Jolourd
of h'uuvocliernlstry. 1976. Vol 26. pp. 135-139 Pergdmon Prcss. Printed in Great Britain.
REDUCTION O F y-AMINOBUTYRIC ACID LEVEL IN THE CAT CEREBRAL CORTEX BY AFFERENT ELECTRICAL STIMULATION D. LICHTSHTEIN and J. DOBKIN DepaLtment of Physiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 28 February 1975. Accepted 23 June 1975)
Abstract-Electrical stimulation for 30 s of one brachial plexus in cat (afferent electrical stimulation = AES) produced a 20% decrease in GABA level of the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex in the same animal. This change in GABA was reversed within a few seconds after cessation of stimulation. Inhibition of GABA catabolism by aminooxyacetic acid elevated considerably the cortical level of GABA but failed to prevent lowering GABA by AES. When AES was performed in preconvulsive condition induced by administration of picrotoxin, the decreasc in GABA was negligible, while similar treatment with pentylenetetrazol had no influence on the decrease in GABA produced by AES. The observed lowering cortical GABA by AES is interpreted as being associated with some mechanism of the inhibitory transmitter inactivation.
THEREis fairly strong evidence that y-aminobutyric acid (GABA) is an inhibitory transmitter a t certain synapses in mammalian brain (for reviews see: CURTIS & JOHNSTON, 1970; KRNJEVI~, 1974).HOWEVER, GABA is compartmented in brain tissue and only a small fraction of the tissue GABA is located within presynaptic nerve terminals and therefore involved in synaptic transmission (BALAZSet al., 1973). Also, since the GABA metabolic flux in brain tissue is only about 8% of the total through the tricarboxylic acid cycle (BALAZSet al., 1970), it is not surprising that the concentration of the inhibitory transmitter in brain appears not to depend o n the state of brain activity. Thus, GABA remains at the normal level in narcosis and hypoxia, it increases only slightly during prolonged seizures induced by general excitants such as picrotoxin and pentylenetetrazol (YOSHINO& ELLIOTT,1970) and is not influenced by electrochock treatment (KAMRIN & KAMRIN, 1961). Our present studies, however, indicate that the steady-state level of GABA in cerebral cortex can be substantially and reversibly reduced by electrical stimulation of sensory nerve fibres (afferent electrical stimulation = AES). To elucidate whether the observed decrease of GABA was due to a change in GABA metabolism or to GABA functioning as a neurotransmitter, the effect of AES was also studied after administration of aminooxyacetic acid (AOAAF-a potent inhibitor of y-aminobutyrate a-ketoglutarate transaminase (GABA-T) which is the only enzyme present in ner& vous tissue capable of catabolizing GABA (HALL KRAVITZ, 1967), or after treatment with picrotoxin Abbreviations used: AES, afferent electrical stimulation; AOAA, aminooxyacetic acid; GAD, L-glutamate decarboxylase; GABA-T y-aminobutyrate ct-ketoglutarate transaminase.
which is a known antagonist of GABA-mediated both pre- and postsynaptic inhibition (DEGROAT,1972 and GALINDO,1969, respectively). The results of the experiments with picrotoxin were compared to those obtained with pentylenetetrazol, since the latter stimulant is not known to interfere with GABA physiological action. MATERIALS AND METHODS The experiments were performed on adult (25-4 kg) unfasted cats of both sexes. The preparation of the animals for monolateral AES, fixation in situ and sampling of the cerebral cortex tissuc were carried out as previously described (DOBKM, 1970). In brief, in cats anaesthetized with pentobarbitone, the nervous trunks of one brachial plexus were exposed and enclosed in a holder containing two electrodes. The skull was removed and the animal left for 1 h for post-operative rest. Care was taken that at the end of this period the animal was maintained under light pentobarbitone anaesthesia, indicated by corneal reflex responsive to stimuli and absence of spontaneous movements. At this point, a metal container packed with dry ice and with added acetone was applied to the surface of the control hemisphere (ipsilateral to the exposed brachial plexus) and 3 s afterwards the opposite hemisphere was stimulated through its contral&ral brachial plexus with square wave pulses (30 V, 2 ms duration at a frequency of 60 per s). After 30 s of stimulation, another freezing container was applied to the timulated hemisphere and 60 s later both frozen cortical areas were excised. It was previously found (DOBKIN, 1970) that freezing one hemisphere for 30 s does not induce measurable change in the temperature of the opposite hemisphere. Unless otherwise stated, the electrical stimulation was continued until after the completion of the excision. The samples thus obtained contained most of the sensory motor cortex, parietal and anterior parts of the occipital lobes. The frozen tissue was freed from most of its white matter to give two samples of 2.2-2.5 mm thickness. Each sample (0.7-0.8 g) was separately extracted with cold 10% trichloroacetic acid using
135
and J. D. LICHTSHTEIN
136
1 ml acid/100 mg tissue. In other experimental groups, AES was applied to cats previously treated with AOAA or picrotoxin or pentylenetetrazol which were used as is indicated in description of the results.
