202
Bra#l Research, 98 (1975) 202-206 (~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Distribution of y-aminobutyric acid (GABA) and glutamate decarboxylase (GAD) activity in the guinea pig hippocampus - - microassay method for the determination of GAD activity
Y A S U H I R O O K A D A AND CH1CACO S H I M A D A
Department of Neurochemistry, Tokyo Metropolitan Institute Jot Neurosciences, Fuchu-shi, Tokyo (Japan) (Accepted July 1st, 1975)
There has been increasing evidence indicating that 7-aminobutyric acid (GABA) is an inhibitory neurotransmitter substance in mammalian CNS 7,8,1°. With regard to the role of GABA in hippocampus, physiological and pharmacological studies showed that G A B A has an inhibitory action on pyramidal cells 4,6,a4, Intrahippocampal laminar distribution of G A B A itself, however, has not been studied because of the limitation of the sensitivity of GABA assay system. In the present paper, G A B A distribution in the layers of hippocampus of the guinea pig was determined using a microchemical GABA assay method 11. Together with the assay of GABA, the glutamate decarboxylase (GAD) activity of each layer in the hippocampus and the area dentata was determined by a new method which enables us to measure G A D activity of a tissue sample below 1 #g in weight. Guinea pigs were decapitated and the brains immediately removed and placed on ice. The hippocampus with the attached area dentata was isolated. Sections transverse to the long axis of the hippocampus were cut at about 5 mm thickness and were immediately frozen in Freon-12 (--150 °C) in liquid nitrogen. The frozen blocks were cut at 20 #m thickness with a microtome in a cryostat (--20 °C). The sections were freeze-dried overnight at - - 3 0 °C in a vacuum. The sections were stored in evacuated tubes at - - 3 5 °C until the assays for G A B A and G A D activity were performed. Each layer of hippocampus was dissected out freehand under a stereomicroscope and the samples, weighing 0.1-0.7 #g, were weighed with a fishpole quartz fiber balance 9. The GABA content of each layer was measured with the method combining the GABase (bacterial enzyme, GABA-transaminase and succinic semialdehyde dehydrogenase) system 13 with the enzymatic N A D P H (nicotinamide adenine dinucleotide phosphate reduced form) cycling method 9. The details of the assay system were as described elsewherO 1, In measuring G A D activity, it has been a common method to measure ~4CO2 produced by the decarboxylation of [x4C]glutamateL However, this method is limited in sensitivity and one has difficulty in getting pure k-[14C]glutamate. In the present paper, the G A D activity was determined by the measurement of G A B A produced during the incubation with G A D assay reagent. The produced GABA was easily
203 measured with the microassay method 11. In this experiment the G A D activity of as little a sample as 0.1 #g tissue could be measured. Increasing the cycling rate of the enzymatic cycling system of N A D P H enabled us to measure the G A D activity of a minute amount of tissue, even as minute as a single cell. Each freeze-dried sample weighing 0.1-0.7/zg was added to 1.7 #1 of GAD assay reaction mixture covered with mineral oil in the oil well (see oil well technique of Lowry and Passonneaug). The G A D assay reagent contained 0.1 M phosphate buffer (pH 6.8), 5 m M glutamate, 250/zM pyridoxal phosphate and 0.4 % mercaptoethanol. After 60 min incubation at 38 °C, 0.6/~1 of 0.25 N HC1 was added to the droplets and they were heated at 60 °C for 10 min to stop the reaction and destroy the endogenous N A D P H in the tissue. After treatment with HCI, 4.3 #1 of GABA assay reaction mixture was added to the droplets and they were incubated at 38 °C for 30 rain. The GABA assay reagent contained 0.3 M Tris.HC1 buffer (pH 8.9), 10 m M a-ketoglutarate, 0.5 m M NADP, 0.01 ~ mercaptoethanol and 0.1 mg protein/ml GABase. At the end of the reaction, 0.6/~1 of 2.5 N NaOH was added to the droplets and they were heated at 60 °C for 20 min to destroy excess N A D P . An aliquot of 4/~1 of the droplets was transferred to 50/zl of N A D P H cycling reagent. The detail of the cycling method has been described elsewhere 11. In this case the cycling rate was adjusted to 500 cycles/30 rain. In each assay at least 6 different GABA standards were run from the first step o f G A D activity assay reagent. The amount of GABA obtained by this method indicates the sum of the endogenous GABA in each sample and the GABA produced after 60 min incubation with G A D assay reagent. Thus the real G A D activity can be obtained by subtracting the content of the endogenous GABA from the
50
~40 ~'~
E
30
o ~20 C_O o 0
0
,
0
0.1
0.2
0.3
0.4
0.5
i
0.6 .ug
Fig. 1. Standard curve for G A D activity against different amounts of tissue samples. A freeze-dried tissue section including hippocampus and area dentata was completely dispersed in water droplets (2 #g//~l) in an oil well. Each 0.05-0.5/~1 aliquot of the droplets, containing 0.1-0.6 Fg of tissue, was added to the G A D assay reagent. For the assay of endogenous GABA, the original water droplet with tissue sample was treated with HC1 (final concentration 0.01 N) and heated at 60 °C for 10 min. Each aliquot of sample (0.1-0.6 pg tissue) was added to the G A D assay reagent. G A D activity was obtained by subtracting the endogenous G A B A from total GABA of aliquots which were not treated with HCI. Ordinate indicates GABA produced after one hour incubation with G A D assay reagent. Details of the method are described in the text.
