Brain Research, 167 (1979) 297-305 ~ Elsevier/North-Holland Biomedical Press
297
T H E R E L A T I O N S H I P B E T W E E N GABA C O N C E N T R A T I O N S IN BRAIN AND CEREBROSPINAL FLUID
PETER BOHLEN*, SYLVIE HUOT and MICHAEL G. PALFREYMAN** Centre de Recherche Merrell International, 16, rue d'Ankara, 67084 Strasbourg Cedex, (France)
(Accepted August 31st, 1978)
SUMMARY GABA concentrations in cerebrospinal fluid (CSF) and brain of rats and cats were determined before and after intraperitoneal injection of three drugs that increase brain G A B A levels. G A B A exists in the CSF in two forms: free and conjugated GABA. In the CSF of untreated animals, there is very little free GABA (65 ± 12 pmol/ml) but considerable amounts of conjugated GABA (2885 -~ 100 pmol/ml). After IP administration &y-vinyl GABA to rats, CSF concentrations of both free and conjugated GABA rise in a dose-dependent manner. There is an exponential correlation (r -- 0.92, P < 0.001) between rat whole brain GABA concentrations and free GABA in the CSF. Concentrations of brain GABA and conjugated CSF GABA are linearly correlated (r -- 0.84, P < 0.001). y-Acetylenic GABA has qualitatively similar effects to 7-vinyl GABA. Treatment with ethanolamine-O-sulfate i.p. at a dose not affecting brain GABA concentrations markedly increases serum GABA, leaves conjugated CSF GABA unchanged and significantly elevates free GABA in the CSF. These findings suggest that total CSF GABA concentrations are related primarily to brain GABA levels and are minimally affected by the changes in the peripheral GABA concentrations. Determination of the levels of free and conjugated GABA in the CSF may be useful for the estimation of brain GABA concentration in patients on therapy intended to alter brain GABA levels.
INTRODUCTION 4-Aminobutyric acid (GABA) has been implicated as a major inhibitory neurotransmitter. Several neurological and psychiatric disorders, e.g. Huntington's disease "1, epilepsy 3 and schizophrenia 11, have been associated with altered brain * Present address: The Salk Institute, P.O. Box 1809, San Diego, Calif. 92112, U.S.A. ** To whom correspondence should be addressed.
298 GABA metabolism. Accordingly, there is much interest in the therapeutic use of drugs that alter brain GABA concentrations. Di-n-propylacetate 25, ethanolamine-O-sulfate 12, aminooxyacetic acid 10, y-acetylenic GABA s, v-vinyl GABA 7 and gabaculine 23 all increase brain GABA concentrations after peripheral administration. Most of them act by inhibiting GABA-transaminase (4-aminobutyric acid: 2-oxoglutarate aminotransferase; EC 2.6.1.19), the enzyme responsible for the catabolism of GABA. Clinical research and therapy with compounds of this type would be facilitated if it were possible to monitor brain GABA levels. Direct tissue sampling is obviously not feasible, but GABA concentrations in cerebrospinal fluid (CSF) can be determined ~, 4 -6. Thus, if CSF GABA concentrations were to reflect brain GABA levels, analysis of CSF samples would provide valuable information on GABA-metabolism in the brain. We have investigated the relationship between GABA concentrations in brain and CSF in animals receiving three agents known to increase brain GABA levels. CSF GABA concentrations were determined as a function of time and of dose and compared to brain concentrations. MATERIALS AND METHODS ),-Vinyl GABA (4-aminohex-5-enoic acid, RMI 71.754) 14, 7-acetylenic GABA (4-aminohex-5-enoic acid, RMI 71.645) 16, and ethanolamine-O-sulfate 15, were synthesized in our laboratories.
CSF, blood and brain sampling Adult female cats (2.2-3.5 kg) were anesthetized with O2/N20/halothane, injected intraperitoneally with 40 mg/kg sodium pentobarbital, mounted in a stereotaxic frame (Kopf Instruments, Tujunga, Calif.) and implanted with a polyethylene cannula in either the lateral or the third ventricle. The cannulae used were 5.2 cm long and had external and internal diameters of 1.57 and 1.14 mm respectively. A small hole was drilled into the skull at the following coordinates26: for the lateral ventricle, 8 mm behind the bregma and 6 mm from the midline; for the third ventricle, 8-10 mm behind the bregma and on the midline. The cannula was lowered into the ventricle and adjusted in depth until CSF rose freely inside the cannula (I-1.2 cm below the external surface of the skull). The first 2{)0-300 #1 of CSF which were contaminated with blood and tissue fragments were discarded. Experiments were only started when clear CSF could be withdrawn from the cannula. Samples (120 #1) were then collected at 30 min intervals and alternately used for analysis of free and conjugated GABA. During the course of the experiment the cats were maintained under anesthesia by supplementary injections of sodium pentobarbital as required. For the determination of serum GABA levels, blood was collected from a femoral vein catheter. At the end of experiments cortex samples were removed for analysis of brain GABA concentrations. Male Sprague-Dawley rats (Charles River, France), body weight 200-400 g, were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and mounted in a stereotaxic frame (Kopf Instruments). For suboccipital puncture of the cisterna magna the neck skin was incised and the muscles cut at the base of the skull. A
299 Hamilton needle (KF 729, 13 ram) connected to a 250 #l Hamilton syringe was inserted through the dura to a depth of 1 mm. Depth was controlled by means of a polyethylene tube stop fitted to the needle. About 120/A of CSF was withdrawn slowly from the cisterna magna. Any CSF contaminated with blood was discarded. Immediately after withdrawal of the CSF the rats were decapitated. Trunk blood was collected and the brain was removed from the skull within 30 sec.
