Unilateral 6-hydroxydopamine lesion of dopamine neurons and subchronic L-DOPA administration in the adult rat alters the expression of the vesicular GABA transporter in different subsets of striatal neurons and in the substantia nigra, pars reticulata

The loss of dopamine neurons combined or not with the subsequent administration of L-DOPA in patients with Parkinson's disease or in experimental models of the disease results in altered GABAergic signaling throughout the basal ganglia, including the striatum and the substantia nigra, pars reticulata. However, the molecular mechanisms involved in altered GABA neurotransmission remain poorly understood. In order to be released from synaptic vesicles, newly synthesized GABA is transported from the cytosol into synaptic vesicles by a vesicular GABA transporter. The objective of this study was to examine the hypothesis that expression of the vesicular GABA transporter (vGAT) is altered in the unilateral 6-hydroxydopamine model of Parkinson's disease. Our results provide evidence that a unilateral 6-hydroxydopamine lesion results in increased and decreased vGAT mRNA levels in striatopallidal and striatonigral neurons, respectively. These two subsets of neurons were identified by the co-expression or lack of co-expression of preproenkephalin, a marker of striatopallidal neurons, using double-labeling in situ hybridization histochemistry. Such changes occurred in the striatum ipsilateral to the 6-hydroxydopamine lesion and were paralleled by decreased vGAT protein levels in the substantia nigra, pars reticulate (SNr). On the other hand, the subchronic systemic administration of L-DOPA increased vGAT mRNA levels in preproenkephalin-negative neurons on the side ipsilateral and, to a lesser extent, the side contralateral to the 6-hydroxydopamine lesion. Systemic L-DOPA also increased vGAT protein levels in the ipsi- and contralateral SNr. As a whole, the results provide original evidence that vGAT expression is altered in the 6-hydroxydopamine model of Parkinson's disease. They also suggest that the behavioral effects induced by a subchronic administration of L-DOPA to 6-hydroxydopamine-lesioned rats involve an increase in the vesicular release of GABA by striatonigral neurons.     

Striatal dopamine and glutamate release: effects of intranigral injections of substance P

The levels of extracellular striatal dopamine and glutamate were measured simultaneously in halothane-anaesthetized rats using microdialysis. Unilateral injections of substance P (0.07 nmol) into the substantia nigra, pars reticulata enhanced the levels of dopamine and glutamate in the ipsilateral striatum. Intranigral injections of neurokinin A (0.09 nmol) enhanced the levels of striatal dopamine, and intranigral injections of γ-aminobutyric acid (300 nmol) or dynorphin A (0.5 nmol) decreased the levels of striatal dopamine, but none of these had any effect on the levels of striatal glutamate. Local perfusion with the dopamine agonists apomorphine (D1/D2), SKF 38393 (DI) or pergolide (D2) (each at 10^-5 M) decreased the levels of striatal dopamine and enhanced the levels of striatal glutamate. In unilateral 6-hydroxydopamine-lesioned rats, basal striatal glutamate levels were decreased bilaterally. Furthermore, on the denervated side intranigral substance P stimulation of striatal glutamate levels was enhanced, while on the intact side intranigral substance P stimulation of striatal dopamine and glutamate levels was similar to that seen in normal rats. These findings suggest that striatonigral substance P provides a stimulatory regulation of ipsilateral striatal glutamate release. Furthermore, it is indicated that striatal glutamate release can also be regulated by dopamine terminals.     

Striatal dopamine and glutamate release: effects of intranigral injections of substance P.

The levels of extracellular striatal dopamine and glutamate were measured simultaneously in halothane-anaesthetized rats using microdialysis. Unilateral injections of substance P (0.07 nmol) into the substantia nigra, pars reticulata enhanced the levels of dopamine and glutamate in the ipsilateral striatum. Intranigral injections of neurokinin A (0.09 nmol) enhanced the levels of striatal dopamine, and intranigral injections of gamma-aminobutyric acid (300 nmol) or dynorphin A (0.5 nmol) decreased the levels of striatal dopamine, but none of these had any effect on the levels of striatal glutamate. Local perfusion with the dopamine agonists apomorphine (D1/D2), SKF 38393 (D1) or pergolide (D2) (each at 10(5) M) decreased the levels of striatal dopamine and enhanced the levels of striatal glutamate. In unilateral 6-hydroxydopamine-lesioned rats, basal striatal glutamate levels were decreased bilaterally. Furthermore, on the denervated side intranigral substance P stimulation of striatal glutamate levels was enhanced, while on the intact side intranigral substance P stimulation of striatal dopamine and glutamate levels was similar to that seen in normal rats. These findings suggest that striatonigral substance P provides a stimulatory regulation of ipsilateral striatal glutamate release. Furthermore, it is indicated that striatal glutamate release can also be regulated by dopamine terminals.     

