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The turnover rate of γ-aminobutyric acid in the substantia nigra following electrical stimulation or lesioning of the strionigral pathways
Brain Research, 155 (1978) 147 152 c~') Elsevier/North-Holland Biomedical Press
147
The turnover rate of 7-aminobutyric acid in the substantia nigra following electrical stimulation or lesioning of the strionigral pathways
C. C. MAO, E. PERALTA*, F. MORIN[** and E. COSTA*** Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint El&abethsHospital, Washington, D.C. 20032 (U.S.A.)
(Accepted June 1st, 1978)
It has long been known that axons from neostriatal neurons innervate the globus pallidus and the substantia nigra2,7,13,17. More recently, some of these strionigral neurons were shown to synthesize, store and secrete gamma-aminobutyric acid (GABA)", 3,5,6,14. These GABAergic axons synapse with dendrites of nigral dopaminergic neurons, thus providing an inhibitory feedback regulation for the nigrostriatal dopaminergic pathway 7. In agreement with this, the iontophoretic application of GABA to substantia nigra inhibits neuronal activity, and this inhibition is nullified by picrotoxin '~. When the head of the nucleus caudatus is stimulated electrically, neuronal firing in substantia nigra is reducedS; this reduction can be blocked by picrotoxin x4. In rats and baboons, a brain hemitransection at the subthalamic level results in a marked decline of the GA BA content and of the glutamic acid decarboxylase (GAD) activity in substantia nigra6,1°; this suggests that, in substantia nigra, GABA is preferentially located in the axon terminals of the descending strionigral pathways. Due to the possible implication of the strionigral pathways in severe neurological disorders such as Parkinson's disease and Huntington's choreaS, 0, biochemical studies to evaluate the regulation of these pathways are of great practical interest. Since the measurement of the steady-state content of GABA in the nigra is of no value in studies of the regulation of GABA neurons, we have explored the limitations and the advantages offered by the estimation of the GABA turnover rate (TRGABA). Thus, the present study was designed to assess whether or not modifications of GABA metabolism in the GABAergic strionigral pathways (through both electrical stimulation and surgical deafferentation) can be detected using TRGABA as an index. It should be pointed out that our estimations of TRGABA probably do not measure with accuracy the absolute value of this parameter, therefore they are valid only for comparative purposesk * Grantee of the Program for Cultural Exchange between Spain and the U.S.A. ** Present Address. Dept. of Pharmacology, University of Florence, Florence, Italy. *** To whom reprint requests should be addressed.
148 Sprague-Dawley rats weighing 110 g were anesthetized with equithesin (0.4 ml/100 g; Jensen Salsberg Labs), and a guide cannula (22-gauge) (Plastic Products. Roanoke, Va.) with an intracranial shaft cut to 2 m m in length was placed stereotaxically in the head of the right nucleus caudatus (AP 7890, L 2600 according t o K6nig and KlippeP~). This cannula was then secured with dental cement and its lumen temporarily occluded with a wire to prevent clogging. Rats were allowed to recover from the surgery for 3 days before proceeding with electrical stimulation experiments. To stimulate the nucleus caudatus, a monopolar electrode (Rhodes Medical Instruments) was inserted into the implanted cannula. The electrode (10 m m shaft length, 0.25 mm shaft diameter) was entirely insulated except for the tip. When the electrode was inserted into the cannula, it protruded by 1 m m into the outer shell of the nucleus caudatus: the reference electrode made contact with the skin. The outer shell of the nucleus caudatus was chosen as the site for the electrode placement. because Bunney and Aghajanian z have shown that the caudate neurons that project to the substantia nigra are located in the outer shell. In other experiments the animals were hemitransected by means of a 3 mm wide knife lowered with an angle of 60 ° at the level of the coronary suture (AP 82; K6nig and KlippePl). The knife was inserted next to the midline and subsequently moved outwards to achieve