Cellular Quantification of Tyrosine Hydroxylase in the Rat Brain by Immunoautoradiography

Abstract: We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a ³⁵S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.     

Glutamate Decarboxylase Distribution in Discrete Motor Nuclei in the Cat Brain

Abstract: The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising γ-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.     

Differential effects after repeated treatment with haloperidol, clozapine, thioridazine and tefludazine on SNC and VTA dopamine neurones in rats

The effects of repeated treatment (21 days) with different antipsychotic compounds (haloperidol, clozapine, thioridazine and tefludazine) on dopamine (DA) neurones in substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) were studied in rats using single unit recording techniques. A dose-dependent decrease in the number of spontaneously active DA neurones in SNC and in VTA was observed with haloperidol. Clozapine showed no significant effect on the activity in SNC while a dose-dependent decrease in the number of active DA neurones in VTA was observed. Thioridazine showed no or weak effect in SNC while repeated treatment induced a marked inhibitory effect on the DA neurones in VTA. Tefludazine, a potential antipsychotic compound, induced a dose-dependent decrease in both SNC and VTA DA activity. However, the effect on the DA neurones in VTA was more pronounced at all doses. Since the classical neuroleptic haloperidol is equally effective in both regions, while the atypical neuroleptics clozapine and thioridazine have selective or predominant effect in the VTA area it has previously been thought that the inhibition of spontaneously active DA neurones in VTA should indicate an antipsychotic effect of a compound while the inhibition of DA neurones in SNC should account for the development of neurological side effects. The data suggests that the potential antipsychotic compound tefludazine should not induce neurological side effects at lower doses but still has an antipsychotic activity while repeated treatment with higher doses of tefludazine might cause extrapyramidal side effects.     

Atypical neuroleptic properties of l-stepholidine -Electrophysiological and behavioral studies

Intravenous administration of l-stepholidine (SPD), a dopamine (DA) receptor antagonist, in-creased the firing rate of DA neurons located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNC) in both anaesthetized and paralyzed rats. However, with the increase of dose, SPD selectively inhibited the fir-ing activity of DA neurons in the VTA but not in the SNC. The inhibition was reversed by the DA agonist apomor-phine (APO), suggesting that it may be via the mechanism of depolarization inactivation (DI). In rats, chronic admin-istration of SPD for 21 d dose-dependently decreased the number of spontaneously active DA neurons in the VTA, of which effect was reversed by APO (i. v. ). In contrast, the same treatment failed to affect the population of DA neu-rons in the SNC. Similarly, the acute treatment of SPD also decreased the number of spontaneously firing DA neurons in the VTA, but not in the SNC. SPD per se only induced very weak catalepsy. Its catalepsy which was not in pro-port     

Dopamine D-1 Receptor and Cyclic AMP-Dependent Phosphorylation in Parkinson''s Disease

D-1 and D-2 receptor densities, evaluated respectively by [3H]SCH 23390 and [3H]spiperone binding, and DARPP-32 (dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein-32K) concentrations, were studied in the brains of control and parkinsonian subjects postmortem. D-2 receptor density was unchanged in the putamen of parkinsonian patients. D-1 receptor density was unchanged in the putamen and substantia nigra pars reticulata (SNR) of parkinsonian patients, but decreased by 28% in the substantia nigra pars compacta (SNC). DARPP-32, which is localized in the same structures as D-1 receptors of which it is thought to represent the intracellular messenger, decreased by 45% in the putamen, 66% in the SNR, and 79% in the SNC. The decrease in D-1 receptors in the SNC may be due to degeneration of pallidonigral GABAergic neurons, but some of the D-1 receptors may be on the nigrostriatal dopaminergic neurons themselves. The dissociation between the alteration of D-1 receptor densities and DARPP-32 concentrations in both the striatum and substantia nigra, which are of the same order in the two structures, may be an index of functional hypoactivity of D-1 neurotransmission.     

