The GABA hypothesis of kindling: Recent assay studies

Neuroscience & BiobehavioralReviews. Vol. 13, pp. 281-288. o Pergamon Press plc. 1989. Printed in the U.S.A.

0149-7634/89 $3.00 + .00

The GABA Hypothesis of Kindling: Recent Assay Studies W. M. B U R N H A M

Department o f Pharmacology, Universit 3, o f Toronto Toronto, Ontario, Canada MSS 1A8

BURNHAM, W. M. The GABA hypothesis of kindling: Recent assay studies. NEUROSCI BIOBEHAV REV 13(4) 281-288, 1989.--The GABA (gamma-aminobutyric acid) hypothesis of kindling suggests that the permanent changes caused by the kindling procedure result from a loss of GABA-mediated inhibition. Pharmacological studies have generally supported this hypothesis: GABA-complex antagonists accelerate (or simulate) kindling, whereas GABA-complex agonists retard (or reverse) it. Assay studies, however, have presented an inconsistent picture. Earlier studies found no GABAergic brain changes after kindling, whereas recent studies have reported postkindling changes in a number of GABA-related parameters. The crucial difference seems to be that earlier studies assayed GABA parameters in "whole tissue," whereas recent studies have concentrated on "synaptic" GABA. As indicated by recent studies, when the "metabolic pool" is excluded, kindled subjects show a variety of persistent abnormalities in the GABA system. These data are generally consistent with the GABA hypothesis of kindling. GABA

Assay studies

Kindling model

Epilepsy

THE GABA HYPOTHESIS

state" (5). In updated form, these suggest that: 1) pharmacological induction of a proposed causal abnormality should accelerate (or simulate) kindling; 2) pharmacological correction of the abnormality should retard (or reverse) kindling; 3) biochemical assays should reveal the abnormality in animals sacrificed at least four weeks after the last seizure. How does the GABA hypothesis measure up to these criteria? As suggested in an earlier review (5), the pharmacological data--related to induction and correction--are generally supportive of the GABA hypothesis. Pharmacological blockade of the GABA system accelerates (and partially simulates) kindling, whereas pharmacological enhancement of GABA activity retards (and reverses) it. Recent experiments with novel drugs and paradigms have simply reinforced this general picture (10,21). With regard to the assay data, however, the story has been far more complex. Early assay data (1978-1985) generally tended to be negative, suggesting that the GABA system was largely unchanged by kindling. Recent assay studies (1985-1988), however, have been more positive, reporting kindling-induced changes in a number of GABAergic parameters.

A number of biochemical hypotheses have been proposed to explain the phenomenon known as "kindling" (2, 5, 12, 24-26, 31, 32). Among these is the GABA hypothesis, which may be stated as follows: "The kindling procedure causes a permanent change in some part of the GABA-A inhibitory system. This change leads to chronic, localized, mild GABAergic hypofunction, and this, in turn, produces all (or part) of the physiological abnormalities which characterize the 'kindled' state." Certain features of this hypothesis deserve discussion, even before the related evidence is considered. First, as presently stated, the hypothesis postulates that kindling-induced GABA hypofunction may be "localized and mild." The idea that hypofunction may be "'localized" stems from studies showing decreased inhibition in some brain areas after kindling, but increased inhibition in others (16). The idea that it may be " m i l d , " stems from the consideration that " s e v e r e " GABAergic hypofunction results in constant seizure activity, as in the bicuculline or picrotoxin models (36). Second, as presently stated, the hypothesis suggests that GABA hypofunction may account for only " p a r t " of the kindled state. This qualification stems from a recent study showing that GABA blockade (in naive animals) simulates part, but not all, of the kindling syndrome (28). In its present formulation, the hypothesis allows for the possibility that defects in several different systems may contribute to the "'kindled state."

