Evaluation of increases in nerve terminal-dependent vs nerve terminal-independent compartments of GABA in vivo

Evaluation of Increases in Nerve TerminalDependent vs Nerve Terminal-Independent Compartments of GABA In Vivo M. J. IADAROLA

AND K. GALE

Department of Pharmacology, Georgetmw University Schools of Medicine and Dentistry, Washington, D.C. 20007

IADAROLA,

M. J. AND K. GALE. Ek3clltctrtion of’incrcwsr.s itt ttcrw forntittrtl-ci~~p~~tt~j~~~tt t’,s ttct’t’c tc,rttti,tct/-i/tc/~~~(,/t~~~,~t~ itt virw. BRAIN RES. BULL. 5: Suppl. 2, 13-19, 1980.-Evaluation of increases in nerve terminal-dependent and nerve te~inal-independent compartments of GABA in vi\*o. Changes in GABA occurring in the absence and in the presence of GABAergic nerve terminals were estimated using rats in which the GABAergic projection to the substantia nigra (SN) was destroyed on one side of the brain. The net increase in GABA conterit of the GABAdenervated SN was compared with that of the intact SN after injection of Gino-oxyaceti~ acid (AOAA), n-dipropylacetate (DPA) and y-vinyl GABA (GVG). One week post-operative, GABA concentration in the SN on the transected hemisphere was IO-2OC% of control. AOAA (30 mgikg) produced a two-fold increase in CABA content in the denervated SN, whereas DPA (300 mgikg) was without effect. Since DPA caused an increase in GABA in the intact SN which was similar to that caused by AOAA (253@%), it appears that the DPA induced elevation of GABA depends upon the presence of GABAergic nerve terminals whereas AOAA primarily elevates GABA in non-nerve-terminal compartments. GVG, an irreversible GABA-T inhibitor, increased GABA levels in both compartments. The predominant effect of GVG was on nerve terminalindependent GABA (4-fold increase after 900 mgikg); the GABA increase associated with nerve terminals was smaller (3(yTo)and did not appear until 60 hr after a single injection of GVG. The ability of DPA, AOAA and GVG to protect against chemically- and electrically-induced seizures was directly correlated with increases in nerve-terminal GABA, and not related to increases in other GABA pools. c,ompcrvlmrrtt,s qf’ GABA

GABA (y-aminobutyric acid) Anticonvulsants Rats

Amino-oxyacetic n-Dipropylacetate Metabolic compa~mentation

RECENT efforts to develop a useful pharmacology of CNS GABAergic neurotransmission have resulted in a number of compounds that selectively act on GABA receptors [4,8,21, 241 or alter the CNS concentration of GABA in the CNS [26, 36, 391. However, we lack adequate information concerning the cellular and anatomic components with which these drugs interact to produce functional effects. For example, GABA exists in a variety of cellular compartments, both neuronal and non-neuronal, and these compartments may be differentially influenced by drug treatments [5, 19, 291. On another level, because GABA appears to function as a neurotransmitter throughout most of the brain [34], it is difficult to identify the particular brain nucleus or system which mediates the changes in function brought about by GABAmimetic drugs. In fact, GABA neurons in one brain area may serve to counteract the influence of GABA neurons in another brain region. These problems of localization are all too familiar complications for studies of CNS GABA neuropharmacology 129, 3 1, 421. In the present report we will focus on one class of GABA-agonist drugs: those that increase brain GABA content. Two aspects of the localization problem will be considered: that of discriminating between the cellular compartments in which an increase in GABA may take place and

Copyright

“’ 1980 ANKHO

International

acid

y-Vinyl GABA

that of comparing GABA increases taking place in different brain regions. Three GABA-elevating agents were used in this study: n-dipropylacetate (DPA, sodium valproate), amino-oxyacetic acid (AOAA) and gamma-vinyl GABA (GVG). These compounds have been reported to elevate brain GABA content by separate mechanisms. AOAA is thought to inhibit GABA-transaminase by interacting with the pyridoxal phosphate cofactor [42]. GVG is a compound that undergoes catalytic conversion by GABA-T to produce a reactive intermediate which then irreversibly inhibits the enzyme [26]. The mechanism whereby DPA increases brain GABA is somewhat controversial but this drug has been reported to inhibit both GABA-T and succinic semialdehyde dehydrogenase, the second enzyme in the GABA catabolic pathway [I, 11, 13, 16, 18, 401. In order to investigate whether the increase in GABA produced by these drugs was taking place in different cellular compartments, we exploited certain unique features of the GABAergic system in the substantia nigra (SN). The SN is a midbrain nucleus that has a very high GABA content [22,32], the majority of which derives from GABAergic projections originating in the ipsilateral striatum [lo, 17, 321. The longdistance nature of this projection allowed us to surgically remove it by making a unilateral hemitransection anterior to