complete hemitransection. The TR6ABA was estimated using a previously described technique 1-, which involves infusion of [13C]glucose through the tail vein and mass fragmentographic determination of the changes with time of the G A B A and glutamic acid enrichment in ~aC. Electrical stimulation of the nucleus caudatus was initiated before the 13C infusion: the experimental protocol required that, regardless of the infusion time with [13C]glucose, the nucleus caudatus was stimulated for 20 min. A detailed account of the stimulation parameters and infusion times is g~ven in the legend for Table [. Homovanillic acid (HVA) in caudate nucleus was assayed by mass fragmentography, using the derivatives described by Dziedzic et al. ~. In preliminary experiments, the nucleus caudatus was stimulated with 2 and 4 V (20 Hz, 0.5 msec pulse duration), but this current intensity failed to increase the TRGABA in either the ipsilateral or contralateral substantia nigra (data not shown). Therefore, the stimulus intensity was increased to 10 V, all other parameters being kept constant. Table I lists the GABA content, rate constant for G A B A efflux (kGABA) and the TRGABA in the substantia nigra and nucleus caudatus ipsilateral and contralateral to the electrically stimulated nucleus caudatus. Calculations were performed using the precursor product relationship at steady-state according to the mathematic model described by Racagni et al. L~ for acetytcholine. The TR6AnA of the nonstimulated side was contrasted with that of the stimulated side: Table 1 also lists the values of kGABA and TRGABA in non-stimulated rats. Fig. 1 shows the ~ C enrichment of glutamm acid and G A B A stored in the substantia nigra ipsitateral and contralaterat to the stimulated nucleus caudatus. It can be seen that the time course of the increment with time of the ~3C enrichment of glutamJc acid is essentially identical in both substantia nigra. In contrast, in the substantia nigra ipsilateral to the stimulus the increment with time of the 1~C enrichment of G A B A is steeper than that of the contralateral substantia nigra.
149
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Fig. 1. Effect of unilateral electrical stimulation of the nucleus caudatus on the ~zC enrichment of GABA and glutamic acid in ipsilateral and contralateral substantia nigra. The intensity o f the electrical stimulation was 8 V, 20 Hz and 0.5 msec for 20 min. * P < 0.005 according to the student paired 't'-test when compared to the laC enrichment of GABA stored in the contralateral substantia nigra.
The GABA and glutamic acid content of substantia nigra or nucleus caudatus remained unchanged following electrical stimulation of nucleus caudatus (see Table I). The kGABA in the substantia nigra of non-stimulated rats or that of the tissue contralateral to the stimulus were similar (see Table I). In contrast, the kGABA of substantia nigra ipsilateral to the stimulus was 2-3-fold greater than that of the substantia nigra of non-stimulated rats or that contralateral to the stimulated nucleus caudatus (see Table I). Since the GABA content did not change, the TRt~ABA in the substantia nigra ipsilateral to the stimulated nucleus caudatus was significantly higher than that of the contralateral side or that of the non-stimulated rats. Fig. 2 shows the TRGABA values in caudatus o f the hemitransected animals one week after surgery. Besides decreasing the TRGABA values, this surgical procedure decreased also the GABA and glutamate content of substantia nigra and the HVA content of striatum (see Table ll). These observations provide direct evidence that electrical activation or surgical lesioning of strionigral GABAergic axons increases and decreases the TRc,~BA, respectively. Since the GABA content failed to increase in sites in which the TRGABa was increased by electrical stimulation, we infer that the increased amount of GABA synthesized was either released and promptly metabolized or was metabolized intraneuronally. Our data indicate that the effects of the stimulus applied to the head of the nucleus caudatus are confined to the ipsilateral substantia nigra, suggesting that the long axons of GABAergic striatal neurons innervate only the ipsilateral dopaminergic neurons of substantia nigra.