Sample processing and GABA determination CSF was immediately deproteinized by the addition of one-third vol. of 5-sulfosalicylic acid (200 g/l) and centrifugation. Free and conjugated GABA were determined in the supernatant. Free GABA was measured by automated high-performance liquid chromatography as previously described 2 but for elution a sodium citrate buffer (0.2 M Na ~, 0.067 M citrate, pH 5.00) containing 1v•°/ethanol was used. The /o addition of ethanol was necessary for adequate separation of GABA from 7-acetylenic GABA. For the determination of conjugated GABA, the acidic supernatant was further diluted with sulfosalicylic acid (50 g/l) and heated in a sealed tube at ! 10 °C for 24 h. This treatment released any GABA present in peptidic or otherwise conjugated forms. Total GABA concentration in the hydrolysate was determined as described above. The concentration of conjugated GABA was defined as the difference between total and free GABA concentrations. Serum, which was immediately prepared from blood samples, was deproteinized with one-third vol. of sulfosalicylic acid (200 g/l). After centrifugation, GABA concentrations in the supernatants were measured as described above. Brain samples were frozen in liquid nitrogen immediately after removal from the skull. The brains were homogenized in 10 vol of cold trichloroacetic acid (150 g/l). The homogenates were allowed to stand at 4 ~'C for I h and were then centrifuged. GABA concentrations in the supernatants were assayed by conventional liquid chromatography 8. Acid extracts of CSF, blood and brain samples were stored at 4 °C until analysis. RESULTS Free GABA was found in the CSF in very small quantities in both cats (61 ~ 13 pmol/ml, mean ± S.E.M., n = 4) and rats (65 ± 12 pmol/ml, n = I 1). In contrast, the CSF of both species contained relatively large amounts of GABA-conjugates, 599 ~_ 268 pmol/ml (n 5) and 2885 ~ 100 pmol/ml (n = 10) for cats and rats, respectively. After intraperitoneal injection ofF-vinyl GABA to cats the levels of free GABA and conjugated GABA in the CSF increased progressively (Fig. 1). Eight hours after injection free and conjugated GABA levels had increased 30- and 8-fold respectively. The corresponding increase of the GABA concentrations in the brain cortex was from 0.7 to 3.8 /~mol/g. GABA concentrations in the serum increased from 0.2 to 1.4 nmol/ml. v-Vinyl GABA also elevates the CSF concentrations of free and conjugated GABA in the rat. This effect is dose-dependent (Fig. 2). As little as 125 mg/kg v-vinyl
300
5
.~/
2E
3
'~ m
2
e
y-VINYL GABA
/
-2
4
Conjugated GABA
I"
O~
U
0 -4 TIME
0
2
6
AFTER ~ - V I N Y L GABA
8 ( hr )
Fig. I. Free and conjugated G A B A concentrations in the CSF of anesthetized cats before and after intraperitoneal injection of),-vinyl G A B A (1000 mg/kg). Intraventricular cannulae were implanted 6-8 h prior to drug administration. Points are means 5- S.E.M. (n ~ 3-4).
GABA causes a significant increase (P < 0.05, Students t-test, 2-tailed) of the brain GABA concentrations, whereas free and conjugated GABA in the CSF are significantly elevated (P < 0.05) only after 250 mg/kg 9,-vinyt GABA. The highest dose ofg,-vinyl GABA used (2000 mg/kg) increases concentrations of brain GABA and conjugated GABA in CSF 5- and 4-fold respectively, but elevates free GABA in the CSF 135-fold over control levels. As the dose of 9,-vinyl GABA is increased the ratio between conjugated and free GABA in CSF changes drastically. This ratio is highest in untreated animals due to the very low levels of free GABA present and is low at high drug doses under which condition large amounts of free GABA are found in the CSF. 9,-Vinyl GABA also elevates serum GABA concentrations (Table I).