Inc.-~341-9~30/80/08~!3-07$01.20/O

14

IADAROLA

the SN without directly damaging tissue within the SN [lo, 12, 17,221. The degeneration of the GAB A containing terminals in the SN of the transected hemisphere is essentially complete by 7-10 days postoperatively [15,32]. At this time we have consistently observed an 82% reduction in GABA content [12,19]. The GABA remaining after these hemitransections represents that primarily associated with the nonnerve terminal compartments composed of neuronal perikarya, glia and non-GABAergic nerve terminals. We will refer to this pool of GABA as the glial-metabolic pool. By comparing a drug-induced increase in GABA in the intact SN (cont~ning both nerve terminal and glial-metabolic pools) with that in the “GABA-dene~ated” SN (primarily the gtial-metabolic pool) we attempted to discriminate the relative contribution of these compartments to the overall GABA elevation produced by the various drug treatments. This compartmental analysis revealed marked differences between the drugs with respect to their influence on nerve terminal versus glial-metabolic GABA. In order to further explore the differences between the actions of these GABA elevating agents, we examined their effects on the GABA content of four additional brain areas containing various steady-state concentrations of GABA. We observed that the differential regional responses to each drug could be, in part, predicted from our compartmental analysis. Our compa~mental analysis also provided information useful for predicting functiona effects of these drugs as anti-seizure agents. METHOD

Animuls

und Drugs

Male Sprague-Dawley rats, housed in groups of 4-6 were used. Animals used in experiments involving surgery weighed 300 g; animals used for testing in experimental seizure models weighed 100-200 g. Drugs were dissolved in distilled water and administered in a volume of 0.5 cc/l00 g. Control animals received vehicle (distilled water) only. Unless otherwise noted injections and other experimental procedures were performed between 11:OOa.m. and 3:00 p.m. DPA (Depakene@) and GVG were gifts from Abbott Labs (Chicago, IL) and the Centre de Recherche, Merrell Tntemational, Strasbourg (France), respectively. AOAA was obtained from IS and K Labs (Plainview, NY); all other chemicals were obtained from Sigma (St. Louis, MO). Lesion

Cerebral hemitransection was performed using a stereotaxic apparatus (David Kopf) with the incisor bar 5 mm above the interaural line. A stainless steel loop, mounted in an electrode carrier, was inclined in the antero-poste~or plane at an angle of 6.3” from vertical with the lower tip directed rostrally. The probe entered the cortex at AP +3.0 1351 and was lowered to intercept the base of the brain l-2 mm anterior to the SN. The probe was moved 5 mm laterally from the midline by manipulating the electrode carrier. The lateral movement was repeated 3-4 times, adjusting the vertical depth as necessary in order to follow the contours of the base of the skull. Transections posterior to the SN were made with the probe in a vertical position; the probe entered the cortex at AP +0.2 and was moved medio-laterally as was done for the anterior transections. Seven to 10 days were allowed postoperatively for degeneration to occur before drugs were administered.

AND GALE

Dis st,ction

The whole brain was removed and placed ventral side up on a cold glass plate. The ventral and lateral extent of the transection could be easily visualized in the cerebral peduncles. A coronal section of tissue between the rostra1 border of the pons and the caudal-most aspect of the mammillary bodes was taken. The SN, visualized as an area of grey matter situated between the medial lemniscus and the cerebral peduncles, was dissected out of the section and placed immediately on dry-ice. The other areas were easily dissected with a small curved forceps or scalpel bfade. The sample of the cerebral cortex was taken from the frontal pole. GA BA Assoy

GABA was extracted from the tissue with0.4N HC10, and measured by the enzymatic-fluorometric method of Okada cut ill. 1341except that the 60” heating step was omitted. Tissue blanks (without GABAase) and reagent blanks (without tissue) were run with each assay and found to be equivalent. Each brain sample was assayed in duplicate along with GABA standards. Protein content of the pellets (resuspended in 0.5 N NaOH) was measured by the method of Lowry c? rfl. 1281.