151
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TRGABA IN SUBSTANTIA NIGRA nrnol/mg prot/hr CONTROL SIDE LESIONED SIDE **
412 +_ 4 4 159 +.t. 3 4 * *
P < 0.01
Fig. 2. Schematic sagittal section showing the location of the knife cut. The following abbreviations are used: HP, h i p p o c a m p u s ; GP, globus pallidus; SN, substantia nigra.
In the ipsilateral and contralateral n. caudatus, the values of the kGABA w e r e not significantly changed by a stimulus that increased the kGABA in the ipsilateral substantia nigra (see Table I). It is known that the long axon GABAergic neurons located in the outer shell of the head of the nucleus caudatus innervate the substantia nigra 2, but the nucleus caudatus also contains a number of small GABAergic interneurons, intrinsic to the caudate which appear to constitute a neuronal population exerting a tonic inhibition of the adjacent neurons t6. The lack of increase in the TABLE II GA BA and glutamate content in substantia nigra and H VA content in n. caudatus o f hemitransected animals Each value represents the mean ± S.E. of at least 7 determinations. Substantia nigra (nmole/mg protein)
Control side Lesion side * P < O.Ol.
Caudate nucleus (pmole/mg protein)
GABA
Glutamate
HVA
69 ± 4.0 26 ± 3.4*
68 ± 2.0 56 ± 1.5"
66 ± 0.66 3l ± 0.34*
152 value o f kGABA in the
stimulated
nucleus
caudatus
suggests t h a t
the
GABA
m e t a b o l i s m in these intrinsic c a u d a t e n e u r o n s was n o t c h a n g e d substantially by our m e t h o d o f stimulation. Perhaps, as indicated in previous experiments t '. the G A B A released by electrical s t i m u l a t i o n causes a transsynaptic inhibition of a n u m b e r o f G A B A e r g i c n e u r o n s : this inhibition and the electrically elicited release o f G A B A equilibrate and the overall values o f the TRGaBA fail to change in the electrically stimulated c a u d a te nucleus. In conclusion, the results presented here d e m o n s t r a t e t h a t in the substantia nigra the TRGaBA increases or decreases when the activity o f the G A B A e r g i c striatonigral p a t h w a y s is e n h a n c e d or suppressed.
1 Bertilsson, L.. Mao. C. C. and Costa, E., Application of principles of steady-state kinetics to the estimation of 7-aminobutyric acid turnover rate in nuclei of rat brain. J. PharmacoL exp. Ther.. 200 (1977) 277-284. 2 Bunney, B. S. and Aghajanian, G. K., The precise localization of nigral afferents in the rat as determined by a retrograde tracing technique, Brain Research, 117 (1976) 423-435. 3 Crossman, A. R., Walker, R. J. and Woodruff, G. N., Pharmacological studies on single neurones in the substantia nigra of the rat, Brit. J. Pharmacol.. 51 (1974) 137P-138P. 4 Dziedzic, S. W., Bertani Dziedzic, L. and Gitlow, S. E., Separation and determination of urinary homovanillic acid and sio-homovanillic acid by gas-liquid chromatography and electron capture detection, J. Lab. clin. Med., 82 (1973) 829-835 5 Feltz, P.. 7-Aminobutyric acid and a caudationigral inhibition, Canad. J. Physiol. Pharmacol., 49 (1971) 1113-1115. 6 Fonnum, F., Grofov~i, 1., Rinvik, E., Storm-Mathisen, J. and Watberg, F., Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Brain Research. 71 (1974) 77-92. 7 Goswell, M. J. and Sedgwick, E. M., Inhibition of the substantia nigra following stimulation of the caudate nucleus, J. Physiol. eLond. ). 218 11971) 327-328. 8 Hornykiewicz, O., Neurochemistry of parkinsonism. In A. Lajtha /Ed.I. Handbook o l Neurochemistry, Vol. 7, Plenum Press, New York, 1972, pp. 465-501. 9 Iversen, L. L., Biochemical psychopharmacology of GABA. In M. A. Lipton, A. DiMascio and K. F. Killam (Eds.), Psychopharmacology: ,4 Generation o f Progress, Raven Press. New York, 1978, pp. 25-38. 10 Kim, J. S., Bak, I. J., Hassler, R and Okada. Y., Role of 7-aminobutyric acid (GABA) in the extrapyramidal motor system. 2. Some evidence for the existence of a type of GABA-rieh strionigral neurons, Exp. Brain Res., 14 (19711 95-104. 