- Email: [email protected]
Uptake and release of γ-aminobutyric acid (GABA) in slices of substantia nigra of rat
214
Brain Research. 4~ (1973) 214-217 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Uptake and release of y-aminobutyric acid (GABA) in dices of substantia • igra of rat
YASUHIRO OKADA* AND ROLF HASSLER Neurobiologische Abteilung, Max-Planck-lnstitut fiir Hirnforschung, ( G.F.R.)
Frankfurt/M., Niederrad
(Accepted October 3rd, 1972)
There is now good evidence indicating that GABA functions as a neurotransmitter in mammalian central nervous system 4,6.In regional studies of the GABA concentration in mammalian CNS, the substantia nigra (Ni) was found to contain the highest amount of GABA in the brain 1,7. Destruction of the striato-nigral fibers produced a marked decrease of the G A B A content in Ni 3. Furthermore, neurophysiological studies of this pathway have demonstrated a distinct inhibitory effect of the caudate nucleus on the Ni 9. In this paper we have studied the uptake and release of [14C]GABA in isolated slices of rat Ni with and without electrical stimulation for the purpose of expanding the evidence that GABA is a neurotransmitter in the nucleus. Albino Dawley rats weighing 150 g were used for the experiment. After decapitation the brain was quickly removed from the skull and about a 3 m m thick cross section was made at the level of the peduncle to expose the Ni. The excised tissue block was put on the cutting table and the slice (350-450 /zm) of the cross section was prepared with a razor blade using a sliding guide in the same manner as reported in the slice of the cortex 5. The Ni was punched out with a small metal cylinder (diameter 1.5 mm) and the white peduncular layer was removed from the slice. After weighing the slice of Ni (about 0.8 mg) with a torsion balance, it was preincubated at 37°C for 15 min in a 1 ml glucose-Krebs-bicarbonate medium which was equilibrated with 95 ~ Oz and 5 ~ COs. Preincubation was started within 3-4 min after the decapitation. After preincubation the tissue was further incubated for 30 rain in a medium containing [14C]GABA and aminooxyacetic a c i d - - A O A A (final concentration 10-SM, purchased from Mann Research Lab.). The final concentration of [14C]GABA (specific activity 200 mCi/mmole, Radiochemical Center Amersham) in the medium was 0.75 × 10-TM. In experiments using [14C]leucine (specific activity 344 mCi/ mmole, Radiochemical Center Amersham) and [14C]glycine (specific activity 108 mCi/mmole, Radiochemical Center Amersham) the final concentrations were 1 × 10-TM and 1.5 x 10-7M, respectively. After incubation, the slice was moved to a * Present address: Dr. Y. Okada, Department of Neurcchemistry, Tokyo Metropolitan Institute for Neurosciences, 2-6, Musashidai, Fuchu-city, Tokyo, Japan.
SHORT
215
COMMUNICATIONS
5.0
4.C
~A B",
=
3.C
~,o
2.C
0
®
I-
/,,,
o
1.0
I..-, IO
,
_
.
,
,
-.-'.-- .--."-_-= o-
•
-iP
I
. . . .
e.
•
3no
20 Minutes
Fig. 1. Release of [14C]GABAfrom Ni slice with and without electrical stimulation. A, (@ - - - - 0) indicates the release of [14C]GABAwith electrical stimulation and B, (m m) without stimulation. In A, onset and period of electrical stimulation are shown by an arrow and black rectangle. Each point is the mean result of 4 slices for A and 5 for B. The S.E.M. of each point is within 12 ~. Ordinate indicates the percentage of released radioactivity against the total radioactivity taken up in the slice during 30 min incubation. In abscissa 0 means the onset of washing. The total radioactivity taken up into the tissue was 110 × 103 5:9 × 103 disint./min/mg wet weight for A and 112 × 10a ± 8.5 × 103 disint./min/mg for B respectively.
