Jortrnal of N e i i i o c h r l s t r ~ ! ,1979. . Vol. 32. pp. 755 to 760. Pergamon Press. Prmied in Great Britain.
METABOLITE LEVELS I N BRAIN FOLLOWING EXPERIMENTAL SEIZURES: THE EFFECTS OF ISONIAZID A N D SODIUM VALPROATE I N CEREBELLAR A N D CEREBRAL CORTICAL LAYERS D. W. MCCANDLESS,' G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20014, U.S.A. (Received 5 June 1978. Accepted 11 September 1978)
Abstract-Levels of glucose, lactate, GABA and cyclic nucleotides were examined in discrete layers of the cerebellum and cerebral cortex of mice following treatment with the anticonvulsant, sodium valproate, and/or the convulsant, isoniazid. The concentrations of the metabolites were essentially uniform among the layers of each region, whether from control or from drug-treated mice. Metabolite concentrations in the isoniazid-treated mice were determined either 30 min after administration (preconvulsive state), or immediatley after the onset of seizures. Glucose and lactate, two markers of energy status in the brain, were only minimally affected by drug treatment. However, the levels of GABA and cyclic nucleotides were markedly different from control values in the drug-treated animals. In the preconvulsive state, GABA levels in cerebellar layers were depressed and the cyclic nucleotides were elevated in most layers of both regions. A t the onset of seizures, the reduction of GABA and the elevation of cyclic AMP in both regions was more pronounced than during the preconvulsive state. While the concentration of cyclic GMP remained elevated in the cerebellar layers at the onset of seizures, the level in the cerebral cortex returned to control values. Valproate elevated GABA in all the layers of both regions and decreased the cyclic G M P in the cerebellar layers. Generally, when valproate was administered in combination with isoniazid, it dampened the isoniazid induced changes in the metabolites. The events leading up to a seizure as well as those that sustain it may be reflected by the disparate responses of the metabolites in the cerebellum and cerebral cortex.
IN THE preceding paper we examined the effects of maximal electroshock (MES) o n the concentrations of selected metabolites in layers of the mouse cerebellum (MCCANDLESS et a/., 1979). Pretreatment with phenytoin suppressed the seizure activity as well as reducing the biochemical changes. The evidence was consistent with a locus of action for phenytoin in the cerebellum. I n the present report we have extended the study o n experimental seizures with the use of a chemical convulsant. Isoniazid was used alone and in combination with the anticonvulsant, sodium valproate. The use of a chemical convulsant allows us to examine those events which lead u p t o the manifestation of seizures. In this study we have extended our investigation t o include the cerebral cortex, which unlike the cerebellum, is an actual site for seizure activity; excesPresent address: Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U S A . Abbreviations used: Layers of Cortex: P, plexiform layer or layer I; OS, outer layer of small pyramidal cells, or layers I1 and 111; IL, inner layer of pyramidal cells or layers IV and V; Po, a polymorphous layer of cells, or layer VI; W, subjacent white matter. MES, maximal electroshock. 755
sive neuronal firing is induced and can persist for prolonged periods in this region. The differential response of the metabolites and the cyclic nucleotides in discrete layers of the two regions of the brain might help to elucidate the neurochemical basis of convulsions; as well as t o explain the interrelationship of the cerebral cortex and cerebellum during convulsions. MATERIALS AND METHODS Male NIH general purpose mice weighing about 25g were deprived of food 12 h prior to experimentation. Following treatment, the mice were frozen in liquid nitrogen and stored at -60°C. A 1-2mm deep wedge of parietal cortex and a portion of dorsal cerebellar vermis corre(1952) were dissponding to areas 6 and 7 after LARSELL sected in a cryostat at -25°C. The tissue samples were then sectioned at 20pm as described (MCCANDLESS et a/., 1979). The layers of the cerebellum were described in the previous paper (MCCANDLESS et a!., 1979).The parietal cerebral cortex was arbitrarily dissected into 5 layers; the plexiform layer (P) or layer I, consisting of myelinated fibers, neuroglia and some neurons; an outer layer of small pyramidal cells ( 0 s ) or layers I1 and 111; an inner layer that contains medium and large pyramidal cells (IL) or layers IV and V; a polymorphous layer (Po) consisting of irregular fusiform and angular cells or layer VI; and the subjacent white matter (W).
