Journal of Neurochenliarry. 1979. Vol. 32. pp. 743 to 753. Pergamon Press. Printed in Great Britain.

METABOLITE LEVELS I N BRAIN FOLLOWING EXPERIMENTAL SEIZURES: THE EFFECTS OF MAXIMAL ELECTROSHOCK A N D PHENYTOIN I N CEREBELLAR LAYERS D. W. MCCANDLESS,'G. K. FEUSSNER, W. D. LUST and J. V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20014, U.S.A. (Received 5 June 1978. Accepted 11 September 1978)

Abstract-The effects of max~malelectroshock (MES) and phenytoin on metabolites and cyclic nucleotides in layers of frozen-dried cerebellum have been investigated. The four layers (molecular, Purkinjecell rich, granular and white matter) had remarkably homogeneous distributions of P-creatine, ATP. glucose, glycogen, lactate, GABA and the cyclic nucleotides. MES caused dramatic decreases in P-creatine, ATP, and glucose at 10s after treatment, followed by a decrease in glycogen at 30s. Lactate levels were elevated, and GABA was unchanged. Cyclic AMP concentrations were increased at 10s and cyclic GMP at 30s. Phenytoin modified most of the MES induced changes in all the layers, although white matter was less affected by MES and/or phenytoin. Lactate concentrations were increased by MES and these effects were not altered when phenytoin was administered. The most dramatic effects of phenytoin were on the changes in cyclic nucleotides. Cyclic AMP concentrations were elevated after MES but the values returned to normal more rapidly when phenytoin was present. The drug almost obliterated the MES induced changes in cyclic GMP. The possible relationship of cyclic nucleotide concentrations and the modulation of seizure activity is discussed.

THEMETABOLIC events in the brain associated with experimental seizures have recently been investigated in our laboratory following various experimental paradigms (LUST& PASSONNEAU, 1976; MCCANDLESS et al., 1978; LUST et al., 1976; LUST et al., 1978). In the present study, maximal electroshock (MES) has been used t o induce seizures in mice. The stimulus is undoubtedly excessive, and does not exactly mimic a n epileptic seizure state. However, the nature and characteristics of the response are highly reproducible and thus afford a useful model for the study of the effects of seizure on the brain. MES results in a massive depolarization in the brain concomitant with neuronal discharge and a tonic-clonic seizure. It has been established in other studies that MES induced neuronal discharge is associated with a rapid increase in the use of energy metabolites (KING et al., 1973; FERRENDELLI & MCDOUGAL, 197 I ; COLLINSet al., 1970; HOWSE& DUFFY, 1975). The expenditure of ATP, P-creatine, glucose and glycogen occurs more rapidly than they can be replenished, resulting in a progressive decrease in net levels. Recent studies from this and other laboratories have examined the effects of M E S and anticonvulsants on energy metabolism and cyclic nucleotide concentrations in the cerebral cortex and cerebellum Present address: Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U.S.A. Abbreuiarjm used: MES, maximal elcctroshock.

of mice (LUSTef al., 1976; LUSTet al., 1978). Results indicate that there is a decrease in high energy phosphate compounds in both cortex and cerebellum following MES, and cyclic nucleotides are increased u p t o 5-fold in both brain regions. In the presence of phenytoin, the effects of MES o n metabolites and cyclic nucleotides are modified to a greater extent in the cerebellum than in the cortex. These observations support the concept that the cerebellum may play an important role in convulsive activity. Since the cerebellum is a layered structure, with each layer containing different cell types. analyses of discrete layers might provide evidence for a more specific locus of action of phenytoin. Accordingly, we have measured energy reserves and cyclic nucleotides in the molecular, Purkinje cell-rich, granular and white layers in the cerebellum foIlowing MES in untreated and phenytoin treated mice. MATERIALS AND METHODS Male NIH general purpose mice weighing 25-27 g were starved overnight before the experimental treatment. Maximal electroshock was administered through corneal electrodes at an intensity of 50mA for 0.2 s. Immediately following electroshock, and 10. 30, 60, and 600s later the mice were fixed with vigorous stirring in liquid N2. In another series of mice, the anticonvulsant phenytoin was administered (25 mg/kg i.p.) 25 min prior to the electroshock. Animals were killcd at the same time interyals as those without phenytoin. Control mice were either not injected, or injected with saline, and otherwisc trcated the same as the experimental mice.

143

1 pl 0.02 N-NaOH, 30min at 60°C.

1 pl 0.1 MNaOH, 30 min at 60°C

Lactate

ATP

5 pl 50 mM-Tris-HCl pH 7.5, 1 m m MgCI,, 0.5 mM-DTT, 0.1 mMglucose, 0.02% BSA 20 p ~ - T P N ' , 2 pg/ml HK, 0.4 pg/ml G-6-PDH.

5 pl 100 mM 2-amine2 methylpropanol pH 9.9, 50 ~ M - D P N 0.02% + BSA, 0.5 mwglutamate, 100 pg/ml LDH, SOpg/ml GOT, 20min

1 pl 0.02 N-NaOH, 30min at 95°C.

Glycogen

.