stimulation chamber (37 °C) which contained a pair of silver wire electrodes (interelectrode distance 0.8 mm) on nylon mesh. Every 2 min 1 ml of [14C]GABA-free incubation medium was introduced into the chamber. Prior to each influx a 1 ml medium sample in which the slice had been washed or stimulated was collected for the determination of radioactivity. After 10 min of washing when the spontaneous ettlux of radioactivity had fallen to a steady level (Fig. 1) rectangular pulse stimulation (0.8 msec, 50 c/sec, 18 V) was applied for 90 sec. During stimulation the level of medium surface was lowered to slice level to have effective stimulation, but it was raised every 15 sec in order to prevent drying of the tissue during stimulation. Ten milliliter of 0.5 ~ PPO (2,5-diphenyloxazole), 1 0 ~ naphthaline in dioxane was added to the aliquot of serial fractions of the collected medium and radioactivity was assayed by liquid scintillation spectrometry (Beckmann PL 200). At the end of the experiment, the tissue slice was dissolved in a vial with 0.5 ml Soluent TM 100 (Packard) and incubated for 20 min at 60 °C. Then 10 ml toluene scintillation solution was added and the radioactivity was assayed. In rat Ni there was a high uptake of [14C]GABA as estimated by the accumulation of radioactivity in the tissue. After incubating the slice for 30 min, the uptake ratio (radioactivity in tissue/mg: radioactivity in medium/#l) was 27.7:1. The ratio of [14C]glycine uptake was 17.0:1, whereas the uptake of [14C]leucine was very low with a ratio of 2.1:1. The high uptake ratio of [14C]GABA of Ni slice is in good agreement with the result of H5kfelt et aL 2 who found the highest uptake of [3H]GABA in Ni in the regional study of rat brain. The release of the radioactivity both with and without stimulation is shown in
216
SHORT COMMUNICATIONS 50
40
¢*
30
u
o
20
n,-
IO
o o
I o
20
30
Minutes
Fig. 2. Effect o f electrical stimulation o n the efflux o f [14C]leucine. Each point is the m e a n result o f 3 slices. Total radioactivity o f the slice was 12.3 × 10 a i 0.8 x 10 3 d i s i n t . / m i n / m g . Experimental procedure was the s a m e as that o f Fig. 1.
Fig. 1. The initial rapid release in the first 2 and 4 min collections probably represents the [14C]GABA of the extracellular space. After 6 min of washing the rate of release steadily decreased. After 30 min of washing, only 15-17 ~ of the total radioactivity taken up during the incubation was released from the tissue. Electrical stimulation for 90 sec after l0 rain washing resulted in 3-fold rise in the etflux of radioactivity (Fig. 1). Repetitive electrical stimulation for 60 sec at 10, 12 and 14 min after the onset of the washing, produced a greater and prolonged increase in [14C]GABA efliux (not shown). The effect of calcium on the release of GABA with stimulation was tested in two slices. The removal of calcium in the washing medium caused no decrease of release after stimulation. The addition of 10 m M EDTA (ethylene-diaminetetraacetic acid) in two experiments showed a slight, probably insignificant decrease in the release of radioactivity. In 3 slices incubated with [14C]leucine the release of radioactivity was extremely rapid compared with that of [14C]GABA (Fig. 2). In the first 2 min 50 ~ of the radioactivity of the total uptake was washed out, and after 30 min washing, 98 ~ of the radioactivity was released. Electrical stimulation failed to produce any increase in the [14C]leucine efftux from the Ni slice (Fig. 2). In 3 of 4 experiments with [14C]glycine, no significant increase in the efflux of radioactivity was found after stimulation, although one of them showed a small increase (1.3-fold). These results indicate that the storage pools for GABA, leucine and glycine may be different and that the release of [14C]GABA after stimulation is not an artifact due to damage of the tissue during stimulation. Incubated tissues and collected mediums with and without electrical stimulation were examined for the metabolites of [14C]GABA by thin-layer electrophoresis in two buffers: Buffer pH 4.8 [pyridine (40 ml) ÷ acetic acid (30 ml) + citric acid (12 g) per liter] and buffer pH 2.1 [acetic acid (12 g) + formic acid (23.7 g) per liter]. Radio-
SHORT COMMUNICATIONS
217
activity was detected on the plates (200/,m Silica Gel G layer) by scanning. With 10-a M A O A A in the medium in both cases approximately 95 ~o of the radioactivity was found in the spots corresponding to GABA. In the case of AOAA-free medium 70 of the radioactivity was present in the G A B A fraction and 30 ~ was found in acidic G A B A metabolites - - on grounds of their electrophoretic mobilities presumably succinate and succinic semialdehyde. Accordingly the high percentage of released radioactivity from incubated tissue samples (see Fig. 1) should be [14C]GABA. Thus we conclude that a high uptake of [14C]GABA and specific release of [14C]GABA after stimulation occurred in the slices of rat Ni. The present results, in connection with evidence that (1) specifically high content of G A B A is found in mammalian Nil, 7, (2) caudato-evoked inhibition in cat Ni is blocked by picrotoxin 8, and (3) there is a marked decrease in the Ni G A B A level after destruction of striatonigral fibers of rat a, provide a credible picture of the function of G A B A as an inhibitory neurotransmitter in mammalian Ni. We wish to thank Dr. Lemmer for his collaboration at the first stage of this study and Dr. N. Seiler and Mr. B. Kn6dgen for their collaboration in the metabolite study of GABA.
