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Irreversible blockade of D2 dopamine receptors by fluphenazine-N-mustard increases glutamic acid decarboxylase mRNA in rat striatum
Neuroscience Letters, 150 (1993) 215-218
215
© 1993 Elsevier Scientific Publishers Ireland Ltd. All fights reserved 0304-3940/93/5 06.00 NSL 09317
Irreversible blockade of D 2 dopamine receptors by fluphenazine-Nmustard increases glutamic acid decarboxylase mRNA in rat striatum Jiang F a n Chen and Benjamin Weiss Division of Neuropsychopharmacology, Department of Pharmacology, The Medical College of Pennsylvania, Philadelphia, PA 19129 (USA) (Received 10 November 1992; Accepted 12 November 1992)
Key words: GABA; Glutamic acid decarboxylase mRNA; DI dopamine receptor; D2 dopamine receptor; Neuroleptic; Fluphenazine-N-mustard; Striatum; In situ hybridization histochemistry; mRNA regulation The influence of dopaminergic activity on the function of GABAergic neurons in striatum was examined by administrating rats the irreversible D2 dopamine receptor antagonist, fluphenazine-N-mustard (FNM), and determining the level of glutamic acid decarboxylase (GAD) mRNA in striatum. Rats were given either an acute single injection or chronic daily injections of FNM (20/.tmol/kg, i.p.) for 6 days. The level of GAD mRNA in striatum was determined by in situ hybridization histochemisl(ry. The results showed that acute treatment with FNM failed to significantly change striatal GAD mRNA. However, chronic FNM treatment significantly increased in the level of striatal GAD mRNA. These results demonstrate that irreversible blockade of D2 dopamine receptors increases the expression of GAD mRNA in rat striatum.
There is substantial evidence suggesting that the interaction of dopamine and y-aminobutyric acid (GABA) systems plays an important role in the control of a variety of motor behaviors [10]. GABA is a major inhibitory neurotransmitter in striatal neurons [10] and glutamic acid decarboxylase (GAD), the rate-limiting enzyme in the production of GABA, is present in a majority of striatal neurons [6]. Morphological studies revealed that dopaminergic axons from the substantial nigra directly synapse with GABAergic neurons in rat striatum [13]. This morphological relationship was further supported by the neurochemical demonstration that lesioning the nigrostriatal pathway with 6-hydroxydopamine increases the content, release and turnover of GABA, the activity of GAD and the level of GAD mRNA in striatum [14, 18, 26]. These results suggest that the nigrostriatal dopaminergic system exerts an inhibitory control on the activity of GABAergic neurons. Examination of the relationship between dopaminergic and GABAergic activity using pharmacological agents failed to produce conclusive results. Chronic administration of dopamine antagonists, such as haloperidol, sulpiride or clozepine, either increased or produced Correspondence: B. Weiss, Division of Neuropsychopharmacology, Department of Pharmacology, The Medical College of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, USA. Fax: (1) (215) 8431515.
no significant change in the content or turnover of GABA, the activity of GAD [1 l, 22] or the levels of GAD mRNA in striatum [2, 17]. The reasons for the differences between these pharmacological studies is unclear. The magnitude and duration of drug action may be important factors in dopaminergic regulation of GABAergic neurons. Inactivation of dopamine receptors by irreversible antagonists provides yet another method for investigating the influence of dopaminergic activity on other neuronal systems. For example, the irreversible antagonist Nethoxycarbonyl-2-ethoxy-l,2,-dihydroquinoline (EEDQ) has been used to study the function of dopaminergic receptors [9]. Other studies have shown that fluphenazine-N-mustard (FNM) irreversibly and selectively inactivates D 2 dopamine receptors and that this action may explain its effects on dopamine-mediated behaviors [25, 27]. In the present study, we examined the effects of irreversible blockade of D2 dopamine receptors by F N M on the expression of the GAD mRNA in rat striatum. Adult male Sprague-Dawley rats were administrated daily intraperitoneal injections of F N M (Res. Biochem. Inc.) (20/gmol/kg) or vehicle (0.1% ascorbic acid) for 5 days and were killed by decapitation 20 hr after the last injection (i.e. 6 days after the initiation of treatment). To study the acute effect of F N M treatment, in separate experiments another group of animals was administrated a single injection of FNM (20 gmol/kg) and was sacrificed
216 3 hr after the injection. The brains were sectioned (10 pro) on a cryostat at the level of the striatum. G A D m R N A levels were determined by in situ hybridization histochemistry using a 36-mer synthetic oligodeoxynucleotide probe (5'-AAG ACC ACC G A G C T G ATG G C A T C T T C C A C T C C T TCG-3') complementary to specific sequences of the G A D m R N A [12]. The probe was radiolabelled with [35S]c~-deoxy-ATP at the 3'-end with terminal deoxynucleotidyl transferase and in situ hybridization histochemistry was performed as described previously [3]. Briefly, the sections were postfixed in 4% paraformaldehyde in 0.1 M of phosphatebuffer (pH 7.4) for 5 min. After a brief rinse with 2X SSC, the sections were overlayed with 300 pl of hybridization buffer, containing 0.4 nM [35S]oligodeoxynucleotide probe, incubated at 37°C overnight and then washed at a final stringency of 0.5 x SSC at 48°C. The hybridization signals of the G A D m R N A were detected by film autoradiography, and the levels of G A D m R N A were quantitated densitometrically using the Drexel University Image Analysis System (DUMAS), as described previously [3]. K o d a k optical density (O.D.) standards were used to construct a curve from which