1 y1 100 mM-Tris-acetate pH 4.5, 3 pg/ml AG, 0.04% BSA, 30 min Then 1 p1 of 100rnMTris-HCI pH 8.8, 15 mM-MgC1, 0.1 mMDTT, 5 0 0 ATP, ~ ~ 100 pm TPN, 5 pg/ml HK, 0.5 pg/ml G6PDH, 20 rnin.

1 p1 0.02 N-HCI, 20min at 60°C.

5 pl 50 mM-Tris-HC1 pH 8.0, 1 mM-MgCl,, 100 pm ATP, 5 prn TPN', 0.02% BSA, I pg/rnl HK, 0.05 pg/ml G6PDH, 20 min

Step 2

Glucose

Step 1

3 pl 0.4 x-NaOH, 30min at 60°C.

Entire sample to 200 pl TPN cycling reagent, 25 pg/ml GDH, 0.8 pg/ rnl G6PDH. 1 h 3 8 T , 2 min at 100°C.

2 pl aliquot, loop1 D P N cycling reagent, 100 pg/ml GDH, 6pg/ml LDH, 1 h at 3 8 T , 2min at 100°C.

2 pI aliquot, 100 pl TPN cycling reagent, 20 pg/ml GDH, 2 pg/rnl G6PDH, 1 h at 3 8 T , 2min at 100.

2 0.15 NNaOH, 20 min at 80"

5 0.2 NNaOH 30min at 60°C.

5 pI aliquot, 100 pl TPN cycling reagent, 5 pg/ml GDH, 1.5 pg/ml G6PDH 1 h at 38°C 2min at 100°C.

Step 4

5p1 0.1 NNaOH 20min at 60°C

Step 3

TABLE1. ANALYTICAL PROCEDURES

1 ml indicator Reagent I, 30 min.

1 ml indicator reagent 11, 20 min, 5 pg/ml G D H in glycerol, 30 min.

1 ml indicator reagent I, 30min.

1 ml indicator reagent I, 30min.

Step 5

.E

P

5 p1 100 mhl-2-amino2-methylpropanol, pH 9.9, 5 mM a-ketoglutarate, 0.1 mM-DTT, 10 p ~ - T P N 2, mM-EGTA, 100 pg/ml GABASE equivalent to 0.06 U/ml, 30 min.

1 0.02 N NaOH, 20min. 60°C.

GABA

5 pl 0.2 N-NaOH, 30min at 60°C.

3 pl 0.4N-NaOH, 30min at 60°C

Entire sample to 200

2 or 4 p1 aliquot, 100 pI TPN cycling reagent, 20 pcglml GDH, 8 pg/ml G-6-PDH

reagent, as for ATP.

pl TPN+ cycling

1 ml indicator reagent I, 30min.

1 ml indicator reagent I, 30 min.

The abbreviations used are as follows: DTT, dithiothreitol; BSA, bovine serum albumin; HK, hexokinase; G6PDH, glucose-6-P-dehydrogenase, GDH, glutamate dehydrogenase; CK, creatine kinase; EGTA, ethyleneglycol-bis (p-amino ethyl ether)-N,N'-tetracetic acid; GABASE, GABA transaminase and succinic semialdehyde dehydrogenase; AG, amyloa-1,4-cc-1,6-glucosidase; GOT, glutamate-oxalacetate transaminase; LDH, lactate dehydrogenase. The TPN cycling reagent contains 100 m~-Tris-HCl pH 8.0, 5 mM a-ketoglutarate. 1 mM-glucose 6-P, 100 pm ADP, 10 mM-ammonium acetate. The D P N cycling reagent contains 100 m~-Tris-HcIpH 8.4, 100 mM-sodium lactate, 300 WM-ADP,5 m~-a-ketogIutarate,2 mM-ammonium acetate. Indicator reagent 1 contains 40 m~-Tris-HCl pH 8.0, 100 PM-EDTA,30 mM-ammonium acetate, 5 m~-MgCl,,50 ~ M - T P N +1 ,pg/ml 6-P-gluconate dehydrogenase. Indicator reagent I1 contains 100 m~-Tris-HCl p H 8.4, 1 mM-DPN+, 100 p ~ - A D P9, mM-H,O,. The ATP was removed from the ADP used in the P-creatine assay according to the method of LOWRY& PASSONNEAU (1972) to minimize the blank. All incubations are at room temperature unless otherwise noted. The final step is read in a ratio Ruorometer. Step 1 serves to destroy native tissue enzymes and substances which might interfere with the specific reaction in Step 2. In the case of glycogen, the high temperature is used to destroy glucose (LOWRYrt al., 1977). Step 3 is required to destroy the substrate levels of T P N + (DPN +) leaving the TPNH (DPNH) which is formed stoichiometrically with the metabolite measured. Step 4 is the amplification procedure used to produce 6-P-gluconate or glutamate in concentrations approx 2000-fold greater than the reduced pyridine nucleotide formed in the specific reaction. Appropriate blanks and standard are carried through the entire procedure.

