absence of modification in gaba and benzodiazepine binding and in choline acetyltransferase activity...

Gen. Pharmac. Vol. 18, No. 6, pp. 593-597, t987 0306-3623/87 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1987 Pergamon Journals Ltd

ABSENCE OF MODIFICATION IN GABA AND BENZODIAZEPINE BINDING AND IN CHOLINE

ACETYLTRANSFERASE ACTIVITY IN BRAIN AREAS OF THE EPILEPTIC MUTANT MOUSE

TOTTERING

C. PSARROPOULOU ~, F. ANGELATOU 1, N. MATSOKIS 2, D. K. VERONIKIS l and G. KOSTOPOULOS L*

'Department of Physiology, and 2Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patras 26100, Greece

[Tel: (061) 992 389]

(Received 23 December 1986)

Abstract--1. In the tottering mutant mouse, which suffers from epilepsy and cerebellar ataxia, we examined whether possible changes in GABA, benzodiazepine receptors and choline acetyltransferase (CHAT) activity are implicated in the pathophysiology of these animals.

2. No alteration in GABA A and GABA B binding could be detected in cerebellar membranes of epileptic mice as compared to normal mice.

3. Benzodiazepine receptor density and affinity showed no statistical difference in cerebellar membranes of epileptic and normal mice.

4. The activity of CHAT determined in the cortices of epileptic and normal mice did not differ significantly between the two groups.

INTRODUCTION

The tottering mouse mutant presents several electro- encephalographic, behavioral and pharmacological similarities to human petit-mal epilepsy (Noebels and Sidman, 1979; Noebels, 1984). Homozygous (tg/tg) mice present an increased terminal arborization of the diffuse noradrenergic projections from locus coeruleus (Levitt and Noebles, 1981) which could conceivably lead to a diffuse cortical hyperexcitability (Waterhouse and Woodward, 1980), a probably necessary condition for the development of generalized epilepsy of this type (Kostopoulos and Gloor, 1982). Recent biochemical experiments, however, reveal other important defects in this mutant, i.e. decreased glycogenolytic response to norepinephrine (Magistrelli et al., 1985), reduction in total cerebellar ganglioside content (Seyfried et al., 1981) and decreased number of muscarinic acetylcholine receptors in cortex and hippocampus (Liles et al., 1986). It appears therefore possible that noradrenergic hyperinnervation is not the primary or the only synaptic defect resulting from the pathological gene. This possibility is also suggested by recent experiments (Phillips and Levitt, 1986) showing a considerable time lag between the completion of the noradrenergic hyperinnervation (14th days) and the appearance of epilepsy (3rd week). Alternatively, it may represent a pathophysiological adaptation, since noradrenergic neurons have remarkable ability for sprouting in response to neuronal loss (Cotman et al., 1981). We have therefore asked whether any of two other

*To whom all correspondence should be addressed.

G.P. 18/6--B

major transmitter systems using, respectively, acetylcholine and GABA are possibly involved in the pathophysiology of tottering mice.

Several lines of evidence implicate a disfunction of GABA mediated inhibition in epileptogenesis (Krnjevic, 1983; Ribak, 1983; Olsen et al., 1984). A decrease in GABA receptor binding has been described in monkey cortex made epileptic by alumina gel (Bakay and Harris, 1981) in human temporal lobe epilepsy (Lloyd et al., 1981), in seizure susceptible mice (Horton et al., 1982) and Mongolian gerbils (Olsen et al., 1984). However, sensitivity to GABA is not impaired in an animal model of generalized epilepsy with spike and wave discharges (Kostopoulos, 1986). It would therefore appear inter- esting to study GABA binding in the brain of the epileptic tottering mice (Noebels and Sidman, 1979). Related is the importance of studying benzodiazepine receptor binding in this mutant, since this binding, taking place at a receptor site modulating the GABA receptor-ionophone complex, correlates well with their clinical anticonvulsant efficacy and is impaired in several animal models of epilepsy (Olsen et al., 1984). In addition to the epileptic symptoms the tottering mouse suffers from severe ataxia of the cerebellar type (Noebels and Sidman, 1979). Other cerebellar mutants show a serious defect in cerebellar GABA (Matsokis and Valcana, 1985) and benzodiazepine binding (Lippa et al., 1978) which appears to correlate with the severity of their ataxia. Cerebellar tissue (Ce) was therefore chosen for our binding studies.