Determination of GABA
Trichloroacetic acid was removed from the extract (5 mi) by passing the solution through a Dowex 1 x 8 acetate W mesh, 1 x 10 cm, followed by washing column of 2 the column with dilute acetic acid. The acidic effluent containing neutral and basic compounds was brought to dryness in a rotary evaporator and the residue redissolved in 4 ml of bidistilled water. GABA was separated from this solution by the method of SANDMAN(1962) based on chromatography on combined columns of Amberlite CG50 and Dowex 50 buffered respectively to pH 5 and 3.1 and then eluting GABA retained by the Dowex 50 W x 8 resin with buffer pH 5.1. This final elution of GABA was made with 20 ml 0 2 M-sodium citrate pH 5.1 instead of 10 ml of the same buffer as is described in the original paper. Aliquots (2ml) of this last eluate were assayed, in duplicate, by the ninhydrin method of MOORE& STEIN (1948) which was used with minor modifications. A blank determination starting with 5 ml 10% TCA and solutions of known quantities of GABA dissolved in 5 ml 10% TCA were included in each series of separation, and the recovery was usually quantitative. The statistical evaluation of the difference in the results between the ipsilateral (control) and contralateral (stimulated) cerebral cortex in the same animal was made with the paired-comparison t-test.
RESULTS I n control experiments on four cats a 30 s interval
was allowed to elapse between the application of the freezing containers to each hemisphere. The content of GABA in the first cortex fixed by freezing was found to be 2.07 0.13 (s.E.M.) and that in the cortex which was frozen 30 s later was 2-08 0.12 p o l / g TABLE1. REDIJCTIONOF
wet weight, thus showing no significant difference between the two. The same was previously found true for the contents of metabolically-associated with GABA glutamic acid and glutamine (DOBKIN, 1970). Electrical stimulation of one brachial plexus for 30 s clearly decreased (by 20.1%) the GABA content of the cortex contralateral to stimulation as compared with the ipsilateral (control) cortex (Table 1). This decrease was not apparent when the fixation by freezing began 5 s after the end of the stimulation (Table 2). When AES was applied to cats that had been treated, 30 min previously with 50 mg/kg of AOAA, the GABA levels in the ipsilateral cortex (Table 3) showed a 1.7-fold increase over the corresponding original values given in Table 1. Elevation of GABA of brain with AOAA is a well-known phenomenon (KURIYAMA et d.,1966; YOSHIN0 & ELLIOIT,1970, and others). It is further seen (Table 3) that the metabolic inhibitor has not prevented the action of AES lowering the cortical level of GABA. In this series the relative decrease from the control value (22.7%) was similar to that obtained without AOAA. Picrotoxin (2 mg/kg) and pentylenetetrazol (65 mg/ kg) induced clonic-tonic convulsions in 6-8 min, and 2-3 rnin after the injection respectively. AES was applied at 5 min after picrotoxin and 1 min after pentylenetetrazol ; at these times the animals appeared aroused and records of their EEG which were taken in separate experiments indicated a preconvulsive condition (Fig. 1). Several animals that happened to start their convulsion already within these known periods after the injection or during the experiment were discarded. It becomes evident from comparing the data in Table 4 with those of Table 1 that the observed decrease in GABA under AES was markedly restored (by 62.8 per cent) by administration of picrotoxin and not in-
CEREBRAL CORTEX LEVEL OF STIMULATION
GABA (Pmolig) Ipsila teral
Contralateral
2.1 7 243 2.17 1.91 2.61 2-20 1.85 1.83 1.84 1.80 2.80 1.42 Mcan S.E.M. 2.08 0.1 I
1.76 1.88 1.63 1.41 2.32 1.82 1.35 1.22 1.63 1.50 2.30 1.02
*
DOBKIN
1.65 2 0.11
GABA
BY AFFERENT ELECTRICAL
Difference (Ipsilateral minus contralateral) 0.41 055 054 0.50 0.29 0.38 0.50 061
0.21 030 0.50 0.40 043
0035*
The control cerebral cortex, ipsilateral to the electrically stimulated brachial plexus, was frozen before the commencement of stimulation. After 30 s of stimulation the contralateral cortex was frozen while stimulation was still under way. * Significantly greater than zero at P > 0.001.