Maximal electroshock seizures (MES) were administered with a Whalquist electroshock apparatus [48]. The stimulus parameters were 150 mA, 0.2 sec. 60 Hz deiivered via saline-moistened cornea1 electrodes. The duration of tonic hindlimb extension (THE) was timed with a stopwatch. All animals were screened prior (24 hr) to drug administration in order to eliminate animals which failed to show THE. Reductions in the duration of this phase were used to assess the anticonvulsant activity 1411. Animals in which THE was blocked completely were scored as having spent zero time in THE. From these measurements the percent reduction in THE duration was calculated for each drug in comparison to a control group of rats run in paratlel. RESULTS

The GABA content of the SN from unoperated controls was equivalent to that of the intact SN of the hemitransected animals: 81 rt 2.7 nmoles/mg protein, shown as the first bar in Fig. 1. Hemitransections caused a decrease in nigral GABA to 18% of control (shown in Fig. 1 by the portion of the first bar that extends below the horizontal axis). In order to assess the reliability of our GABA-denervation procedure, a series of operated rats (n=44) was examined for GABA content. Nigral GABA was reduced to less than 25% of control in the majority (88%) of the animals. In addition, to explore the possibility that causal brain areas might contribute GABA-con~ning terminals to the SN, unilateral transections were placed posterior to the SN (n=5). No significant change in the GABA content of the SN on either the lesioned or intact hemispheres was observed 7-10 days after the posterior hemitransections. In Fig. 1, the 2nd, 3rd and 4th bars represent the nigral GABA levels at three time periods following a single dose of GVG (900 mg/kg). The entire length of each bar represents the GABA level of the intact SN. A large increase in GABA in the intact SN can be seen at 12 hr (160 * 8.1 nmoles/mg protein) and 36 hr (1.50 * 8.8) and this began to decrease by 60 hr (127 2 5.3) after treatment. The portion of each bar in

NERVE TERMINAL 12

Time(hrs) CONTROL

Drug

DosePwW loo-

AND NON-NERVE 36

60

&VlNVL-GABA

60-

z

40200 20 : --

2 =

40 60

0.5

2

DPA

ADAA

INCREASES

900

80 -

z ._ s % M 5 ul Y 5

TERMINAL

L

L

80 100 120

FIG. 1. The effect of GVG, DPA and AOAA on GABA content of the intact and GABA-denervated SN. In this figure the total length of a bar represents the GABA content of the intact SN. The portion of the bar that extends below the horizontal axis represents the GABA content of the GABA-denervated SN. The GABA content on the denervated SN was subtracted from that on the intact SN to obtain the nerve terminal associated GABA level. The control level of GABA in the nerve terminal and non-nerve compartments is shown in the first bar and the dotted lines. The net increase due to drug treatment, in both compartments is indicated by the shaded portion of each bar. Each group represents the mean of at least 6 animals. Animals were sacrificed by focused microwave irradiation of the head for 4 set 121.All drug induced increases in GABA in the intact and denervated SN were significantly different from the respective controls except for DPA in the denervated SN. Criterion for significance was ~~0.05. Comparisons were made by analysis of variance and Duncan’s new multiple range test.