1l K~Snig.J. F. R. and Klippel, R. A., The Rat Brain: ,4 Stereotaxic Atlas, R. E. Krieger Publishing, 1967. 12 Mao, C. C., Marco, E., Revuelta, A., Bertilsson, L. and Costa, E., The turnover rate of F-aminobutyric acid in the nuclei of telencephalon: implications in the pharmacology of antipsychotics and of a minor tranquilizer, Biol. Psychiat., 12 (1977) 359-371. 13 Niimi, K., Ikeda, T., Kawamura, S. and Inoshita, H., Efferent projections of the head of the caudate nucleus in the cat, Brain Research, 21 (1970) 327-343 14 Precht. W. and Yoshida, M., Blockage of caudate-evoked inhibition of neurons m the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. 15 Raeagni, G., Cheney, D. L., Trabucchi, M.. Wang, C. and Costa, E., Measurement of acetylcholine turnover rate in discrete areas or rat brain, Life Sci., 15 (1974) 1961-1975. 16 Roberts, E., Disinhibition as an organizing principle in the nervous system the role of the GA BA system. Application to neurologic and psychiatric disorders. In E. Roberts, T. N. Chase and D. B. Tower (Eds.), G,4B,4 in the Nervous System Function, Raven Press, New York, 1976, pp. 532-534. 17 Vogt, C. und Vogt, O., Sitz un Wesen der Krankheiten im Lichte der topistischen Hirnforschung und des Variierens de Tiere. I. Befunde der topistischen Hirnforschung als Beitrag zur Lehre yon Krankheitssitz, J. Psychol. Neurol. (Lpz. J. 47 (1937) 237 457.
147
The turnover rate of 7-aminobutyric acid in the substantia nigra following electrical stimulation or lesioning of the strionigral pathways
C. C. MAO, E. PERALTA*, F. MORIN[** and E. COSTA*** Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint El&abethsHospital, Washington, D.C. 20032 (U.S.A.)
(Accepted June 1st, 1978)
It has long been known that axons from neostriatal neurons innervate the globus pallidus and the substantia nigra2,7,13,17. More recently, some of these strionigral neurons were shown to synthesize, store and secrete gamma-aminobutyric acid (GABA)", 3,5,6,14. These GABAergic axons synapse with dendrites of nigral dopaminergic neurons, thus providing an inhibitory feedback regulation for the nigrostriatal dopaminergic pathway 7. In agreement with this, the iontophoretic application of GABA to substantia nigra inhibits neuronal activity, and this inhibition is nullified by picrotoxin '~. When the head of the nucleus caudatus is stimulated electrically, neuronal firing in substantia nigra is reducedS; this reduction can be blocked by picrotoxin x4. In rats and baboons, a brain hemitransection at the subthalamic level results in a marked decline of the GA BA content and of the glutamic acid decarboxylase (GAD) activity in substantia nigra6,1°; this suggests that, in substantia nigra, GABA is preferentially located in the axon terminals of the descending strionigral pathways. Due to the possible implication of the strionigral pathways in severe neurological disorders such as Parkinson's disease and Huntington's choreaS, 0, biochemical studies to evaluate the regulation of these pathways are of great practical interest. Since the measurement of the steady-state content of GABA in the nigra is of no value in studies of the regulation of GABA neurons, we have explored the limitations and the advantages offered by the estimation of the GABA turnover rate (TRGABA). Thus, the present study was designed to assess whether or not modifications of GABA metabolism in the GABAergic strionigral pathways (through both electrical stimulation and surgical deafferentation) can be detected using TRGABA as an index. It should be pointed out that our estimations of TRGABA probably do not measure with accuracy the absolute value of this parameter, therefore they are valid only for comparative purposesk * Grantee of the Program for Cultural Exchange between Spain and the U.S.A. ** Present Address. Dept. of Pharmacology, University of Florence, Florence, Italy. *** To whom reprint requests should be addressed.