Effect of afferent stimulation on cortical GABA level
TABLE 2. REVERSIBILITY OF REDUCTION
OF CEREBRAL CORTEX LEVFL OF
137
GABA
AFTER CESSATION
OF AFFERENT ELECTRICAL STIMULATION
GABA (mol/g)
No. of cats 8
Ipsilateral 2.07
0.14
Contralateral 2.11
Difference (Ipsilateral minus contralateral) -0.04 _+ 0.09*
013
Conditions were the same as in Table 1 except that after 30 s of stimulation the current was switched off and freezing of contralateral (stimulated) cortex was initiated 5 s later. Data are means k S.E.M. * Not significantly differcnt from zero. TABLE3. ErFECT OF
AFFERENT ELECTRICAL STIMULATION ON THE CORTICAL LEVEL OF
GABA
IN
AMINOOXYACETIC ACID-TREATED CATS
GABA (Pmol/g) No. of cats
Ipsilateral
12
3.74 f 0.15
Contralateral 2.89
0.14
Difference (Ipsilateral minus contralateral) 0.85
0,10*
AES was performed as indicated in Table I , 30 min after AOAA (50 mgikg) had been injected i.v. Results are expressed as means f S.E.M.
* Significantly greater
than zero at P < 0.001
fluenced by pentylenetetrazol administration, in which case the decrease was even slightly but not significantly greater. Neither convulsant drug produced at this stage of its action any significant change in the level of GABA in the cerebral cortex (cf. YOSHINO & ELLIOTT,1970), though in both cases the level showed a slight downward trend. DISCUSSION
Our results clearly demonstrate that unilateral AES of 30 s produces a decrease of GABA content in the stimulatcd cortex, amounting to about one fifth of the total. This change in GABA was not prevented
FIG.1. Elcctrocorticographic effects of picrotoxin and pentylenetetrazol in the anaesthetized cat. A, control; BI, 5 min after injection of picrotoxin (2 m a g ) ; CI, 1 min after injection and pentylenetetrazol (65 mg/kg). BII and CII, beginning of clonic tonic convulsions 6-8 mia after picrotoxin and 2-3 min after pentylenetetrazol respectively.
by inhibition of GABA-T with AOAA, therefore it could not be due to increased metabolic utilization of GABA through the dominating pathway-i.e. GABA transaminating with a-ketoglutarate to form succinic semialdehyde which is subsequently oxidized to succinic acid. The observations that the AES-evoked decrease in GABA was largely inhibited by an antagonist of GABA physiological action-picrotoxin but not by pentylenetetrazol, dissociate once more this decrease from the overall metabolism of this amino acid in the brain and clearly relate it to the minor part of tissue GABA which functions as a neurotransmitter. It may, therefore, be assumed that the mechanism by which AES lowered the amount of GABA in cortical tissue involves release of GABA from inhibitory nerve terminals. This view is supported by previous reports that GABA is released when brain tissue is stimulated in uivo (OBATA& TADEKA, 1969; MITCHELL & SRINIVASAN, 1969) and also when cerebral cortex slices in uitro are subjected to depolarizing stimulation (SRINIVASAN et al., 1969; G T z et a[., 1969; MACHIYAMA et al., 1970; ARNFRED& HERTZ, 1971) and this in uitro efflux of GABA is supposed to be an approximate model of in uiuo release of GABA at inhibitory synapses (MACHIYAMAet al., 1970; JOHNSTON & MITCHELL, 1971). In particular, the electrically-evoked release of GABA by brain cortex slices in uitro is also prevented by picrotoxin and not influenced by pentylenetetrazol (JOHNSTON & MITCHELL,1971) so that in these respects it closely resembles the present decrease in GABA under AES. The amount of GABA located within nerve terminal is supposed to be 25-30% of the y-aminobutyrate in brain tissue (MACHIYAMAet al., 1970; BALLZS
D. LICHTSHTEIN and J. DOBKIN
138
TABLE 4. THEEFFECTS
OF PICROTOXIN AND PENTYLENETETRAZOLON THE DECREASE IN CORTICAL INDUCED BY AFFERENT ELECTRICAL STIMULATION
GABA
GABA (pmol/g) Treatment
Difference (Ipsilateral minus contralateral)
No. of cats
Ipsilateral
12
197 f 0.09
1.81
0.09
0-16 f 0.05*
1.87
1.35 f 008
0.52 f 009t
Contralateral
~
Picrotoxin Pentylenetetrazol
8
004