D. W. MCCANDLESS, G. K . FEUSSNER, W. D. LUST and J. V. PASSONNEAU
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treatments. The glucose concentrations in the plexiform layer (P) and the layer of inner large pyramidal cells (IL) were significantly elevated in animals treated with valproate and isoniazid. The glucose levels in bination of both. Mice were frozen 30min after injection the white matter (Wj were depressed in the two with the exception of one isoniazid group, which was grdups of mice treated with only isoniazid. In the frozen at the onset of seizures (40 k 3 min after administracerebellum, valproate alone or given with isoniazid tion). There was no cvidence of hyperexcitability in the isoniazid-treated mice frozen at 30 min. Control mice were appeared to cause an elevation of glucose in all given an equivalent volume of saline and were otherwise layers; however, the changes were only significant in the white layer (Wj (Fig. 1Bj. Isoniazid alone did not treated as the experimental group. Materials for the cyclic nucleotide assay were purchased affect the glucose concentrations in the cerebellar from Schwarz/Mann (Orangeburg, NY). Enzymes were layers. The minimal changes in glucose levels indicate obtained from Boehringer/Mannheim (Indianapolis, IN) that glucose metabolism was not substantially and substrates, cofactors and isoniazid from Sigma Chemi- affected by the various treatments. Surely, if the seizcal Company (St. Louis, MO). Sodium valproate was a ure state had been allowed to progress, the changes gift from Abbott Laboratories (Chicago, 1L). in glucose would have been more substantial. Statistical significance was determined using Student’s The levels of lactate, the end-product of anaerobic t-test (STUDENT, 1907). glycolysis, should indicate any changes in metabolic demands imposed by the various agents. The changes in lactate following isoniazid are in contrast to the RESULTS large increases observed following MES (LUSTet a/., The control values for the metabolites and cyclic 1978; MCCANDLESS t t a[., 1979). In the cerebral cortinucleotides in the cerebellar and cerebral cortical cal layers, the lactate levels were either unchanged layers are given in Table 1. The calculated values on or depressed (Fig. 2Aj. The low lactate concentrations a lipid-free basis are given in parentheses. It has pre- in the presence of valproate might be attributed to viously been demonstrated that after MES glucose the fact that at the dose level used, the mice were is rapidly consumed to meet the additional energy somewhat sedated; a condition which could decrease demands resulting from the excitable state. The effects the metabolic flux. In the cerebellum, the lactate levels of isoniazid on glucose concentrations in the layers are not affected by isoniazid but are depressed in all of the cerebral cortex alone or in combination with layers by valproate, and in all but the Purkinje cellvalproate are shown in Fig. 1A. There were small rich layer after the combination of valproate and changes in most of the layers following the various isoniazid (Fig. 2B). Many of the seizure-induced metaDrug treatment. Valproate and isoniazid were dissolved
in isotonic saline at concentrations of 40 and 20mg/ml, respectively. Mice were given either 400 mg/kg ip. of sodium valproate, or 200mg/kg sc of isoniazid, or a comI
TABLEl. CONCENTRATIONS OF METABOLITES AND M
Cerebellar layers P G
CYCLIC
W
NUCLEOTIDES I N CEREBELLAR AND CEREBRAL LAYERS
Cerebral layers IL
0s
P
Po
W
nrnol/mg dry wt Glucose
5.87 k0.96 (10.8) 25.0 k2.0 (46.5) 5.98 k0.50 (10.5)
Lactate GABA
5.47 k0.68 (9.77) 24.0 k2.0 (42.9) 6.04 k0.74 (10.8)
5.83 (9.90) 23.4 k1.8 (39.7) 6.35 k0.50 (10.79)
3.92 k0.32 (11.2) 15.6 k0.81 (44.5) 7.16 k0.3 (20.5)
4.59 k0.57 (7.78) 2.02 k0.51 (3.42) 59
4.61 k0.17 (13.2) 0.88 k0.20 (2.51) 35
k1.11
6.48 k0.37 (10.1) 19.2 If- 1.4 (30.0) 7.53 k0.2 (11.7)
6.67 k0.44 (10.1) 24.3 k 2.4 (37.8) 7.35 0.2 (11.1)
6.29 k0.25 (9.83) 24.7 k2.6 (38.7) 7.16 kO.1 (11.2)
5.67 k0.75 (8.60) 19.9 k 1.8 (30.2) 5.91 k0.5 (8.97)
8.10 k0.62 (18.4) 25.9 k 1.87 (58.8) 5.36 k0.75 (12.2)
8.42 k0.77 (12.8) 0.195 k0.026 (0.295) 66
6.53 k1.02 (10.2) 0.187 k0.024 (0.292) 64
7.15 k0.92 (10.8) 0.196 k0.026 (0.297) 66
4.53 k0.33 (10.3) 0.236 k0.075 (0.536) 44
*
pmol/mg dry wt Cyclic AMP Cyclic GMP Protein
(x of dry
5.38 k0.46 (9.96) 2.24 k0.25 (4.15) 54
-
56
8.92 k0.72 (13.9) 0.24 1 k0.037 (0.376) 64
wt.)