ENZYMES ASSOCIATED WITH THE METABOLISM OF CATECHOLAMINES, ACETYLCHOLINE AND GABA IN HUMAN CONTROLS AND PATIENTS WITH PARKINSON''S DISEASE AND HUNTINGTON''S CHOREA

—Tyrosine hydroxylase (TH), dopa decarboxylase (DDC), glutamic acid decarboxylase (GAD), choline acetyltransferase (CAT), and acetylcholinesterase (AChE) were measured in 18–55 areas of brain from humans post mortem. Individuals meeting sudden and unexpected death (22), patients dying in hospital with non–neurological illness (6), Parkinson's disease (12), Huntington's chorea (8), terminal coma (6) or head injury (2) were included in the series. The absolute values obtained compared favourably with some previous human studies where high values for these enzymes were obtained, as well as with monkey and baboon data. The regional distributions of the enzymes were also comparable to those previously reported in human and animal studies. A number of important points with regard to human tissue seemed to emerge from the study. The mode of death was not a factor in enzyme levels in non–neurological and non-coma cases. Post mortem delay did not seem to be a major factor either even though a substantial decline in GAD, TH and DDC could be demonstrated in rats left several hours between sacrifice and removal of the brain for assay. Age had a highly significant effect in certain areas of brain. The decline typically followed a curvilinear pattern (activity = A/age + B with the sharpest drops being in the younger age groups). DDC seemed to be the enzyme most severely affected by age but all the enzymes showed declines in certain brain areas, while in other areas there was no significant decline. All the enzymes were very depressed by coma from illness except AChE. TH and DDC in the brain stem were, however, not affected in the head injury cases. The Parkinsonian cases showed a sharply decreased TH activity in the substantia nigra, caudate and putamen. There were decreases in GAD in the globus pallidus (GP) and substantia nigra with marginal decreases in the neostriatum. CAT levels in the extrapyramidal nuclei were normal. In Huntington's chorea there was a substantial decrease in GAD in all the extrapyramidal structures. There was a patchy loss of CAT in the neostriatum and locus coeruleus.     

Atypical neuroleptic properties ofl-stepholidine

Intravenous administration ofl-stepholidine (SPD), a dopamine (DA) receptor antagonist, increased the firing rate of DA neurons located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNC) in both anaesthetized and paralyzed rats. However, with the increase of dose, SPD selectively inhibited the firing activity of DA neurons in the VTA but not in the SNC. The inhibition was reversed by the DA agonist apomorphine (APO), suggesting that it may be via the mechanism of depolarization inactivation (DI). In rats, chronic adrninistration of SPD for 21 d dose-dependently decreased the number of spontaneously active DA neurons in the VTA, of which effect was reversed by APO (i. v.). In contrast, the same treatment failed to affect the population of DA neurons in the SNC. Similarly, the acute treatment of SPD also decreased the number of spontaneously firing DA neurons in the VTA, but not in the SNC. SPD per se only induced very weak catalepsy. Its catalepsy which was not in proportion to dosage was only observed in the dose range of 10–40 mg/kg and lasted 15 min. SPD effectively antagonized the APO (2 mg/kg, i. p.)-induced stereotypy.The above-mentioned results suggest that SPD selectively inactivates the DA neurons in the VTA not in the SNC. SPD may associate with a low incidence of extrapyramidal side-effects and may be ranked as a promising compound for searching for a new kind of atypical neuroleptics.     

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.     

Heterogeneous Distribution of Kir3 Potassium Channel Proteins Within Dopaminergic Neurons in the Mesencephalon of the Rat Brain

1. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of the ventral mesencephalon play an important role in the regulation of the parallel basal ganglia loops. 2. We have raised affinity-purified polyclonal rabbit antibodies specific for all four members of the Kir3 family of inwardly rectifying potassium channels (Kir3.1–Kir3.4) to investigate the distribution of the channel proteins in the dopaminergic neurons of the rat mesencephalon at light and electron microscopic level. In addition, immunocytochemical double labeling with tyrosine hydroxylase (TH), a marker of dopaminergic neurons, were performed. 3. All Kir3 channels were present in this region. However, the individual proteins showed differential cellular and subcellular distributions. 4. Kir3.1 immunoreactivity was found in SNc fibers and some neurons of the substantia nigra pars reticulata (SNr). Few Kir3.3-positive neurons were found in the SNc. However, a strong Kir3.3 signal was identified in the SNr neuropil. Weak Kir3.4 staining was detected in neuronal somata as well as in dendritic fibers of both parts of the SN. 5. In the VTA, Kir3.1, Kir3.3, and Kir3.4 showed only weak staining of neuropil structures. The distribution of the Kir3.2 channel protein was especially striking with strong labeling in the SNc and in the lateral but not central VTA. 6. Our results suggest that the heterogeneously distributed Kir3.2 channel proteins could help to discriminate the dopaminergic neurons of VTA and SNc.     