AES was performed as indicated for Table 1 5 min after i.v. injection of picrotoxin (2 mg/kg) or 1 min after injection of pentylenetetrazol (65 mg/kg). At each time the animals appeared aroused but general convulsions had not begun. Results are expressed as mean S.E.M. * Significantly greater than zero ( P < 0.02). t Significantly greater than zero ( P < 0401b e t al., 1973), so that also from a quantitative point of view the observed 207( reduction of cortical GABA caused by AES may well be related to synapticallyreleased GABA. Inhibition of GABA-T by AOAA increased by 1-7 times the cortical GABA content and doubled the amount of GABA reduced by AES. (Table 3 versus Table 1) According to BALLZSe t al., (1973) the nerve terminal compartment of GABA accounts for about 50% of the total GABA formation flux and therefore, in this compartment the proportion of the tricarboxylic acid cycle flux channelled through GABA may be considerably larger than in the brain tissue as a whole. It follows from these considerations that the inhibition of GABA catabolism which occurred in our experiments with AOAA has presumably elevated GABA within nerve terminals to an even greater extent than the observed 1.7-fold increase in the cortical tissue as a whole, and this is reflected in the relatively large amount of GABA that has been released by AES. Clearly, the main question that emerges from the present results concerns the mechanism by which AES caused the apparent disappearance of synapticalIy-released GABA from the cortical tissue, or, in other words, what was the mechanism responsible for inactivation of the inhibitory transmitter after it has been released at synaptic cleft in response to afferent stimulation. It has recently been postulated (e.g. IVERSEN, 1971; KELLYet al., 1973) that amino acids which function as neurotransmitters, particularly GABA, terminate their postsynaptic action by a specific process of re-uptake into presynaptic nerve terminals. This type of inactivation would not, by itself, alter the GABA concentration in the tissue and thus would not provide an explanation for the observed decrease in GABA. Since the possibility of inactivation by enzymic degradation is also excluded by the results of the experiments with AOAA, some other mechanisms of transmitter inactivation may be considered (for review see WERMAN,1966). Thus GABA could be removed from synaptic cleft by diffusion into CSF or be transferred into the blood stream. Actually,
the latter possibility is not likely, since recent experiments in our laboratory showed that AES does not result in any leakage of GABA into cerebral venous blood. Apart from extracellular diffusion GABA could react in an enzymatic anabolic process to form one or more of its products (such as y-aminobutyrylcholine, homocarnosine and others) known to be present in mammalian brain (for review see BAXTER, 1970). The restoration of GABA to its control level after cessation of the stimuli was at a fairly rapid rate of 0.43 pnol/5s/g., a value which exceeds many times those reported for the synthesis of GABA by GAD (e.g. SZE& L~VELL, 1970, found the rate of 35 pmol/h/ g in the mouse brain). Since following the application of the freezing containers it takes brain cortex some 4 s to cool to 0°C at a depth of 1-2 mm (DOBKIN, 1970) it may well be that the activity of GAD continued during the first seconds of cooling. Nevertheless, we have to assume that a part of the missing GABA was recovered by a process other than synthesis de nouo, as, for instance by liberation from a chemically bound form. In any case, our results indicate that after cessation of cerebral stimulation, there exists a mechanism which rapidly restores the cortical level of this inhibitory neurotransmitter. Acknowledgement-This work was supported in part by a grant from the Hebrew University-Hadassah Medical School.
REFERENCES ARNFRED T . & HERTZ L. (1971) J. Neurochem. 18. 259-265. BALAZS R., MACHIYAMA Y., HAMMONDB. J., JULIAN T., & TICHTER D. (1970) Biochem. J. 116. 445461. BAIAZSR., MACHIYAMY.& PATEL A. J. (1973) in Metabolic Compartmentation in the brain (BALAZS R . & CREMER J. E., eds.) pp. 57-70. MacMillan, London. BAXTERC. F. (1970) in Handbook of Neurochemistry (LAJTHA A., ed.). Vol. 3 pp. 289-353. Plenum Press, New York. CURTISD. R. & JOHNSTON G. A. R. (1970) in Handbook of Neurochemistry (LAITHA A,, ed.) Vol. 4, pp. 115-134. Plenum Press, New York.
Effect of afferent stimulation on cortical GABA level DE GROATW. A. (1972) Brain Res. 38. 429432. DOBKIN J. (1970) J . Neurochem. 17, 237-246. GALINDO A. (1969) Brain Res. 14. 763-767. HALLZ. W. & KRAVITLE. A. (1967) J. Neurochem. 14. 45-54. IVERSEN L. L. (1971) Br. J . Pharmac. 41. 571-591. JOHNSTON G. A. R. & MITCHELL J. F. (1971) J. Neurochem. 18. 2441-2446. KAMRIN A. 0. & KAMRWA. A. (1961) J . Neurochem. 6, 219-225. KATZR. I., CHASE T. N. & KOHNI. J. (1969) J . Neurochem. 16. 961-967. KELLYJ. S., IVERSEN L. L., MINCHIN M. & SCHONF. (1973) Abst. of 4th Meeting of International Society for Neurochemistry, Tokyo, p. 27. KRNJEVICK. (1974) Physiol. Rev. 54. 418-540.