Evidence that neurokinin A (substance K) neurons project from the striatum to the substantia nigra in rats

The striatonigral neurokinin A (NKA) pathway in rats was examined with an antiserum specific to the N-terminal region of NKA. Thermal lesions of the striatum decreased both substance P-like immunoreactivity (SP-LI) and NKA-like immunoreactivity (NKA-LI) in the substantia nigra (SN). The releases of SP-LI and NKA-LI in the SN were measured by a push pull superfusion technique. Addition of 50 mM K+ to the superfusion medium increased the releases of both SP-LI and NKA-LI to 1.5-2.0 times the basal values. These results suggest that NKA neurons as well as SP neurons in the striatum project to the SN in rats.     

Fast sodium channel dependency of the somatodendritic release of dopamine in the rat's brain.

Somatodendritic release of dopamine (DA) was studied by microdialysis. The basal release of endogenous extracellular DA in the rat's substantia nigra was 1.0 +/- 0.14 fmol/min (mean +/- S.E.M.; n = 6). Compared with the basal extracellular level of DA found in the striatum (12.9 +/- 0.94; n = 12), the former value was about 13 times smaller than the latter value. The addition to the Ringer solution of the fast sodium channel inhibitor, tetrodotoxin (TTX), at a concentration of 1 microM, produced the total disappearance of DA in the substantia nigra. When TTX was removed from the Ringer solution, the DA extracellular level came back slowly to the control value. This result suggests that the somatodendritic basal endogenous DA release measured by microdialysis is dependent on the sodium channel conductance.     

Synaptosomal uptake and release of dopamine in substantia nigra: effects of gamma-aminobutyric acid and substance P.

Nigral tissue prepared as synaptosomes demonstrates both high and low affinity uptake of [3H]dopamine. Recently accumulated [3H]dopamine is releasable by 35 mN K+. Substance P increases both uptake and release of dopamine by nigral synaptosomes; gamma-aminobutyric acid (GABA) inhibits release with no effect on uptake at concentrations less than 10-(4) M. In the striatum, substance P inhibits both uptake and release of dopamine. The results support the existence of dopamine-containing terminals in substantia nigra tissue. The differences in response to substance P and GABA found between nigra and striatum may reflect structural differences in dopamine-containing processes in these areas, related to their proposed origin as dendtritic (substantia nigra) and axonal (striatal) terminal.     

Influence of R-type (Cav2.3) and t-type (Cav3.1-3.3) antagonists on nigral somatodendritic dopamine release measured by microdialysis

The release of dopamine from soma and dendrites of dopaminergic neurons in substantia nigra has been reported to be calcium-dependent, but it remains to be determined which calcium channels mediate this effect. We have used in vivo microdialysis in rat substantia nigra and striatum to investigate the effect of Ca(v)3.1-3.3 (T-type) and Ca(v)2.3 (R-type) calcium channel antagonists on somatodendritic and terminal dopamine release. Local reverse dialysis administration of 0.1-10 microM of the Ca(v)2.3 inhibitor SNX-482, or 100 microM of mibefradil, decreased the concentrations of dopamine and its metabolites in dialysate from substantia nigra, whereas 1 microM mibefradil or 40-80 microM nickel(II) induced an increase in nigral dialysate dopamine concentrations. Dopamine concentrations in striatal dialysates were decreased only by 10 microM of SNX-482 or 100 microM of mibefradil. Nickel(II) induced an increase in striatal dialysate dopamine concentration similar to that in substantia nigra. The results indicate a role for Ca(v)2.3 (R-type) voltage sensitive calcium channels in the calcium dependency of somatodendritic dopamine release, but argue against a calcium dependency mediated substantially by Ca(v)3.1-3.3 (T-type) channels.     