Fig. 1 that lies below the horizontal axis represents the GABA content of the GABA-denervated SN. In this tissue, the large increase in GABA (from 15 _t 2.0 to 112 * 7.3 nmolesimg protein) observed at 12 hr was significantly diminished at 36 hr (71 t 3.8 nmoles/mg protein) and 60 hr (39 * 3.1 nmolesimg protein). The net increase in GABA due to drug treatments are indicated by the shaded areas in Fig. 1 (i.e., the portions of each bar which lie outside the control values obtained from animals not treated with drugs). At 12 hr the net increase in GABA in the SN on the denervated side was at least as great as the net increase measured in the SN on the intact side. Since the GABA increase on the denervated side can account for the entire GABA increase on the intact side, this observation indicates that the increase in nigral GABA occurring 12 hr following GVG treatment

IN GABA

15

does not depend upon the presence of nigral GABAergic nerve terminals. At 36 hr these relationships began to change and the SN on the intact side exhibited a greater net increase in GABA than can be accounted for by the net increase in GABA in the SN from the denervated hemisphere. The net increase in GABA which depends upon the presence of nerve terminals is depicted in Fig. 1 by the shaded portions on the upper half of the graph. It can be seen that the increase in nerve-terminal dependent GABA after GVG became even larger at 60 hr; at this time it appears that the total net increase in GABA was equally distributed between the nerve terminal and non-nerve-terminal compartments. The GABA-denervated SN was also used to evaluate the compartmental distribution of GABA increases after DPA and AOAA (last two bars of Fig. 1). In the SN on the intact hemisphere, treatment with DPA (300 mg/kg IP) and AOAA (30 mg/kg IP) each produced significant increases in GABA which were of similar magnitude (net increase=32 and 22 nmolesiprotein, respectively). In the SN on the denervated side, AOAA produced a significant increase in GABA (net increase= 18 nmoles/mg protein) which was equivalent to more than 80% of the net increase on the intact side. Consequently, only a small portion of the overall increase in GABA after AOAA could be attributed to the nerve-terminal dependent compartment. In contrast DPA failed to significantly increase the GABA content of the denervated SN; more than 90% of the GABA increase after DPA was found to be dependent upon the presence of the GABA nerve terminals. Thus, in the relative absence of GABAergic nerve terminals the GABA-elevating effect of DPA appears to be lost, whereas the GABA-elevating effect of AOAA is retained. To determine whether the biochemical changes in GABA in a particular compartment might be correlated with functional effects, we examined the anticonvulsant activity of DPA, AOAA and GVG in the maximal electroshock (MES) seizure test. In this test, a reduction in the duration of the tonic hindlimb extension phase of the seizure was used to quantify the anticonvulsant activity. The time course of action of GVG in the MES test is shown in Table 1. At 12 hr after GVG no blockade or reduction in duration of THE was observed. At 36 hr a slight reduction in THE duration was seen and by 60 hr the reduction in THE was significant. At 60 hr the dose-response relationship for GVG in the MES test was determined. The ED,,, was found to be approximately 933 mg/kg and complete protection was observed with 1600 mg/kg. The delayed appearance of anticonvulsant activity with GVG prompted us to reconsider other studies [38,39] which had shown GVG to be inactive against seizures induced by pentylenetetrazol and bicuculline. These latter studies had been conducted within the first 12 hr after GVG treatment. When we tested animals 60 hr after GVG (900 mgikg) we found them to be completely protected from clonic seizures induced by an ED,,,,, dose of bicuculline (0.25 mgikg IV) and 65% were protected from seizures induced by twice this dose. At 60 hr after administration, GVG (900 mgikg) provided partial protection (50%) from clonic seizures induced by pentylenetetrazol (100 mgikg SC). AOAA was also capable of suppressing THE in the MES test with an ED,,, of 40 mgikg. Thirty mg/kg (the dose employed in our biochemical studies) reduced the THE duration by 18%. The ED,,, for DPA was 140 mg/kg and a 9m reduction in the duration was obtained with 300 mg/kg. Our data suggested that functional augmentation of GABAergic synaptic transmission may be correlated with changes in

1ADAROLA AND GALE TABLE

1

RELATIONSHIP BETWEEN SUPRESSION OF THE IN THE MES TEST AND INCREASES IN GABA IN THE INTACT AND DEVERVATED SN AND THE NERVE TERMINAL COMPARTMENT

Treatment DPA

GVG (900 m&d

(300 mgikg)

AOAA

(30

mdkg)

Time after treatment 12 hr % Reduction in duration

60 hr

0.5 hr

2 hr

THE

% Increase in nerve terminal GABA %

36 hr

Increase in intact SN

% Increase in non-nerve terminal GABA

0

28

45

90

18

0

19

33

39

7

98

85

57

36

25

648

373

160

28

100

Data from Fig. 1were used to calculate the percent increases over control in the various compartments. Percent reductions in THE duration are from studies done in this laboratory with GVG. DPA and AOAA.