139
KURIYAMA K., ROBER,I.S E. & RUBINSTEIN M. K. (1966) Biochem. Pharmac. 15. 221-236. MACHIYAMA Y., BALAZSR., HAMMOND B. J., JULIANT. & RICHTERD. (1970) Biochem. J . 116. 469-481. MITCHELL J. F. & SRMIVASAN V. (1969) Nature, Lond. 224. 663-666. MOORES . & STEINW. H. (1948) J . biol. Chem. 176. 367-388. ORATAK. & TAREDA K. (1969) 1. Neurochem. 16. 10431047. SANDMAN R. P. (1962) Anulyt. Biochem. 3. 158-163. SRINIVASAN V., NEALM. J, & MITCHELLJ. F. (1969) J . Neurochem. 16. 1235-1244. SZEP. Y.& LOVELLR. A. (1970) J . Neurochem. 17. 16571664. WERMAN R. (1966) Comp. Biochem. Physiol. 18. 745-766. YOSHINOY. & ELLIOTTK. A. C. (1970) Can. J . Biochem. 48, 228--235.
of h'uuvocliernlstry. 1976. Vol 26. pp. 135-139 Pergdmon Prcss. Printed in Great Britain.
REDUCTION O F y-AMINOBUTYRIC ACID LEVEL IN THE CAT CEREBRAL CORTEX BY AFFERENT ELECTRICAL STIMULATION D. LICHTSHTEIN and J. DOBKIN DepaLtment of Physiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel (Received 28 February 1975. Accepted 23 June 1975)
Abstract-Electrical stimulation for 30 s of one brachial plexus in cat (afferent electrical stimulation = AES) produced a 20% decrease in GABA level of the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex in the same animal. This change in GABA was reversed within a few seconds after cessation of stimulation. Inhibition of GABA catabolism by aminooxyacetic acid elevated considerably the cortical level of GABA but failed to prevent lowering GABA by AES. When AES was performed in preconvulsive condition induced by administration of picrotoxin, the decreasc in GABA was negligible, while similar treatment with pentylenetetrazol had no influence on the decrease in GABA produced by AES. The observed lowering cortical GABA by AES is interpreted as being associated with some mechanism of the inhibitory transmitter inactivation.
THEREis fairly strong evidence that y-aminobutyric acid (GABA) is an inhibitory transmitter a t certain synapses in mammalian brain (for reviews see: CURTIS & JOHNSTON, 1970; KRNJEVI~, 1974).HOWEVER, GABA is compartmented in brain tissue and only a small fraction of the tissue GABA is located within presynaptic nerve terminals and therefore involved in synaptic transmission (BALAZSet al., 1973). Also, since the GABA metabolic flux in brain tissue is only about 8% of the total through the tricarboxylic acid cycle (BALAZSet al., 1970), it is not surprising that the concentration of the inhibitory transmitter in brain appears not to depend o n the state of brain activity. Thus, GABA remains at the normal level in narcosis and hypoxia, it increases only slightly during prolonged seizures induced by general excitants such as picrotoxin and pentylenetetrazol (YOSHINO& ELLIOTT,1970) and is not influenced by electrochock treatment (KAMRIN & KAMRIN, 1961). Our present studies, however, indicate that the steady-state level of GABA in cerebral cortex can be substantially and reversibly reduced by electrical stimulation of sensory nerve fibres (afferent electrical stimulation = AES). To elucidate whether the observed decrease of GABA was due to a change in GABA metabolism or to GABA functioning as a neurotransmitter, the effect of AES was also studied after administration of aminooxyacetic acid (AOAAF-a potent inhibitor of y-aminobutyrate a-ketoglutarate transaminase (GABA-T) which is the only enzyme present in ner& vous tissue capable of catabolizing GABA (HALL KRAVITZ, 1967), or after treatment with picrotoxin Abbreviations used: AES, afferent electrical stimulation; AOAA, aminooxyacetic acid; GAD, L-glutamate decarboxylase; GABA-T y-aminobutyrate ct-ketoglutarate transaminase.