EARLY (NEGATIVE)ASSAYDATA Starting in 1978, a series of assay studies began to appear which suggested that the GABA-A system was "unchanged" in kindled brains. These are summarized in Table 1. As indicated by the table, these reports suggested that GAD (glutamic acid decarboxylase) was normal in kindled brains (19); that GABA levels were normal (7, 8, 18-20); GABA-T (gamma-aminobutyric acid transaminase) activity was normal (19); that GABA release and response were normal (1,20); that GABA-related binding was unchanged or increased (27,37); and so forth. In contrast to these

EVIDENCERELATEDTO THE GABAHYPOTHESIS:GENERALSURVEY Several years ago, Burnham et al. proposed a set of criteria to be used in evaluating biochemical hypotheses of the "kindled

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TABLE 1 STUDIES WHICH FAILED TO DEMONSTRATE POSTKINDLING DEFICITS

Experimenters

Subjects

Preparation and Assay

Results (Kindled vs. Control)

GAD Activity Reedy et al. (33) University of British Columbia

Lemer-Natoli et al.

(19) Laboratory of Experimental Medicine, Montpellier

Rats (m, R.V.H), amygdalakindled (daily) to 5 Stage 5, 24 hr to sacrifice

whole-tissue homogenates

1) unchanged: (regions unspecified)

Rats (m, Sprague-Dawley) amygdala kindled (daily) to 34 stimulations, I week to sacrifice

whole-tissue homogenates (radiometric assay)

1) unchanged: AMYG, CB, CX, HPC, STR

GABA Release Liebowitz et al. (20)

Stanford University

Rats (m, SpragueDawley), entorhinal kindled (daily) to 5-7 "generalized convulsions," 24 hr to sacrifice

hippocampal slices (radioactive dansylation assay)

l) unchanged: spontaneous release 2) increased: K+ stimulated release (60%) HPC (ipsi and contra)

GABA Levels Liebowitz et al. (20)

Stanford University

Fabisiak and Schwark (7) Cornell University

Fabisiak and Schwark (8) Cot'nell University

Rats (m, SpragueDawley) entorhinal kindled (daily) to 5-7 "generalized convulsions," 24 hr to sacrifice

hippocampal slices (radioactive dansylation assay)

1) unchanged: HPC (bilat?)

Rats (m, Sprague-. Dawley) amygdala kindled (daily) to "Stage 5 seizures," 1 week to sacrifice

wholetissue homogenates (assay by cation exchange chromatography)

1) unchanged: BS, CB, CX (frontal)

Rats (m, SpragueDawley) metrazol kindled (21 administrations), 1 week to sacrifice

whole-tissue homogenates (assay by cation exchange chromatography)

1) unchanged: BS, CB, CX (frontal), CX, limbic lobes, midbrain

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283

TABLE 1 CONTINUED

Experimenters Leach et al. (18) Wellcome Research Laboratories

Lerner-Natoli et al. (19)

Laboratory of Experimental Medicine, Montpellier Schmutz et al. (34)

Ciba-Geigy Ltd., Basel

Subjects

Preparation and Assay

Results (Kindled vs. Control

Rats (m, Wistar) CX kindled (daily) to 3 "kindled responses," then (weekly) for 3 months, 1 week to sacrifice

whole-tissue homogenates (spectrofluorometric assay)

I) unchanged: CX

Rats (m, Sprague-Dawley) amygdala kindied (daily) to 34 stimulations, 1 week to sacrifice

whole-tissue homogenates (gas chromatographic assay)

I) unchanged: (ipsi and contra) AMYG, CX, HPC, OLF BULB STR

Rats (m, Tif: RAIf SPF) amygdala "kindled (daily) to Stages 0, 1 or 4, 20 min to sacrifice

whole-tissue homogenates (HPLC used for assay)

1) no "significant enhancement:" AMYG CX, HPC (at Stage 4)

(Note: Significant differences may have been observed at Stage 2, but these were seen "rarely" at Stage 4 and were not reported fully.) GABA Receptor Binding (GABA-A and Benzodiazepine) Data Presented in Burnham (2) (See Text) GABA-T Activity Lerner-Natoli et al. (19)

Laboratory of Experimental Medicine, Montpellier

Rats (m, Sprague-Dawley) amygdala kindled (daily) to 34 stimulations, 1 week to sacrifice

whole-tissue homogenates (spectrophotometric assay)