Depletion in substance P- and neurokinin A-like immunoreactivity in substantia nigra after ibotenate-induced lesions of striatum

Changes in substance P (SP)- and neurokinin A (NKA)-like immunoreactivity (LI) in the rat substantia nigra (SN) after striatal lesion produced by ibotenate were studied using immunohistochemical methods. Compared to the SN on the control side, both SP- and NKA-LI were dramatically depleted in the SN on the lesion side. These findings are consistent with the existence of SP- and/or NKA-containing neurons in the striatum projecting to the SN.     

Aspartate, glutamate and GABA levels in pallidum, substantia nigra, center median and dorsal raphe nuclei after cylindric lesion of caudate nucleus in cat

Aspartate, glutamate and GABA levels were determined in afferent and efferent projection nuclei of the striatum after unilateral cylindric lesion in the head of the caudate nucleus in cats. Two and four weeks after operation, GABA content was significantly reduced in substantia nigra and pallidum ipsilateral to the lesion. Glutamate (GLU) level was decreased in substantia nigra and pallidum only 4 weeks after lesion, whereas aspartate content in substantia nigra decreased significantly already after 2 weeks. No changes in the contents of these amino acids were detected in the dorsal raphe nucleus, which receives a projection from the caudate, as well as in the center median nucleus, which projects to the striatum. These experiments using longer survival times substantiate the role of GABA in caudato-pallidal and caudatonigral projections. The possibility is discussed that aspartate (ASP) could function as transmitter of cortico-nigral fibers.     

Nigral neurotensin receptor regulation of nigral glutamate and nigroventral thalamic GABA transmission: a dual-probe microdialysis study in intact conscious rat brain

Dual-probe microdialysis in the awake rat was employed to investigate the effects of intranigral perfusion with the tridecapeptide neurotensin on local dialysate glutamate and GABA levels in the substantia nigra pars reticulata and on dialysate GABA levels in the ventral thalamus. Intranigral neurotensin (10-300nM, 60min) dose-dependently increased (+29+/-3% and +46+/-3% vs basal for the 100 and 300nM concentrations, respectively) local dialysate glutamate levels, while the highest 300nM concentration of the peptide exerted a long-lasting and prolonged reduction in both local and ventral thalamic (-20+/-4% and -22+/-2%, respectively) GABA levels. Intranigral perfusion with the inactive neurotensin fragment neurotensin(1-7) (10-300nM, 60min) was without effect. Furthermore, the non-peptide neurotensin receptor antagonist SR 48692 (0.2mg/kg) and tetrodotoxin (1microM) fully counteracted the intranigral neurotensin (300nM)-induced increase in local glutamate. SR 48692 (0.2mg/kg) also counteracted the decreases in nigral and ventral thalamic GABA release induced by the peptide. In addition, intranigral perfusion with the dopamine D(2) receptor antagonist raclopride (1microM) fully antagonized the neurotensin (300nM)-induced decreases in nigral and ventral thalamic GABA levels. The ability of nigral neurotensin receptor activation to differently influence glutamate and GABA levels, whereby it increases nigral glutamate and decreases both nigral and ventral thalamic GABA levels, suggests the involvement of neurotensin receptor in the regulation of basal ganglia output at the level of the nigra.     

Relative involvement of globus pallidus and subthalamic nucleus in the regulation of somatodendritic dopamine release in substantia nigra is dopamine-dependent