1 FAHN, S., AND C~Tt~, L. J., Regional distribution of ~,-aminobutyric acid (GABA) in brain of Rhesus monkey, d. Neurochem., 15 (1968) 209-213. 2 HOKFELT,T., JOHNSON, G., AND LJUNGDAHL,A., Regional uptake and subcellular localization of (aH)-gamma-aminobutyric acid (GABA) in rat brain slices, Life Sci., 9 (1970) 203-212. 3 KIM,J. S., BAK,I. J., HASSLER,R., AND OKADA, Y., Role of),-aminobutyric acid in extrapyramidal motor system. II. Some evidence for existence of a type of GABA-rich strionigral neurons, Exp. Brain Res., 14 (1972) 95-104. 4 KRNJEVI~,K., AND SCHWARTZ, S., The action of 7-aminobutyric acid on cortical neurons, Exp. Brain Res., 3 (1967) 320-336. 5 MCILWAIN,H., AND RODNIGHT, R., Practical Neurochemistry, Churchill, London, 1962, pp. 109-120. 60BATA, K., ITO, M., OCHI,R., ANDSATO,N., Pharmacological properties of the postsynaptic inhibition of Purkinje cell axons and the action of ~,-aminobutyric acid on Deiters' neurones, Exp. Brain Res., 4 (1967) 43-57. 70KADA, Y., NITSCH-HASSLER,C., KIM, J. S., BAK,I. J., ANDHASSLER,R., Role of ),-aminobutyric acid (GABA) in the extrapyramidal motor system. 1. Regional distribution of GABA in rabbit, rat, guinea pig and baboon CNS, Exp. Brain Res., 13 (1971) 514-518. 8 PRECHT,W., ANDYOSHIDA,M., Blockade of caudato-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. 9 YOSHIDA,M., AND PRECHT,W., Monosynaptic inhibition of neurons of the substantia nigra by caudato-nigral fibers, Brain Research, 32 (1971) 225-228.
Brain Research. 4~ (1973) 214-217 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Uptake and release of y-aminobutyric acid (GABA) in dices of substantia • igra of rat
YASUHIRO OKADA* AND ROLF HASSLER Neurobiologische Abteilung, Max-Planck-lnstitut fiir Hirnforschung, ( G.F.R.)
Frankfurt/M., Niederrad
(Accepted October 3rd, 1972)
There is now good evidence indicating that GABA functions as a neurotransmitter in mammalian central nervous system 4,6.In regional studies of the GABA concentration in mammalian CNS, the substantia nigra (Ni) was found to contain the highest amount of GABA in the brain 1,7. Destruction of the striato-nigral fibers produced a marked decrease of the G A B A content in Ni 3. Furthermore, neurophysiological studies of this pathway have demonstrated a distinct inhibitory effect of the caudate nucleus on the Ni 9. In this paper we have studied the uptake and release of [14C]GABA in isolated slices of rat Ni with and without electrical stimulation for the purpose of expanding the evidence that GABA is a neurotransmitter in the nucleus. Albino Dawley rats weighing 150 g were used for the experiment. After decapitation the brain was quickly removed from the skull and about a 3 m m thick cross section was made at the level of the peduncle to expose the Ni. The excised tissue block was put on the cutting table and the slice (350-450 /zm) of the cross section was prepared with a razor blade using a sliding guide in the same manner as reported in the slice of the cortex 5. The Ni was punched out with a small metal cylinder (diameter 1.5 mm) and the white peduncular layer was removed from the slice. After weighing the slice of Ni (about 0.8 mg) with a torsion balance, it was preincubated at 37°C for 15 min in a 1 ml glucose-Krebs-bicarbonate medium which was equilibrated with 95 ~ Oz and 5 ~ COs. Preincubation was started within 3-4 min after the decapitation. After preincubation the tissue was further incubated for 30 rain in a medium containing [14C]GABA and aminooxyacetic a c i d - - A O A A (final concentration 10-SM, purchased from Mann Research Lab.). The final concentration of [14C]GABA (specific activity 200 mCi/mmole, Radiochemical Center Amersham) in the medium was 0.75 × 10-TM. In experiments using [14C]leucine (specific activity 344 mCi/ mmole, Radiochemical Center Amersham) and [14C]glycine (specific activity 108 mCi/mmole, Radiochemical Center Amersham) the final concentrations were 1 × 10-TM and 1.5 x 10-7M, respectively. After incubation, the slice was moved to a * Present address: Dr. Y. Okada, Department of Neurcchemistry, Tokyo Metropolitan Institute for Neurosciences, 2-6, Musashidai, Fuchu-city, Tokyo, Japan.
