Direct Transfer into Nitrocellulose and Quantitative Radioautographic Anatomical Determination of Brain Tyrosine Hydroxylase Protein Concentration

An improved quantitative immunochemical determination of brain tyrosine hydroxylase (TH) concentrations was designed using direct transfer into nitrocellulose from 20-microns thick brain sections, followed by immunodetection and quantitative radioautography in three reference brain structures (locus ceruleus, substantia nigra, and ventral tegmental area). Results obtained by this methodology were similar to those obtained after extraction and Western blotting of the TH protein in control and reserpine-treated animals. Moreover, this methodology allows the combination of high sensitivity and high anatomical resolution in the study of the distribution of pharmacological effects. The locus ceruleus exhibited a significant posteroanterior distribution of TH protein concentration in control and reserpine-treated animals.     

Long-Term Effects of RU24722 on Tyrosine Hydroxylase of the Rat Brain

The effects of RU24722 (14,15-dihydro-20,21-dinoreburnamine-14-ol) on tyrosine hydroxylase in central catecholaminergic neurons were studied in rats treated with different quantities of the molecule, and a time course was done for the minimal dose that gave the maximal effect. RU24722 induced increases in tyrosine hydroxylase activities and specific protein content in noradrenergic cells of the locus ceruleus and decreased all these parameters in dopaminergic neurons of the substantia nigra and ventral tegmental area. The results pointed out that the specific activity of newly synthesized tyrosine hydroxylase in the loci cerulei was potentially greater but was not expressed "in vivo" except 7 days after injection. The phenotypic specificity and the time course pattern of the action could be considered as a consequence of an induction mechanism. The comparison of long-term change in tyrosine hydroxylase values after piperoxane, RU24722, clonidine, and combined RU24722-clonidine treatment demonstrated that an activation during a few hours did not induce tyrosine hydroxylase in central noradrenergic neurons. Clonidine antagonized the activating effect of RU24722 following its injection but did not affect its long-term induction properties.     

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.     

Similar Time Course Changes in Striatal Levels of Glutamic Acid Decarboxylase and Proenkephalin mRNA Following Dopaminergic Deafferentation in the Rat

An immunoblot procedure was developed to quantify the amount of tyrosine hydroxylase protein in homogenate of small brain regions. With the use of this method we have studied the variations in tyrosine hydroxylase activity and protein levels in some catecholaminergic neurons at different times following a single reserpine injection (10 mg/kg s.c.) and reevaluated the anatomical specificity of tyrosine hydroxylase induction by this drug. Reserpine administration provoked a long-lasting increase in both tyrosine hydroxylase activity and protein levels within locus ceruleus neurons. This effect culminated at day 4 after injection. At this time, the enzyme activity and protein levels in treated animals were respectively 2.7 and 2.6 times that measured in vehicle-treated animals. Both parameters varied in parallel so that tyrosine hydroxylase specific activity did not change over time. In contrast, reserpine did not cause any changes in tyrosine hydroxylase activity in the dopaminergic neurons of the substantia nigra, but provoked a moderate increase in tyrosine hydroxylase protein level. This latter effect was maximal (1.5 times) 4 days after treatment. In the adjacent dopaminergic area, i.e., the ventral tegmental area, a small decrease in the enzyme activity was recorded at day 2 without any significant change in the level of the protein. In conclusion, first, our data show the capacity of our method to assay tyrosine hydroxylase protein amounts in small brain catecholaminergic nuclei. Second, our results confirm and extend previous studies on the effect of reserpine on the regulation of tyrosine hydroxylase level within brain noradrenergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Micro topography of Tyrosine Hydroxylase, Glutamic Acid Decarboxylase, and Choline Acetyltransferase in the Substantia Nigra and Ventral Tegmental Area of Control and Parkinsonian Brains

Tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD), and choline acetyl transferase (CAT) were used as markers for catecholamine, gamma-aminobutyric acid, and acetylcholine containing neurons in human mesencephalon. Their rostrocaudal, mediolateral, and dorsoventral distribution was investigated within the substantia nigra pars compacta (SNC) and pars reticulata (SNR) and in the ventral tegmental area (VTA). TH activity was highest in the caudal, medial, and ventral SNC and in the middle of VTA medio-ventrally. The enzyme activity in SNR was low and uniformly distributed. In SNC as well as SNR, GAD activity was high and greater laterally and in the middle of the rostro-caudal extent. No particular pattern of distribution was observed in VTA. an area with low GAD content. In the substantia nigra, CAT activity was low. A characteristic medio-ventral distribution with a peak of high enzyme activity in the middle of the rostrocaudal extent was observed. In VTA, enzyme levels were high and also concentrated medio-ventrally and in the middle of the area. In parkinsonian brains, the distribution of TH was uniformly affected throughout the rostro-caudal extent. In VTA the enzyme activity was not as reduced as in SNC and SNR; the CAT pattern was only disrupted in a very localized part of SNC but not in SNR and VTA. In all three areas, GAD activity was reduced to a uniformly low distribution.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

Morphine and Cocaine Exert Common Chronic Actions on Tyrosine Hydroxylase in Dopaminergic Brain Reward Regions

We studied levels of tyrosine hydroxylase immunoreactivity and phosphorylation state in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in an effort to understand better the mechanisms by which these brain reward regions are influenced by opiates and cocaine. In the VTA, chronic, but not acute, administration of either morphine or cocaine increased levels of tyrosine hydroxylase immunoreactivity by 30-40%, with no change observed in the relative phosphorylation state of the enzyme. In the NAc, chronic, but not acute, morphine and cocaine treatments decreased the phosphorylation state of tyrosine hydroxylase, without a change in its total amount. In contrast, morphine and cocaine did not regulate tyrosine hydroxylase in the substantia nigra or caudate/putamen, brain regions generally not implicated in drug reward. Morphine and cocaine regulation of tyrosine hydroxylase could represent part of a common biochemical basis of morphine and cocaine addiction and craving.     