3 pI from ATP step 2. I pI 0.125 N-HCI, 10 min, 4 p1 200 mM-TrisHC1 pH 8.6, 80 p~ ADP, 0.02%. BSA, 4 pg/ ml HK, 0.8 pglml G6PDH. 100pg/ml CK

Same sample as ATP

P-Creatine

g.

0

g2

2.

Y,

B

U CT

Bn

r

K 8

2

e

I

z

746

D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU

Following sacrifice, mice were stored at -60°C. Subsequently. the dorsal cerebellar vermis corresponding to areas 6 and 7 after LARSELL (1952) were dissected in a cryostat at -25°C. The following procedures are described (1972). Briefly, the in detail by LQWRY & PASSONNEAU, small brain pieces were mounted in wooden holders, and sectioned in an International LT-1 cryostat at -20°C. The resultant 20 pm sections were transferred to pre-chilled specially designed aluminum holders. These holders were placed in glass vacuum tubes (Ace Glass Co., Vineland, NJ) and dried under vacuum, 0.01 mm Hg, for a minimum of 24 h at -45°C. After freeze-drying, the scctions were stored under vacuum at -60°C. When ready for assay, sections were brought to room temperature under vacuum in the glass tubes. Sections were then removed from the holders and dissected free-hand into molecular, Purkinje cell-rich, granular, and white layers. Dissections were done in a room with relative humidity maintained at 50% or less. Stained sections from the samqmaterial served to aid in identification of the regions. Dissection was accomplished using specially constructed microscalpels, and visualized through a stereo dissecting microscope. Following separation of sections into the 4 regions, samples (0.2-1 p g ) were weighed on a quartz fibre fish-pole balance, and transferred to oil well racks. Samples were pushed through the oil with hair points, until contact was made with the droplet (1 pl) of the appropriate reagent of the first assay step. The assay conditions for the individual metabolite determinations are outlined in Table 1. Technical details are (1972). For cyclic nudescribed in LOWRY& PASSONNEAU cleotide analysis, the dried sections (10-20 p g of pooled samples of the discrete layers) were extracted in 50p1 of 0.025 N-HCl, and heated at 60°C for 10 min. The extracts were neutralized with 20 pl of 0.125 ~-Tris-HCl buffer, pH 8.6, and were acetylated according to the method of & BROOKER (1975). The cyclic nucleotides were HARPER then measured by radioimmunoassay as described by STEINER et al. (1972). Extracts prepared with HCI and with O.~N-PCAwere compared for cyclic nucleotide concentrations. The ratios for HCI vs PCA for cyclic AMP in cerebellum and cortex were 1.05 and 1.02 respectively while those for cyclic G M P were 1.10 and 0.89. Substrates and cofactors wete purchased from Sigma Chemical Co. (St. Louis, MO), enzymes from Boehringer Mannheim (Indianapolis, IN) and materials for the cyclic nucleotide assays from Schwarz-Mann (Orangeburg, .NY). Significance was calculated using Student’s t-test (STUDENT, 1907). RESULTS The reproducible response of the mice t o MES consisted of a 1.6s latency and initial tonic flexion, followed by a 13 s tonic extension. Subsequently, there occurred a 7.6s intermittent clonus, followed by a postictal depressive period. In mice pretreated with the anticonvulsant phenytoin, the response t o M E S was modified so that the tonic extension was prevented, and was replaced temporally by a pronounced bilateral clonic seizure. I n a preliminary series of assays, ATP, P-creatine and GABA were measured in extracts of frozen cerebellar vermis, in frozen-dried sections of the undis-

sected vermis, and in frozen-dried sections of cerebellar layers to determine the reliability of the preparative techniques for these labile metabolites (Table 2). The values for whole vermis and for frozen-dried cross-sections are expressed as nmol/mg protein, whereas the microsection data is expressed as nmol/mg fat-free dry wt. T h c non-protein component of the fat-free tissue sections is minor consisting primarily of glycogen, which is about 0.5% of the dry wt. The values as given are thus nearly equivalent but would tend to be lower. While there is some variability, generally the levels of metabolites are preserved during the various procedures. The control values for the metabolites and cyclic nucleotides for the 4 layers examined are given in Table 3, with the calculated values for concentration based on lipid-free dry weight given in parentheses. Although the layers appear different in dissection and obviously contain different cell types, the distribution of metabolites is buit,e homogcncous (see Discussion). After the administration of phenytoin alone, P-creatine was significantly elevated in the granular and white layers of the cercbellum (Fig. 1,C). It has been shown that phenytoin depresses cerebral rneta& NELSON, bolism a t a dose of 250mg/kg (BRODDLE 1968). Whether there is a metabolic effect of phenytoin to account for elevated P-creatine a t the present dose is not clear. P-creatine levels decreased maximally a t 10s after M E S in all layers. The changes were similar in the molecular, Purkinje cell-rich, and granular layers and were less than 20% of control values, while the decrease in white matter was only to 70% of control. The recovery t o normal levels or greater occurred by 60 s in all layers except the white matter. In the presence of phenytoin, the P-creatine was still significantly depressed by M E S when compared with unshocked animals; however, the changes were significantly less than in nontreated animals. P-creatine was also restored by 60s after MES in the presence of the drug. TABLE2. THECONCENTRATIONS