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297
T H E R E L A T I O N S H I P B E T W E E N GABA C O N C E N T R A T I O N S IN BRAIN AND CEREBROSPINAL FLUID
PETER BOHLEN*, SYLVIE HUOT and MICHAEL G. PALFREYMAN** Centre de Recherche Merrell International, 16, rue d'Ankara, 67084 Strasbourg Cedex, (France)
(Accepted August 31st, 1978)
SUMMARY GABA concentrations in cerebrospinal fluid (CSF) and brain of rats and cats were determined before and after intraperitoneal injection of three drugs that increase brain G A B A levels. G A B A exists in the CSF in two forms: free and conjugated GABA. In the CSF of untreated animals, there is very little free GABA (65 ± 12 pmol/ml) but considerable amounts of conjugated GABA (2885 -~ 100 pmol/ml). After IP administration &y-vinyl GABA to rats, CSF concentrations of both free and conjugated GABA rise in a dose-dependent manner. There is an exponential correlation (r -- 0.92, P < 0.001) between rat whole brain GABA concentrations and free GABA in the CSF. Concentrations of brain GABA and conjugated CSF GABA are linearly correlated (r -- 0.84, P < 0.001). y-Acetylenic GABA has qualitatively similar effects to 7-vinyl GABA. Treatment with ethanolamine-O-sulfate i.p. at a dose not affecting brain GABA concentrations markedly increases serum GABA, leaves conjugated CSF GABA unchanged and significantly elevates free GABA in the CSF. These findings suggest that total CSF GABA concentrations are related primarily to brain GABA levels and are minimally affected by the changes in the peripheral GABA concentrations. Determination of the levels of free and conjugated GABA in the CSF may be useful for the estimation of brain GABA concentration in patients on therapy intended to alter brain GABA levels.
INTRODUCTION 4-Aminobutyric acid (GABA) has been implicated as a major inhibitory neurotransmitter. Several neurological and psychiatric disorders, e.g. Huntington's disease "1, epilepsy 3 and schizophrenia 11, have been associated with altered brain * Present address: The Salk Institute, P.O. Box 1809, San Diego, Calif. 92112, U.S.A. ** To whom correspondence should be addressed.
298 GABA metabolism. Accordingly, there is much interest in the therapeutic use of drugs that alter brain GABA concentrations. Di-n-propylacetate 25, ethanolamine-O-sulfate 12, aminooxyacetic acid 10, y-acetylenic GABA s, v-vinyl GABA 7 and gabaculine 23 all increase brain GABA concentrations after peripheral administration. Most of them act by inhibiting GABA-transaminase (4-aminobutyric acid: 2-oxoglutarate aminotransferase; EC 2.6.1.19), the enzyme responsible for the catabolism of GABA. Clinical research and therapy with compounds of this type would be facilitated if it were possible to monitor brain GABA levels. Direct tissue sampling is obviously not feasible, but GABA concentrations in cerebrospinal fluid (CSF) can be determined ~, 4 -6. Thus, if CSF GABA concentrations were to reflect brain GABA levels, analysis of CSF samples would provide valuable information on GABA-metabolism in the brain. We have investigated the relationship between GABA concentrations in brain and CSF in animals receiving three agents known to increase brain GABA levels. CSF GABA concentrations were determined as a function of time and of dose and compared to brain concentrations. MATERIALS AND METHODS ),-Vinyl GABA (4-aminohex-5-enoic acid, RMI 71.754) 14, 7-acetylenic GABA (4-aminohex-5-enoic acid, RMI 71.645) 16, and ethanolamine-O-sulfate 15, were synthesized in our laboratories.
CSF, blood and brain sampling Adult female cats (2.2-3.5 kg) were anesthetized with O2/N20/halothane, injected intraperitoneally with 40 mg/kg sodium pentobarbital, mounted in a stereotaxic frame (Kopf Instruments, Tujunga, Calif.) and implanted with a polyethylene cannula in either the lateral or the third ventricle. The cannulae used were 5.2 cm long and had external and internal diameters of 1.57 and 1.14 mm respectively. A small hole was drilled into the skull at the following coordinates26: for the lateral ventricle, 8 mm behind the bregma and 6 mm from the midline; for the third ventricle, 8-10 mm behind the bregma and on the midline. The cannula was lowered into the ventricle and adjusted in depth until CSF rose freely inside the cannula (I-1.2 cm below the external surface of the skull). The first 2{)0-300 #1 of CSF which were contaminated with blood and tissue fragments were discarded. Experiments were only started when clear CSF could be withdrawn from the cannula. Samples (120 #1) were then collected at 30 min intervals and alternately used for analysis of free and conjugated GABA. During the course of the experiment the cats were maintained under anesthesia by supplementary injections of sodium pentobarbital as required. For the determination of serum GABA levels, blood was collected from a femoral vein catheter. At the end of experiments cortex samples were removed for analysis of brain GABA concentrations. Male Sprague-Dawley rats (Charles River, France), body weight 200-400 g, were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and mounted in a stereotaxic frame (Kopf Instruments). For suboccipital puncture of the cisterna magna the neck skin was incised and the muscles cut at the base of the skull. A
299 Hamilton needle (KF 729, 13 ram) connected to a 250 #l Hamilton syringe was inserted through the dura to a depth of 1 mm. Depth was controlled by means of a polyethylene tube stop fitted to the needle. About 120/A of CSF was withdrawn slowly from the cisterna magna. Any CSF contaminated with blood was discarded. Immediately after withdrawal of the CSF the rats were decapitated. Trunk blood was collected and the brain was removed from the skull within 30 sec.