204 R.superior
R.inferior
>-
Area dentata
T I1"1
T
I
!1
Fig. 2. Distribution of GABA in hippocampal regio superior, regio inferior and area dentata. The zones dissected from regio superior were: A, alveus; O, stratum oriens (divided into O1 and 02); P, stratum pyramidale; R, stratum radiatum (divided into R1, Rz, Ra) and M, stratum moleculare. From area dentata: M, stratum moleculare (divided into M1, M2, M3); Gl, stratum granulare; G2, M4 and Ms were ventral part of granular and molecular layer and H, hilus fasciae dentatae. From regio inferior: mr, mossy fiber layer; O, stratum oriens (divided into O~ and 02); P, stratum pyramidale and F, fimbria. Nomenclature according to Blackstad~. GABA concentration is expressed as mmoles/kg by dry weight. Vertical bars on each histogram show the S.E.M. Samples from each layer were at least 8 samples from 3 animals.
total GABA of each sample measured after the incubation. Fig. 1 shows a standard curve of G A D activity obtained in this manner. The possibility of the degradation of the GABA produced during the incubation with the G A D activity assay reagent was negligible since the pH 6.8 of G A D assay reaction mixture was far from the optimal pH of G A B A degrading enzymes 13. The distribution of GABA in the regio superior, regio inferior and the area dentata is shown in Fig. 2. The GABA concentration was highest in the area dentata. The GABA content in the regio inferior was higher than that in regio superior. In the regio superior, the laminar distribution showed a not so remarkable difference in GABA concentration in each layer, though the pyramidal cell layer and the stratum moleculare contained a somewhat higher level of GABA. However, in the regio inferior GABA was concentrated in the vicinity of the pyramidal cell layer. It is interesting to note that the GABA concentration o f the pyramidal cell layer in regio inferior was 21.0 mmoles/kg by dry weight and was defnitely higher than that in regio superior, whose GABA concentration was 13.6 mmoles/kg. The regions of the alveus and the middle of stratum radiatum (,Re) in regio superior contained low concentrations of GABA. The fimbria, in which major afferent and efferent pathway is included, contained the lowest level of GABA in the hippocampus. The distribution of G A D in the hippocampus and area dentata is shown in Fig. 3. As observed in the case of GABA distribution, G A D activity was the highest in area dentata. In regio superior G A D was accumulated in the vicinity of the pyramidal layer and in the stratum moleculare. The alveus had the lowest activity of GAD. In
205
R.superior
R, inferior
Area dentata
.~125
i _
• :.,oo
~
~o
i IP M Fig. 3. Distribution of GAD activity in hippocampus and area dentata. GAD activity is expressed as GABA mmoles/kg/h dry weight. Samples from each layer were at least 20 pieces from 5 animals. The nomenclature is the same as Fig. 2.