SHORT
215
COMMUNICATIONS
5.0
4.C
~A B",
=
3.C
~,o
2.C
0
®
I-
/,,,
o
1.0
I..-, IO
,
_
.
,
,
-.-'.-- .--."-_-= o-
•
-iP
I
. . . .
e.
•
3no
20 Minutes
Fig. 1. Release of [14C]GABAfrom Ni slice with and without electrical stimulation. A, (@ - - - - 0) indicates the release of [14C]GABAwith electrical stimulation and B, (m m) without stimulation. In A, onset and period of electrical stimulation are shown by an arrow and black rectangle. Each point is the mean result of 4 slices for A and 5 for B. The S.E.M. of each point is within 12 ~. Ordinate indicates the percentage of released radioactivity against the total radioactivity taken up in the slice during 30 min incubation. In abscissa 0 means the onset of washing. The total radioactivity taken up into the tissue was 110 × 103 5:9 × 103 disint./min/mg wet weight for A and 112 × 10a ± 8.5 × 103 disint./min/mg for B respectively.
stimulation chamber (37 °C) which contained a pair of silver wire electrodes (interelectrode distance 0.8 mm) on nylon mesh. Every 2 min 1 ml of [14C]GABA-free incubation medium was introduced into the chamber. Prior to each influx a 1 ml medium sample in which the slice had been washed or stimulated was collected for the determination of radioactivity. After 10 min of washing when the spontaneous ettlux of radioactivity had fallen to a steady level (Fig. 1) rectangular pulse stimulation (0.8 msec, 50 c/sec, 18 V) was applied for 90 sec. During stimulation the level of medium surface was lowered to slice level to have effective stimulation, but it was raised every 15 sec in order to prevent drying of the tissue during stimulation. Ten milliliter of 0.5 ~ PPO (2,5-diphenyloxazole), 1 0 ~ naphthaline in dioxane was added to the aliquot of serial fractions of the collected medium and radioactivity was assayed by liquid scintillation spectrometry (Beckmann PL 200). At the end of the experiment, the tissue slice was dissolved in a vial with 0.5 ml Soluent TM 100 (Packard) and incubated for 20 min at 60 °C. Then 10 ml toluene scintillation solution was added and the radioactivity was assayed. In rat Ni there was a high uptake of [14C]GABA as estimated by the accumulation of radioactivity in the tissue. After incubating the slice for 30 min, the uptake ratio (radioactivity in tissue/mg: radioactivity in medium/#l) was 27.7:1. The ratio of [14C]glycine uptake was 17.0:1, whereas the uptake of [14C]leucine was very low with a ratio of 2.1:1. The high uptake ratio of [14C]GABA of Ni slice is in good agreement with the result of H5kfelt et aL 2 who found the highest uptake of [3H]GABA in Ni in the regional study of rat brain. The release of the radioactivity both with and without stimulation is shown in
216
SHORT COMMUNICATIONS 50
40
¢*
30
u
o
20
n,-
IO
o o
I o
20
30
Minutes
Fig. 2. Effect o f electrical stimulation o n the efflux o f [14C]leucine. Each point is the m e a n result o f 3 slices. Total radioactivity o f the slice was 12.3 × 10 a i 0.8 x 10 3 d i s i n t . / m i n / m g . Experimental procedure was the s a m e as that o f Fig. 1.
