Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions

The subcellular distribution of pyridoxal phosphate (PLP) was studied in mouse brain, as well as the effect of pyridoxal phosphate-γ-glutamyl hydrazone (PLPGH—a convulsant drug which decreases both PLP levels and glutamate decarboxylase activity [GAD] in whole brain) upon both the PLP concentration and the GAD activity in subcellular fractions. An electron microscopic evaluation of the subcellular particles of control and PLPGH-treated animals was also carried out. The main findings were the following: (1) PLP was localized mainly in the supernatant and crude mitochondrial fractions; two-thirds of the amount present in the latter were located in the subfraction containing pure mitochondria, and the remainder was in the synaptosomal fraction. After osmotic disruption of synaptosomes, PLP was found in both the intrasynaptosomal mitochondria and the synaptoplasm. (2) Treatment of mice with PLPGH decreased levels of PLP in several brain fractions, this effect being much more notable in the soluble fractions than in the particulate fractions. After osmotic disruption of the synaptosomes, a specific decrease of PLP in the synaptoplasm was observed. (3) Treatment with PLPGH produced also an inhibition of GAD activity in most of the fractions studied, when this enzyme was assayed in the absence of PLP. In general, the inhibition was greater in those fractions in which levels of PLP were also affected. In synaptosomes, this correlation between the decreased levels of PLP and decreased activity of GAD occurred only in the synaptoplasm. (4) The activation of GAD by PLP added to incubation mixtures was much greater in those fractions from PLPGH-treated animals which displayed extensive inhibition of GAD, in comparison to the corresponding fractions from control animals. (5) No ultrastructural changes were detected in the subcellular fractions from treated animals. Our results show that the decreases of both the levels of PLP and the activity of GAD (as previously found in whole brain) actually occur in the synaptosomes, a finding that supports the hypothesis that the role of PLP in the mechanisms controlling excitability can be explained, at least in part, by its regulatory action on GAD activity, which in turn determines the rate of GABA synthesis at the nerve endings.     

Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-γ-glutamyl hydrazone in the developing rat

We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate--glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B₆ depletion, but alterations of other B₆-dependent biochemical pathways cannot be discarded.     

Regulation of GABA Metabolism in Discrete Rabbit Brain Regions under Methoxypyridoxine—Regional Differences in Cofactor Saturation and the Preictal Activation of Glutamate Decarboxylase Activity1

Abstract: The activities of the enzymes of the GABA system, glutamate decarboxylase (GAD) and GABA-transaminase, were measured in discrete regions of the rabbit brain before the onset and during the course of sustained epileptiform seizures induced by the vitamin B₆, analogue methoxypyridoxine (MP). GAD activities were measured in a reaction mixture alternatively containing the cofactor pyridoxal-5′-phosphate (PLP) in excess or containing no PLP (holoenzyme of GAD). A comparison between these two estimations showed that the apoenzyme of GAD is only partially saturated with cofactor and that the degree of saturation varied from brain area to brain area, being highest in cerebellar cortex and lowest in substantia nigra. Holoenzyme activity fell steeply after administration of 100 mg/kg MP. The regional degree of enzyme inhibition by MP was a function of the saturation of the apoenzyme with cofactor; i.e., a low rate of saturation resulted in a high degree of inhibition, and vice versa. That GAD from the regio inferior of the hippocampus did not fit into the scheme (strong inhibition is present although the degree of saturation is high) is discussed in view of the role of the hippocampus in seizure genesis and generalization. Inhibition of GAD activity by MP was completely reversible in vitro by excess PLP. Before the onset of seizures but not during their course, apoenzyme activity surpassed control levels. This preictal activation is significant in regio inferior of hippocampus, in superior colliculus, and in cerebellar cortex. GABA-transaminase activities were not significantly altered. The present study demonstrates that only investigation during the preictal period and in regional brain areas can reveal changes specific for the drug and perhaps representing the cause for seizure development, without being masked by additional alterations resulting from the severe functional and metabolic derangement during the ictal events. Thereby, it was disclosed that a decrease in vivo in the level of the enzyme product, GABA, is able to activate GAD.     