Impairment of cholinergic synapses has also been implicated in epileptogenesis (Maynert et al., 1975).

593

594 C. PSARROPOULOU et al.

Exper iments with ano the r animal model have indicated tha t reduced activity of a cholinergic ascending reticular ac t ivat ion might set the permissive state for deve lopment of generalized epilepsy with spike and wave discharges ( G o o b e r m a n and Gloor , 1974; Kos topoulos and Gloor , 1982). Since muta t ions result ing in defective choline acetyl transferase (CHAT) have been described (Rand and Russell, 1984), we decided to compare the activity of this enzyme in affected and normal mice.

MATERIALS AND METHODS

The animals used were epileptic (tg/tg) and normal ( t g / + , + / + ) of the strain C57BL/6J, obtained from Jack- son Laboratories, Boston. In each experiment we compared epileptic to normal brains using three months old littermates of the same sex. For the determination of [3H]GABA and [3H]Flunitrazepam ([3H]FIu) binding the cerebella (Ce) of epileptic (E) and normal (N) animals were used.

To determine the GABA A and GABA B binding, the synaptosomal membrane fraction was prepared as described by Zukin et al. (1974) and Toffano et al. (1978). The tissue was homogenized in ice-cold 0.32 M sucrose. The homoge- nate was centrifuged at 1000 g for 20 min. The nuclear pellet was discarded and the supernatant was centrifuged at 48,000g for 20 min. The pellet was resuspended in 20 vol of distilled H20, homogenized and centrifuged at 48,000g for 20 min. The pellet was frozen at -20°C for 12-18 hr. The pellet was dispersed again and centrifuged at 8000g for I0 min. The mitochondria free supernatant of this step was centrifuged again at 48,000 g for 20 min. The pellet after this centrifugation was resuspended in 50 vol 50 mM Tris-HC1 pH 7.4 including 2.5 mM CaC12. The resulting membrane preparation (crude synaptosomal membranes) was used for GABAu binding. For further purification, the crude synaptosomal membranes which were kept at -20°C for 12-18hr, were thawed, suspended in 50vol 50mM Tris-citrate buffer (pH 7.1) containing 0.02% Triton X-100 and incubated at 37°C for 30 min. At the end of the incubation the preparations were centrifuged twice at 48,000g for 20 min, the pellets were dispersed in 50 vol 50 mM Tris-citrate buffer pH 7.1. The resulting membrane preparation was used for GABA A binding.

GABA A binding was determined according to the method of Toffano et al. (1978). Samples of membranes treated by Tr i ton X-100 containing 100-200/~g protein, were incubated at 4°C for 15 min in a final volume of 1 ml at 50 mM Tris-citrate buffer pH 7.1 in the presence of 20nM [3H]GABA.

GABA B binding was determined according to method of Hill and Bowvery (1981). Samples of crude synaptosomal membranes containing 100-50 # g protein, were incubated at 25°C for 20 min in a final volume 1 ml at 50 mM Tris-HCI buffer pH 7.4 in presence of 5 mM CaC12, 10/zm iso- guvacine and 100 nM [3H]GABA. To determine the non- specific binding of [3H]GABA binding, parallel incubations were carried out in the presence at [3H]GABA and 1000 M excess of non-radioactive GABA. At the end of incubation period the assay tubes were centrifuged at 48,000g for 30min at 4°C. The supernatant was removed and the radioactivity of the pellet was determined with a liquid scintillation counter (LS-7000, Beckman) using the follow- ing mixture of scintillation fluid: 60 g napthanol, 2 g PPO, 0.35 g POPOP, 100 ml ethylene-glycol, 100 ml methanol and dioxan to a final volume of 1 litre.