w

E 60 c_ c t

I

10 Percent

Increase

I

20 in Nerve

L

30 Terminal

I

1

40 GAB

50 A

FIG. 2. Relationship between percent reduction in THE duration in the MES test and percent increase in nerve terminal associated GAB A. The graph was derived from the data of Table 1. Three drug treatments are represented: GVG 900 mg/kg at 12 hr (origin), 36 hr (2nd point), 60 hr (3rd point); AOAA 30 mgikg at 2 hr (1st point); and DPA 300 mgikg at 0.5 hr (4th point). The correlation coefftcient between the two parameters was 0.93.

nerve-terminal related GABA and not simply with gross increases in brain tissue levels of GABA. To further explore this relationship we compared the anticonvulsant activity of the three drugs in the MES test with the increases in GABA in the SN as a whole and the nerve-terminal and non-nerveterminal compartments (Table 1). Comparison of the percent increase in GABA in the nerve terminal compartment reveals that a positive correlation (r=O.93) exists between increases in this compartment and protection from electroshock seizures. This correlation is presented graphically in Fig. 2 for DPA, AOAA, and over time for GVG. In contrast, the anticonvulsant activity was not positively correlated with increases in GABA in the nigra as a whole (intact SN) or in the non-nerve terminal pool. Our hemitransection experiments have indicated that differences exist between AOAA, DPA and GVG with respect to their ability to elevate nerve terminal dependent GABA. Since it is well known that regional variation exists in brain with respect to the density of GABAergic nerve terminals, it might be expected that the regional responses of GABA levels to drug treatment might help to define the GABA compartment most affected by a particular drug. Accordingly, we examined the effect of GVG, AOAA and DPA on four brain areas in addition to the SN: superior and inferior colliculus, caudate nucleus and cerebral cortex. The results of this experiment are shown in Fig. 3 in which the net increases in GABA after treatment with each of the three drugs are plotted for each of the five brain areas. The five brain areas are placed in rank order from highest to lowest with respect to glutamic acid decarboxylase (GAD) activity 19, 32, 33, 43, 441 and GABA levels. The normal GABA levels (without drug) for each area are shown in the small inset in Fig. 3; these values are closely correlated with the GAD activity for each area. Data from the GABAdenervated SN is also included. The net increases in GABA

NERVE TERMINAL

AND NON-NERVE

TERMINAL

INCREASES

seen with DPA are not uniform

but vary from a high of 45 nmolesimg protein in the SN to a low of 6 nmolesimg protein in the caudate nucleus. Comparison with the control profile of GABA reveals that DPA is the only drug in which the rank order of the net GABA increases closely corresponds to the normal control GAD activity or GABA content of the five areas. In contrast. AOAA and GVG (12 hr after injection) produce net increases in GABA which are fairly uniform across regions and appear to be independent of the regional GAD activity or GABA levels. Furthermore, for both AOAA and GVG the net increase in GABA obtained in the various brain regions are similar to the increase obtained in the GABA-denervated SN. DISCUSSION

The data obtained with GVG in the GABA-denervated SN indicate that this drug is capable of elevating GABA independently of the presence of GABAergic nerve terminals. At early time periods after GVG administration the increase in GABA occurred almost exclusively in the nonnerve-terminal ~ompa~ment and it was only after a considerable delay that an effect upon nerve-terminal dependent GABA was observed. These observations suggest that some second order phenomenon, such as a redistribution of GABA, may be responsible for the increase in nerve terminal GABA. AOAA was also found to produce an increase in GABA in the relative absence of GABAergic nerve terminals and this nerve terminal independent increase could account for almost all of the increase in GABA observed in the intact SN. Thus, AOAA like GVG, appears to exert its effect predominantly on the glial-metabolic pool of GABA. AOAA-induced

