesion time. GSH related enzyme activities and GSH content from other areas outside the lesioned striatum were not affected. GST activation could represent a beneficial compensatory response to neutralize some of the oxidant agents generated by the lesion. However, this effect together with the reduction in GRD activity could be the cause or a contributing factor to the observed QA-induced deficit in GSH availability and, consequently, further disrupt the oxidant homeostasis of the injured striatal tissue. Therefore, these results provide evidence that the in vivo excitotoxic injury to the brain might affect oxidant/antioxidant equilibrium by eliciting changes in glutathione metabolism.     

Ischemia induced changes in expression of the astrocyte glutamate transporter GLT1 in hippocampus of the rat

Changes in cellular uptake of glutamate following transient cerebral ischemia is of possible importance to ischemia induced cell death. In the present study, we employed in situ hybridization and immunohistochemistry to investigate the influence of cerebral ischemia on expression of mRNA and protein of the astrocyte glutamate transporter GLT1, and of glial fibrillary acidic protein. Different subfields of CA1 and CA3 of the rat hippocampus were studied at various time-points after ischemia (days 1, 2, 4, and 21). In CA1, GLT1-mRNA was decreased at all time-points after ischemia except from day 2, whereas in CA3, decreases were seen only on day 1. Expression of GLT1-protein in CA1 was unchanged during the initial days after ischemia, but decreased markedly from day 2 to 4. In CA3, GLT1-protein increased progressively throughout the observation period after ischemia. Following the degeneration of CA1 pyramidal cells, a positive correlation between the number of CA1 pyramidal cells and expression of either GLT1-mRNA or -protein was evident selectively in CA1. Increases in expression of mRNA and protein of glial fibrillary acidic protein were present from day 2, most notable in CA1. The present data provide evidence that expression of GLT1 in CA1 of the hippocampus is not decreased persistently before the degeneration of CA1 pyramidal cells, but is downregulated in response to loss of these neurons. Since the reduction in GLT1 expression evolved concomitantly with the degeneration of CA1 pyramidal cells, it may contribute to the severity of CA1 pyramidal cell loss. A progressive postischemic increase in GLT1 expression in CA3 may be linked to the resistance of CA3 neurons to ischemic cell damage.     

Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

Ceftriaxone-induced up-regulation of cortical and striatal GLT1 in the R6/2 model of Huntington's disease

Background  

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by cortico-striatal dysfunction and loss of glutamate uptake. At 7 weeks of age, R6/2 mice, which model an aggressive form of juvenile HD, show a glutamate-uptake deficit in striatum that can be reversed by treatment with ceftriaxone, a β-lactam antibiotic that increases GLT1 expression. Only at advanced ages (> 11 weeks), however, do R6/2 mice show an actual loss of striatal GLT1. Here, we tested whether ceftriaxone can reverse the decline in GLT1 expression that occurs in older R6/2s.     

N-Acetyl-aspartyl-glutamate in the rat dorsal striatum: topographical distribution and effect of sensorimotor cortex lesions

The dipeptide N-acetyl-aspartyl-glutamate (NAAG) has been proposed as putative neurotransmitter of some corticostriatal projections. To further explore this possibility, endogenous NAAG levels were measured in various microdissected striatal regions in normal animals and in those with bilateral lesion of sensorimotor cortex. In intact rats there was a rostro-caudal gradient for NAAG, with highest concentrations in the more caudal portions of the striatum without significant differences between the medial and lateral regions. Decortication induced no significant changes in peptide concentration in any of the striatal regions or in the respective crude synaptosomal (P2) fractions. However, decorticated animals showed a large degree of deafferentation as evidenced by a marked and significant decrease in [³H]glutamate uptake as well as in glutamate levels measured in striatal homogenates or in crude synaptosomal fractions. No changes in striatal dopamine levels were observed in lesioned animals. Thus, these findings are not in favor of the existence of corticostriatal projections arising from the sensorimotor cortex using NAAG as neurotransmitter.     