O.D. values could be determined from gray levels of the signal after subtracting the film background. Optical density measurements were taken over the entire striatum (excluding nucleus accumbens) in both coronal and sagittal sections. The sections from representative striata of vehicle- and FNM-treated animals were analyzed at the same rostrocaudal level of striatum (bregrna 0.70 m m for coronal sections and lateral 3.40 m m for sagittal sections, according to Paxinos and Watson [19]). To minimize the experimental variation in the in situ hybridization studies, each experiment consisted of a pair of rat brains: one vehicle-treated and one F N M treated animal. These brains were mounted on the same stage, sectioned, mounted on the same slides, and processed for in situ hybridization at the same time. The results from 3 adjacent striatal sections were averaged to determine the mean O.D. value for each rat in each individual experiment, and the data are expressed as a percentage of the values of vehicle-treated animals. A paired t-test was used to determine the statistical significance of differences. The specificity of the oligodeoxynucleotide probe was assessed in preliminary studies as follows: (i) Northern analysis of R N A from mouse striatum using this probe detected one major band at 3.7 k D a corresponding to the molecular size of the G A D m R N A (GAD67) [5, 12]; (ii) the hybridization signal of the G A D m R N A using this probe was specifically displaced by the unlabelled oligonucleotide probe added in excess. The results showed that specific labelling of the G A D
m R N A was consistently seen over neurons of turn, nucleus accumbens, lateral septal nuclei, tubercle and pyriform cortex (Fig. IA). This distribution of G A D m R N A was similar to the previous investigations [7, 16].
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Fig. 1. Effect of FNM treatment on GAD mRNA in rat striatum. Rats were treated with a single injection of FNM (20 pmol/kg, i.p.) and were sacrificed 3 h later, or were given daily injections of FNM (20 ¢tmol/kg, i.p.) for 5 days and were sacrificed 20 h later. The GAD mRNA was determined in coronal sections at the level of the striatum by in situ hybridization histochemistry, using an oligodeoxynucleotideprobe as described in the text. The hybridization signal was detected by film autoradiography. A shows a representative autoradiogram, illustrating that specific labelling of GAD mRNA was detected in striatum (ST), nucleus accumbens (AC), lateral septal nuclei (LS), olfactory tubercle (OT) and pyriform cortex (CX). The figure shows that FNM treatment for 6 days but not for 3 h increased the expression of the GAD mRNA in striatum. I B shows a quantitative analysis of these results using the DUMAS image analysis system. Each value is the mean of 6 experiments. Vertical brackets indicate the standard error. *P < 0.01 compared to the vehicle-treated group using a paired t-test.
217
Fig. 1 also shows that 3 h after an acute injection of FNM, there was little change in GAD m R N A in striatum. By contrast, treating rats with F N M chronically for 6 days resulted in a significant increase in striatal GAD m R N A (Fig. 1A). A quantitative analysis of six similar experiments, using the DUMAS image system, showed that F N M treatment increased striatal GAD mRNA by about 40% (Fig. 1B). A examination of other brain areas showed that chronic F N M treatment had no significant effect on GAD mRNA in parietal cortex, and that although this treatment tended to increase the levels of GAD mRNA in nucleus accumbens, these changes failed to reach statistical significance (data not shown). A similar differential effect on GAD mRNA levels in different brain regions had also been noted earlier following 6hydroxydopamine treatment [14]. The present study demonstrates that irreversible blockade of dopamine receptors for 6 days significantly induced the expression of GAD mRNA in rat striatum. The results suggest that the dopaminergic system exerts an inhibitory effect on the expression of GAD mRNA in GABAergic neurons in striatum. This is in agreement with studies in which the nigrostriatal pathway was lesioned with 6-hydroxydopamine [14, 18, 26] and those in which animals were treated chronically with sulpiride [2]. Our results are also consistent with results showing that the 6-hydroxydopamine-induced increase in striatal GAD mRNA was partially reversed by implanting dopamine-containing fetal grafts in striatum [23]. On the other hand, the present results are in contrast to the recent report showing that neuroleptics, such as haloperidol and clozapine, failed to produce any significant change of the GAD mRNA in striatum [17]. The difference may, in part, be explained by the fact that F N M produces an irreversible blockade of dopamine receptors. The F N M treatment paradigm employed in this study resulted in a persistent and profound inactivation of dopamine receptors, whereas dopamine antagonists, such as haloperidol and clozapine, produce a reversible inactivation of dopamine receptors. The results showing that a single acute treatment with FNM failed to significantly increase striatal GAD mRNA suggests that prolong blockade of dopamine receptors is required to induce the expression of GAD mRNA in striatum. Therefore, the magnitude and duration of inhibition of dopaminergic input by dopamine antagonists may be important factors in regulating target gene expression. This conclusion is consistent with that of O'Connor et al. [18] who showed that local infusion with dopamine for 3 h attenuated the potassium-stimulated GABA release, but had no significant effects on the GAD mRNA in deafferented striatum. This result, taken together with ours, indicates that dopaminergic regula-