Sample processing and GABA determination CSF was immediately deproteinized by the addition of one-third vol. of 5-sulfosalicylic acid (200 g/l) and centrifugation. Free and conjugated GABA were determined in the supernatant. Free GABA was measured by automated high-performance liquid chromatography as previously described 2 but for elution a sodium citrate buffer (0.2 M Na ~, 0.067 M citrate, pH 5.00) containing 1v•°/ethanol was used. The /o addition of ethanol was necessary for adequate separation of GABA from 7-acetylenic GABA. For the determination of conjugated GABA, the acidic supernatant was further diluted with sulfosalicylic acid (50 g/l) and heated in a sealed tube at ! 10 °C for 24 h. This treatment released any GABA present in peptidic or otherwise conjugated forms. Total GABA concentration in the hydrolysate was determined as described above. The concentration of conjugated GABA was defined as the difference between total and free GABA concentrations. Serum, which was immediately prepared from blood samples, was deproteinized with one-third vol. of sulfosalicylic acid (200 g/l). After centrifugation, GABA concentrations in the supernatants were measured as described above. Brain samples were frozen in liquid nitrogen immediately after removal from the skull. The brains were homogenized in 10 vol of cold trichloroacetic acid (150 g/l). The homogenates were allowed to stand at 4 ~'C for I h and were then centrifuged. GABA concentrations in the supernatants were assayed by conventional liquid chromatography 8. Acid extracts of CSF, blood and brain samples were stored at 4 °C until analysis. RESULTS Free GABA was found in the CSF in very small quantities in both cats (61 ~ 13 pmol/ml, mean ± S.E.M., n = 4) and rats (65 ± 12 pmol/ml, n = I 1). In contrast, the CSF of both species contained relatively large amounts of GABA-conjugates, 599 ~_ 268 pmol/ml (n 5) and 2885 ~ 100 pmol/ml (n = 10) for cats and rats, respectively. After intraperitoneal injection ofF-vinyl GABA to cats the levels of free GABA and conjugated GABA in the CSF increased progressively (Fig. 1). Eight hours after injection free and conjugated GABA levels had increased 30- and 8-fold respectively. The corresponding increase of the GABA concentrations in the brain cortex was from 0.7 to 3.8 /~mol/g. GABA concentrations in the serum increased from 0.2 to 1.4 nmol/ml. v-Vinyl GABA also elevates the CSF concentrations of free and conjugated GABA in the rat. This effect is dose-dependent (Fig. 2). As little as 125 mg/kg v-vinyl
300
5
.~/
2E
3
'~ m
2
e
y-VINYL GABA
/
-2
4
Conjugated GABA
I"
O~
U
0 -4 TIME
0
2
6
AFTER ~ - V I N Y L GABA
8 ( hr )
Fig. I. Free and conjugated G A B A concentrations in the CSF of anesthetized cats before and after intraperitoneal injection of),-vinyl G A B A (1000 mg/kg). Intraventricular cannulae were implanted 6-8 h prior to drug administration. Points are means 5- S.E.M. (n ~ 3-4).
GABA causes a significant increase (P < 0.05, Students t-test, 2-tailed) of the brain GABA concentrations, whereas free and conjugated GABA in the CSF are significantly elevated (P < 0.05) only after 250 mg/kg 9,-vinyt GABA. The highest dose ofg,-vinyl GABA used (2000 mg/kg) increases concentrations of brain GABA and conjugated GABA in CSF 5- and 4-fold respectively, but elevates free GABA in the CSF 135-fold over control levels. As the dose of 9,-vinyl GABA is increased the ratio between conjugated and free GABA in CSF changes drastically. This ratio is highest in untreated animals due to the very low levels of free GABA present and is low at high drug doses under which condition large amounts of free GABA are found in the CSF. 9,-Vinyl GABA also elevates serum GABA concentrations (Table I).
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