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.     

Inhibitors of crayfish glutamic acid decarboxylase

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN^– and Cl^–), and some cations (e.g. Zn⁺²). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.     

Quantum chemistry studies of the catalysis mechanism differences between the two isoforms of glutamic acid decarboxylase

The production of gamma-aminobutyric acid (GABA) is catalyzed by two isoforms of glutamic acid decarboxylase (GAD), using pyridoxal 5′-phosphate (PLP) as the cofactor. Between the two enzymes, GAD67 accounts for normal GABA requirement, while GAD65 stays inactive until emergent demand for GABA. Recent crystal structure findings revealed that the distinct conformation of a common catalytic loop of the enzymes may account for their different functions (Fenalti et al Nat Struct Mol Biol, 14:280-286, 2007). Enlightened by their inferences, we studied the underlying reaction mechanism of the two GAD isoforms using density functional theory (DFT). A rather complete reaction pathway is identified, including nine transition state (TS) structures and 14 intermediate (IM) structures. The rate limiting step occurs early during the reaction and involves a proton transfer. In the late stage, there are two pathways that involve C_4’ and C_α protonation by Tyr or Lys. Our calculations show that the reaction barriers corroborate the conjecture made by Fenalti et al.

THE CONVULSANT ACTION OF METHYLDITHIOCARBAZINATE: A COMPARISON WITH OTHER SULPHUR-CONTAINING HYDRAZIDES

—The convulsant action of methyldithiocarbazinate (MDTC), thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) has been studied in mice. The relationship between dose and time to convulsions indicated that MDTC has a dual action and is more potent than TSC. Pretreatment of mice with pyridoxal phosphate (0.25 mmol/kg) protected against convulsions and death produced by low doses of MDTC or TCH, and low or high doses of TSC. Pretreatment with pyridoxine hydrochloride (0.25 mmol/kg) protected mice against TSC but not against TCH. It protected against low doses of MDTC (0.12 mmol/kg), but shortened the latency to convulsions after intermediate doses of MDTC (0.37 mmol/kg). Glutamate decarboxylase activity (GAD, EC 4.1.1.15) in whole brain homogenates from mice killed at the onset of seizures, was significantly reduced by all 3 drugs at all doses. This inhibition did not exceed 30% after any dose of TSC or TCH, but was 64% in mice killed 4 min after the injection of MDTC (0.98 mmol/kg). The addition of pyridoxal phosphate to brain homogenates abolished GAD inhibition after MDTC but not after TCH. In vitro brain GAD was 50% inhibited by 10⁻⁴m -MDTC, 18% by 10⁻⁴m -TSC and 8% by 10 ⁻⁴m -TCH. Kinetic studies suggested that at low concentrations MDTC inhibits by competing with pyridoxal phosphate. At the onset of convulsions the cerebral content of pyridoxal phosphate was reduced after low or high doses of TSC (0.27 and 2.2 mmol/kg) and after high doses of MDTC (0.98 mmol/kg). All three drugs (at 10⁻⁵−10⁻⁴m ) inhibited pyridoxal phosphokinase (EC 2.7.1.35) in vitro. Short latency convulsions after MDTC (0.37–0.98 mmol/kg) very probably arise from inhibition of cerebral GAD, due to competition for coenzymic sites and/or unavailability of coenzyme. Long-latency convulsions after MDTC (0.12–0.37 mmol/kg) are comparable to those seen after TSC (0.27–2.2 mmol/kg) and may depend on a mechanism additional to inhibition of GAD.     

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.     