17

in GABA have also been reported for neural tissues which are not known to contain GABA nerve terminals such as posterior pituitary [61, pineal gland and superior cervical ganglion, 1451. Furthermore, in subcellular fractionation studies the predominant increase in GABA after in riro administration of AOAA was observed in a high speed supernatant (i.e., soluble) fraction (471. The fact that GABA elevations in the nerve-terminal compartment with AOAA are seen only after other pools are elevated severalfold may help to explain why relatively large increases in GABA are necessary to observe GABA-related functional effects with this drug [7, 14, 231. The well characterized ability of GVG and AOAA to inhibit GABA-transaminase (GABA-T) can account for the observed increase in brain GABA after treatment with these drugs 126,421. Biochemical and immunocytochemical studies have localized GABA-T largely to neuronal perikarya and glial cells 13.251. As our data demonstrate, changes in GABA which occur in these non-nerve terminal pools may actually conceal the degree of change in GABA in the functionally relevant (nerve terminal) compartment. The data obtained with DPA demonstrate that increases in nerve terminal GABA can be obtained in the absence of large increases in other compa~ments of GABA. This observation is supported by the subcellular fractionation studies of Sarhan and Seller 1371 which demonstrated a selective effect of DPA on synaptosomal GABA. Thus, although the total GABA increase after DPA is modest (in contrast to AOAA and GVG), it appears to be almost exclusively nerve terminal associated. The experiments reported here indicate that the increase in nerve-terminal dependent GABA produced by DPA is sufficient to predict marked anticonvulsant activity in the MES seizure model. The relationship between the DPA induced increase in GABA and the presence of GABAergic nerve terminals is further supported by the effect of this drug on GABA in different brain areas. Since GAD activity has been suggested to be a marker for GABAergic nerve terminals, we chose areas that represent a range of GAD activities, with the intact SN the highest, the denervated SN the lowest and the other areas falling in between. DPA was observed to increase GABA in direct proportion to the GAD activity and GABA level of each area. This observation is consistent with a nerve terminal locus of action. It is also consistent with the failure of DPA to elevate GABA in the absence of GABA nerve terminals. In contrast to DPA, the profiles of net increases in GABA with AOAA and GVG show only small variations across brain areas and the magnitude of the change is independent of the normal steady-state GABA content. Furthermore the net increase in most areas is equivalent to the nerveterminal-independent increase in GABA in the denervated SN. This compa~son further supports the inte~retation that AOAA and GVG exert their predominant effect on a pool of GABA that is not dependent upon GABA nerve terminals. Although we have considered the relationship between drug action and steady-state concentrations of GABA in various brain regions, we have not examined the possibility that the rate of GABA turnover may influence the drug effect. Steady-state levels of GABA may reflect the density of GABAergic nerve terminals but they do not necessarily reflect the rate of neuronal activity. It is possible that certain brain areas with a low steady-state concentration of GABA may have a high rate of neuronal activity and therefore a high rate of GABA turnover. At the same time. the drug induced increases

FIG. 3. Net increase in GABA after DPA, AOAA and GVG in five areas of rat brain: substantia nigra, superior colliculus (SC), inferior colliculus (ICY),caudate nucleus (CN) and cerebral cortex (CC). The last bar for each drug treatment represents the net increase in the GABA-denervated SN (DSN). The areas are rank ordered from highest to lowest according to the control GAD activity. GAD activity was found to be directly proportional to the control GABA levels across the different areas. The control GABA levels of the different areas is shown in the small insert at the top of the graph. Druginduced increases in GABA in all areas were significantly different from control @
IN GABA

IX

IADAROLA

elevation of GABA may itself affect turnover. For example, the GABA increase in the caudate was disproportionately low relative to other brain regions as well as with respect to the steady-state concentration of GABA in this nucleus. A possible explanation for this is suggested by studies of GABA turnover in this nucleus as reported by Mao (~1crl. 1301. These investigators found that activation of GABA receptors caused a marked depression of the normally high turnover rate of GABA in the caudate nucleus: this effect was not as pronounced in the other brain regions studied. It is possible that the GABA-elevating agents used in our study may likewise cause a depression in the turnover rate of GABA; this in turn might decrease the rate of accumulation of GABA subsequent to GABA-T inhibition. Some unique feature of the neuronal circuitry in the caudate may therefore influence the metabolism of GABA in this nucleus after drug treatment. This raises the possibility that synaptic activity in local neural circuits may influence both the regional metabolism of GABA and the ability of drugs to alter this metabolism. The possible interaction between synaptic activation and GABA turnover raises an additional question concerning the relationship between neuronal and glial GABA metabolism. It is possible that the density of GABA nerve terminals may influence glial GABA metabolism. In particular, it is possible that in areas in which the density of GABA nerve terminals is low (either normally as in cortex or as a result of a lesion as in our denervated SN), GABA metabolism in the glialmetabolic compartment may be different from that in areas with a high density of GABA nerve terminals. Although we