tion of GABA neurons in striatum may include both a short-term effect at the level of GABA release independent of the level of GAD mRNA, and a long-term modulation at the level of GAD gene expression. It has been suggested that D1 and D2 dopamine receptors differentially regulate GABAergic events. For example, the D1 dopamine agonist SKF38393 facilitated GABA release whereas the D2 dopamine agonist RU24926 and pergolide inhibited it [8, 21]. Moreover, Caboche et al. [1] showed that the D1 antagonist SCH23390 decreased the level of GAD mRNA and GAD activity in striatum, and the D2 agonist quinpirole further reinforced the decrease of GAD mRNA expression and GAD activity induced by SCH23390. Consistent with these results are those showing that the D2 dopamine antagonist sulpiride significantly increased the level of GAD mRNA in striatum [2]. That F N M acts primarily on D2 dopamine receptors was demonstrated in earlier studies which showed that treating mice with FNM irreversibly inactivates dopamine receptors of striatum, with a relatively selective action on D2 rather than D1 dopamine receptors [25, 27]. More recent studies examining brain sections adjacent to those used in the present study showed that the F N M treatment paradigm employed in the present experiments (i.e. ether acute or repeated administration) profoundly inactivated dopamine receptors. These receptor autoradiographic studies showed that chronic F N M treatment inhibited D2 dopamine receptors by more than 85% but inhibited D~ dopamine receptors by only 20% [4]. These results support the hypothesis that dopamine exerts its inhibitory effects on GAD activity and GAD mRNA through the activation of D 2 dopamine receptors. The results showing that irreversible blockade of dopaminergic activity increases GAD mRNA may explain the increased GAD activity in the striatum following 6-hydroxydopamine lesions and neuroleptic treatments. Since GAD activity was measured in the presence of saturating concentrations of substrate and co-factors, the increased GAD activity suggests an increased de novo synthesis of GAD enzymes. The present study supports this notion. The mechanism underlying the FNM-induced increase in GAD mRNA in striatum may involve both direct and indirect effects of blockade of D z dopamine receptors in striatal neurons. Direct effects involve blockade of postsynaptic D2 dopamine receptors in striatal GABAergic neurons [10]. An indirect action of D2 dopamine receptors on corticostriatal terminals may also contribute to the observed effects. Blockade of D2 dopamine receptors at corticostriatal terminals may result in an increased release of glutamate from the cerebral cortex [15]. This action may lead to an increased activity of stri-
218
atal neurons, which, in turn, may increase the expression of GAD mRNA in striatum. The possible clinical implications of these studies are still unclear. There are reports that the level and uptake of GABA are decreased in schizophrenia [20], and that the release of GABA and the activity of GAD is reduced in synaptosomes from schizophrenic patents [24]. This decrease could be partially reversed by pre-incubating synaptosomes with haloperidol [24]. These results suggested the hypothesis of a GABA deficiency in schizophrenia and that neuroleptics restore the GABA function in this disorder. The present results suggest that one of the consequences of neuroleptic treatment may involve recovery of the function of striatal GABAergic neurons by increasing the expression of the transcript encoding the rate-limiting enzyme GAD, and thereby, increasing the activity of GAD and the levels of GABA. 1 Caboche, J., Vernier, P., Julien, J.-F., Rogard, M., Mallet, J. and Besson, M.-J., Parallel decrease of glutamic acid decarboxylase and preproenkephalin mRNA in the rat striatum following chronic treatment with a dopaminergic D~ antagonist and D2 agonist, J. Neurochem., 56 (1991) 428435. 2 Caboche, J., Vernier, P., Rogard, M., Julien J.-F., Mallet, J. and Besson M.-J., Role of dopaminergic D2 receptors in the regulation of glutamic acid decarboxylase messenger RNA in the striatum of the rat, Eur. J. Neurosci., 4 (1992) 438-447. 3 Chen, J.F., Qin, Z.H., Szele, F., Bai, G. and Weiss, B., Neuronal localization and modulation of the D2 dopamine receptor mRNA in brain of normal mice and mice lesioned with 6-hydroxydopamine, Neuropharmacology, 30 (1991) 927-941. 4 Chen, J.F., Qin, Z.H. and Weiss, B., Irreversible blockade of dopamine receptors by fluphenazine-N-mustard increases D2 dopamine receptor mRNA and proenkephalin mRNA in rat striatum, Soc. Neurosci. Abstr., 18 (1992) 398. 5 Erlander, M.G., Tillakaratne, N.J.K., Feldblum, S., Patel, N. and Tobin, A.J., Two genes encode distinct glutamate decarboxylases, Neuron, 7 (1991) 91-100. 6 Erlander, M.G. and Tobin, A.J., The structural and functional heterogeneity of glutamic acid decarboxylase: A review, Neurochem. Res., 16 (1991) 215~26. 7 Ferraguti, F., Zoli, M., Aronsson, M., Agnati, L.F., Goldstein, M., Filer, D. and Fuxe, K., Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain, J. Chem. Neuroanat., 3 (1990) 377-396. 8 Girault, J.A., Spampinato, U., Glowinski, J. and Besson, M.J., In vivo release of [3H]-y-aminobutyric acid in the rat neostriatum II. Opposing effects of D1 and D2 dopamine receptor stimulation in the dorsal caudate putamen, Neuroscience, 19 (1986) 1109 1117. 9 Goodale, D.B., Jacobi, A.M., Seyfried, D.M. and Weiss, B., Selective protection from the inhibition by EEDQ of Dj and D 2 dopamine agonist-induced rotational behavior in mice, Pharmacol. Biochem. Behav., 30 (1987) 457~,62. 10 Graybiel, A.M., Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci., 13 (1990) 244-254. 11 Itoh, M., Effects of haloperidol on glutamate decarboxylase activity in discrete brain areas of the rat, Psychopharmacology, 79 (1983) 169 172.