which is a known antagonist of GABA-mediated both pre- and postsynaptic inhibition (DEGROAT,1972 and GALINDO,1969, respectively). The results of the experiments with picrotoxin were compared to those obtained with pentylenetetrazol, since the latter stimulant is not known to interfere with GABA physiological action. MATERIALS AND METHODS The experiments were performed on adult (25-4 kg) unfasted cats of both sexes. The preparation of the animals for monolateral AES, fixation in situ and sampling of the cerebral cortex tissuc were carried out as previously described (DOBKM, 1970). In brief, in cats anaesthetized with pentobarbitone, the nervous trunks of one brachial plexus were exposed and enclosed in a holder containing two electrodes. The skull was removed and the animal left for 1 h for post-operative rest. Care was taken that at the end of this period the animal was maintained under light pentobarbitone anaesthesia, indicated by corneal reflex responsive to stimuli and absence of spontaneous movements. At this point, a metal container packed with dry ice and with added acetone was applied to the surface of the control hemisphere (ipsilateral to the exposed brachial plexus) and 3 s afterwards the opposite hemisphere was stimulated through its contral&ral brachial plexus with square wave pulses (30 V, 2 ms duration at a frequency of 60 per s). After 30 s of stimulation, another freezing container was applied to the timulated hemisphere and 60 s later both frozen cortical areas were excised. It was previously found (DOBKIN, 1970) that freezing one hemisphere for 30 s does not induce measurable change in the temperature of the opposite hemisphere. Unless otherwise stated, the electrical stimulation was continued until after the completion of the excision. The samples thus obtained contained most of the sensory motor cortex, parietal and anterior parts of the occipital lobes. The frozen tissue was freed from most of its white matter to give two samples of 2.2-2.5 mm thickness. Each sample (0.7-0.8 g) was separately extracted with cold 10% trichloroacetic acid using
135
and J. D. LICHTSHTEIN
136
1 ml acid/100 mg tissue. In other experimental groups, AES was applied to cats previously treated with AOAA or picrotoxin or pentylenetetrazol which were used as is indicated in description of the results.
Determination of GABA
Trichloroacetic acid was removed from the extract (5 mi) by passing the solution through a Dowex 1 x 8 acetate W mesh, 1 x 10 cm, followed by washing column of 2 the column with dilute acetic acid. The acidic effluent containing neutral and basic compounds was brought to dryness in a rotary evaporator and the residue redissolved in 4 ml of bidistilled water. GABA was separated from this solution by the method of SANDMAN(1962) based on chromatography on combined columns of Amberlite CG50 and Dowex 50 buffered respectively to pH 5 and 3.1 and then eluting GABA retained by the Dowex 50 W x 8 resin with buffer pH 5.1. This final elution of GABA was made with 20 ml 0 2 M-sodium citrate pH 5.1 instead of 10 ml of the same buffer as is described in the original paper. Aliquots (2ml) of this last eluate were assayed, in duplicate, by the ninhydrin method of MOORE& STEIN (1948) which was used with minor modifications. A blank determination starting with 5 ml 10% TCA and solutions of known quantities of GABA dissolved in 5 ml 10% TCA were included in each series of separation, and the recovery was usually quantitative. The statistical evaluation of the difference in the results between the ipsilateral (control) and contralateral (stimulated) cerebral cortex in the same animal was made with the paired-comparison t-test.
RESULTS I n control experiments on four cats a 30 s interval
was allowed to elapse between the application of the freezing containers to each hemisphere. The content of GABA in the first cortex fixed by freezing was found to be 2.07 0.13 (s.E.M.) and that in the cortex which was frozen 30 s later was 2-08 0.12 p o l / g TABLE1. REDIJCTIONOF
wet weight, thus showing no significant difference between the two. The same was previously found true for the contents of metabolically-associated with GABA glutamic acid and glutamine (DOBKIN, 1970). Electrical stimulation of one brachial plexus for 30 s clearly decreased (by 20.1%) the GABA content of the cortex contralateral to stimulation as compared with the ipsilateral (control) cortex (Table 1). This decrease was not apparent when the fixation by freezing began 5 s after the end of the stimulation (Table 2). When AES was applied to cats that had been treated, 30 min previously with 50 mg/kg of AOAA, the GABA levels in the ipsilateral cortex (Table 3) showed a 1.7-fold increase over the corresponding original values given in Table 1. Elevation of GABA of brain with AOAA is a well-known phenomenon (KURIYAMA et d.,1966; YOSHIN0 & ELLIOIT,1970, and others). It is further seen (Table 3) that the metabolic inhibitor has not prevented the action of AES lowering the cortical level of GABA. In this series the relative decrease from the control value (22.7%) was similar to that obtained without AOAA. Picrotoxin (2 mg/kg) and pentylenetetrazol (65 mg/ kg) induced clonic-tonic convulsions in 6-8 min, and 2-3 rnin after the injection respectively. AES was applied at 5 min after picrotoxin and 1 min after pentylenetetrazol ; at these times the animals appeared aroused and records of their EEG which were taken in separate experiments indicated a preconvulsive condition (Fig. 1). Several animals that happened to start their convulsion already within these known periods after the injection or during the experiment were discarded. It becomes evident from comparing the data in Table 4 with those of Table 1 that the observed decrease in GABA under AES was markedly restored (by 62.8 per cent) by administration of picrotoxin and not in-
CEREBRAL CORTEX LEVEL OF STIMULATION
GABA (Pmolig) Ipsila teral
Contralateral
2.1 7 243 2.17 1.91 2.61 2-20 1.85 1.83 1.84 1.80 2.80 1.42 Mcan S.E.M. 2.08 0.1 I
1.76 1.88 1.63 1.41 2.32 1.82 1.35 1.22 1.63 1.50 2.30 1.02
*
DOBKIN
1.65 2 0.11
GABA
BY AFFERENT ELECTRICAL
Difference (Ipsilateral minus contralateral) 0.41 055 054 0.50 0.29 0.38 0.50 061
0.21 030 0.50 0.40 043
0035*
The control cerebral cortex, ipsilateral to the electrically stimulated brachial plexus, was frozen before the commencement of stimulation. After 30 s of stimulation the contralateral cortex was frozen while stimulation was still under way. * Significantly greater than zero at P > 0.001.