The tissues were prepared and analyzed as described in Materials and Methods. Cerebellar lipid-free dry weight is from BERCER et ( I / . (1977). Cerebral cortical lipid-free dry wcight was determined by extracting the dried tissue samples with hexane and absolute ethanol. The cerebellar layers are M. molecular; P, Purkinje cell-rich; G, granular; and W,white matter. The cerebral cortical layers are P. plexiform; 0s. outer small; IL, inner large; Po, polyrnorphous and W, white as described in Materials and Methods. Values represent the mean S.E.M.for 9-15 determinations from 3 mice.
Metabolite distribution after isoniazid and/or valproate bolic events, particularly in MES, are associated with hypoxia which results in elevated lactate levels. The low brain lactate in this study indicates that there is no hypoxic component to be considered. Fluctuations in GABA concentrations in the brain are thought to play a role in the regulation of seizure activity; GABA has been shown to be increased by some anticonvulsants, and decreased by some convulsants. In the present study we have examined GABA concentrations in the cerebral cortical layers as well as in the layers of the cerebellum. Following the administration of valproate, GABA was significantly elevated in all regions ofthe cortex except the polymorphous layer (Po) (Fig. 3A). When valproate and isoniazid were both given, the GABA levels were unchanged from those of control animals. Isoniazid given alone had no effect 30 min after injection; however at the onset of seizures at 40min, GABA was significantly decreased in the outer small cell layer (OS), in the inner large cell layer (IL) and in the subjacent white matter (W). As previously seen in measurements in the whole cerebellum, GABA increased significantly following administration of valproate; the effect was seen in all 4 layers. While this effect was partially antagonized
1-
A.
CEREBRAL CORTEX
P I
0s
Po
IL
I
I
I
w I
8.
CEREBELLUM M
P
G
W
I
I
1
I
FIG. 1. Glucose concentrations in 5 layers of the cerebral cortex (A) and 4 layers of the cerebellum (B) 30min after treatment with (400 mg/kg, IP) valproate, (0);treatment with (200 mg/kg, SC), isoniazid (A); treatment with both drugs, ( 0 )or approximately 40 min after treatment with isoniazid at the onset of seizure activity (0).The layers of each brain region are described in Materials and Methods. The shaded area indicates control values s.E.M., the filled symbols indicate differences significant from control values (P < 0.05). There were 9 determinations for each treatment from 3 animals (a total of at least 15 mice)
I57
~
(LACTATEI
A. 4
2'
c
e
'0
P
E -m E
I
\4
CEREBRAL CORTEX IL Po
0s I
I
I
w I
8.