12 Katarova, Z., Szabo, G., Mugnaini, E. and Greenspan, R.J., Molecular identification of the 62 kd form of glutamic acid decarboxylase from the mouse, Eur. J. Neurosci., 2 (1990) 190-202. 13 Kubota, Y., Inagaki, S., Kito, S. and Wu, J.-Y., Dopaminergic axons directly make synapses with GABAergic neurons in the rat neostriatum, Brain Res., 406 (1987) 147-156. 14 Lindefors, N., Brene, S., Herrera.-Marschitz, M. and Persson, H., Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain, Exp. Brain Res., 77 (1989) 611-620. 15 Maura, G., Carbone, R. and Raiteri, M., Aspartate-releasing nerve terminals in rat striatum possess D-2 dopamine receptors mediating inhibition of release, J. Pharmacol. Exp. Ther., 251 (1989) 11421146. 16 Mercugliano, M., Soghomonian, J..-J., Qin, Y., Nguyen, H.Q., Feldblum, S., Erlander, M.G., Tobin, A.J. and Chesselet, M..-F., Comparative distribution of messenger RNAs encoding glutamic acid decarboxylase (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat, J. Comp. Neurol., 318 (1992) 245-254. 17 Mercugliano, M., Sailer, C.F., Salama, A.I., U'Prichard, D.C. and Chesselet, M.-F., Clozapine and haloperidol have differential effects on glutamic acid decarboxylase mRNA in the pallidal nuclei of the rat, Neuropsychopharmacology, 6 (1992) 179- 187. 18 O'Connor, W.T., Lindefors, N., Brene, S., Herrera.-Marschitz, M., Persson, H. and Ungerstedt, U., Short-term dopaminergic regulation of GABA release in dopamine deafferented caudate-putamen is not directly associated with glutamic acid decarboxylase gene expression, Neurosci. Lett., 128 (1991) 66-70. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, 1986. 20 Perry, T.L., Kish, S.J., Buchanan, J. and Hansen, S., Gamma-aminobutyric acid deficiency in brain of schizophrenic patients, Lancet, 1 (1979) 237--239. 21 Reid, S.M., O'Connor, W.T., Herrera-Marschitz, M. and Ungerstedt, U., The effects of intranigral GABA and dynorphin A injections on striatal dopamine and GABA release: evidence that dopamine provides inhibitory regulation of striatal GABA neurons via D z receptors, Brain Res., 519 (1990) 255-260. 22 Rupniak, N.M.J., Prestwich, S.A., Horton, R.W., Jenner, P. and Marsden, C.D.r Alterations in cerebral glutamic acid decarboxylase and 3H-flunitrazepam binding during continuous treatment of rats for up to 1 year with haloperidol, sulpiride or clozapine, J. Neural Transm., 68 (1987) 113-125. 23 Segovia, J., Castro, R., Notario, V. and Gale, K., Transplants of fetal substantia nigra regulate glutamic acid decarboxylase gene expression in host striatal neurons, Mol. Brain Res., 10 (1991) 359362. 24 Sherman, A.D., Davidson, A.T., Baruah, S., Hegwood, T.S. and Waziri, R., Evidence of glutamatergic deficiency in schizophrenia, Neurosci. Lett., 121 (1991) 77 80. 25 Thermos, K., Winkler, J.D. and Weiss, B., Comparison of the effects of fluphenazine-N-mustard on dopamine binding sites and on behavior induced by apomorphine in supersensitive mice, Neuropharmacology, 26 (1987) 1473-1480. 26 Vernier, P., Julien, J.-F., Rataboul, P., Fourrier, O., Feuerstein, C. and Mallet, J., Similar time course changes in striatal levels of glutamic acid decarboxylase and proenkephalin mRNA following dopaminergic deafferentation in the rat, J. Neurochem., 51 (1988) 1375-1380. 27 Winkler, J.D., Thermos, K. and Weiss, B., Differential effects of fluphenazine-N-mustard on calmodulin activity and on D~ and D2 dopaminergic responses, Psychopharmacology, 92 (1987) 285-291.