[3H]Flunitrazepam ([3H]Flu] binding was determined according to the methods of M6hler and Okada (1978) and Speth et al. (1978). Samples of crude synaptosomal membranes containing 90-200 # g protein, were incubated at 4°C for 30 min in a final volume of 1 ml of 50 mM Tris--eitrate buffer, pH7.2 and various concentrations of [3H]FIu

(0.1-6 nM) for the determination of total [3H]FIu binding. To determine the non-specific binding at each concentration of [3H]Flu, parallel incubations were carried out in the presence of [3H]FIu and 1 mM excess of clonazepam (La Roche). The incubation was terminated by filtration through a Whatman GF/C filter, under reduced pressure. The filter was washed twice with 5 ml, 50 mM Tris--citrate, pH 7.2 and the radioactivity of the filter papers was determined as described above. Specific binding was analyzed according to Scatchard (1949).

For the determination of ChAT activity the cerebral cortex of epileptic and normal animal was used. ChAT activity expressed in nmol of acetylcholine (ACh) formed/mg protein/hr was determined in the extract of cerebral cortex according to Fonnum (1975a). Homogenates of brain cortex were incubated in triplicate for 15 min at 37°C is sodium phosphate buffer (pH 7.4) containing 8 mM choline bromide, 0.2mM [3H]acetyl-CoA and 0.1mM eserine sulfate. The [3H]acetyl-CoA (0.55Ci/mmol from Amersham) was diluted 1 : 10 with the unlabeled compound. The reaction was terminated by extraction of ACh into an organic phase containing scintillation fluid. For the determination of the kinetic constants choline bromide was used in 12 concentrations ranging from 0.2 to 8 mM, and the [3H]acetyl-CoA at a concentration of 0.2 mM.

Statistical analysis was carried Out by the Student's t-test. The protein concentration was determined by the method of Lowry et al. (1951).

RESULTS

The G A B A binding in cerebellar m e m b r a n e s to indentify the GABAA and GABAB binding sites is shown in Table 1. The GABAA binding in epileptic Ce in the presence of 20 n M [3H]GABA does not differ significantly f rom the normal . Sca tchard analysis for GABAA binding in a pre l iminary exper iment showed two b inding componen t s with h igh and low affinity, respectively, and K d and Bm~ values which did no t differ significantly in no rma l and epileptic Ce (data no t shown here). The difference in GABAB binding between normal and epileptic Ce was no t statistically significant ei ther (Table 1). Sca tchard analysis of [3H]GABA binding to crude cerebellar synaptic membranes , to indentify G A B A e b inding sites, showed only one c o m p o n e n t with similar Kd and Bm~ values in normal and epileptic Ce (prel iminary experiment , da ta no t shown here).

Figure 1 presents the kinetic analysis o f [3H]FIu b inding (range 0 . 1 - 6 n M ) , in the crude m e m b r a n e f ract ion of normal and epileptic mouse Ce. The kinetic parameters as de termined by Sca tchard analysis (Table 2) show tha t there is no significant difference either in the affinity [Kd = 1.88 + 0.06 (E)

Table 1. Specific [3H]GABA binding to crude synaptosomal membranes from cerebellum of normal

and epileptic mice

GABA^* GABAst (fmols/mg) protein

Normal 306 _+ 742~ (3) 240 + 56 (3) Epileptic 283 _+ 63 (3) 317 + 74 (3)

*The concentration of [3H]GABA in incubation mixture was 20 nM.

tThe concentration of [3H]GABA in incubation mixture was I00 nM.

:~Values arc m e a n + SEM of the experiments indicated in parentheses.