Previously, we have shown that GABA(A) receptors and glutamate receptors in substantia nigra play distinct roles in the regulation of somatodendritic dopamine release. GABAergic input to substantia nigra was found to be the primary determinant of the level of spontaneous somatodendritic dopamine release. In contrast, acute blockade of dopamine receptors by systemic haloperidol administration produced an increase in somatodendritic dopamine release in substantia nigra that was found to be dependent exclusively upon activation of nigral glutamate receptors. The focus of the present study was to identify anatomical structures that may participate in the differential regulation of somatodendritic dopamine release by GABA and glutamate under these two conditions. To this end, we pharmacologically inhibited the activity of either globus pallidus or subthalamic nucleus using microinfusion of the GABA(A) receptor agonist muscimol. The effects of these manipulations on spontaneous efflux of somatodendritic dopamine and on increases in this measure produced by systemic haloperidol administration were determined in ipsilateral substantia nigra using in vivo microdialysis. As observed previously, administration of haloperidol (0.5 mg/kg, i.p.) significantly increased extracellular dopamine in substantia nigra. Microinfusion of muscimol (400 ng/200 nl) into globus pallidus also produced a significant increase in somatodendritic dopamine efflux. When haloperidol was administered systemically in conjunction with microinfusion of muscimol into globus pallidus, an increase in nigral dopamine efflux was observed that was significantly greater than that which was produced singly by muscimol microinfusion into globus pallidus or by systemic haloperidol administration. The additive nature of the increases in somatodendritic dopamine release produced by these two manipulations indicates that independent neural circuitries may be involved. Inactivation of subthalamic nucleus by microinfusion of muscimol (200 ng/100 nl) had no effect on spontaneous somatodendritic dopamine efflux. Muscimol application into subthalamic nucleus, however, completely abolished the stimulatory effect of systemic haloperidol on dendritic dopamine efflux in substantia nigra.The present data extend our previous findings by demonstrating: 1) an important involvement of globus pallidus efferents in the GABAergic regulation of somatodendritic dopamine efflux in substantia nigra under normal conditions and, 2) an emergent predominant role of subthalamic nucleus efferents in the glutamate-dependent increase in somatodendritic dopamine efflux observed after systemic haloperidol administration. Thus, the relative influence of globus pallidus and subthalamic nucleus in the determination of the level of somatodendritic dopamine release in substantia nigra qualitatively varies as a function of dopamine receptor blockade. These findings are relevant to current models of basal ganglia function under both normal and pathological conditions, e.g. Parkinson's disease.     

Effects of GABA and L-glutamic acid on the potassium-evoked in vitro release of substance P- and neurokinin A-like immunoreactivities are different in the rat striatum and substantia nigra

Slices of the rat substantia nigra and striatum were superfused in vitro to measure release of tachykinins (TKs). Potassium (30 and 60 mM) infusion caused a 3- to 10-fold outflow of both substance P-like immunoreactivity (SP-LI) and neurokinin A-like immunoreactivity (NKA-LI) in the substantia nigra as well as in the striatum as measured by radioimmunoassay. The potassium-evoked release of SP-LI and NKA-LI was significantly, but not completely (by 25-70%) inhibited by simultaneous perfusion with L-glutamic acid (50 microM) and gamma-aminobutyric acid (GABA, 50 microM) in the substantia nigra. No significant inhibition was, however, observed in the striatum. The present data indicate a differential regulation of tachykinins in the striatum and substantia nigra by L-glutamic acid and GABA. The presynaptic regulation of TK release may therefore differ in the dendritic and terminal region of the striatonigral pathway.     

帕金森病大鼠模型纹状体中谷氨酸、γ-氨基丁酸与多巴胺之间的关系

目的:了解帕金森病大鼠模型纹状体内谷氨酸(glutamate,Glu)、γ-氨基丁酸(gama-aminobutyric acid,GABA)和多巴胺(dopamine,DA)之间的关系,从而进一步探讨帕金森病的发病机制。方法:动物分为溶剂对照组、假手术组和帕金森模型组。大脑右侧黑质致密部和前脑内侧束两点注射6-羟基多巴胺(6-hydroxydopamine,6-OHDA)建立帕金森病大鼠模型,溶剂对照组注入生理盐水,假手术组不注射任何药物,采用脑微透析术于建模后第3,4,5,6周连续动态透析大鼠毁损侧纹状体,结合高效液相色谱(HPLC)动态监测各组谷氨酸、GABA和多巴胺的变化。结果:(1)PD组纹状体内多巴胺含量到第5周仅为溶剂对照组和假手术组的1/5;(2)谷氨酸含量随建模时间逐渐升高,到第6周PD组是溶剂对照组和假手术组的1倍以上;(3)GABA含量呈下降趋势,到第6周约降至溶剂对照组、假手术组的1/2。结论:帕金森病大鼠模型纹状体内谷氨酸的变化与多巴胺分泌可能存在某种联系;GABA含量随建模时间的增加而下降。     