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© 1993 Elsevier Scientific Publishers Ireland Ltd. All fights reserved 0304-3940/93/5 06.00 NSL 09317
Irreversible blockade of D 2 dopamine receptors by fluphenazine-Nmustard increases glutamic acid decarboxylase mRNA in rat striatum Jiang F a n Chen and Benjamin Weiss Division of Neuropsychopharmacology, Department of Pharmacology, The Medical College of Pennsylvania, Philadelphia, PA 19129 (USA) (Received 10 November 1992; Accepted 12 November 1992)
Key words: GABA; Glutamic acid decarboxylase mRNA; DI dopamine receptor; D2 dopamine receptor; Neuroleptic; Fluphenazine-N-mustard; Striatum; In situ hybridization histochemistry; mRNA regulation The influence of dopaminergic activity on the function of GABAergic neurons in striatum was examined by administrating rats the irreversible D2 dopamine receptor antagonist, fluphenazine-N-mustard (FNM), and determining the level of glutamic acid decarboxylase (GAD) mRNA in striatum. Rats were given either an acute single injection or chronic daily injections of FNM (20/.tmol/kg, i.p.) for 6 days. The level of GAD mRNA in striatum was determined by in situ hybridization histochemisl(ry. The results showed that acute treatment with FNM failed to significantly change striatal GAD mRNA. However, chronic FNM treatment significantly increased in the level of striatal GAD mRNA. These results demonstrate that irreversible blockade of D2 dopamine receptors increases the expression of GAD mRNA in rat striatum.
There is substantial evidence suggesting that the interaction of dopamine and y-aminobutyric acid (GABA) systems plays an important role in the control of a variety of motor behaviors [10]. GABA is a major inhibitory neurotransmitter in striatal neurons [10] and glutamic acid decarboxylase (GAD), the rate-limiting enzyme in the production of GABA, is present in a majority of striatal neurons [6]. Morphological studies revealed that dopaminergic axons from the substantial nigra directly synapse with GABAergic neurons in rat striatum [13]. This morphological relationship was further supported by the neurochemical demonstration that lesioning the nigrostriatal pathway with 6-hydroxydopamine increases the content, release and turnover of GABA, the activity of GAD and the level of GAD mRNA in striatum [14, 18, 26]. These results suggest that the nigrostriatal dopaminergic system exerts an inhibitory control on the activity of GABAergic neurons. Examination of the relationship between dopaminergic and GABAergic activity using pharmacological agents failed to produce conclusive results. Chronic administration of dopamine antagonists, such as haloperidol, sulpiride or clozepine, either increased or produced Correspondence: B. Weiss, Division of Neuropsychopharmacology, Department of Pharmacology, The Medical College of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, USA. Fax: (1) (215) 8431515.
no significant change in the content or turnover of GABA, the activity of GAD [1 l, 22] or the levels of GAD mRNA in striatum [2, 17]. The reasons for the differences between these pharmacological studies is unclear. The magnitude and duration of drug action may be important factors in dopaminergic regulation of GABAergic neurons. Inactivation of dopamine receptors by irreversible antagonists provides yet another method for investigating the influence of dopaminergic activity on other neuronal systems. For example, the irreversible antagonist Nethoxycarbonyl-2-ethoxy-l,2,-dihydroquinoline (EEDQ) has been used to study the function of dopaminergic receptors [9]. Other studies have shown that fluphenazine-N-mustard (FNM) irreversibly and selectively inactivates D 2 dopamine receptors and that this action may explain its effects on dopamine-mediated behaviors [25, 27]. In the present study, we examined the effects of irreversible blockade of D2 dopamine receptors by F N M on the expression of the GAD mRNA in rat striatum. Adult male Sprague-Dawley rats were administrated daily intraperitoneal injections of F N M (Res. Biochem. Inc.) (20/gmol/kg) or vehicle (0.1% ascorbic acid) for 5 days and were killed by decapitation 20 hr after the last injection (i.e. 6 days after the initiation of treatment). To study the acute effect of F N M treatment, in separate experiments another group of animals was administrated a single injection of FNM (20 gmol/kg) and was sacrificed
216 3 hr after the injection. The brains were sectioned (10 pro) on a cryostat at the level of the striatum. G A D m R N A levels were determined by in situ hybridization histochemistry using a 36-mer synthetic oligodeoxynucleotide probe (5'-AAG ACC ACC G A G C T G ATG G C A T C T T C C A C T C C T TCG-3') complementary to specific sequences of the G A D m R N A [12]. The probe was radiolabelled with [35S]c~-deoxy-ATP at the 3'-end with terminal deoxynucleotidyl transferase and in situ hybridization histochemistry was performed as described previously [3]. Briefly, the sections were postfixed in 4% paraformaldehyde in 0.1 M of phosphatebuffer (pH 7.4) for 5 min. After a brief rinse with 2X SSC, the sections were overlayed with 300 pl of hybridization buffer, containing 0.4 nM [35S]oligodeoxynucleotide probe, incubated at 37°C overnight and then washed at a final stringency of 0.5 x SSC at 48°C. The hybridization signals of the G A D m R N A were detected by film autoradiography, and the levels of G A D m R N A were quantitated densitometrically using the Drexel University Image Analysis System (DUMAS), as described previously [3]. K o d a k optical density (O.D.) standards were used to construct a curve from which O.D. values could be determined from gray levels of the signal after subtracting the film background. Optical density measurements were taken over the entire striatum (excluding nucleus accumbens) in