Effect of afferent stimulation on cortical GABA level
TABLE 2. REVERSIBILITY OF REDUCTION
OF CEREBRAL CORTEX LEVFL OF
137
GABA
AFTER CESSATION
OF AFFERENT ELECTRICAL STIMULATION
GABA (mol/g)
No. of cats 8
Ipsilateral 2.07
0.14
Contralateral 2.11
Difference (Ipsilateral minus contralateral) -0.04 _+ 0.09*
013
Conditions were the same as in Table 1 except that after 30 s of stimulation the current was switched off and freezing of contralateral (stimulated) cortex was initiated 5 s later. Data are means k S.E.M. * Not significantly differcnt from zero. TABLE3. ErFECT OF
AFFERENT ELECTRICAL STIMULATION ON THE CORTICAL LEVEL OF
GABA
IN
AMINOOXYACETIC ACID-TREATED CATS
GABA (Pmol/g) No. of cats
Ipsilateral
12
3.74 f 0.15
Contralateral 2.89
0.14
Difference (Ipsilateral minus contralateral) 0.85
0,10*
AES was performed as indicated in Table I , 30 min after AOAA (50 mgikg) had been injected i.v. Results are expressed as means f S.E.M.
* Significantly greater
than zero at P < 0.001
fluenced by pentylenetetrazol administration, in which case the decrease was even slightly but not significantly greater. Neither convulsant drug produced at this stage of its action any significant change in the level of GABA in the cerebral cortex (cf. YOSHINO & ELLIOTT,1970), though in both cases the level showed a slight downward trend. DISCUSSION
Our results clearly demonstrate that unilateral AES of 30 s produces a decrease of GABA content in the stimulatcd cortex, amounting to about one fifth of the total. This change in GABA was not prevented
FIG.1. Elcctrocorticographic effects of picrotoxin and pentylenetetrazol in the anaesthetized cat. A, control; BI, 5 min after injection of picrotoxin (2 m a g ) ; CI, 1 min after injection and pentylenetetrazol (65 mg/kg). BII and CII, beginning of clonic tonic convulsions 6-8 mia after picrotoxin and 2-3 min after pentylenetetrazol respectively.
by inhibition of GABA-T with AOAA, therefore it could not be due to increased metabolic utilization of GABA through the dominating pathway-i.e. GABA transaminating with a-ketoglutarate to form succinic semialdehyde which is subsequently oxidized to succinic acid. The observations that the AES-evoked decrease in GABA was largely inhibited by an antagonist of GABA physiological action-picrotoxin but not by pentylenetetrazol, dissociate once more this decrease from the overall metabolism of this amino acid in the brain and clearly relate it to the minor part of tissue GABA which functions as a neurotransmitter. It may, therefore, be assumed that the mechanism by which AES lowered the amount of GABA in cortical tissue involves release of GABA from inhibitory nerve terminals. This view is supported by previous reports that GABA is released when brain tissue is stimulated in uivo (OBATA& TADEKA, 1969; MITCHELL & SRINIVASAN, 1969) and also when cerebral cortex slices in uitro are subjected to depolarizing stimulation (SRINIVASAN et al., 1969; G T z et a[., 1969; MACHIYAMA et al., 1970; ARNFRED& HERTZ, 1971) and this in uitro efflux of GABA is supposed to be an approximate model of in uiuo release of GABA at inhibitory synapses (MACHIYAMAet al., 1970; JOHNSTON & MITCHELL, 1971). In particular, the electrically-evoked release of GABA by brain cortex slices in uitro is also prevented by picrotoxin and not influenced by pentylenetetrazol (JOHNSTON & MITCHELL,1971) so that in these respects it closely resembles the present decrease in GABA under AES. The amount of GABA located within nerve terminal is supposed to be 25-30% of the y-aminobutyrate in brain tissue (MACHIYAMAet al., 1970; BALLZS
D. LICHTSHTEIN and J. DOBKIN
138
TABLE 4. THEEFFECTS
OF PICROTOXIN AND PENTYLENETETRAZOLON THE DECREASE IN CORTICAL INDUCED BY AFFERENT ELECTRICAL STIMULATION
GABA
GABA (pmol/g) Treatment
Difference (Ipsilateral minus contralateral)
No. of cats
Ipsilateral
12
197 f 0.09
1.81
0.09
0-16 f 0.05*
1.87
1.35 f 008
0.52 f 009t
Contralateral
~
Picrotoxin Pentylenetetrazol
8
004