regio inferior the pyramidal layer showed the peak G A D activity which was 1.3 times higher than that in the same layer of regio superior. The fimbria showed the lowest G A D activity in the hippocampus. The distribution pattern of G A D was in good agreement with that of GABA in the hippocamus, although GABA concentration in the pyramidal layer in regio superior did not show as sharp a peak as G A D did in the same region. Storm-Mathisen and Fonnum15,16 measured G A D activity in rat hippocampus by the method measuring 14CO2 production from [14C]glutamate. The present results obtained by our new method agree well with the G A D distribution reported by them. High concentration of GABA and high G A D activity in the pyramidal cell layer, especially in the regio inferior and the granular layer in area dentata, may indicate that GABA is concentrated in basket cell structures which inhibit the pyramidal cells in the hippocampus 2,6 and in the terminal of basket cells located in the vicinity of granular layer 3. Biscoe and Straughan 4 and Salmoiraghi and Stefanis 14 observed that GABA inhibits pyramidal cells in the hippocampus, and bicuculline, a specific GABA antagonist, blocks the synaptic inhibition of hippocampal pyramidal cells. The decrease in GABA concentration in the hippocampal slice in vitro induced a typical depolarization shift and seizure discharge in pyramidal cells in the hippocampus 12. High concentration of GABA and high G A D activity in the pyramidal cell layer in the hippocampus, together with the reports mentioned above, provides credible support to the hypothesis that GABA functions as an inhibitory neurotransmitter in this area of the hippocampus.
ALBERTS,R. W., AND BRADY, R. O., The distribution of glutamic decarboxylase in the nervous system of rhesus monkey, J. biol. Chem., 234 (1959) 926-928. 2 ANDERSEN,P., ECCLES,J. C., AMDLOYNtNG,Y., Pathway of postsynaptic inhibition in the hippocampus, J. NeurophysioL, 27 (1964) 608-619. 1
206 3 ANDERSEN, P., HOLMQVIST, B., AND BOORHOEVE, P. E., Entorhinal activation of dentate granule cells, Acta physiol, scand., 66 (1966) 448-460. 4 BISCOE,T. J., AND STRANGHAN, D. W., Micro-electrophoretic studies of neurones in the cat hippocampus, J. PhysioL (Lond.), 183 (1966) 341-359. 5 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp, Neurol., 105 (1959) 417-521. 6 CURTIS, D. R., FELIX, D., AND MCLENNEAN, H., GABA and hippocampal inhibition, Brit. J. Pharmacol., 40 (1970) 881-883. 7 CURTIS, D. R., DUGGAN, A. W., AND FELIX, D., GABA and inhibition of Deiters' neurons, Brain Research, 23 (1970) 117-120. 8 KRNJEVIC, K., AND SCHWARTS, S., The action of 7,,-aminobutyric acid on cortical neurons, Exp. Brain Res., 3 (1967) 320-336. 9 LOWRy, O. H., AND PASSONNEAU,J. V., A Flexible System o f Enzymatic Analysis, Academic Press, New York, 1972. 10 OBATA, K., ITO, M., Ocm, R., AND SATO, N., Pharmacological properties of the postsynaptic inhibition of Purkinje cell axons and the action of y-aminobutyric acid on Deiters neurons, Exp. Brain Res., 4 (1967) 43-57. 11 OKADA, Y., Distribution of y-aminobutyric acid (GABA) in the layers of superior colliculus of the rabbit, Brain Research, 75 (1974) 326-365. 12 OZAWA, S., AND OKADA, Y., Decrease of GABA levels and the appearance of a depolarization shift in thin hippocampal slices in vitro. In E. ROBERTS, C. THOMAS AND D. B. TOWER (Eds.), GABA in Nervous System Function, Raven Press, in press. 13 SCOTT, E. M., AND JACOBY, W. B., Soluble y-aminobutyric-glutamic transaminase from Pseudomonas fluorescens, J. biol. Chem., 234 (1959) 932-936. 14 SALMOIRAGHI,G. S., AND STEFANIS, C. N., A critique of iontophoretic studies of central nervous system neurons, Int. Rev. Neurobiol., l0 0967) 1-30. 15 STORM-MATHISEN,J., AND FONNUM, F., Quantitative histochemistry of glutamate decarboxylase in the rat hippocampal region, J. Neurochem., 18 (1971) 1105-1111. 16 STORM-MATH1SEN,J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fibre systems. Evidence that the enzyme is localized in intrinsic neurones, Brain Research, 40 (1972) 215-235.