both coronal and sagittal sections. The sections from representative striata of vehicle- and FNM-treated animals were analyzed at the same rostrocaudal level of striatum (bregrna 0.70 m m for coronal sections and lateral 3.40 m m for sagittal sections, according to Paxinos and Watson [19]). To minimize the experimental variation in the in situ hybridization studies, each experiment consisted of a pair of rat brains: one vehicle-treated and one F N M treated animal. These brains were mounted on the same stage, sectioned, mounted on the same slides, and processed for in situ hybridization at the same time. The results from 3 adjacent striatal sections were averaged to determine the mean O.D. value for each rat in each individual experiment, and the data are expressed as a percentage of the values of vehicle-treated animals. A paired t-test was used to determine the statistical significance of differences. The specificity of the oligodeoxynucleotide probe was assessed in preliminary studies as follows: (i) Northern analysis of R N A from mouse striatum using this probe detected one major band at 3.7 k D a corresponding to the molecular size of the G A D m R N A (GAD67) [5, 12]; (ii) the hybridization signal of the G A D m R N A using this probe was specifically displaced by the unlabelled oligonucleotide probe added in excess. The results showed that specific labelling of the G A D
m R N A was consistently seen over neurons of turn, nucleus accumbens, lateral septal nuclei, tubercle and pyriform cortex (Fig. IA). This distribution of G A D m R N A was similar to the previous investigations [7, 16].
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the striaolfactory neuronal results of
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Fig. 1. Effect of FNM treatment on GAD mRNA in rat striatum. Rats were treated with a single injection of FNM (20 pmol/kg, i.p.) and were sacrificed 3 h later, or were given daily injections of FNM (20 ¢tmol/kg, i.p.) for 5 days and were sacrificed 20 h later. The GAD mRNA was determined in coronal sections at the level of the striatum by in situ hybridization histochemistry, using an oligodeoxynucleotideprobe as described in the text. The hybridization signal was detected by film autoradiography. A shows a representative autoradiogram, illustrating that specific labelling of GAD mRNA was detected in striatum (ST), nucleus accumbens (AC), lateral septal nuclei (LS), olfactory tubercle (OT) and pyriform cortex (CX). The figure shows that FNM treatment for 6 days but not for 3 h increased the expression of the GAD mRNA in striatum. I B shows a quantitative analysis of these results using the DUMAS image analysis system. Each value is the mean of 6 experiments. Vertical brackets indicate the standard error. *P < 0.01 compared to the vehicle-treated group using a paired t-test.
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Fig. 1 also shows that 3 h after an acute injection of FNM, there was little change in GAD m R N A in striatum. By contrast, treating rats with F N M chronically for 6 days resulted in a significant increase in striatal GAD m R N A (Fig. 1A). A quantitative analysis of six similar experiments, using the DUMAS image system, showed that F N M treatment increased striatal GAD mRNA by about 40% (Fig. 1B). A examination of other brain areas showed that chronic F N M treatment had no significant effect on GAD mRNA in parietal cortex, and that although this treatment tended to increase the levels of GAD mRNA in nucleus accumbens, these changes failed to reach statistical significance (data not shown). A similar differential effect on GAD mRNA levels in different brain regions had also been noted earlier following 6hydroxydopamine treatment [14]. The present study demonstrates that irreversible blockade of dopamine receptors for 6 days significantly induced the expression of GAD mRNA in rat striatum. The results suggest that the dopaminergic system exerts an inhibitory effect on the expression of GAD mRNA in GABAergic neurons in striatum. This is in agreement with studies in which the nigrostriatal pathway was lesioned with 6-hydroxydopamine [14, 18, 26] and those in which animals were treated chronically with sulpiride [2]. Our results are also consistent with results showing that the 6-hydroxydopamine-induced increase in striatal GAD mRNA was partially reversed by implanting dopamine-containing fetal grafts in striatum [23]. On the other hand, the present results are in contrast to the recent report showing that neuroleptics, such as haloperidol and clozapine, failed to produce any significant change of the GAD mRNA in striatum [17]. The difference may, in part, be explained by the fact that F N M produces an irreversible blockade of dopamine receptors. The F N M treatment paradigm employed in this study resulted in a persistent and profound inactivation of dopamine receptors, whereas dopamine antagonists, such as haloperidol and clozapine, produce a reversible inactivation of dopamine receptors. The results showing that a single acute treatment with FNM failed to significantly increase striatal GAD mRNA suggests that prolong blockade of dopamine receptors is required to induce the expression of GAD mRNA in striatum. Therefore, the magnitude and duration of inhibition of dopaminergic input by dopamine antagonists may be important factors in regulating target gene expression. This conclusion is consistent with that of O'Connor et al. [18] who showed that local infusion with dopamine for 3 h attenuated the potassium-stimulated GABA release, but had no significant effects on the GAD mRNA in deafferented striatum. This result, taken together with ours, indicates that dopaminergic regula-