0
2
M
CEREBELLUM P G
w
I
cortex (A) and 4 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are the same as for Fig. 1. There were 9-15 determinations for each treatment from at least 3 animals. by isoniazid in the molecular (M), Purkinje (P) and granular layers (G), the depression of GABA was most dramatic in the white matter (W) (Fig. 3B). Isoniazid alone significantly depressed GABA levels in all cerebellar layers except the molecular (M) and a further decrease in GABA was evident at the onset of seizures. The two GABAergic agents do affect the GABA levels in the layers as would be predicted by studies on the whole cerebellum and cerebral cortex; however, the results d o not reflect a specific locus of action and in fact it would appear that there is generalized diffuse effect on GABA at multiple sites. Cyclic AMP levels in the cerebral cortex in mice treated with valproate alone or in combination with isoniazid were not significantly different from control values (Fig. 4A). When isoniazid was given, there were significant increases in cyclic A M P in the outer small cell layer (OS), inner large cell layer (IL) and the white matter (W). At the onset of seizures, there was an additional increase in cyclic AMP in all layers of the cerebral cortex. As in the cerebral cortex, cerebellar cyclic A M P was unaffected by valproate (Fig. 4B). Isoniazid alone caused a significant increase in the granular layer (G) and the increase was not prevented by valproate. There were dramatic increases in cyclic A M P (2.5-fold) at the onset of seizures in all the layers of both the cerebellum and cerebral cortex. A similar cyclic A M P effect in the molecular (M) and granular (G) layers of the cerebellum has been reported by RUBIN& FERRENDELLI (1977) at the onset of a pentylenetetrazol-induced seizure.
D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU
758
' . CEREBRAL CORTEX v E
P
QS
IL
Po
w
D
I
I
I
I
I
10 '
5 CEREBELLUM M
P
G
W
I
FIG. 3. GABA'concentrations in 5 layers of the cerebral cortex (A) and 4 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are as for Fig. 1. There were 9-15 determinations for each treatment from at least 3 animals.
Cyclic GMP was significantly elevated in the middle 3 layers of the cerebral cortex following the administration of valproate (Fig. 5A). Treatment with isoniazid significantly increased cyclic GMP in all but the white layer (W). Surprisingly, valproate and isoniazid given in tandem resulted in no significant changes in cyclic GMP from control values. Furthermore, the isoniazid-induced increase in cortical cyclic GMP disappeared with the onset of convulsions. The effects of valproate and isoniazid in the cerebellar layers mimicked those in an earlier report on the whole cerebellum (LUST et al., 1976). Valproate significantly decreased cyclic GMP in all layers examined, and isoniazid produced an increase. Valproate prevented the increase in cyclic GMP when given with isoniazid. In contrast to the cerebral cortex, the elevated cyclic GMP in the cerebellum persisted after the onset of seizures. Unlike the other metabolites measured after treatment, the cyclic GMP response in the cerebral cortex is markedly different in several respects from that in the cerebellum. These differences between the two regions may offer some clue as to the physiological interrelationships of the cerebral cortex and cerebellum during seizures.
be derived from using chemical convulsants like isoniazid. Certainly, the investigation of the metabolic events in the preconvulsive state is fundamental to understanding the biochemical perturbations that lower seizure threshold. For this type of study, MES is not useful. MES decreased glucose significantly in et al., 1979) and has all cerebral layers (MCCANDLESS been shown to decrease glucose in cerebral cortical tissue as well (KINGet al., 1973; FERRENDELLI & MCDOUGAL,1971). Following the administration of isoniazid even at the onset of seizures, glucose was depressed only in white matter of cortex. There were no changes in glucose elicited in the cerebellum by isoniazid. Lactate was significantly elevated by MES in the cerebellar layers, and this response was not modified by phenytoin. Other reports have shown that lactate is increased in the cortex by electroconvulsive shock (LUST et al., 1978; KINGet al., 1973; FERRENDELLI & MCDOUGAL,1971). In the cerebral cortical layers, lactate was less than control values in all instances. While valproate appeared to have some sedative effect which might account for the decrease in lactate, it was surprising that even in animals which had begun to convulse, the lactate levels were not elevated. In the cerebellum, lactate was also decreased in the presence of valproate while isoniazid had no significant effect. It appears that metabolism of these two brain regions is not compromised to any
pmmq
A.
P
0s
M
CEREBRAL CORTEX IL Po
CEREBELLUM G
W
W
DISCUSSION It is of interest to compare the effects of MES and isoniazid on the metabolic events in brain. Although the convulsive behavior after isoniazid is less predictable than that for MES, certain advantages are to
FIG.4. Cyclic AMP concentrations in 5 layers of the cerebral cortex (A) and 3 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are as for Fig. i. There were 9-15 determinations of each treatment from at least 3 animals.