148 Sprague-Dawley rats weighing 110 g were anesthetized with equithesin (0.4 ml/100 g; Jensen Salsberg Labs), and a guide cannula (22-gauge) (Plastic Products. Roanoke, Va.) with an intracranial shaft cut to 2 m m in length was placed stereotaxically in the head of the right nucleus caudatus (AP 7890, L 2600 according t o K6nig and KlippeP~). This cannula was then secured with dental cement and its lumen temporarily occluded with a wire to prevent clogging. Rats were allowed to recover from the surgery for 3 days before proceeding with electrical stimulation experiments. To stimulate the nucleus caudatus, a monopolar electrode (Rhodes Medical Instruments) was inserted into the implanted cannula. The electrode (10 m m shaft length, 0.25 mm shaft diameter) was entirely insulated except for the tip. When the electrode was inserted into the cannula, it protruded by 1 m m into the outer shell of the nucleus caudatus: the reference electrode made contact with the skin. The outer shell of the nucleus caudatus was chosen as the site for the electrode placement. because Bunney and Aghajanian z have shown that the caudate neurons that project to the substantia nigra are located in the outer shell. In other experiments the animals were hemitransected by means of a 3 mm wide knife lowered with an angle of 60 ° at the level of the coronary suture (AP 82; K6nig and KlippePl). The knife was inserted next to the midline and subsequently moved outwards to achieve complete hemitransection. The TR6ABA was estimated using a previously described technique 1-, which involves infusion of [13C]glucose through the tail vein and mass fragmentographic determination of the changes with time of the G A B A and glutamic acid enrichment in ~aC. Electrical stimulation of the nucleus caudatus was initiated before the 13C infusion: the experimental protocol required that, regardless of the infusion time with [13C]glucose, the nucleus caudatus was stimulated for 20 min. A detailed account of the stimulation parameters and infusion times is g~ven in the legend for Table [. Homovanillic acid (HVA) in caudate nucleus was assayed by mass fragmentography, using the derivatives described by Dziedzic et al. ~. In preliminary experiments, the nucleus caudatus was stimulated with 2 and 4 V (20 Hz, 0.5 msec pulse duration), but this current intensity failed to increase the TRGABA in either the ipsilateral or contralateral substantia nigra (data not shown). Therefore, the stimulus intensity was increased to 10 V, all other parameters being kept constant. Table I lists the GABA content, rate constant for G A B A efflux (kGABA) and the TRGABA in the substantia nigra and nucleus caudatus ipsilateral and contralateral to the electrically stimulated nucleus caudatus. Calculations were performed using the precursor product relationship at steady-state according to the mathematic model described by Racagni et al. L~ for acetytcholine. The TR6AnA of the nonstimulated side was contrasted with that of the stimulated side: Table 1 also lists the values of kGABA and TRGABA in non-stimulated rats. Fig. 1 shows the ~ C enrichment of glutamm acid and G A B A stored in the substantia nigra ipsitateral and contralaterat to the stimulated nucleus caudatus. It can be seen that the time course of the increment with time of the ~3C enrichment of glutamJc acid is essentially identical in both substantia nigra. In contrast, in the substantia nigra ipsilateral to the stimulus the increment with time of the 1~C enrichment of G A B A is steeper than that of the contralateral substantia nigra.
149
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I 3 6 . GLUCOSE INFUSION
Fig. 1. Effect of unilateral electrical stimulation of the nucleus caudatus on the ~zC enrichment of GABA and glutamic acid in ipsilateral and contralateral substantia nigra. The intensity o f the electrical stimulation was 8 V, 20 Hz and 0.5 msec for 20 min. * P < 0.005 according to the student paired 't'-test when compared to the laC enrichment of GABA stored in the contralateral substantia nigra.