OF LABILE METABOLITES IN

FRESH TISSUE A N D FROZEN-DRIED SECTIONS

ATP

Whole vermis Frozen dried sections of whole vermis

Microsections Molecular (0.54) Purkinje cell-rich (0.56) Granular (0.59) White (0.35)

P-creatine GABA

28.1

(nmol/mg protein) 35.0 36.6

12.9 17.3

25.8 21.5 19.2 21.5

(nmol/mg fat-free dry weight) 37.2 33.0 24.4 13.3

18.8 14.1 9.4 16.9

25.1

The tissues were prepared and analyzed as described in Materials and Methods. The values for lipid-free dry weight are given in parentheses for each cerebellar layer et al., 1977). (BERGER

Maximal electroshock and phenytoin effects on cerebellar layers

747

1 P-CREATINE I

SECONDS AFTER MES

SECONDS AFTER

MES

FIG. I. The concentrations of P-creatine in 4 layers of the cerebellar cortex following MES. Each point represents the mean value k S.E.M. for measurements made on 8-18 sections from 3 different animals, a total of 36 animals. C = control animals, untreated or saline injected; the experimental animals were treated as described in Materials and Methods. Filled symbols indicate values of the electroshocked mice, treated and untreated, which are significantly different from those of control and the asterisks (*) represent values of phenytoin-treated animals significantly diffcrcnt from untreated animals ( P < 0.05).

The decrease in ATP elicited by MES was maximal at 10s, and was greater in the molecular, Purkinje cell-rich and granular layers than in the white matter, similar to the results with P-creatine (Fig. 2). ATP was restored to normal values by 1 m except in the granular layer. In the presence of phenytoin, the fall TABLE3. THE CONCENTRATIONS Molecular layer

in ATP concentrations after MES was less in all layers, although the effect was only significant in the granular layer at 10s. The decrease in the white layer was only evident at 60 and 600 s. With that exception, ATP was restored in the 3 other layers at the same rate as in the absence of the drug.

OF METABOLITES IN FOUR CEREBELLAR LAYERS OF CONTROL MICE

Purkinje cell-rich layer

Granular layer

White matter

~

P-creatine

ATP Glycogen Glucose Lactate GABA Cyclic A M P Cyclic G M P

18.9 k 1.1 (34.9) 12.6 k 1.1 (23.3) 12.5 k 0.81 (23.0) 5.05 k 0.37 (9.3) 11.7 ? 0.75 (21.6) 7.97 k 1.6 (14.7) 3.32 f 0.40 (6.14) 3.37 & 0.86 (6.24)

* P < 0.05 different from other

(nmol/mg dry wt) 17.8 f 0.62 (31.8) 10.8 1.2 (19.3) 12.5 f 1.2 (22.4) 4.14 0.49 (7.4) 13.1 1.2 (23.5) 6.60 0.4 (11.8) (pmol/mg dry

-

15.5 0.9t (26.2) 11.3 k 0.5 (19.1) 12.6 f 0.8 (21.3) 5.14 f 0.85 (8.7) 12.1 k 1.0 (20.6) 5.55 k 0.311 (9.4) wt) 3.56 & 0.42 (6.03) 2.10 f 0.39 (3.56)

7.10 k 1.4* (20.3) 7.66 0.62* (21.9) 12.1 f 1.1 (34.7) 4.78 & 0.71 (13.6) 12.3 f 1.6 (35.2) 5.94 k 0.37 ( I 7.0)

+

3.64 f 0.42 (10.4) 2.06 i 0.56 (5.88)

3 layers. molecular and Purkinje cell-rich layers. Purkinje cell-rich layer. The tissues were prepared and analyzed as described in Materials and Methods. The valucs represent the mean f S.E.M. for 8-18 samples from 3 mice. Values given in parentheses arc calculated for lipid-free weight (BEKGEK ef a/., 1977).

t P i0.05 different from 1P < 0.05 different from

748

D. W. MCCANDLESS, G . K. FEUSSNER, W. D. L ~ J Sand T J. V. PASSONNEAU

MOLECULAR LAYER I

I

I

n

PURKINJE CELL LAYER 1

GRANULAR LAYER 1

I

0

30

1

I

I

1

. "

1

-

1

WHITE LAYER

-

1

1

I

0

C

60 ' 600 SECONDS AFTER MES

30 60 SECONDS AFTER

1

60(

"

MES

FIG.2. The concentrations of ATP in 4 layers of the cerebellar cortex following MES. The determinations were made on the same tissue section as P-creatine. Abbreviations and symbols are as for Fig. 1.

Glucose concentrations were depressed in all layers of the cerebellum 10 and 30s subsequent to MES (Fig. 3). When phenytoin was administered prior to the stimulation, there were no significant changes in glucose during the convulsive period. However, in both groups of animals, glucose was significantly elevated 10min after MES.