Epileptic mutant mouse tottering

4 * Normot l ~ e o EpiLeptic ~e

o

o °

° \ I I I " . \ .5

0 200 400 600 ~0

Bound ( f m o t e s / m g prote in) ' - I x T_ 4

Fig, I. Scatchard analysis of [3H]Flu binding to cerebellar membranes of epileptic (O) and normal mice (O). The ~. results are derived from one experiment and are representative of all three similar experiments conducted. Correlation ~ 3 factor was r =0.99 for both plots. Normal Ce had " Ka = 1.8 nM and Bm~ = 735 fmol/mg protein. Epileptic Ce ~ I

had K d = 1.9 nM and Br~x = 729 fmol/mg protein. ~ 2 Q 0

and K d -- 1.97 +_ 0.07 (hi)] or in the number of binding E sites [Bma x = 713 + 79 fmol/mg protein (E) and - 1 Bma x = 708 + 54 (N)] between epileptic and normal -T> mouse Ce. These results expressed as specific binding - j per total tissue or per mg of wet tissue are shown in /

Table 2. The protein content in normal and epileptic o Ce was the same and the total binding per mg wet tissue was the same as well.

The time course of ACh synthesis [Fig. 2(A)] shows that the reaction reaches an equilibrium after 40 min of incubation. According to this curve, we used in our experiments an incubation time of 15 min (when the rate of ACh synthesis is linear) in order to compare ChAT activity in epileptic and normal mouse. The ChAT activity determined at 8 mM of choline bromide in cerebral cortex of epileptic mouse showed no significant difference compared to the normal (Table 3). In order to exclude the possibility of any alteration in the enzyme concentration (Vm~) or substrate affinity (Kin) in the epileptic cortex, that could not be detected by estimation of ChAT activity at 8 mM of choline bromide we determined the kinetic characteristics of the enzyme by testing the effects of different concentrations on the enzyme activity.

The values of V~, and K~ were estimated from these assays according to Lineweaver and Burk analysis (1934). As can be seen in Fig. 2(B) and Table 3, neither Vm~ nor K~ showed any significant difference between epileptic and normal animals. These results confirmed the first estimation of ChAT activity in the cortices. Preliminary experiments (n = 2) in hippocampus additionally showed that the

/ e / e

II/

I I I I 20 40 60 80

T (rain)

595

.oE /

B / / / _ e N

I I I I I 1 2 3 4 5

( ~ ) (raM) - t

Fig. 2. (A) The time course of acetylcholine synthesis in normal mice cerebral cortex expressed in nmol of acetyb choline formed/mg of protein. (B) Double reciprocal plots comparing, according to Lineweaver and Burk 0934), ChAT activity in cerebral cortex of epileptic (O) and normal (O) mouse. The results are derived from one experiment and are representative of all four similar experiments conducted. The plots were fitted to a straight line by the method of least squares. Correlation factor r = 0.99 for both plots. Epileptic cortex had /~,= 1.8mM, Vm~= 228.18nmol/mg protein/hr. Normal cortex had

K, -- 1.66 mM and V ~ = 235.6 nmol/mg protein/hr.

ChAT activity in epileptic (89.78 +_ 0.66 nmol/mg protein/hr) does not differ significantly from the normal (101.19 _+ 8.89 nmol/mg protein/hr).

DISCUSSION

The hypothesis tested in this study was that the tottering mutation resulted primarily in a defect in

• the G A B A receptor-ionophone complex. Such a defect--if generalized in the brain could have pre- sumably contributed to both major symptoms char- acterizing the tottering mutant; generalized epilepsy

Table 2. Binding constants and maximal binding concentration of [3H]Fiu binding to crude cerebellar m e m b r a n e s and protein content of normal and epileptic mice cerebellum

Kd Bin, (fmol/mg Total protein per fmoi/mg (riM) protein) C,e (mg) pmol/Ce wet tissue

Normal 1.97 + 0.07* 708 + 54 5.38 + 0.41 3.811 ± 293 54 ± I Epileptic 1.88 + 0.06 713 ± 79 5.36 ± 0.60 3.821 ± 419 53 ± 6

*Values are mean + SEM of three parallel experiments.

596 C. PSARROPOULOU et al.

Table 3. Choline acetyltransferase activity and its kinetic parameters in cerebral cortex of normal and epileptic mice

ChAT activity* K m Vma x Normal 147.99 _+ 13.7 (9) 1.67 + 0.93 (3) 171.06 + 27.87 (4) Epileptic 158.36 + 13.2 (10) 1.66 + 0.11 (3) 179.5 __. 32.2 (4)

Units are: ChAT activity = nmol acetylcholine/mg protein/hr; K m = mM; Vma x = nmol acetylcholine/mg protein/hr. Protein was determined according to Lowry et al. (1951). Values are mean +SEM of the number of animals indicated in parentheses.