Striatal grafts in rats with unilateral neostriatal lesions--II. In vivo monitoring of GABA release in globus pallidus and substantia nigra

GABA release was recorded in vivo by push-pull perfusion from the globus pallidus and substantia nigra of control rats, rats with unilateral ibotenic acid lesions of the neostriatum, and rats with embryonic striatal tissue grafts implanted in the lesioned striatum. The lesions reduced baseline levels of GABA release to 5% of control levels in the globus pallidus and to 13% of control levels in the substantia nigra pars reticulata. GABA release was substantially restored in both the globus pallidus and substantia nigra of the grafted rats, to 34 and 60%, respectively. Peripheral injection of the dopaminergic stimulant methamphetamine induced a short (lasting approximately 20 min) 4-5 fold increase in GABA release in the intact globus pallidus and a longer (lasting longer than 80 min) increase in the substantia nigra. The stimulatory effect of methamphetamine on GABA release was completely abolished in both sites by the strial lesions, suggesting that the effect was mediated via a direct or indirect dopaminergic action on striatal output neurons. The grafts reinstated methamphetamine-induced stimulation of GABA release in striatal output targets to a level (as a proportion of baseline) that was similar to that seen in the control rats. The results support the view that activation of the dopaminergic inputs to the striatum is functionally excitatory on the major striatal output projections to the globus pallidus and substantia nigra pars reticulata. The results also support the hypothesis that striatal grafts have the capacity to become functionally incorporated by reciprocal graft-host connections into the neural circuitry of the host brain.     

γ-Aminobutyric acid infusion in substantia nigra pars reticulata in rats inhibits ascorbate release in ipsilateral striatum

A relatively high level of extracellular ascorbate in the striatum, which is known to modulate impulse flow in striatal neurons, originates primarily from glutamate-containing corticostriatal afferents. Increasing evidence suggests that ascorbate release from these fibers is regulated by a multisynaptic loop that includes γ-aminobutyric acid (GABA) mechanisms in the substantia nigra. To assess the role that nigral GABA plays in striatal ascorbate release, extracellular ascorbate was monitored voltammetrically in the striatum during infusions of GABA into the substantia nigra pars reticulata (SNr) of awake, unrestrained rats. Compared to vehicle infusions, intranigral GABA lowered striatal ascorbate by >50%. In contrast, intranigral application of picrotoxin, a GABA antagonist, had the opposite effect. Neither GABA nor picrotoxin altered striatal 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite. Collectively, these results indicate that intranigral GABA exerts a tonic inhibitory influence on ascorbate release in the striatum.     

Estrogen rapidly potentiates amphetamine-induced striatal dopamine release and rotational behavior during microdialysis

Ovariectomy (OVX) of female rats results in a decreased behavioral response to stimulation of the mesostriatal dopamine (DA) system and decreased striatal DA release in vitro. Estrogen replacement restores both behavioral and neurochemical responsiveness. In this report, microdialysis in freely moving rats is used to simultaneously study the behavioral and neurochemical effects of systemic estradiol. OVX rats received a unilateral 6-hydroxy-dopamine lesion of the substantia nigra and then underwent microdialysis of the intact striatum. Thirty min after a single injection of 5 micrograms estradiol benzoate, amphetamine-induced rotational behavior and striatal DA release are both potentiated, relative to the response of oil-treated control animals.     