AES was performed as indicated for Table 1 5 min after i.v. injection of picrotoxin (2 mg/kg) or 1 min after injection of pentylenetetrazol (65 mg/kg). At each time the animals appeared aroused but general convulsions had not begun. Results are expressed as mean S.E.M. * Significantly greater than zero ( P < 0.02). t Significantly greater than zero ( P < 0401b e t al., 1973), so that also from a quantitative point of view the observed 207( reduction of cortical GABA caused by AES may well be related to synapticallyreleased GABA. Inhibition of GABA-T by AOAA increased by 1-7 times the cortical GABA content and doubled the amount of GABA reduced by AES. (Table 3 versus Table 1) According to BALLZSe t al., (1973) the nerve terminal compartment of GABA accounts for about 50% of the total GABA formation flux and therefore, in this compartment the proportion of the tricarboxylic acid cycle flux channelled through GABA may be considerably larger than in the brain tissue as a whole. It follows from these considerations that the inhibition of GABA catabolism which occurred in our experiments with AOAA has presumably elevated GABA within nerve terminals to an even greater extent than the observed 1.7-fold increase in the cortical tissue as a whole, and this is reflected in the relatively large amount of GABA that has been released by AES. Clearly, the main question that emerges from the present results concerns the mechanism by which AES caused the apparent disappearance of synapticalIy-released GABA from the cortical tissue, or, in other words, what was the mechanism responsible for inactivation of the inhibitory transmitter after it has been released at synaptic cleft in response to afferent stimulation. It has recently been postulated (e.g. IVERSEN, 1971; KELLYet al., 1973) that amino acids which function as neurotransmitters, particularly GABA, terminate their postsynaptic action by a specific process of re-uptake into presynaptic nerve terminals. This type of inactivation would not, by itself, alter the GABA concentration in the tissue and thus would not provide an explanation for the observed decrease in GABA. Since the possibility of inactivation by enzymic degradation is also excluded by the results of the experiments with AOAA, some other mechanisms of transmitter inactivation may be considered (for review see WERMAN,1966). Thus GABA could be removed from synaptic cleft by diffusion into CSF or be transferred into the blood stream. Actually,
the latter possibility is not likely, since recent experiments in our laboratory showed that AES does not result in any leakage of GABA into cerebral venous blood. Apart from extracellular diffusion GABA could react in an enzymatic anabolic process to form one or more of its products (such as y-aminobutyrylcholine, homocarnosine and others) known to be present in mammalian brain (for review see BAXTER, 1970). The restoration of GABA to its control level after cessation of the stimuli was at a fairly rapid rate of 0.43 pnol/5s/g., a value which exceeds many times those reported for the synthesis of GABA by GAD (e.g. SZE& L~VELL, 1970, found the rate of 35 pmol/h/ g in the mouse brain). Since following the application of the freezing containers it takes brain cortex some 4 s to cool to 0°C at a depth of 1-2 mm (DOBKIN, 1970) it may well be that the activity of GAD continued during the first seconds of cooling. Nevertheless, we have to assume that a part of the missing GABA was recovered by a process other than synthesis de nouo, as, for instance by liberation from a chemically bound form. In any case, our results indicate that after cessation of cerebral stimulation, there exists a mechanism which rapidly restores the cortical level of this inhibitory neurotransmitter. Acknowledgement-This work was supported in part by a grant from the Hebrew University-Hadassah Medical School.
REFERENCES ARNFRED T . & HERTZ L. (1971) J. Neurochem. 18. 259-265. BALAZS R., MACHIYAMA Y., HAMMONDB. J., JULIAN T., & TICHTER D. (1970) Biochem. J. 116. 445461. BAIAZSR., MACHIYAMY.& PATEL A. J. (1973) in Metabolic Compartmentation in the brain (BALAZS R . & CREMER J. E., eds.) pp. 57-70. MacMillan, London. BAXTERC. F. (1970) in Handbook of Neurochemistry (LAJTHA A., ed.). Vol. 3 pp. 289-353. Plenum Press, New York. CURTISD. R. & JOHNSTON G. A. R. (1970) in Handbook of Neurochemistry (LAITHA A,, ed.) Vol. 4, pp. 115-134. Plenum Press, New York.
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