Rapid alterations in corticostriatal ensemble coordination during acute dopamine-dependent motor dysfunction

Dopaminergic dysregulation can cause motor dysfunction, but the mechanisms underlying dopamine-related motor disorders remain under debate. We used an inducible and reversible pharmacogenetic approach in dopamine transporter knockout mice to investigate the simultaneous activity of neuronal ensembles in the dorsolateral striatum and primary motor cortex during hyperdopaminergia ( approximately 500% of controls) with hyperkinesia, and after rapid and profound dopamine depletion (<0.2%) with akinesia in the same animal. Surprisingly, although most cortical and striatal neurons ( approximately 70%) changed firing rate during the transition between dopamine-related hyperkinesia and akinesia, the overall cortical firing rate remained unchanged. Conversely, neuronal oscillations and ensemble activity coordination within and between cortex and striatum did change rapidly between these periods. During hyperkinesia, corticostriatal activity became largely asynchronous, while during dopamine-depletion the synchronicity increased. Thus, dopamine-related disorders like Parkinson's disease may not stem from changes in the overall levels of cortical activity, but from dysfunctional activity coordination in corticostriatal circuits.     

Extracellular Striatal Dopamine and Glutamate After Decortication and Kainate Receptor Stimulation, as Measured by Microdialysis

Abstract: Disruption of corticostriatal glutamate input in the striatum decreased significantly extracellular striatal glutamate and dopamine levels. Local administration of 300 µ M concentration of excitatory receptor agonist kainic acid increased significantly extracellular striatal dopamine in intact freely moving rats. These findings support the hypothesis that glutamate exerts a tonic facilitatory effect on striatal dopamine release. The effect of kainic acid on extracellular striatal glutamate concentration in intact rats was a biphasic increase. The first glutamate increase can be explained by stimulation of presynaptic kainate receptors present on corticostriatal glutamatergic nerve terminals; the second increase is probably the result of a continuous interaction of the different striatal neurotransmitters after disturbance of their balance. Release of dopamine and glutamate was modulated differently in the intact striatum and in the striatum deprived of corticostriatal input. Dopamine release in the denervated striatum after kainate receptor stimulation was significantly lower than in intact striatum, confirming the so-called cooperativity between glutamate and kainic acid. Loss of presynaptic kainate receptors on the glutamatergic nerve terminals after decortication resulted in a loss of effect of kainic acid on glutamate release in denervated striatum. Aspartate showed no significant changes in this study.     

Aberrant Resting-State Corticostriatal Functional Connectivity in Cirrhotic Patients with Hyperintense Globus Pallidus on T1-Weighted MR Imaging

Neurobiological and neuroimaging studies have emphasized the structural and functional alterations in the striatum of cirrhotic patients, but alterations in the functional connections between the striatum and other brain regions have not yet been explored. Of note, manganese accumulation in the nervous system, frequently reflected by hyperintensity at the bilateral globus pallidus (GP) on T1-weighted imaging, has been considered a factor affecting the striatal and cortical functions in hepatic decompensation. We employed resting-state functional magnetic resonance imaging to analyze the temporal correlation between the striatum and the remaining brain regions using seed-based correlation analyses. The two-sample t-test was conducted to detect the differences in corticostriatal connectivity between 44 cirrhotic patients with hyperintensity at the bilateral GP and 20 healthy controls. Decreased connectivity of the caudate was detected in the anterior/middle cingulate gyrus, and increased connectivity of the caudate was found in the left motor cortex. A reduction in functional connectivity was found between the putamen and several regions, including the anterior cingulate gyrus, right insular lobe, inferior frontal gyrus, left parahippocampal gyrus, and anterior lobe of the right cerebellum; increased connectivity was detected between the putamen and right middle temporal gyrus. There were significant correlations between the corticostriatal connectivity and neuropsychological performances in the patient group, but not between the striatal connectivity and GP signal intensity. These alterations in the corticostriatal functional connectivity suggested the abnormalities in the intrinsic brain functional organiztion among the cirrhotic patients with manganese deposition, and may be associated with development of metabolic encephalopathy. The manganese deposition in nervous system, however, can not be an independent factor predicting the resting-state brain dysfunction in real time.