1) unchanged: whole brain (homogenized in entirety)

(Note: Activity of succinic semialdehyde was also unchanged in whole brains.) GABA Response Burchfiel et al. (I)

Harvard Medical School

Rats (m, SpragueDawley) fornix "kindled" to one or two discharges (hourly?), 0 4 hr to test

extracellular unit recording in whole brain (acute expts.) plus iontophoresis

1) unchanged: HPC (CA 1)

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BURNHAM

TABLE 2 STUDIES WHICH DEMONSTRATED POSTKINDLING DEFICITS Experimenters

Subjects

Moiseev et al. (29)

Mice, metrazol kindled (daily) for 3 weeks, 24 hr to sacririce

Preparation

Results

GAD Activity

Pirogov Odessa Medical Institute

brain sections (cytophotometric assay allowed separate study of neurons and glia)

1) reduced: CX (sensori-motor) (18%)

(Note: Reductions were observed in both neurons and glia.) Loscher and Schwark (22) Free University of Berlin

Rats (f, Wistar) amygdala kindled (daily) to 10+ Stage 5, I + week to sacrifice

Synaptosomes (fluorimetric assay)

1) unchanged: AMYG, CB, CX (frontal), HPC, HYPO, MED, OLF BULB, PONS, STR, TECT, THAL 2) reduced: SN (3O--40%)

(Note: IPSI and CONTRA were homogenized together for all areas except the AMYG, which was IPSI only. Thirty to forty percent reductions were observed in the AMYG and STR as well, but they were not significant. ) Loscher and Schwark (23) Free University of Berlin

Rats (f, Wistar) amygdala kindled (daily) to 10+ Stage 5, l + week to sacririce

Synaptosomes (fluorimetric assay)

1) reduced: AMYG (40%), STR (50%), SN (45%) 2) unchanged: CB, CX, HPC, HYPO, MED, OLF B, PONS

(Note: IPSI and CONTRA were homogenized together for all areas except the AMYG, which was IPSI only.) GABA Levels Loscher and Schwark (23) Free University of Berlin

Rats (f, Wistar) amygdala kindled (daily) to 10+ Stage 5, 1 + week to sacririce

Synaptosomes (radioreceptor assay)

1) reduced: AMYG (35%) 2) unchanged: CB, CX, HPC, HYPO, MED, OLF B, PONS SN, STR, THAL

(Note: IPSI and CONTRA were homogenized together for all areas except the AMYG, which was IPSI only. Nonsignificant reductions of 20% and 40% were observed in the STR and SN, respectively.) Receptor Binding (GABA-A and Benzodiazepine) Majority of Data Presented in Burnham (2) (See Text) Loscher and Schwark (23) Free University of Berlin

Rats (f, Wistar) amygdala kindled (daily) to 10+ Stage 5, 1 + week to sacririce

Washed membranes (centrifugation assay)

1) increased: STR (107%) 2) reduced: AMYG (63%), SN (45%)

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285

TABLE 2 CONTINUED Experimenters

Subjects

Preparation

Results

GABA Re-Uptake Chaudieu et al. (6)

Laboratory of Experimental Medicine, Montpellier

Rats (m, Sprague-Dawley) amygdala kindled (daily) to 14 or 37 stimulations, 8 or 75 days to sacrifice

Synaptosomes (assay of 3H-GABA uptake)

1) Increased: comb. HPC (10-20%), increase is in maximal uptake, not K m

GABA-T Activity Moiseey et al. (29) Pirogov Odessa Medical Institute

Mice, metrazol "kindled (daily) for 3 weeks, 24 hr to sacririce

brain sections (cytophotometric assay allowed separate study of neurons and glia)

1) reduced: CX (sensori-motor) (12%)

(Note: Reductions were observed in neurons but not glia.) Itagaki and Kimura ( I 1)

Shiga University of Medical Science

Rats (m, Sprague-Dawley) amygdala kindled (daily) to 5+ consecutive Stage 5, 7-21 days to sacrifice

whole-tissue homogenates after treatment with gabaculine (fluorometric enzymatic assay at 16 or 48 hours)