Regional differences in cofactor saturation of glutamate decarboxylase (GAD) in discrete brain nuclei of the rat

Glutamate decarboxylase (GAD) activities with and without added pyridoxal-5-phosphate were determined in discrete brain nuclei of freeze-dried samples. The distribution of GAD holoenzyme activity as well as the cofactor saturation, was found to be uneven in the discrete nuclei. In addition, it was found that repeated haloperidol treatment reduced GAD holoenzyme activity in the substantia nigra pars reticulata.     

DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE WITHIN THE SUBSTANTIA NIGRA AND IN OTHER BRAIN REGIONS FROM CONTROL AND PARKINSONIAN PATIENTS

—The distribution of choline acetyltransferase (ChAc, EC 2.3.1.6) and l -glutamate 1-carboxylyase (glutamate decarboxylase, GAD, EC 4.1.1.15) was studied in serial frontal slices of the substantia nigra (SN) (pars compacta, PC; pars reticulata, PR; an intermediate region, IR) as well as in other brain areas from post mortem tissue of control and Parkinsonian patients. Within the SN from control brain ChAc and GAD activities showed a distinctive distribution: ChAc activity in PC was higher than in PR and IR by 427% and 253% respectively and within PC the enzyme activity in the rostral part exceeded that in the control part by 353%. The GAD activity in PC was higher by 41% than that in PR and within PC seemed to be higher in the caudal than in the rostral part. For both enzyme activities there were no significant differences between PR and IR or within these regions. In Parkinsonian brain both ChAc and GAD activities were reduced to 15-25% of controls in all 3 regions of the SN. The distinctive distribution of ChAc and GAD activity found in the SN of control brain was abolished: no difference was observed between the 3 regions. However, within PC the ChAc activity was lower in the medial than in the rostral part. Since nigral ChAc is possibly located in interneurons, the decrease in enzyme activity may be connected with the cell loss observed in the SN of Parkinsonian brain. By contrast, nigral GAD is probably contained in terminals of strio-nigral neurons and the decrease in enzyme activity in Parkinson's disease in the absence of striatal cell loss, may reflect a change in the functional state of these GABA neurons. Among various areas of control brains ChAc activity was highest in caudate nucleus and putamen while GAD was highest in SN. caudate nucleus, putamen and cerebral cortex. In Parkinsonian brain the most severe reduction in ChAc and GAD activities was found in the SN.     

POST MORTEM STABILITY AND STORAGE IN THE COLD OF BRAIN ENZYMES

Tyrosine hydroxylase (TH), glutamate-decarboxylase (GAD) and choline acetyltransferase (CAT) were estimated in the striatum of rat brains kept at 20°C or 4°C for various periods of time up to 48 h after death. At 20°C TH and GAD activities decreased up to 4&50% of controls after 48 h; CAT activity was not affected. Maintenance of dead animals at 4°C completely (GAD and CAT) or partially (TH) prevented the decrease in enzyme activities. In a second series of experiments, TH, G A D and CAT activities were measured in striata (tissue or homogenate) stored immediately after death at different temperatures (4°C; -35°C; -70°C) for various time intervals up to 3 months. Storage of striata at 4°C induced a rapid decrease of all enzyme activities with time (GAD > CAT > TH). TH, GAD and CAT activities in striata kept at -35°C or -70°C were fairly stable. However, CAT activity was slightly decreased when the dissected striata were not homogenized; GAD activity was substantially reduced after 3 months at -35°C. Stability of TH, GAD and CAT activities were confirmed in homogenates of human caudate nucleus stored at -70°C for 1 month. If human enzymes behave similarly to the rat enzymes the following conclusions should be drawn: (1) brains should be obtained at autopsy within 8 h after death; (2) placement of dead bodies in the refrigerator should be done as soon as possible; (3) dissected brain structures (preferably as homogenates) should be stored at -70°C.     