Metabolite distribution after isoniazid and/or valproate
759
Valproate caused significant increases in GABA in all layers of the cerebral cortex except that containing polymorphous cells. However, GABA concentrations were not decreased in the cortical layers by isoniazid as they were in the cerebellum. Only in animals at the onset of seizures were GABA levels significantly decreased in 3 of the 5 cortical layers examined. The difference in response to isoniazid between the two O 0 23 I brain regions may be significant in relation to seizures. The triggering event could be in part the reducCEREBRAL CORTEX tion of GABA in the cerebellar layers, with conse0: :1 9" y quent changes in the cerebellar influence on cortical activity. Using these GABAergic agents, the initiation of, as well as the prevention of seizures is associated with derangements in the steady state levels of GABA. However, other seizures both chemically and electrically-induced apparently occur independent of GABA levels. Thus, it would appear that perturbation of GABA metabolism is only one of several vulnerable CEREBELLUM sites capable of eliciting seizures. The changes in cyclic nucleotide concentrations may play a key role in modulating neuronal activity. Valproate had no effect on cyclic AMP concentrations in either the cortex or the cerebellum, nor FIG.5. Cyclic GMP concentrations in 5 layers of the cere- did it have much effect on the isoniazid induced bral cortex (A) and 3 layers of the cerebellum (B) from changes. Isoniazid alone caused some increases in the same experiment as Fig. 1. The abbreviations and sym- both brain regions, and at the onset of seizures, the bols are as for Fig. 1. There were 9-15 determinations increases were magnified. The increase in cyclic AMP contrasts with our previous studies in whole cerebelof each treatment from at least 3 animals. lum and cerebral cortex; increases in cyclic AMP great extent by isoniazid or the resultant limited seiz- were never observed during seizures with the excepures. The glucose-lactate changes are in general mir- tion of MES (LUST et al., 1976; LUST et al., 1978). ror images of one another; the agents which elicited Cyclic AMP has been shown to have an inhibitory increases in glucose caused decreases in lactate con- effect on pyramidal tract neurons as well as on Purcentrations. The contrast to the effect of MES is kinje cells (BLOOM,1975). The elevation in cyclic AMP sharp; the metabolic events concerned with energy in both brain regions could result in opposing effects; use may, in part, be dissociated from a direct role a decrease in the inhibitory output from the cerebelin seizure activity. The pronounced imbalance of lum with a consequent effect on the cerebral cortex energy production and utilization which occurs dur- to sustain seizures; and a suppression of the seizure ing MES is certainly not a factor in the present study. activity in the cortex by the local elevated concenBy limiting the development of the seizures, the dele- tration of cyclic AMP. terious effects of energy depletion and anoxia which Cyclic G M P changes in the cerebellar layers were are central to the electroshock studies can be disre- like those seen in the whole cerebellum and have been garded. discussed in the preceding paper (MCCANDLESS et a/., Both isoniazid and valproate have previously been 1979). The net effect is that the anticonvulsant valshown to affect GABA metabolism and it is through proate decreases cyclic G M P and permits increased this GABAergic component that these agents are output of the Purkinje cells, while the convulsant thought to exert their effects (MELDRUM 1975; LUST isoniazid increases cyclic AMP and thus counteracts et a/., 1976). GABA levels in the cerebellar layers were this effect. Cyclic G M P has been shown to stimulate increased following valproate and decreased following pyramidal tract neurons. Surprisingly, the anticonvulisoniazid; these changes confirmed previous observa- sant valproate increased cyclic G M P in the layers of tions in the whole cerebellum (LUSTet a)., 1978). Val- the cerebral cortex containing neurons. Isoniazid also proate has been shown to prevent decreases in GABA elevated cyclic G M P in cerebral cortical layers, which due to isoniazid in the whole cercbellum; in the cere- might be expected. However, the two agents given bellar layers there was a similar antagonism in all simultaneously had no effect on cyclic G M P concenlayers except the white matter. Since valproate was trations. Such disparate results are hard to reconcile. effcctive in preventing seizures, the lack of effect in The decreased cyclic G M P in the cerebral cortical the white layer is of no consequence, as would be layers once seizures have begun may play a role in expected if the primary action of GABA is at the the modulation of seizure activity. The predicted effect of decreased cyclic G M P would be a decrease synapses.