There were small but significant decreases in glycogen in all cerebellar layers, which were not evident until 30s following MES (Fig. 4). Glycogen concentrations returned to control values by 600s in all regions except the white matter. In the presence of phenytoin, there were no decreases in glycogen concentrations, and rather surprisingly there were signifi-

[GLUCOSE I

MOLECULAR LAYER

I I

PLRKINJE CELL LAYER

,a,,''

-

I

t 1I I

1

1

0

30

1

-

I

1

I

1

60"600

SECONDS AFTER MES

I

1

1

I

C

0

30

1

-

1

1

..

1

-

6 0 6 O c

SECONDS AFTER MES

FIG. 3. The concentrations of glucose in 4 layers of the cerebellar cortex following MES. Each point represents the mean f S.E.M. for measurements made on 9-21 sections from the same animals as Fig. 1. Abbreviations and symbols are as for Fig. 1.

Maximal electroshock and phenytoin. effects on cerebellar layers

749

(GLYCOGEN]

GRANULAR LAYER 1

I

I

WHITE LAYER

-

1

cant increases in the molecular and Purkinje cell-rich layers at 30s. The reason for such increases could not be accounted for in the present investigation. Lactate concentrations increased in all layers following MES to peak values at 30s (Fig. 5). Lactate remained significantly elevated over control values for at least 60 s in all layers except the Purkinje cell-rich layer, and for 600 s in the molecular and white layers.

I

1

I

.

1

.

In this case, the changes were as great in white matter as in the other layers. The administration of phenytoin had no significant effect on the MES-induced increase in lactate except in the white matter, and only in animals frozen immediately after the shock. In both the granular and white layers, the return to normal values was facilitated in the presence of phenytoin (600 s).

MOLECULAR LAYER

PURKlNJE CELL LAYER

GRANULAR LAYER

WHITE LAYER

t

c

I

o

I

I

33

60

-

SECONDS AFTER MES

I

600

C

1

I

I

1

0

30

60

600

.

SECONDS AFTER MES

FIG. 5. The concentrations of lactate in 4 layers of the cerebellar cortex following MES. Each point represents the mcan & S.E.M. for measurements made on 6-15 sections from the same animals as Fig. 1. Abbreviations and symbols are as for Fig. 1.

750

D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU

1

I

I

PURKINJE CELL LAYER

MOLECULAR LAYER

GRANULAR LAYER

WHITE LAYER

o

0

I

c

I

1

I

30 60 600 SECONDS AFTER MES

”

I

I

I

..

I

6 0 SECONDS AFTER MES

30

,

600

FIG. 6. The concentrations of GABA in 4 cerebellar layers following MES. Each point represents the mean & S.E.M. for 8-9 sections from the same animals as Fig. 1. Abbreviations and symbols are as for Fig. 1.

The concentrations of the putative neurotransmitter GABA were not altered by MES except for a significant decrease in the white layer 10 s after the stimulus (Fig. 6). There were increased concentrations of GABA in all layers %,hen phenytoin was given in conjunction with MES. The measurement of cyclic nucleotides required 1&ZO pg of tissue, consequently these compounds were measured in only the molecular, granular and white layers. The Purkinje cells were included in the molecular and granular layers. Cyclic AMP increased very rapidly to maxima concentrations 10 or 30s following MES (Fig. 7). By 60s the nucleotide levels were normal. When phenytoin was administered, the increases in cyclic AMP were diminished and the return to normal values was accelerated. Cyclic G M P concentrations were elevated following MES, the increases were greater and occurred later than those of cyclic AMP (Fig. 8). The peak levels were reached at 60s and had returned to normal by 10 min. Phenytoin had a dramatic effect; there were only slight increases in all 3 layers which were significantly different from the concentrations following MES alone. DISCUSSION

It is increasingly evident that the cerebellum, although not a site for paroxysmal activity, is intimately involved in the regulation of seizures. Previous biochemical studies indicated that the ability of phenytoin to prevcnt tonic extension following electroshock was expressed, not in the cerebral cortex. but in the cerebellum (LUST et al., 1978). The data

[CYCLIC AMP]

15

t

GRANULAR LAYER

0

E ’

1

I

I

C

0

I

I

I . .

I

I

”

I

6 0 6 0 0 SECONDS AFTER MES

30

FIG. 7. The concentrations of cyclic AMP in 3 cerebellar layers following MES. Each point represents measurements made on 6-18 samples from the same animals as Fig. 1. Abbreviations and symbols are as for Fig. 1.