AND GALE

have assumed, for present purposes, that the glial-metabolic compartment in the denervated SN is representative of the normal glial-metabolic compartment in the intact SN, we realize that this is only an approximation. We are currently investigating these issues using GABA-denervation procedures in other areas of the brain. In conclusion, we feel that our data may help to explain some of the apparent paradoxes observed when drugs which elevate brain GABA are examined for functional effects. One paradox concerns the relationship between the magnitude of the GABA elevations produced by AOAA and DPA and protection from experimental seizures. The antiseizure effect of AOAA is associated with much larger increases in brain GABA than those seen with anticonvulsant doses of DPA. However, our data suggest that DPA interacts with a pool of GABA that seems to be directly linked to synaptic transmission, whereas with AOAA much larger increases in total GABA are needed in order to affect this pool of GABA. Another paradox concerns the time course of anticonvulsant effects and GABA increases after GVG. At early time periods after GVG no effect on MES seizures was observed despite maximal elevations of brain GABA. Conversely, at a time when the total GABA was declining, seizure protection became evident. However, our analysis of compartmentalized changes in GABA after GVG revealed that nerve-terminal related GABA increased in parallel with the development of anticonvulsant activity. These data demonstrate that changes in nerve terminal associated GABA may serve as a predictor for functional effects such as seizure protection.

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and P. Jacobsen. GABA upstructurally related “pro-drugs”. In: GABA-Neurofransmitters, edited by P. Krogsgaard-Larsen, J. Scheel-Kruger and H. Kofod. Copenhagen: Munksgaard, 1978, pp. 247-262. take

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9. DiChiara, G., M. L. Porceddu, M. Morelli, M. L. Mulas and G. L. Gessa. Strio-nigral and nigro-thalamic GABAergic neurons as output pathways for striatal responses. In: GABAedited by P. Krogsgaard-Larsen, J. Neltrotronsmittc,rs. Scheel-Kruger and H. Kofod. Copenhagen: Munksgaard, 1978, pp. 465-481. 10. Fonnum, F., Z. Gottesfeld and I. Grofova. Distribution of glutamate decarboxylase, choline acetyltransferase and aromatic amino acid decarboxylase in the basal ganglia of normal and operated rats. Evidence for striatopahidal, striatoentopeduncular and striatonigral GABAergic fibers. Brain Rrs. 143: 125-138, 1978. 11. Fowler, L. J., J. Beckford and R. A. John. An analysis of the kinetics of the inhibition of rabbit brain y-aminobutyric aminotransferase by sodium-n-dipropylacetate and some other simple carboxvlic acids. Biochem. Phurmac. 24: 1267-1270, 1975. 12. Gale, K. and M. Iadarola. GABAergic denervation of rat substantia nigra: functional and pharmacological properties. Bruin Rc\. 183: 217-223, 1980. 13. Godin, Y., L. Heiner, J. Mark and P. Mandel. Effects of di-npropylacetate, an anticonvulsive compound, on GABA metabolism. /. Neurochem. 16: 869-873, 1969. 14. Gottesfeld, Z., J. S. Kelly and L. P. Renaud. The in viw neuropharmacology of amino-oxyacetic acid in the cerebral cortex of the cat. Bruin Res. 42: 3lS335, 1972. 15. Grofova, I. and E. Rinvik. An experimental electron microscopic study on the striatonigral projection in the cat. Expl Bruin Res. 11: 249-262, 1970. 16. Harvey, P. K. P., H. F. Bradford and A. N. Davison. The inhibitory effect of sodium-dipropylacetate on the degradative enzymes of the GABA shunt. FEES Lett. 52: 251-254, 1975.

NERVE TERMINAL

AND NON-NERVE

TERMINAL

INCREASES

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