Taurine Biosynthesis in Rat Brain In Vivo: Lack of Relationship with Cysteine Sulfinate Decarboxylase Glutamate Decarboxylase-Associated Activity (GAD/CSDII)

Two distinct forms of cysteine sulfinate decarboxylase (CSD), respectively, CSDI and CSDII, have already been separated in rat brain. One of them, CSDII, appeared to be closely associated with glutamate decarboxylase (GAD). We have investigated whether the taurine concentration in brain was dependent on CSDII activity in vivo. CSDI and CSDII activities were specifically measured in crude brain extracts after selective immunotrapping. After 4 days of chronic treatment of mice with gamma-acetylenic gamma-aminobutyric acid, a drastic and identical decrease in CSDII and GAD activities was observed in the brain. Taurine concentration and CSDI activities were not significantly altered. Following striato-nigral pathway lesioning in the rat brain, GAD and CSDII show an identical 80% decrease in the substantia nigra. In contrast, CSDI activity and taurine concentration in the substantia nigra were similarly but only slightly affected with an about 30% decrease. Our results provide further evidence that GAD and CSDII are indeed the same enzyme. They show that CSDII does not play any role in the biosynthesis of taurine in vivo. Our findings suggest that CSDI might be the biosynthetic enzyme for taurine in vivo and that there might be some endings projecting into the substantia nigra that contain CSDI and taurine.     

The localization of GABA-Transaminase in the striato-nigral system

The localization of gamma-aminobutyric acid transaminase (GABA-T), the degrading enzyme for γ-aminobutyric acid, was examined in the striatum and substantia nigra using biochemical techniques. Selective destruction of the nigrostriatal dopaminergic system with 6-hydroxydopamine had no effect on the activity of GABA-T in either the striatum or the substantia nigra, although striatal tyrosine hydroxylase activity was reduced by half. Intrastriatal injection of kainic acid in adult rats resulted in a significant dose-dependent decrease in GABA-T activity in both the striatum and the substantia nigra. The decrease in both of these regions was significantly correlated with the decrease in the GABA synthetic enzyme glutamate decarboxylase (GAD). The intrastriatal injection of kainic acid in ten day old rats did not affect striatal GAD or GABA-T activities, although striatal choline acetyl-transferase activity was reduced by half.It is concluded that the GABA-T activity in the striatum is predominantly localized in neuronal elements, although not, apparently, in cholinergic neurons. Some GABA-T activity is also present in the terminals of the striatonigral neurons. However, the dopaminergic nigrostriatal neurons do not appear to contain GABA-T. It is suggested that high GABA-T activity may be characteristic of GABA neurons.     

SLC35D3 increases autophagic activity in midbrain dopaminergic neurons by enhancing BECN1-ATG14-PIK3C3 complex formation

Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons.     

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Abstract: Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17β-estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17β-estradiol (10 μg/0.2 ml), progesterone (0.72 mg/0.2 ml). 17β-estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [³H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 ± 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16-23%. Chronic treatment of ovariectomized rats with 17β-estradiol progesterone, or their combination increased to the same extent and uniformly [³H]-GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17β-estradiol and/ or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.     

甲基苯丙胺中毒大鼠相关脑区多巴胺能神经毒性研究

目的：探讨MA中毒多巴胺能神经毒性的损伤机制。方法：将Wistar大鼠40只,随机分成对照组10只和实验组30只（实验组分成三个亚组,分为末次给药后1天组、4天组和7天组,n=10）。实验组给予20mg/kg的MA腹腔注射,对照组给予同样剂量的生理盐水,每天注射一次,注射时间为20：00,连续注射4天。分别于末次给药后1天,7天,14天处死实验大鼠,用免疫组织化学染色法（S-P法）和荧光分光光度计法检测大鼠中脑黑质致密区（SNC）、中脑腹侧被盖区（VTA）、前额叶皮质（PFC）以及纹状体（CPu）四个脑区的多巴胺神经元细胞的形态和数量的变化,对神经纤维进行灰度值分析。结果：1、黑质致密区和腹侧被盖区TH阳性细胞图像分析结果与细胞计数分析结果一致：与对照组相比,各实验组TH免疫反应阳性降低,差异具显著性（P〈0.05）,d1组开始降低（P〈0.05）,d7组达到低谷（P〈0.01）,d14天组黑质致密区和腹侧被盖区TH免疫反应阳性有不同程度的恢复（P〈0.05）。2、纹状体和前额叶皮质TH阳性纤维图像定量分析结果：各实验组TH免疫反应阳性均减低（P〈0.05）,d7组阳性反应最弱（P〈0.01）,d14组仍未恢复（P〈0.05）。3、黑质致密区、腹侧被盖区、纹状体及前额叶皮质荧光分光光度计检测DA递质含量结果：与上述免疫组化结果基本一致。结论：1、大鼠各脑区TH阳性表达和DA含量,均出现不同程度的减低。2、MA中毒大鼠各脑区DA递质含量的变化与TH的变化结果基本一致。     

Short- and Long-Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive-Induced Seizures

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.