Fig. 1. The initial rapid release in the first 2 and 4 min collections probably represents the [14C]GABA of the extracellular space. After 6 min of washing the rate of release steadily decreased. After 30 min of washing, only 15-17 ~ of the total radioactivity taken up during the incubation was released from the tissue. Electrical stimulation for 90 sec after l0 rain washing resulted in 3-fold rise in the etflux of radioactivity (Fig. 1). Repetitive electrical stimulation for 60 sec at 10, 12 and 14 min after the onset of the washing, produced a greater and prolonged increase in [14C]GABA efliux (not shown). The effect of calcium on the release of GABA with stimulation was tested in two slices. The removal of calcium in the washing medium caused no decrease of release after stimulation. The addition of 10 m M EDTA (ethylene-diaminetetraacetic acid) in two experiments showed a slight, probably insignificant decrease in the release of radioactivity. In 3 slices incubated with [14C]leucine the release of radioactivity was extremely rapid compared with that of [14C]GABA (Fig. 2). In the first 2 min 50 ~ of the radioactivity of the total uptake was washed out, and after 30 min washing, 98 ~ of the radioactivity was released. Electrical stimulation failed to produce any increase in the [14C]leucine efftux from the Ni slice (Fig. 2). In 3 of 4 experiments with [14C]glycine, no significant increase in the efflux of radioactivity was found after stimulation, although one of them showed a small increase (1.3-fold). These results indicate that the storage pools for GABA, leucine and glycine may be different and that the release of [14C]GABA after stimulation is not an artifact due to damage of the tissue during stimulation. Incubated tissues and collected mediums with and without electrical stimulation were examined for the metabolites of [14C]GABA by thin-layer electrophoresis in two buffers: Buffer pH 4.8 [pyridine (40 ml) ÷ acetic acid (30 ml) + citric acid (12 g) per liter] and buffer pH 2.1 [acetic acid (12 g) + formic acid (23.7 g) per liter]. Radio-
SHORT COMMUNICATIONS
217
activity was detected on the plates (200/,m Silica Gel G layer) by scanning. With 10-a M A O A A in the medium in both cases approximately 95 ~o of the radioactivity was found in the spots corresponding to GABA. In the case of AOAA-free medium 70 of the radioactivity was present in the G A B A fraction and 30 ~ was found in acidic G A B A metabolites - - on grounds of their electrophoretic mobilities presumably succinate and succinic semialdehyde. Accordingly the high percentage of released radioactivity from incubated tissue samples (see Fig. 1) should be [14C]GABA. Thus we conclude that a high uptake of [14C]GABA and specific release of [14C]GABA after stimulation occurred in the slices of rat Ni. The present results, in connection with evidence that (1) specifically high content of G A B A is found in mammalian Nil, 7, (2) caudato-evoked inhibition in cat Ni is blocked by picrotoxin 8, and (3) there is a marked decrease in the Ni G A B A level after destruction of striatonigral fibers of rat a, provide a credible picture of the function of G A B A as an inhibitory neurotransmitter in mammalian Ni. We wish to thank Dr. Lemmer for his collaboration at the first stage of this study and Dr. N. Seiler and Mr. B. Kn6dgen for their collaboration in the metabolite study of GABA.
1 FAHN, S., AND C~Tt~, L. J., Regional distribution of ~,-aminobutyric acid (GABA) in brain of Rhesus monkey, d. Neurochem., 15 (1968) 209-213. 2 HOKFELT,T., JOHNSON, G., AND LJUNGDAHL,A., Regional uptake and subcellular localization of (aH)-gamma-aminobutyric acid (GABA) in rat brain slices, Life Sci., 9 (1970) 203-212. 3 KIM,J. S., BAK,I. J., HASSLER,R., AND OKADA, Y., Role of),-aminobutyric acid in extrapyramidal motor system. II. Some evidence for existence of a type of GABA-rich strionigral neurons, Exp. Brain Res., 14 (1972) 95-104. 4 KRNJEVI~,K., AND SCHWARTZ, S., The action of 7-aminobutyric acid on cortical neurons, Exp. Brain Res., 3 (1967) 320-336. 5 MCILWAIN,H., AND RODNIGHT, R., Practical Neurochemistry, Churchill, London, 1962, pp. 109-120. 60BATA, K., ITO, M., OCHI,R., ANDSATO,N., Pharmacological properties of the postsynaptic inhibition of Purkinje cell axons and the action of ~,-aminobutyric acid on Deiters' neurones, Exp. Brain Res., 4 (1967) 43-57. 70KADA, Y., NITSCH-HASSLER,C., KIM, J. S., BAK,I. J., ANDHASSLER,R., Role of ),-aminobutyric acid (GABA) in the extrapyramidal motor system. 1. Regional distribution of GABA in rabbit, rat, guinea pig and baboon CNS, Exp. Brain Res., 13 (1971) 514-518. 8 PRECHT,W., ANDYOSHIDA,M., Blockade of caudato-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Research, 32 (1971) 229-233. 9 YOSHIDA,M., AND PRECHT,W., Monosynaptic inhibition of neurons of the substantia nigra by caudato-nigral fibers, Brain Research, 32 (1971) 225-228.