Bra#l Research, 98 (1975) 202-206 (~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Distribution of y-aminobutyric acid (GABA) and glutamate decarboxylase (GAD) activity in the guinea pig hippocampus - - microassay method for the determination of GAD activity
Y A S U H I R O O K A D A AND CH1CACO S H I M A D A
Department of Neurochemistry, Tokyo Metropolitan Institute Jot Neurosciences, Fuchu-shi, Tokyo (Japan) (Accepted July 1st, 1975)
There has been increasing evidence indicating that 7-aminobutyric acid (GABA) is an inhibitory neurotransmitter substance in mammalian CNS 7,8,1°. With regard to the role of GABA in hippocampus, physiological and pharmacological studies showed that G A B A has an inhibitory action on pyramidal cells 4,6,a4, Intrahippocampal laminar distribution of G A B A itself, however, has not been studied because of the limitation of the sensitivity of GABA assay system. In the present paper, G A B A distribution in the layers of hippocampus of the guinea pig was determined using a microchemical GABA assay method 11. Together with the assay of GABA, the glutamate decarboxylase (GAD) activity of each layer in the hippocampus and the area dentata was determined by a new method which enables us to measure G A D activity of a tissue sample below 1 #g in weight. Guinea pigs were decapitated and the brains immediately removed and placed on ice. The hippocampus with the attached area dentata was isolated. Sections transverse to the long axis of the hippocampus were cut at about 5 mm thickness and were immediately frozen in Freon-12 (--150 °C) in liquid nitrogen. The frozen blocks were cut at 20 #m thickness with a microtome in a cryostat (--20 °C). The sections were freeze-dried overnight at - - 3 0 °C in a vacuum. The sections were stored in evacuated tubes at - - 3 5 °C until the assays for G A B A and G A D activity were performed. Each layer of hippocampus was dissected out freehand under a stereomicroscope and the samples, weighing 0.1-0.7 #g, were weighed with a fishpole quartz fiber balance 9. The GABA content of each layer was measured with the method combining the GABase (bacterial enzyme, GABA-transaminase and succinic semialdehyde dehydrogenase) system 13 with the enzymatic N A D P H (nicotinamide adenine dinucleotide phosphate reduced form) cycling method 9. The details of the assay system were as described elsewherO 1, In measuring G A D activity, it has been a common method to measure ~4CO2 produced by the decarboxylation of [x4C]glutamateL However, this method is limited in sensitivity and one has difficulty in getting pure k-[14C]glutamate. In the present paper, the G A D activity was determined by the measurement of G A B A produced during the incubation with G A D assay reagent. The produced GABA was easily
203 measured with the microassay method 11. In this experiment the G A D activity of as little a sample as 0.1 #g tissue could be measured. Increasing the cycling rate of the enzymatic cycling system of N A D P H enabled us to measure the G A D activity of a minute amount of tissue, even as minute as a single cell. Each freeze-dried sample weighing 0.1-0.7/zg was added to 1.7 #1 of GAD assay reaction mixture covered with mineral oil in the oil well (see oil well technique of Lowry and Passonneaug). The G A D assay reagent contained 0.1 M phosphate buffer (pH 6.8), 5 m M glutamate, 250/zM pyridoxal phosphate and 0.4 % mercaptoethanol. After 60 min incubation at 38 °C, 0.6/~1 of 0.25 N HC1 was added to the droplets and they were heated at 60 °C for 10 min to stop the reaction and destroy the endogenous N A D P H in the tissue. After treatment with HCI, 4.3 #1 of GABA assay reaction mixture was added to the droplets and they were incubated at 38 °C for 30 rain. The GABA assay reagent contained 0.3 M Tris.HC1 buffer (pH 8.9), 10 m M a-ketoglutarate, 0.5 m M NADP, 0.01 ~ mercaptoethanol and 0.1 mg protein/ml GABase. At the end of the reaction, 0.6/~1 of 2.5 N NaOH was added to the droplets and they were heated at 60 °C for 20 min to destroy excess N A D P . An aliquot of 4/~1 of the droplets was transferred to 50/zl of N A D P H cycling reagent. The detail of the cycling method has been described elsewhere 11. In this case the cycling rate was adjusted to 500 cycles/30 rain. In each assay at least 6 different GABA standards were run from the first step o f G A D activity assay reagent. The amount of GABA obtained by this method indicates the sum of the endogenous GABA in each sample and the GABA produced after 60 min incubation with G A D assay reagent. Thus the real G A D activity can be obtained by subtracting the content of the endogenous GABA from the
50
~40 ~'~
E
30
o ~20 C_O o 0
0
,
0
0.1
0.2
0.3
0.4
0.5
i
0.6 .ug
Fig. 1. Standard curve for G A D activity against different amounts of tissue samples. A freeze-dried tissue section including hippocampus and area dentata was completely dispersed in water droplets (2 #g//~l) in an oil well. Each 0.05-0.5/~1 aliquot of the droplets, containing 0.1-0.6 Fg of tissue, was added to the G A D assay reagent. For the assay of endogenous GABA, the original water droplet with tissue sample was treated with HC1 (final concentration 0.01 N) and heated at 60 °C for 10 min. Each aliquot of sample (0.1-0.6 pg tissue) was added to the G A D assay reagent. G A D activity was obtained by subtracting the endogenous G A B A from total GABA of aliquots which were not treated with HCI. Ordinate indicates GABA produced after one hour incubation with G A D assay reagent. Details of the method are described in the text.