The GABA and glutamic acid content of substantia nigra or nucleus caudatus remained unchanged following electrical stimulation of nucleus caudatus (see Table I). The kGABA in the substantia nigra of non-stimulated rats or that of the tissue contralateral to the stimulus were similar (see Table I). In contrast, the kGABA of substantia nigra ipsilateral to the stimulus was 2-3-fold greater than that of the substantia nigra of non-stimulated rats or that contralateral to the stimulated nucleus caudatus (see Table I). Since the GABA content did not change, the TRt~ABA in the substantia nigra ipsilateral to the stimulated nucleus caudatus was significantly higher than that of the contralateral side or that of the non-stimulated rats. Fig. 2 shows the TRGABA values in caudatus o f the hemitransected animals one week after surgery. Besides decreasing the TRGABA values, this surgical procedure decreased also the GABA and glutamate content of substantia nigra and the HVA content of striatum (see Table ll). These observations provide direct evidence that electrical activation or surgical lesioning of strionigral GABAergic axons increases and decreases the TRc,~BA, respectively. Since the GABA content failed to increase in sites in which the TRGABa was increased by electrical stimulation, we infer that the increased amount of GABA synthesized was either released and promptly metabolized or was metabolized intraneuronally. Our data indicate that the effects of the stimulus applied to the head of the nucleus caudatus are confined to the ipsilateral substantia nigra, suggesting that the long axons of GABAergic striatal neurons innervate only the ipsilateral dopaminergic neurons of substantia nigra.
151
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TRGABA IN SUBSTANTIA NIGRA nrnol/mg prot/hr CONTROL SIDE LESIONED SIDE **
412 +_ 4 4 159 +.t. 3 4 * *
P < 0.01
Fig. 2. Schematic sagittal section showing the location of the knife cut. The following abbreviations are used: HP, h i p p o c a m p u s ; GP, globus pallidus; SN, substantia nigra.
In the ipsilateral and contralateral n. caudatus, the values of the kGABA w e r e not significantly changed by a stimulus that increased the kGABA in the ipsilateral substantia nigra (see Table I). It is known that the long axon GABAergic neurons located in the outer shell of the head of the nucleus caudatus innervate the substantia nigra 2, but the nucleus caudatus also contains a number of small GABAergic interneurons, intrinsic to the caudate which appear to constitute a neuronal population exerting a tonic inhibition of the adjacent neurons t6. The lack of increase in the TABLE II GA BA and glutamate content in substantia nigra and H VA content in n. caudatus o f hemitransected animals Each value represents the mean ± S.E. of at least 7 determinations. Substantia nigra (nmole/mg protein)
Control side Lesion side * P < O.Ol.
Caudate nucleus (pmole/mg protein)
GABA
Glutamate
HVA
69 ± 4.0 26 ± 3.4*
68 ± 2.0 56 ± 1.5"
66 ± 0.66 3l ± 0.34*
152 value o f kGABA in the
stimulated
nucleus
caudatus
suggests t h a t
the
GABA
m e t a b o l i s m in these intrinsic c a u d a t e n e u r o n s was n o t c h a n g e d substantially by our m e t h o d o f stimulation. Perhaps, as indicated in previous experiments t '. the G A B A released by electrical s t i m u l a t i o n causes a transsynaptic inhibition of a n u m b e r o f G A B A e r g i c n e u r o n s : this inhibition and the electrically elicited release o f G A B A equilibrate and the overall values o f the TRGaBA fail to change in the electrically stimulated c a u d a te nucleus. In conclusion, the results presented here d e m o n s t r a t e t h a t in the substantia nigra the TRGaBA increases or decreases when the activity o f the G A B A e r g i c striatonigral p a t h w a y s is e n h a n c e d or suppressed.
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