tion of GABA neurons in striatum may include both a short-term effect at the level of GABA release independent of the level of GAD mRNA, and a long-term modulation at the level of GAD gene expression. It has been suggested that D1 and D2 dopamine receptors differentially regulate GABAergic events. For example, the D1 dopamine agonist SKF38393 facilitated GABA release whereas the D2 dopamine agonist RU24926 and pergolide inhibited it [8, 21]. Moreover, Caboche et al. [1] showed that the D1 antagonist SCH23390 decreased the level of GAD mRNA and GAD activity in striatum, and the D2 agonist quinpirole further reinforced the decrease of GAD mRNA expression and GAD activity induced by SCH23390. Consistent with these results are those showing that the D2 dopamine antagonist sulpiride significantly increased the level of GAD mRNA in striatum [2]. That F N M acts primarily on D2 dopamine receptors was demonstrated in earlier studies which showed that treating mice with FNM irreversibly inactivates dopamine receptors of striatum, with a relatively selective action on D2 rather than D1 dopamine receptors [25, 27]. More recent studies examining brain sections adjacent to those used in the present study showed that the F N M treatment paradigm employed in the present experiments (i.e. ether acute or repeated administration) profoundly inactivated dopamine receptors. These receptor autoradiographic studies showed that chronic F N M treatment inhibited D2 dopamine receptors by more than 85% but inhibited D~ dopamine receptors by only 20% [4]. These results support the hypothesis that dopamine exerts its inhibitory effects on GAD activity and GAD mRNA through the activation of D 2 dopamine receptors. The results showing that irreversible blockade of dopaminergic activity increases GAD mRNA may explain the increased GAD activity in the striatum following 6-hydroxydopamine lesions and neuroleptic treatments. Since GAD activity was measured in the presence of saturating concentrations of substrate and co-factors, the increased GAD activity suggests an increased de novo synthesis of GAD enzymes. The present study supports this notion. The mechanism underlying the FNM-induced increase in GAD mRNA in striatum may involve both direct and indirect effects of blockade of D z dopamine receptors in striatal neurons. Direct effects involve blockade of postsynaptic D2 dopamine receptors in striatal GABAergic neurons [10]. An indirect action of D2 dopamine receptors on corticostriatal terminals may also contribute to the observed effects. Blockade of D2 dopamine receptors at corticostriatal terminals may result in an increased release of glutamate from the cerebral cortex [15]. This action may lead to an increased activity of stri-
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atal neurons, which, in turn, may increase the expression of GAD mRNA in striatum. The possible clinical implications of these studies are still unclear. There are reports that the level and uptake of GABA are decreased in schizophrenia [20], and that the release of GABA and the activity of GAD is reduced in synaptosomes from schizophrenic patents [24]. This decrease could be partially reversed by pre-incubating synaptosomes with haloperidol [24]. These results suggested the hypothesis of a GABA deficiency in schizophrenia and that neuroleptics restore the GABA function in this disorder. The present results suggest that one of the consequences of neuroleptic treatment may involve recovery of the function of striatal GABAergic neurons by increasing the expression of the transcript encoding the rate-limiting enzyme GAD, and thereby, increasing the activity of GAD and the levels of GABA. 1 Caboche, J., Vernier, P., Julien, J.-F., Rogard, M., Mallet, J. and Besson, M.-J., Parallel decrease of glutamic acid decarboxylase and preproenkephalin mRNA in the rat striatum following chronic treatment with a dopaminergic D~ antagonist and D2 agonist, J. Neurochem., 56 (1991) 428435. 2 Caboche, J., Vernier, P., Rogard, M., Julien J.-F., Mallet, J. and Besson M.-J., Role of dopaminergic D2 receptors in the regulation of glutamic acid decarboxylase messenger RNA in the striatum of the rat, Eur. J. Neurosci., 4 (1992) 438-447. 3 Chen, J.F., Qin, Z.H., Szele, F., Bai, G. and Weiss, B., Neuronal localization and modulation of the D2 dopamine receptor mRNA in brain of normal mice and mice lesioned with 6-hydroxydopamine, Neuropharmacology, 30 (1991) 927-941. 4 Chen, J.F., Qin, Z.H. and Weiss, B., Irreversible blockade of dopamine receptors by fluphenazine-N-mustard increases D2 dopamine receptor mRNA and proenkephalin mRNA in rat striatum, Soc. Neurosci. Abstr., 18 (1992) 398. 5 Erlander, M.G., Tillakaratne, N.J.K., Feldblum, S., Patel, N. and Tobin, A.J., Two genes encode distinct glutamate decarboxylases, Neuron, 7 (1991) 91-100. 6 Erlander, M.G. and Tobin, A.J., The structural and functional heterogeneity of glutamic acid decarboxylase: A review, Neurochem. Res., 16 (1991) 215~26. 7 Ferraguti, F., Zoli, M., Aronsson, M., Agnati, L.F., Goldstein, M., Filer, D. and Fuxe, K., Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain, J. Chem. Neuroanat., 3 (1990) 377-396. 8 Girault, J.A., Spampinato, U., Glowinski, J. and Besson, M.J., In vivo release of [3H]-y-aminobutyric acid in the rat neostriatum II. Opposing effects of D1 and D2 dopamine receptor stimulation in the dorsal caudate putamen, Neuroscience, 19 (1986) 1109 1117. 9 Goodale, D.B., Jacobi, A.M., Seyfried, D.M. and Weiss, B., Selective protection from the inhibition by EEDQ of Dj and D 2 dopamine agonist-induced rotational behavior in mice, Pharmacol. Biochem. Behav., 30 (1987) 457~,62. 10 Graybiel, A.M., Neurotransmitters and neuromodulators in the basal ganglia, Trends Neurosci., 13 (1990) 244-254. 11 Itoh, M., Effects of haloperidol on glutamate decarboxylase activity in discrete brain areas of the rat, Psychopharmacology, 79 (1983) 169 172.