Electron microscopic study of the substantia nigra and the strio-nigral projection in the rat

Summary The normal ultrastructure of rat substantia nigra was investigated. Special attention was paid to the different types of boutons, and the mode of termination of the striatonigral connection was examined. Two different types of nigra neurons with deep nuclear membrane indentations were observed. The synaptic organization of rat substantia nigra is very complex. Six types of boutons, with many characteristics, could be differentiated: pleomorphicvesicle boutons (40%), elongated-vesicle boutons (10%), small-round-vesicle boutons (10%), large-round-vesicle boutons (20%), clear-terminal (15%) and dense-core-vesicle boutons (about 5%). Vesicle-containing dendrites were observed entering dendro-dendritic synapses. One to four days after large unilateral striatal lesions were made, a great number of degenerating boutons were seen in the ipsilateral substantia nigra, while the contra-lateral substantia nigra was entirely free of degeneration. The newly defined pleomorphic-vesicle boutons were found almost exclusively in the process of degeneration. The neuronal origin of the other bouton types is discussed.On leave of absence from Anatomical Institut, Semmelweis University, Budapest.     

Experimental Parkinson's disease in monkeys. Effect of ergot alkaloid derivative on lipid peroxidation in different brain areas

The effects of the Parkinsonism induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were evaluated in four different monkey brain areas (frontal and occipital cortex, caudate putamen, substantia nigra). The basal and stimulated lipid peroxidation and the reduced glutathione (GSH) concentration were evaluated in three groups of maleMacaca fascicularis monkeys (6 animals/group): (a) controls; (b) MPTP-treated animals; (c) animals treated with MPTP and -dihydroergocryptine (DEK; ergot alkaloid characterized by a dopaminergic agonist action). In MPTP-treated animals the GSH concentration was unchanged or decreased in a non-significant way in the frontal and occipital cortex, and in substantia nigra. The basal thiobabituric acid reactive substance (TBARS) concentrations were significantly higher in the caudate putamen and substantia nigra of MPTP-treated animals. In the MPTP-treated monkeys the DEK administration induced a restoration of basal TBARS values to nearly normal ones. By incubating tissue from different brain areas with FeSO₄ plus ascorbic acid, the stimulation of lipid peroxidation decreased the TBARS production in the substantia nigra of the MPTP-treated animals. These results, taken together, may indicate that an increased lipid peroxidation could possibly play a role in producing the Parkinson-line syndrome by MPTP and that a free radical excess could be responsible for the degeneration of the substantia nigra. The treatment with an ergot alkaloid (i.e., -dihydroergocryptine) partially antagonizes the MPTP-induced increase in basal TBARS concentration in caudate putamen.     

Signal alterations of the basal ganglia in the differential diagnosis of Parkinson’s disease: a retrospective case-controlled MRI data bank analysis

Background

Based upon the acquainted loss of dopaminergic neurons in the substantia nigra in Parkinson’s disease (PD), we hypothesised changes in magnetic resonance imaging signal intensities of the basal ganglia to be useful as an additional technical tool in the diagnostic work-up.

Methods

Region-of-interest analyses (substantia nigra and globus pallidus internus) of T2-weighted scans were performed in seventy subjects with PD, 170 age- and gender-matched controls and 38 patients with an atypical form of neurodegenerative Parkinsonian syndrome (N?=?11 multisystem atrophy, N?=?22 progressive supranuclear palsy, N?=?5 corticobasal syndrome).

Results

In patients with PD, significant changes in signal intensities within the substantia nigra were observed compared to controls at p?<?0.001. For the globus pallidus internus, signal alterations in PD and progressive supranuclear palsy were found to be significant (p?<?0.001) if compared to controls. Furthermore, signal changes of substantia nigra correlated with signal intensities of globus pallidus internus in the ipsilateral hemisphere in both groups. Sensitivity was 86% and specificity was 90% for the combined analysis of substantia nigra and globus pallidus internus in the complete patient sample versus controls.

Conclusions

Signal alterations of substantia nigra and globus pallidus internus in routine magnetic resonance imaging were useful to distinguish patients with PD from controls. In addition, signal changes in globus pallidus internus could be used to differentiate progressive supranuclear palsy patients from controls. These analyses have the potential to serve as an additional non-invasive technical tool to support the individual differential diagnosis of PD.

     

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.     