Maximal electroshock and phenytoin effects on cerebellar layers

ICYCLICGMPI

751

electrophysiological studies that Purkinje cell discharge rate in seizing cats is enhanced following the administration of phenytoin. Some controversy exists, 301 MOLECULAR LAYER however, as PIERI& HAEFELY (1976) were unable to repeat this finding. Despite some conflicting evidence, our previous results on the effects of MES and phenytoin in the cerebellum, strengthened by the above studies, emphasized the need for additional work. Localization of the biochemical response within the cerebellum is one approach to understanding the structure-function relationships during convulsions. In this respect, the cerebellum is a layered structure which can be dissected with relative ease into 4 discrete layers, molecular, granular, Purkinje cell-rich and white. Each layer can be characterized by the predominant cellular elements present. In studies where the experimental and control values for a given layer are compared, the data can be expressed in 3ob WHITE LAYER terms of dry weight. However, a comparison of metabolite values among the different layers must take into account certain variables, such as; (1) lipid content, (2) the extracellular volume, (3) the ratio of the intracellular volume to the amount of protein and (4)the compartmentation of metabolites (it., extracellular vs. intracellular, glial vs. neuronal). While the last three parameters are difficult to assess, the variSECONDS AFTER MES able lipid content can be eliminated by converting FIG. 8. The concentrations of cyclic GMP in 3 layers of the values to fat-free dry weight using the lipid to the cerebellar cortex following MES. Each point represents protein ratio reported by BERGERet al. (1977). The the measurement on 4 16 samples from the same animals as Fig. 1. Abbreviations and symbols are as for Fig. 1. ATP and P-creatine levels were significantly lower in the white layer on the basis of dry weight (Table 1). By converting to lipid-free dry weight, the levels of favor the concept that phenytoin either attenuated the the high-energy phosphates approximate those in the electroshock signal or suppressed the normal re- other layers. Such an adjustment, however, results in sponse elicited by MES by a direct effect on the meta- higher levels of glycogen, glucose, GABA, lactate and bolic machinery. It was proposed that the pharmaco- cyclic AMP in white matter than in the other layers. logically-induced protection from seizures is, in part, While the elevation of these metabolites in the white exerted by enhancing the inhibitory influence of the layer may be real, such a conclusion should be treated with caution, since other parametcrs such as the intracerebellum. Additional support for such a role for the cerebel- cellular-extracellular distribution of the metabolites lum is substantial. Thc major output of the cerebel- may be a factor. The data on the intermediary metalum is derived from the Purkinje cells, and is inhibi- bolites suggest a fairly homogeneous concentration tory. The well known corticepurkinje-dentate-tha- in the layers of the cerebellum. Other investigators have reported an enrichment lamo loop provides one anatomical example of a direct inhibitory role of Purkinje cells on cortical out- of certain metabolites in the different fayers of the (1977) have put. In fact, it has been shown that stimulation of cerebellum. RUBIN & FERRENDELLI the cerebellum during cerebral cortical seizure activity demonstrated a cyclic G M P but not a cyclic AMP produces a decrease in seizure duration (COOPERet gradient in the layers of the cerebellum; the cyclic al., 1973). Similarly, destruction of cerebellar tissue G M P values in the molecular layers were 2- and has an opposite effect; it tends to augment seizure 5-fold greater than in the granular and white layers, respectively. In contrast, we found concentrations to activity @OW et al., 1962). A number of investigators have also shown an as- be comparable in the white and granular layer, and sociation between the cerebellum and phenytoin. about 1.5 times as much in the molecular layer. Our et al. (1965) have demonstrated that pheny- analyses required only an HCI extraction and conseKOKENGE toin appears t o be preferentially accumulated in the quently fewer manipulations; otherwise we have no cerebellum. Another group has reported a reduction explanation for these differences. Using immumohisin the anticonvulsive activity of phenytoin in the cere- tochemistry, cyclic G M P has been localized at sites bellar cortectomized animal (GABREELS, 1972). associated with fibers and membranes; whereas cyclic Another strong line of evidence comes from the work AMP was found primarily in the cytosol of the Puret al., 1977; BLOOM of JULIEN& HALPERN(1972) who demonstrated in kinje and granular cell (CUMMINC I

i

N.C. 3213-E

752

- -

-- ., -~ u. -_. w. MCLANDLESS, cr. K. ~ E U S S N I ; R W. D. LUSTand J. I

et a/., 1972). These results should be treated with some caution since an anoxic artefact due to decapitation could account for the fluorescent response. The distribution of GABA in the cerebellum has been reported by two other groups (BERGERet a/., 1977; NADI et a/., 1977). Although NADIet al. (1977) reported some minor fluctuations, the GABA concentrations were relatively constant in all layers when based on lipidfree dry wt. Our results, in concert with the above reports, suggest that substantial differences of the metabolite concentrations among the layers are not evident. Thus, the discrete anatomical layers of the cerebellum are not representative of a discrete biochemical localization. Maximal electroshock elicited a reproducible seizure response characterized by tonic extension, intermittent clonic movements (excitable stage) followed by post-ictal depression (quiescent stage). As observed in the whole cerebellum, there was a significant drop in glucose and high-energy phosphates and a build-up of lactate in all layers during the excitable stages. The decrease in the energy status can be attributed to (1) the increased energy demands due to the massive depolarization and (2) the decreased energy production due to the hypoxia sociated with tonic extension. Administration of phenytoin prevented the tonic extension and thereby should improve the ratio of energy consumption to energy production. The attenuated response in ATP, P-creatine, and glucose after MES in phenytoin treated mice supported this contention. The effect of the drug cannot be attributed solely to a reduction in hypoxia since the accumulation of lactate was not diminished in the presence of phenytoin. The increases in cyclic G M P after MES, and the prevention of these increases by phenytoin are further evidence that the effect of phenytoin is not simply an amelioration of the hypoxic state. Cyclic G M P has been shown to decrease rather than increase during hypoxia (STEINER et al., 1972; LUST et a/., 1978). The elevation of glucose after 10min may reflect a decrease in glucose utilization subsequent to the massive insult and has been observed after other treatments which affect brain metabolism (KOBAYASHI et al., 1977). Other possibilities could be postictal hyperglycemia and/or increased cerebral blood flow. Specificity in the energy metabolite response among the regions was not evident and may be due to the supramaximal stimulus used in these experiments. GABA did not change during or after seizures in agreement with previous observations in whole cerebellum (LUSTet a/., 1978). The cyclic nucleotide response in the layers was also similar to the results in whole cerebellum; the onset in the cyclic AMP increase was rapid, and cyclic AMP peaked at 30s, while cyclic G M P levels were maximal at 60s after MES. The reduction in the cyclic nucleotide response after MES in the phenytoin-pretreated mice was more pronounced in the layers than in the whole cerebellum. Perhaps the presence of deeper structures as well