12 Katarova, Z., Szabo, G., Mugnaini, E. and Greenspan, R.J., Molecular identification of the 62 kd form of glutamic acid decarboxylase from the mouse, Eur. J. Neurosci., 2 (1990) 190-202. 13 Kubota, Y., Inagaki, S., Kito, S. and Wu, J.-Y., Dopaminergic axons directly make synapses with GABAergic neurons in the rat neostriatum, Brain Res., 406 (1987) 147-156. 14 Lindefors, N., Brene, S., Herrera.-Marschitz, M. and Persson, H., Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain, Exp. Brain Res., 77 (1989) 611-620. 15 Maura, G., Carbone, R. and Raiteri, M., Aspartate-releasing nerve terminals in rat striatum possess D-2 dopamine receptors mediating inhibition of release, J. Pharmacol. Exp. Ther., 251 (1989) 11421146. 16 Mercugliano, M., Soghomonian, J..-J., Qin, Y., Nguyen, H.Q., Feldblum, S., Erlander, M.G., Tobin, A.J. and Chesselet, M..-F., Comparative distribution of messenger RNAs encoding glutamic acid decarboxylase (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat, J. Comp. Neurol., 318 (1992) 245-254. 17 Mercugliano, M., Sailer, C.F., Salama, A.I., U'Prichard, D.C. and Chesselet, M.-F., Clozapine and haloperidol have differential effects on glutamic acid decarboxylase mRNA in the pallidal nuclei of the rat, Neuropsychopharmacology, 6 (1992) 179- 187. 18 O'Connor, W.T., Lindefors, N., Brene, S., Herrera.-Marschitz, M., Persson, H. and Ungerstedt, U., Short-term dopaminergic regulation of GABA release in dopamine deafferented caudate-putamen is not directly associated with glutamic acid decarboxylase gene expression, Neurosci. Lett., 128 (1991) 66-70. 19 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, 1986. 20 Perry, T.L., Kish, S.J., Buchanan, J. and Hansen, S., Gamma-aminobutyric acid deficiency in brain of schizophrenic patients, Lancet, 1 (1979) 237--239. 21 Reid, S.M., O'Connor, W.T., Herrera-Marschitz, M. and Ungerstedt, U., The effects of intranigral GABA and dynorphin A injections on striatal dopamine and GABA release: evidence that dopamine provides inhibitory regulation of striatal GABA neurons via D z receptors, Brain Res., 519 (1990) 255-260. 22 Rupniak, N.M.J., Prestwich, S.A., Horton, R.W., Jenner, P. and Marsden, C.D.r Alterations in cerebral glutamic acid decarboxylase and 3H-flunitrazepam binding during continuous treatment of rats for up to 1 year with haloperidol, sulpiride or clozapine, J. Neural Transm., 68 (1987) 113-125. 23 Segovia, J., Castro, R., Notario, V. and Gale, K., Transplants of fetal substantia nigra regulate glutamic acid decarboxylase gene expression in host striatal neurons, Mol. Brain Res., 10 (1991) 359362. 24 Sherman, A.D., Davidson, A.T., Baruah, S., Hegwood, T.S. and Waziri, R., Evidence of glutamatergic deficiency in schizophrenia, Neurosci. Lett., 121 (1991) 77 80. 25 Thermos, K., Winkler, J.D. and Weiss, B., Comparison of the effects of fluphenazine-N-mustard on dopamine binding sites and on behavior induced by apomorphine in supersensitive mice, Neuropharmacology, 26 (1987) 1473-1480. 26 Vernier, P., Julien, J.-F., Rataboul, P., Fourrier, O., Feuerstein, C. and Mallet, J., Similar time course changes in striatal levels of glutamic acid decarboxylase and proenkephalin mRNA following dopaminergic deafferentation in the rat, J. Neurochem., 51 (1988) 1375-1380. 27 Winkler, J.D., Thermos, K. and Weiss, B., Differential effects of fluphenazine-N-mustard on calmodulin activity and on D~ and D2 dopaminergic responses, Psychopharmacology, 92 (1987) 285-291.