Iron accumulation in the striatum predicts aging-related decline in motor function in rhesus monkeys

Changes in the nigrostriatal system may be involved with the motor abnormalities seen in aging. These perturbations include alterations in dopamine (DA) release, regulation and transport in the striatum and substantia nigra, striatal atrophy and elevated iron levels in the basal ganglia. However, the relative contribution of these changes to the motor deficits seen in aging is unclear. Thus, using the rhesus monkey as a model, the present study was designed to examine several of these key alterations in the basal ganglia in order to help elucidate the mechanisms contributing to age-related motor decline. First, 32 female rhesus monkeys ranging from 4 to 32 years old were evaluated for their motor capabilities using an automated hand-retrieval task. Second, non-invasive MRI methods were used to estimate brain composition and to indirectly measure relative iron content in the striatum and substantia nigra. Third, in vivo microdialysis was used to evaluate basal and stimulus-evoked levels of DA and its metabolites in the striatum and substantia nigra of the same monkeys. Our results demonstrated significant decreases in motor performance, decreases in striatal DA release, and increases in striatal iron levels in rhesus monkeys as they age from young adulthood. A comprehensive statistical analysis relating age, motor performance, DA release, and iron content indicated that the best predictor of decreases in motor ability, above and beyond levels of performance that could be explained by age alone, was iron accumulation in the striatum. This suggests that striatal iron levels may be a biomarker of motor dysfunction in aging; and as such, can be monitored non-invasively by longitudinal brain MRI scans. The results also suggest that treatments aimed at reducing accumulation of excess iron in the striatum during normal aging may have beneficial effects on age-related deterioration of motor performance.     

Dopamine release from the rat substantia nigra in vitro. Effect of raphe lesions and veratridine stimulation

In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [³H]dopamine from the partially denervated substantia nigra was determined in vitro and found to be very similar to the release observed from slices of control substantia nigra. These results lend further support to the theory that the release of exogenously applied [³H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5-hydroxytryptamine-containing neurons.A veratridine-induced release of [³H]dopamine from the pars reticulata of the substantia nigra is also described. An almost complete blockade of veratridine (3.0 μM) stimulation was observed with 100 nM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [³H]dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.     

Extracellular levels of amino acids in striatum and globus pallidus of 6-hydroxydopamine-lesioned rats measured with microdialysis

Extracellular aspartate, glutamate, glutamine, taurine and GABA concentrations were measured by microdialysis in the rat striatum and globus pallidus after a unilateral 6-hydroxydopamine lesion of the dopamine system. The basal and potassium-evoked overflow of GABA was increased in the ipsilateral striatum, but the evoked overflow was decreased in both contralateral striatum and pallidum. Both basal and evoked overflow of glutamate was increased in ipsilateral striatum. The basal overflow of aspartate was significantly increased in the ipsilateral side. Basal glutamine on the other hand was decreased in the ipsilateral side. Taurine remained unchanged in both regions. These results suggest that dopamine is involved in the regulation of transmission by GABA and glutamate. Since glutamine might be the precursor to glutamate, the change in glutamate might affect the glutamine level. The changed aspartate level has no obvious explanation.     

Oxidative stress and dopamine depletion in an intrastriatal 6-hydroxydopamine model of Parkinson's disease

Although the etiology of Parkinson's disease (PD) is unknown, a common element of most theories is the involvement of oxidative stress, either as a cause or effect of the disease. There have been relatively few studies that have characterized oxidative stress in animal models of PD. In the present study a 6-hydroxydopamine (6-OHDA) rodent model of PD was used to investigate the in vivo production of oxidative stress after administration of the neurotoxin. 6-OHDA was injected into the striatum of young adult rats and the production of protein carbonyls and 4-hydroxynonenal (HNE) was measured at 1, 3, 7, and 14 days after administration. A significant increase in both markers was found in the striatum 1 day after neurotoxin administration, and this increase declined to basal levels by day 7. There was no significant increase found in the substantia nigra at any of the time points investigated. This same lesion paradigm produced dopamine depletions of 90-95% in the striatum and 63-80% in the substantia nigra by 14-28 days post-6-OHDA. Protein carbonyl and HNE levels were also measured in middle-aged and aged animals 1 day after striatal 6-OHDA. Both protein carbonyl and HNE levels were increased in the striatum of middle-aged and aged animals treated with 6-OHDA, but the increases were not as great as those observed in the young adult animals. Similar to the young animals, there were no increases in either marker in the substantia nigra of the middle-aged and aged animals. There was a trend for an age-dependent increase in basal amounts of oxidative stress markers when comparing the non-lesioned side of the brains of the three age groups. These results support that an early event in the course of dopamine depletion following intrastriatal 6-OHDA administration is the generation of oxidative stress.