V. PASSONNEAU

as the cerebellar hemispheres in the previous experiments diluted out the phenytoin-induced effects that occurred in the cortical layers of the vermis. The possible relationship of the cyclic nucleotides to neuronal activity and seizures is of interest. The iontophoretic application of cyclic AMP has been shown to inhibit the firing rate of Purkinje cells in the cerebellum (BLOOM,1975). The initial rise in cyclic AMP after MES would inhibit the Purkinje cells and thus have a permissive effect on seizure activity. The diminished cyclic AMP response in the cerebellum in the presence of phenytoin would favor the suppression of seizures. Since an iontophoretic effect of cyclic G M P has yet to be established in the cerebellum, the role that the elevated cyclic G M P plays with respect to seizure activity is less clearly defined. Of note, however, is that most anticonvulsants decrease and convulsants increase cyclic G M P levels in the cerebellum. If protection against seizures is partially a function of the cerebellum, then the levels of cerebellar cyclic G M P may be an indicator of the inhibitory output of the cerebellum; cyclic G M P levels being inversely proportional to the net inhibitory influence of the cerebellum. The elevated cyclic G M P would be permissive to the seizure state and prevention of the cyclic G M P increase by phenytoin would suppress the convulsions. Of course, the cyclic G M P increase was maximal during postictal depression which would favor the response being a product of seizures, not a cause. The nature of the electroshock-induced convulsion, however, is unique in that the events normally occurring in the preconvulsive state may be imposed on the seizure itself. Even with the microanalytical procedures, the use of microgram quantities of tissue may mask significant changes occurring in smaller regions. The methodology is available for the measurement of enzyme activities and metabolites in at least the larger cells of the CNS, such as pyramidal cells and Purkinje cells (PASSONNEAU & LOWRY,1971 ; KATO& LOWRY, 1973; BERCER et a/., 1977). Studies at the cellular level would provide a more localized analysis of metabolic events and are currently in progress in our laboratory. REFERENCES

BERGER S. J., CARTERJ. G. & LOWRY0. H. (1977) The distribution of glycine, GABA, glutamate and aspartate in rabbit spinal cord, cerebellum and hippocampus. J . Neurochem. 28, 149-158. BLOOMF. E. (1975)The role of cyclic nucleotides in central synaptic function. Rev. Physiol. Biochem. Pharmac. 74,

1-81. BLQOM F. E., HOFFERB. J., BATTENBERG E. R., SICGINS G. R., STtlNER A. L., PARKER C. W. & WEDNEKH. J. (1972) Adenosine 3'. 5'-monophosphate is localized in

cerebellar

neurons:

immunofluorescence evidence.

Science 177, 436438. BRODDLCW. & NELSONS. R. (1968) The effect of diphenylhydantoin on energy levels in brain. Fedn Proc. Fedn Am. Sacs exp. Biol. 27, 751.