204 R.superior
R.inferior
>-
Area dentata
T I1"1
T
I
!1
Fig. 2. Distribution of GABA in hippocampal regio superior, regio inferior and area dentata. The zones dissected from regio superior were: A, alveus; O, stratum oriens (divided into O1 and 02); P, stratum pyramidale; R, stratum radiatum (divided into R1, Rz, Ra) and M, stratum moleculare. From area dentata: M, stratum moleculare (divided into M1, M2, M3); Gl, stratum granulare; G2, M4 and Ms were ventral part of granular and molecular layer and H, hilus fasciae dentatae. From regio inferior: mr, mossy fiber layer; O, stratum oriens (divided into O~ and 02); P, stratum pyramidale and F, fimbria. Nomenclature according to Blackstad~. GABA concentration is expressed as mmoles/kg by dry weight. Vertical bars on each histogram show the S.E.M. Samples from each layer were at least 8 samples from 3 animals.
total GABA of each sample measured after the incubation. Fig. 1 shows a standard curve of G A D activity obtained in this manner. The possibility of the degradation of the GABA produced during the incubation with the G A D activity assay reagent was negligible since the pH 6.8 of G A D assay reaction mixture was far from the optimal pH of G A B A degrading enzymes 13. The distribution of GABA in the regio superior, regio inferior and the area dentata is shown in Fig. 2. The GABA concentration was highest in the area dentata. The GABA content in the regio inferior was higher than that in regio superior. In the regio superior, the laminar distribution showed a not so remarkable difference in GABA concentration in each layer, though the pyramidal cell layer and the stratum moleculare contained a somewhat higher level of GABA. However, in the regio inferior GABA was concentrated in the vicinity of the pyramidal cell layer. It is interesting to note that the GABA concentration o f the pyramidal cell layer in regio inferior was 21.0 mmoles/kg by dry weight and was defnitely higher than that in regio superior, whose GABA concentration was 13.6 mmoles/kg. The regions of the alveus and the middle of stratum radiatum (,Re) in regio superior contained low concentrations of GABA. The fimbria, in which major afferent and efferent pathway is included, contained the lowest level of GABA in the hippocampus. The distribution of G A D in the hippocampus and area dentata is shown in Fig. 3. As observed in the case of GABA distribution, G A D activity was the highest in area dentata. In regio superior G A D was accumulated in the vicinity of the pyramidal layer and in the stratum moleculare. The alveus had the lowest activity of GAD. In
205
R.superior
R, inferior
Area dentata
.~125
i _
• :.,oo
~
~o
i IP M Fig. 3. Distribution of GAD activity in hippocampus and area dentata. GAD activity is expressed as GABA mmoles/kg/h dry weight. Samples from each layer were at least 20 pieces from 5 animals. The nomenclature is the same as Fig. 2.