1) reduced: ipsi and contra AMYG, BST, CX, HPC, THAL, (reductions seen at 16 hours, when assay reflects activity in neurons)

(Note: no kindled/control differences seen in nongabaculine subjects or in subjects assayed 48 hours after gabaculine, when staining mostly reflects activity in glia and neuropil.) GABA-Stimulated Chloride Flux Burnham et al. (3)

University of Toronto

Rats, (m, Royal Victoria hooded) entorhinal kindled (daily) to 6 Stage 5, 24 hr or 28 days to sacrifice

synaptoneurosomes (scintilation counting of 36 chloride uptake)

1) unchanged (at either time period): CX, CB 2) reduced (at both times) BS (50%)

GABA Immunoreactivity Kamphuis et al. (14)

University of Amsterdam

Rats (m, Wistar) Schaefer collateral kindled (3/day) to 6+ Stage 5, 24 days to sacririce*

brain slices, stained with a GABA antiserum and studied via camera lucida

(*Note: a single Stage 5 seizure was administered the day before sacrifice.)

1) reduced: HPC (CA I). lpsi (35%). Contra nonsig, trend

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BURNHAM

TABLE 2 CONTINUED Experimenters

Subjects

Preparation

Results

Kamphuis et al. (15)

Rats (m, Wistar) Schaefer collateral kindled to 6, or 14 afterdischarges or 7-12 Stage 5 convulsions, 24 hr to sacrifice

brain slices, stained with a GABA antiserum and studied via camera lucida and light microscope

1) increased: HPC (CA 1). Ipsi: 38% rise after 14 AD's disappears after Stage 5 convulsions. Contra: 22% rise after Stage 5 convulsions

University of Amsterdam

Abbreviations: AMYG, amygdala; BS, brain stem; CB. cerebellum; CX, cortex; HPC, hippocampus; HYPO, hypothalmus; MED, medulla; OLF, olfactory; SN, substantia nigra; STR, striatum; TECT, tectum; THAL, thalamus.. Note: Material in book chapters and in abstracts has been omitted whenever the same material appears in journal articles. % to nearest 5%. negative results, only a few positive studies appeared during this period, i.e., reports that benzodiazepine binding was low in discrete areas (4), and that kindled animals showed a differential sensitivity to GABA-related ionophore blockers (13). Taken together, these largely negative data seemed to present a major problem for the GABA hypothesis.

changes which occur in the "synaptic pool." This explains why the early studies failed to find postkindling GABAergic changes, whereas the recent studies have found a wide variety. Taken together, the combined data seem to suggest that kindling has little or no effect on " m e t a b o l i c " GABA, but that it causes definite and long-lasting changes in " s y n a p t i c " GABA.

RECENT (POSITIVE) ASSAY DATA

RECEPTOR BINDING STUDIES: A CONTINUING PROBLEM

Starting in 1984, however, a second series of assay studies began to appear. These studies, summarized in Table 2, reported positive findings, suggesting the existence of postkindling abnormalities in a number of different GABAergic parameters. Among them are reports that GAD activity is decreased in certain areas of kindled brains (22, 23, 29); that GABA levels are selectively decreased (23); that GABA-T synthesis is abnormal (11); that GABA binding is decreased (23); that GABA-mediated chloride flux is decreased (3); and so forth. Taken together, these studies seem to indicate that the GABA system is profoundly changed by kindling, and that some of the changes are very long lasting. EARLY AND RECENT STUDIES COMPARED Why have the early and recent studies produced such different results? In a few cases, it seems possible that the early researchers were looking for changes at the wrong place or the wrong time. Studies limited to certain areas of the hippocampus, or to the first few hours after kindling, may miss important changes (2). In most cases, however, the difference between the early and the late studies seems to relate to the types of assay involved. Without exception, the later (positive) studies have used assays which concentrated on "synaptic" GABA. The earlier (negative) studies primarily concentrated on "whole tissue" GABA parameters. The importance of this difference relates to the fact that the GABA in the brain exists in two separate "'pools," one devoted to synaptic transmission and the other to oxidative metabolism. " M e t a b o l i c " GABA, which is found primarily in glia and cell bodies, constitutes at least half, and possibly as much as 90% of the GABA found in "whole tissue" brain homogenates (9,17). Because of this, assays which utilize whole tissue may well miss