Maximal electroshock and phenytoin effects on cerebellar layers

COLLINS R. C., POSNER J. B. & PLUMF. (1970) Cerebral energy metabolism during electroshock seizures in mice. Am. J . Physiol. 218, 943-950. E. & AMIN I. (1973) Clinical and COOPER1. S., CRTGHIL physiological effects of stimulation of the palec-cerebellum in humans. J. Am. Geriat. Soc. 21, 4C-43. A. (1977) ImmunoCUMMING R., ECCLESTON D. & STEINER histochemical localization of cyclic G M P in rat cerebellum. J . Cyclic Nucleot. Res. 3, 275-282. A. & MANNI E. Dow R. S., FERNANDEZ-GUARDIOLA (1962) The influence of the cerebellum on experimental epilepsy. Electroenceph. Clin. Neurophysiol. 14, 383-398. FERRENDELLI J. A. & MCDOUGALD. B., Jr. (1971) The effect of electroshock on regional CNS energy reserves in mice J . Neurochem. 18, 1197-1205. GABREELS F. J. M. (1972) De involed van phenytoin op de Purkinje-cel van de rat. Epilepsy Ahst. 5, 110. HARPERJ. F. & BRWKERG. (1975) Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic G M P after 2 0 acetylation by acetic anhydride in aqueous solution. J . Cyclic Nucleot. Res. 1, 207-218. HOWSED. C. & DUFFYT. E. (1975) Control of redox state of the pyridine nucleotides in rat cerebral cortex. Effect of electroshock induced seizures. J . Neurochem. 24, 935-940. JULIEN R. M. & HALPERN L. M. (1972) Effect of diphenylhydantoin and other antiepileptic drugs on epileptiform activity and Purkinje cell discharge rates. Epilepia 13, 387400. KING L. J.. CARLJ. L. & LAO L. (1973) Carbohydrate metabolism in brain during convulsions and its modification by phenobarbitone. J . Neurochem. 20, 477485. KINGL. J., LOWRY0. H., PASSONNEAU J. V. & VENSON V. (1967) Effects of convulsants on energy reserves in the cerebral cortex. J . Neurochem. 14, 599-611. J. V. (1977) KOBAYASHI M., LUSTW. D. & PASSONNEAU Concentrations of energy metabolites and cyclic nucleotides during and after bilateral ischemia in the gerbil cerebral cortex. J . Neurochem. 29, 53-59. F.L(1965) NeurologiKOKENGE R., KUTTH. & M C D ~ W E L cal sequelae following Dilantin overdose in a patient and experimental animals. Neurology 15, 823429. 0.(1952) The morphogenesis and adult pattern LARSELL of the lobules and fissues of the cerebellum of the white rat. J . comp. Neurol. 97, 281-356. S . J., CHI M. M.-Y., CARTER J. G., LOWRY0. H., BERGER A. & OUTLAW W. (1977) Diversity of metaBLACKSHAW bolic patterns in human brain tumors. I: High energy phosphate compounds and basic composition. J . Neurochem. 29, 959-977. LOWRY0. H. & PASSONNEAU J. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York.

753

N. R., LEINERK. Y., Wu M.-L., LOWRY0. H., ROBERTS FARRA. L. & ALRERS R. W. (1954) The quantitative histochemistry of the brain. 111: Ammon’s Horm. J . b i d . Chem. 207, 3949. LUSTW. D., KUPFERBERG H. J., PASSONNEAU J. V. & PENRY J. K. (1976) On the mechanism of action of sodium valproate: the relationship of GABA and cyclic G M P levels to anticonvulsant activity. In Clinical and Pharmacological Aspects of Sodium Valproate (Epilim) in the Treatment of Epilepsy (LEGGN. J. ed.) pp. 123-129. MCS Consultants, Tunbridge Wells, England. H. J., YONEKAWAW. D., PENRY LUSTW. D., KUPPERBERC J. K., PASSONNEAU J. V. & WHEATONA. D. (1978) Changes in brain metabolites induced by convulsants or electroshock: effects of anticonvulsant agents. Molec. Pharmac. 14, 347-356. J. V. (1976) Cyclic nucleotides LUSTW. D. & PASSONNEAU in murine brain: effect of hypothermia on adenosine 3’. 5‘ monophosphate, glycogen phosphorylase, glycogen synthase and metabolites following maximal electroshock or decapitation. J . Neurochem. 26, 11-16. G. K. & PASMCCANDLESS D. W., LUSTW. D., FEUSNER SONNEAU J. V. (1978) Mechanism of action of phenytoin: evidence for a cerebellar locus. Adv. Eehav. Biol. (in press). NADIN. S., MCBRIDE W. J. & APRISONM. H. (1977) Distribution of several amino acids in regions of the cerebellum of the rat. J . Neurochem. 28, 453-455. J. V. & LOWRY0. H. (1971) Metabolite flux PASSONNEAU in single neurons during ischemia and anesthesia. In Recent Advances in Quantitative Histo-and Cyto-chemisU. C. & SCHMIDTU., eds.) pp. 198-209. try, (DUBACH Hans Huber, Bern, Switzerland. PIERI L. & HAEFELY W. (1976)The effect of diphenylhydantoin, diazepam and clonazepam on the activity of Purkinje cells in the rat cerebellum. Naunyn-Schmiedebt.rg’s Arch. Pharmac. 296, 1 4 . RUBINE. H. & FERRENDELLI J. A. (1977) Distribution and regulation of cyclic nucleotide levels in cerebellum. in viva J . Neurochem. 29, 43-51. A. L., FERRENDELLI J. A. & KIPNISD. M. (1972) STEINER Radioimmunoassay for cyclic nucleotides. Effect of ischemia, changes during development and regional distribution of adenosine 3’, 5’-monophosphate and guanosine 3‘, 5’-monophosphate in mouse brain. J . hiol. Chem. 247, 1121-1 124. STEINER A. L., WEHMANN R. E., PARKER C. W. & KIPNIS D. M. (1972) Radioimmunoassay for the measurement of cyclic nucleotides. In Advances in Cyclic Nucleotide Research, (GREENGARD P. & ROBINSON G. A., eds.) pp. 51-61. Raven Press, New York. (COSSET W. S.) (1907) On the error of counting STUDENT with a hemocytometer. Biometrika 5, 351-360.