I1 I
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D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU
in the stimulus t o the cortex; a t the same time there is a n increase in cyclic AMP, with a predicted inhibitory effect. The combination of events might influence the course of seizure activity and ultimately result in its cessation. REFERENCES BLOOM F. E. (1975) The role of cyclic nucleotides in central synaptic function. Rev. Physiol. Biochem. Pharmac. 14, 1-81. FERRENDELLI J. A. & MCDOUGALD. B., JR. (1971) The effect of electroshock on regional CNS energy reserves in mice. J. Neurochem. 18, 1197-1205. KINGL. J., CARLJ. L. & LAO L. (1973) Carbohydrate metabolism in brain during convulsions and its rnodification by phenobarbitone. J . Neurochem. 20, 477485. LARSELL 0. (1952) The morphogenesis and adult pattern of the lobules and fissues of the cerebellum of the white rat. J . Comp. Neurol. 97, 281-356. H. J., PASSONNEAU J. V. & LUST W. D., KUPFERBERG 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 Yafproate (Epipilim) in the Treatment ofEpilepsy (LEGGN. J., ed.), pp. 123129. MCS Consultants, Tunbridge Wells, England. Hr J., YONEKAWA W. D., PEmv LUSTW. D., KUPFERBERG J. K., PASSONNEAU J. V. & WHEATONA. D. (1978) Changes in brain metabolites induced by convulsants or electroshock: effects of anticonvulsant agents. Mol. Pharmac. 14, 347-356. G. K., LUSTW. D. & PASMCCANDLES D. W., FEUSSNER SONNEAU J. V. (1979) Metabolite levels in brain following experimental seizures: the effects of maximal electroshock and phenytoin in cerebellar layers. J. Neurochem. 32, 743-753. MELDRUM B. S. (1975) Epilepsy and gamma-aminobutyric acid-mediated inhibition. In International Review of Neurobiology (PFEIFFERC. C . & SMYTHIES J. R., eds.), pp. 1-36. Academic Press, New York. RUBIN E. H. & FERRENDELLI J. A. (1977) Distribution and regulation of cyclic nucleotide levels in cerebellum in vivo. J . Neurochem. 29, 4351. STUDENT (GOSSETW. S.) (1907) O n the error of counting with a hemocytometer. Biometrika 5, 351-360.
METABOLITE LEVELS I N BRAIN FOLLOWING EXPERIMENTAL SEIZURES: THE EFFECTS OF ISONIAZID A N D SODIUM VALPROATE I N CEREBELLAR A N D CEREBRAL CORTICAL LAYERS D. W. MCCANDLESS,' G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20014, U.S.A. (Received 5 June 1978. Accepted 11 September 1978)
Abstract-Levels of glucose, lactate, GABA and cyclic nucleotides were examined in discrete layers of the cerebellum and cerebral cortex of mice following treatment with the anticonvulsant, sodium valproate, and/or the convulsant, isoniazid. The concentrations of the metabolites were essentially uniform among the layers of each region, whether from control or from drug-treated mice. Metabolite concentrations in the isoniazid-treated mice were determined either 30 min after administration (preconvulsive state), or immediatley after the onset of seizures. Glucose and lactate, two markers of energy status in the brain, were only minimally affected by drug treatment. However, the levels of GABA and cyclic nucleotides were markedly different from control values in the drug-treated animals. In the preconvulsive state, GABA levels in cerebellar layers were depressed and the cyclic nucleotides were elevated in most layers of both regions. A t the onset of seizures, the reduction of GABA and the elevation of cyclic AMP in both regions was more pronounced than during the preconvulsive state. While the concentration of cyclic GMP remained elevated in the cerebellar layers at the onset of seizures, the level in the cerebral cortex returned to control values. Valproate elevated GABA in all the layers of both regions and decreased the cyclic G M P in the cerebellar layers. Generally, when valproate was administered in combination with isoniazid, it dampened the isoniazid induced changes in the metabolites. The events leading up to a seizure as well as those that sustain it may be reflected by the disparate responses of the metabolites in the cerebellum and cerebral cortex.