*Determined at 8 mM choline bromide.

resulting from a disinhibition induced hyperexcitability in cortex and ataxia resulting from a disturbance of GABAergic transmission in Ce. Our data demonstrating a non-impaired binding capacity to both GABAA and GABAB and benzodiazepine receptors indicate that the pathophysiology of tottering mutation involves primarily a mechanism other than an impairment of GABA receptors. The possibility that GABA receptors are affected specifically in brain regions other than the Ce cannot be ruled out. However, the severity of ataxia in this mutant sug- gests that the genetic defect is not sparing Ce. The GABAergic disinhibition hypothesis is certainly attainable in several models of epilepsy (Krnjevic, 1983; Ribak, 1983; Olsen et al., 1984). However, generalized epilepsy with spike and wave discharges apparently develops in the tottering mouse as well as other epileptic models (Kostopoulos, 1986) by some neuronal mechanisms which demand an intact sensitivity to GABA. GABA administration enhances spike and wave discharges (Farriello and Ticku, 1983; Vergnes et al., 1984) and GABA release is actually increased during conditions which promote generalized epilepsy of the petit-mal type, i.e. somnolence and EEG synchronization (Jasper et al., 1965). It is therefore tempting to speculate that petit-mal type epilepsy may not be consistent with a decreased GABA mediated activity (Myslobodsky, 1984; Kostopoulos, 1986).

The K m value of ChAT activity determined in cortices of the normal animals (1.6 mM) is higher than the one determined by Ryan and McClure (1980) for rat brain (1.0 mM). Differences in species and assay methods used could possibly account for this discrepancy. Liles et al. (1986) demonstrated a decrease in muscarinic receptor density in adult tg/tg mice. They further suggested that this decrease is not the cause but the result of sustained depolarization of hippocampal and cortical neurons, underlying epilepsy in this mutant. Alternatively an increased pro- duction of acetylcholine, due to an increased ChAT activity, could have produced such strong depolarization leading to epilepsy (Benardo and Prince, 1982) as well as to a down regulation of muscarinic receptors. On the other hand, the proliferation of noradrenergic terminals (Levitt and Noebels, 1981) could conceivably be an adaptational response (Cotman et al., (1981) to a lack of cholinergic terminals in the mutant's cortex. The present study demonstrating normal ChAT activity--the best indi- cator of functioning cholinergic terminals (Fonnum, 1975b)--in epileptic mice tends to exclude both the above alternative mechanisms among the ones possibly underlying epileptogenesis in tottering mice. The question of cholinergic involvement in this mutant 's

epileptogenesis remains unresolved. Comparative studies of the tottering brain's ability to liberate acetylcholine as well as its cholinesterase activity might help in this direction.

In conclusion, our studies have failed to implicate the examined aspects of cholinergic and GABAergic transmission in the pathophysiology of the tottering mutation. Thus, indirectly they support the hypothesis (Noebels and Sidman, 1979) that the noradrenergic hyperinnervation of both cortex and Ce may be at the cause of, respectively, generalized epilepsy and ataxia in this mutant. The data on GABA binding are in accord with recent attempts in emphasizing the differences in the neuronal mechanisms underlying models of generalized epilepsy of the petit-mal type as opposed to other types of epilepsy (Kostopoulos and Gloor, 1982; Farriello and Ticku, 1983; Myslobodsky, 1984; Kostopoulos, 1986).

Acknowledgements--C.P. is a fellow of the State Fellowship Foundation (I.K.Y.). The study was supported by a grant from the Ministry of Research and Technology to G. K. Last but not least we would like to thank Mrs C. Psilou for her valuable secretarial assistance.

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absence of modification in gaba and benzodiazepine binding and in choline acetyltransferase activity...

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