A Transgenic Mouse Model to Study Transsynaptic Regulation of Tyrosine Hydroxylase Gene Expression

Abstract: Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5' flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type-specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity-dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down-regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg, s.c.), which up-regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X-gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine-treated transgenic animals. These data showed that the 9-kb sequence also mediates cell type-specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.     

Further Characterization of the Long-Term Effect of RU24722 on Tyrosine Hydroxylase in the Rat Locus Coeruleus

Abstract: Recent data have indicated that the long-lasting increase in tyrosine hydroxylase (TH) protein could be differently expressed in the anterior and posterior locus coeruleus (LC) after a single intraperitoneal injection of RU24722, which has been proposed as a potent activator of catecholaminergic systems. In the present study, we have evaluated the dose and time course responses and the effect of a repeated treatment with RU24722 at 3-day intervals on TH protein level in the anterior and posterior rat LC. The results showed that RU24722 induces a long-lasting increase of TH protein level in the anterior and posterior LC that was maximal 3 days following a single injection of 30 mg/kg. The increase in TH protein was maintained at a constant level after repeated administrations of RU24722 at 3-day intervals. Furthermore, we have investigated whether the effect of the drug on TH protein could be modulated via several hormonal systems. The long-term increase of TH steady-state content after RU24722 was still observed 15 days after castration, adrenalectomy, hypophysectomy, and thyroidectomy. The initial steady-state TH protein level was significantly higher in the anterior LC of thyroid- or hypophysectomized and in the posterior LC of hypophysectomized rats. However, this increase was reversed when animals were housed at 28°C.     

Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Tyrosine Hydroxylase mRNA Concentration in Midbrain Dopaminergic Neurons Is Differentially Regulated by Reserpine

Tyrosine hydroxylase (TH)-mRNA, assayed by in situ hybridization combined with TH immunocytochemistry, showed a selective increase in the ventral tegmental area (A-10) but not in the substantia nigra (A-9) midbrain dopaminergic (DAergic) neurons 3 days after reserpine treatment. TH-mRNA in locus ceruleus noradrenergic (A-4) neurons was increased by reserpine, as confirmed by RNA blot hybridization. These findings show that TH-mRNA is differentially regulated in midbrain DAergic neurons in response to reserpine.     

Elevated Tyrosine Hydroxylase in the Locus Coeruleus of Suicide Victims

Abstract: The amounts of tyrosine hydroxylase protein in locus coeruleus from nine pairs of antidepressant-free suicide victims and age-matched, sudden-death control cases were determined by quantitative blot immunolabeling of cryostat-cut sections from the caudal portion of the nucleus. In each of the nine age-matched pairs, the concentration of tyrosine hydroxylase was greater in the sample from the suicide victim, with values ranging from 108 to 172% of the matched control value (\-x = 136%). By contrast, there were no differences in the concentrations of neuron-specific enolase protein in the same set of samples. Similarly, the number of neuromelanin-containing cells, counted in sections of locus coeruleus adjacent to those taken for blot immunolabeling analyses, did not differ between the two groups. These data indicate that locus coeruleus neurons from suicide victims contain higher than normal concentrations of tyrosine hydroxylase, thus raising the possibility that the expression of tyrosine hydroxylase in locus coeruleus may be relevant in the pathophysiology of suicide.     

大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.     

Dale's principle and glutamate corelease from ventral midbrain dopamine neurons

Summary. While direct application of dopamine modulates postsynaptic activity, electrical stimulation of dopamine neurons typically evokes excitation. Most of this excitation appears to be due to activation of collateral pathways; however, several lines of evidence have suggested that there is a monosynaptic component due to glutamate corelease by dopamine neurons. Recently, more direct evidence obtained in culture has shown that ventral midbrain dopamine neurons release both dopamine and glutamate. Moreover, they appear to do so from separate release sites, calling into question recent modifications of Dale's Principle. The neurochemical phenotype of a given synapse may be determined by subcellular neurotransmitter levels, uptake, or storage. However, the relationship between dopamine and glutamate release from dopamine neuron synapses in the intact brain – and the mechanisms involved – has yet to be resolved. Received August 31, 1999 Accepted September 20, 1999     

Subcellular Distribution of Tyrosine Hydroxylase in Some Catecholaminergic Rat Brain Areas Determined by a Quantitative Immunoblot Assay

The subcellular distribution of the protein tyrosine hydroxylase (TH) after fractionation of rat brain tissue was studied by a sensitive technique of immunoblot quantification in the dopaminergic nigrostriatal and the dorsal noradrenergic pathways and in the ventrolateral medulla. This repartition indicates that in all catecholaminergic regions of the cell bodies studied, the contribution of the nerve endings to the total TH amount is very low (less than 7%), in contrast to that observed in the terminal fields. The correlative subcellular determination of the TH amount and activity in the same tissue could be a useful approach for studying experimentally induced mechanisms of catecholamine synthesis modulation in different brain catecholaminergic pathways.