regio inferior the pyramidal layer showed the peak G A D activity which was 1.3 times higher than that in the same layer of regio superior. The fimbria showed the lowest G A D activity in the hippocampus. The distribution pattern of G A D was in good agreement with that of GABA in the hippocamus, although GABA concentration in the pyramidal layer in regio superior did not show as sharp a peak as G A D did in the same region. Storm-Mathisen and Fonnum15,16 measured G A D activity in rat hippocampus by the method measuring 14CO2 production from [14C]glutamate. The present results obtained by our new method agree well with the G A D distribution reported by them. High concentration of GABA and high G A D activity in the pyramidal cell layer, especially in the regio inferior and the granular layer in area dentata, may indicate that GABA is concentrated in basket cell structures which inhibit the pyramidal cells in the hippocampus 2,6 and in the terminal of basket cells located in the vicinity of granular layer 3. Biscoe and Straughan 4 and Salmoiraghi and Stefanis 14 observed that GABA inhibits pyramidal cells in the hippocampus, and bicuculline, a specific GABA antagonist, blocks the synaptic inhibition of hippocampal pyramidal cells. The decrease in GABA concentration in the hippocampal slice in vitro induced a typical depolarization shift and seizure discharge in pyramidal cells in the hippocampus 12. High concentration of GABA and high G A D activity in the pyramidal cell layer in the hippocampus, together with the reports mentioned above, provides credible support to the hypothesis that GABA functions as an inhibitory neurotransmitter in this area of the hippocampus.
ALBERTS,R. W., AND BRADY, R. O., The distribution of glutamic decarboxylase in the nervous system of rhesus monkey, J. biol. Chem., 234 (1959) 926-928. 2 ANDERSEN,P., ECCLES,J. C., AMDLOYNtNG,Y., Pathway of postsynaptic inhibition in the hippocampus, J. NeurophysioL, 27 (1964) 608-619. 1
206 3 ANDERSEN, P., HOLMQVIST, B., AND BOORHOEVE, P. E., Entorhinal activation of dentate granule cells, Acta physiol, scand., 66 (1966) 448-460. 4 BISCOE,T. J., AND STRANGHAN, D. W., Micro-electrophoretic studies of neurones in the cat hippocampus, J. PhysioL (Lond.), 183 (1966) 341-359. 5 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp, Neurol., 105 (1959) 417-521. 6 CURTIS, D. R., FELIX, D., AND MCLENNEAN, H., GABA and hippocampal inhibition, Brit. J. Pharmacol., 40 (1970) 881-883. 7 CURTIS, D. R., DUGGAN, A. W., AND FELIX, D., GABA and inhibition of Deiters' neurons, Brain Research, 23 (1970) 117-120. 8 KRNJEVIC, K., AND SCHWARTS, S., The action of 7,,-aminobutyric acid on cortical neurons, Exp. Brain Res., 3 (1967) 320-336. 9 LOWRy, O. H., AND PASSONNEAU,J. V., A Flexible System o f Enzymatic Analysis, Academic Press, New York, 1972. 10 OBATA, K., ITO, M., Ocm, R., AND SATO, N., Pharmacological properties of the postsynaptic inhibition of Purkinje cell axons and the action of y-aminobutyric acid on Deiters neurons, Exp. Brain Res., 4 (1967) 43-57. 11 OKADA, Y., Distribution of y-aminobutyric acid (GABA) in the layers of superior colliculus of the rabbit, Brain Research, 75 (1974) 326-365. 12 OZAWA, S., AND OKADA, Y., Decrease of GABA levels and the appearance of a depolarization shift in thin hippocampal slices in vitro. In E. ROBERTS, C. THOMAS AND D. B. TOWER (Eds.), GABA in Nervous System Function, Raven Press, in press. 13 SCOTT, E. M., AND JACOBY, W. B., Soluble y-aminobutyric-glutamic transaminase from Pseudomonas fluorescens, J. biol. Chem., 234 (1959) 932-936. 14 SALMOIRAGHI,G. S., AND STEFANIS, C. N., A critique of iontophoretic studies of central nervous system neurons, Int. Rev. Neurobiol., l0 0967) 1-30. 15 STORM-MATHISEN,J., AND FONNUM, F., Quantitative histochemistry of glutamate decarboxylase in the rat hippocampal region, J. Neurochem., 18 (1971) 1105-1111. 16 STORM-MATH1SEN,J., Glutamate decarboxylase in the rat hippocampal region after lesions of the afferent fibre systems. Evidence that the enzyme is localized in intrinsic neurones, Brain Research, 40 (1972) 215-235.