Use of Inhibitors of γ-Aminobutyric Acid (GABA) Transaminase for the Estimation of GABA Turnover in Various Brain Regions of Rats: A Reevaluation of Aminooxyacetic Acid

The technique of estimating gamma-aminobutyric acid (GABA) turnover by inhibiting its major degrading enzyme GABA-T (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) and measuring GABA accumulation has been used repeatedly, but, at least in rats, its usefulness has been limited by several difficulties, including marked differences in the degree of GABA-T inhibition in different brain regions after systemic injection of GABA-T inhibitors. In an attempt to improve this type of approach for measuring GABA turnover, the time course of GABA-T inhibition and accumulation of GABA in 12 regions of rat brain has been studied after systemic administration of aminooxyacetic acid (AOAA), injected at various doses and with different routes of administration. A total and rapidly occurring inhibition of GABA-T in all regions was obtained with intraperitoneal injection of 100 mg/kg AOAA, whereas after lower doses, marked regional differences in the degree of GABA-T inhibition were found, thus leading to underestimation of GABA synthesis rates, e.g., in substantia nigra. The activity of the GABA-synthesizing enzyme GAD (L-glutamate-1-decarboxylase; EC 4.1.1.15) was not reduced significantly at any time after intraperitoneal injection of AOAA, except for a small decrease in olfactory bulbs. Even the highest dose of AOAA tested (100 mg/kg) was not associated with toxicity in rats, but induced motor impairment, which was obviously related to the marked GABA accumulation found with this dose. The increase in GABA concentrations induced with intraperitoneal injection of 100 mg/kg AOAA was rapid in onset, allowing one to estimate GABA turnover rates from the initial rate of GABA accumulation, i.e., during the first 30 min after AOAA injection. GABA turnover rates thus determined were correlated in a highly significant fashion with the GAD activities determined in brain regions, with highest turnover rates measured in substantia nigra, hypothalamus, olfactory bulb, and tectum. Pretreatment of rats with diazepam, 5 mg/kg i.p., 5-30 min prior to AOAA, reduced the AOAA-induced GABA accumulation in all 12 regions examined, most probably as a result of potentiation of postsynaptic GABA function. The data indicate that AOAA is a valuable tool for regional GABA turnover studies in rats, provided the GABA-T inhibitor is administered in sufficiently high doses to obtain complete inhibition of GABA degradation.     

HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity

Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild‐type (WT ) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R ₂ relaxation rate within the substantia nigra of the paraquat‐treated mice compared to their saline‐treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R ₂ relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L‐ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT . Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease modifier for Parkinson's disease. Moreover, the finding that the HFE mutant mice are resistant to paraquat may provide a model in which to study resistant mechanisms to neurotoxicants.

     

REDUCTION OF LEVEL OF L-GLUTAMIC ACID DECARBOXYLASE BY γ-AMINOBUTYRIC ACID IN MOUSE BRAIN1

Abstract— The effects of accumulated endogenous GABA on the activity of L-glutamic acid decarboxylase (GAD) were studied in mouse brain. When the content of GABA in the brain was increased after administration in vivo of aminooxyacetic acid (AOAA), there was a reduction of GAD activity which could not be reversed by the addition of pyridoxal-5′-phosphate (PLP). Since inhibition of GAD activity by AOAA could be readily reversed by PLP, the reduction of GAD activity measured in the presence of added PLP indicated a decrease in the level of GAD apoenzyme. Similarly, increase of GABA content by hydrazine was also accompanied by a reduction in the level of GAD. Thiosemicarbazide and hydroxylamine did not affect the content of GABA appreciably, and in both cases levels of GAD remained unchanged when measured in the presence of added PLP. The correlation of the reduction in the levels of GAD with the increases in content of GABA suggests that GABA may regulate its own synthesizing enzyme by feedback repression.     

THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO-NIGRAL PATHWAY

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices. Unilateral transection of the striato-nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1-¹⁴C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U-¹⁴C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U-¹⁴C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side. Incubation of slices from substantia nigra with β-mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.     