While the synaptic/metabolic distinction explains a number of conflicts in the assay field, it does not explain the conflicts which continue to exist in the receptor-binding data. These data--which are by definition "'synapse s p e c i f i c " - - a r e not included in Tables l and 2, because they are very extensive, and because they have been the subject of a recent, full-scale review (2). (Note: Loscher and Schwark (23) has been included in Table 2 because it is not included in (2). The major results--and conflicts--may be summarized as follows: With regard to benzodiazepine binding, McNamara and his co-workers have done a large number of studies, using both homogenate and autoradiographic assays. Their final conclusion seems to be that benzodiazepine binding is normal in most areas of kindled brain, but transiently and bilaterally up-regulated (Bronx) in the fascia dentata of the hippocampal formation. These upregulations are seen at 24 hours but not at 28 days (35). Several other groups have offered similar, though slightly variant results. Tuff et al. for instance, have reported bilateral up-regulations (Bmax) in the amygdalae at two weeks, and bilateral downregulations (Bronx) in the striatum (37). These differences perhaps relate to differences in kindling site; the overall impression remains that binding is unchanged or up-regulated. Burnham's group, however, have reported a long-lasting (2 weeks, 2 months) down-regulation of benzodiazepine binding (Bma ~) in the cortex and hypothalamus, with no changes in amygdala, hippocampus or a variety of other areas (4,30). They have been able to relate this conflict in data to differences in homogenate preparation: upregulations are seen in the hippocampus and amygdala on/y when "washed membranes" are employed, and down-regulations are seen in the cortex and hypothalamus on/.,,, when "crude homogenate" is employed. It appears then, that the data obtained in

GABA AND KINDLING

287

benzodiazepine experiments depends on the homogenate used. Further research will be required to interpret these odd findings. [For references and full details of binding studies, see (2)]. The GABA-A receptor results have been equally complex. McNamara's group, using autoradiography, reported up-regulation in the fascia dentata and " n o change" in the cortex or substantia nigra. Like the benzodiazepine up-regulation, this change was seen at 24 hours but not at 28 days. Tuff et al., working in a homogenate assay, reported " n o change" in amygdala, cortex, hippocampus or striatum at 2 weeks, suggesting that McNamara's transient elevations were gone by that time (37). Loscher and Schwark, however, working at one week, have recently reported significant down-regulations in the amygdala and substantia nigra, and a very large and significant up-regulation in the striatum (23). None of these changes were observed in the previous studies. Conceivably, these conflicts, like the conflicts in the benzodiazepine-binding data, will eventually be related to technical differences. In summary, there is no general agreement in the receptorbinding field. Some experiments report transient and localized up-regulations which seem unlikely to relate to the "'kindled state," while others report down-regulations (some long lasting) which might well be involved. Since there is good reliability

within the work of various groups, it seems probable that technical differences between groups are crucial. At present, however, there is no firm conclusion to be drawn from the receptor assay studies. THE PRESENTSTATE OF THE GABA HYPOTHESIS No present conclusions can be drawn from the binding field-except that more work is needed. In a variety of other areas, however, recent "synapse specific" assays have revealed striking postkindling GABA-A complex abnormalities. As yet, no general pattern has emerged, but it is clear that the abnormalities are found in a number of brain areas, and that some of them are long lasting and, therefore, possible contributors to the kindled state. These recent studies reverse the early impression that the GABA complex is unchanged after kindling. In combination with the pharmacological data, they provide strong support for the GABA hypothesis, which must now be considered one of the strongest of the biochemical hypotheses of kindling. ACKNOWLEDGEMENTS The author would like to thank Dr. Georgia Cottrell for help in preparation of the manuscript.

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