EFFECTS OF AMINOOXYACETIC ACID AND l-GLUTAMIC ACID-γ-HYDRAZIDE ON GABA METABOLISM IN SPECIFIC BRAIN REGIONS

Abstract— Aminooxyacetic acid (AOAA) administration produced an increase in γ-aminobutyric acid (GABA) levels in regions of cerebral cortex, subcortex and cerebellum. In some cortical areas studied, the maximal effect was observed with 25 mg/kg AOAA; in other regions GABA levels were increased further with 50 and 75 mg/kg AOAA. Pretreatment with 25 mg/kg AOAA effectively inhibited GABA:2-oxoglutarate aminotransferase (GABA-T) and partially inhibited glutamic acid decarboxylase (GAD) activity in regions of cerebral cortex. However, this dose did not affect GAD activity in substantia nigra while GABA-T in the nigra and in the cerebellum was only partially inhibited. In both cortical and subcortical areas, the increase in GABA produced by 25 mg/kg of AOAA was linear. In contrast, l -glutamic acid-hydrazide (GAH) had no effect in the pyriform and cingulate cortex for the first 60 min after injection, and produced a biphasic GABA increase in caudate and substantia nigra over a 4 h period. Results suggest that GAH and AOAA affect regional GABA metabolism differentially and that there are several problems associated with estimating absolute GABA synthesis rates by measuring the rate or GABA accumulation after inhibition of GABA catabolism with these agents. This approach, however, may provide an easily obtainable indication of whether drugs or other manipulations are altering GABA synthesis in a given region.     

Effect of exercise on hyperactivity,impulsivity and dopamine D2 receptor expression in the substantia nigra and striatum of spontaneous hypertensive rats

[Purpose]

Attention-deficit/hyperactivity disorder (ADHD) is a heritable, chronic, neurobehavioral disorder that is characterized by hyperactivity, inattention, and impulsivity. It is commonly believed that the symptoms of ADHD are closely associated with hypo-function of the dopamine system. Dopamine D₂ receptor activation decreases the excitability of dopamine neurons, as well as the release of dopamine. Physical exercise is known to improve structural and functional impairments in neuropsychiatric disorders. We investigated the therapeutic effect of exercise on ADHD.

[Methods]

Open field task and elevated-plus maze task were used in the evaluation of hyperactivity and impulsivity, respectively. Dopamine D₂ receptor expression in the substantia nigra and striatum were evaluated by western blotting.

[Results]

The present results indicated that ADHD rats showed hyperactivity and impulsivity. Dopamine D₂ receptor expression in the substantia nigra and striatum were increased in ADHD rats. Exercise alleviated hyperactivity and impulsivity in ADHD rats. Furthermore, dopamine D₂ receptor expression in ADHD rats was also decreased by exercise.

[Conclusion]

We thus showed that exercise effectively alleviates ADHD-induced symptoms through enhancing dopamine D₂ expression in the brain.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Time Course and Localization of the Effects of Estrogen on Glutamic Acid Decarboxylase Activity1

Abstract: Two approaches were used in an attempt to characterize the effect of estrogen on glutamic acid decarboxylase (GAD) [EC 4.1.1.15] activity in ovariectomized rats. In the first experiment, estradiol-17β (E₂) was unilaterally implanted in one of five different brain areas. After 3 days of estrogen exposure, the animals were sacrificed, and GAD activity in the substantia nigra (SN) and ventral tegmental region (VTR) was measured. Estrogen implanted into the preoptic area and the ventromedial nucleus was ineffective, as were implants of cholesterol, regardless of implant site. However, GAD activity was decreased in the SN when E₂ was implanted into the caudate nucleus or amygdala and in the VTR when implanted into the nucleus accumbens septi. Furthermore, this decrease in GAD activity occurred only in the implanted side. In the second experiment, the time course of changes in GAD activity was measured in ovariectomized rats given a single systemic injection of either 8μg estradiol benzoate (EB) or oil. Rats were sacrificed at 0, 12, 29, or 53 h postinjection. It was found that GAD activity in the SN was maximally suppressed 29 h after EB, whereas decreased GAD activity in the VTR was apparent 12 h after EB but had returned to normal by 29 h. Oil injections had no significant effect on GAD activity. These results suggest that there may be two separate and distinct γ-aminobutyric acid pathways, which are differentially responsive to estrogen.