IN THE preceding paper we examined the effects of maximal electroshock (MES) o n the concentrations of selected metabolites in layers of the mouse cerebellum (MCCANDLESS et a/., 1979). Pretreatment with phenytoin suppressed the seizure activity as well as reducing the biochemical changes. The evidence was consistent with a locus of action for phenytoin in the cerebellum. I n the present report we have extended the study o n experimental seizures with the use of a chemical convulsant. Isoniazid was used alone and in combination with the anticonvulsant, sodium valproate. The use of a chemical convulsant allows us to examine those events which lead u p t o the manifestation of seizures. In this study we have extended our investigation t o include the cerebral cortex, which unlike the cerebellum, is an actual site for seizure activity; excesPresent address: Department of Neurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston, TX 77025, U S A . Abbreviations used: Layers of Cortex: P, plexiform layer or layer I; OS, outer layer of small pyramidal cells, or layers I1 and 111; IL, inner layer of pyramidal cells or layers IV and V; Po, a polymorphous layer of cells, or layer VI; W, subjacent white matter. MES, maximal electroshock. 755
sive neuronal firing is induced and can persist for prolonged periods in this region. The differential response of the metabolites and the cyclic nucleotides in discrete layers of the two regions of the brain might help to elucidate the neurochemical basis of convulsions; as well as t o explain the interrelationship of the cerebral cortex and cerebellum during convulsions. MATERIALS AND METHODS Male NIH general purpose mice weighing about 25g were deprived of food 12 h prior to experimentation. Following treatment, the mice were frozen in liquid nitrogen and stored at -60°C. A 1-2mm deep wedge of parietal cortex and a portion of dorsal cerebellar vermis corre(1952) were dissponding to areas 6 and 7 after LARSELL sected in a cryostat at -25°C. The tissue samples were then sectioned at 20pm as described (MCCANDLESS et a/., 1979). The layers of the cerebellum were described in the previous paper (MCCANDLESS et a!., 1979).The parietal cerebral cortex was arbitrarily dissected into 5 layers; the plexiform layer (P) or layer I, consisting of myelinated fibers, neuroglia and some neurons; an outer layer of small pyramidal cells ( 0 s ) or layers I1 and 111; an inner layer that contains medium and large pyramidal cells (IL) or layers IV and V; a polymorphous layer (Po) consisting of irregular fusiform and angular cells or layer VI; and the subjacent white matter (W).
D. W. MCCANDLESS, G. K . FEUSSNER, W. D. LUST and J. V. PASSONNEAU
756
treatments. The glucose concentrations in the plexiform layer (P) and the layer of inner large pyramidal cells (IL) were significantly elevated in animals treated with valproate and isoniazid. The glucose levels in bination of both. Mice were frozen 30min after injection the white matter (Wj were depressed in the two with the exception of one isoniazid group, which was grdups of mice treated with only isoniazid. In the frozen at the onset of seizures (40 k 3 min after administracerebellum, valproate alone or given with isoniazid tion). There was no cvidence of hyperexcitability in the isoniazid-treated mice frozen at 30 min. Control mice were appeared to cause an elevation of glucose in all given an equivalent volume of saline and were otherwise layers; however, the changes were only significant in the white layer (Wj (Fig. 1Bj. Isoniazid alone did not treated as the experimental group. Materials for the cyclic nucleotide assay were purchased affect the glucose concentrations in the cerebellar from Schwarz/Mann (Orangeburg, NY). Enzymes were layers. The minimal changes in glucose levels indicate obtained from Boehringer/Mannheim (Indianapolis, IN) that glucose metabolism was not substantially and substrates, cofactors and isoniazid from Sigma Chemi- affected by the various treatments. Surely, if the seizcal Company (St. Louis, MO). Sodium valproate was a ure state had been allowed to progress, the changes gift from Abbott Laboratories (Chicago, 1L). in glucose would have been more substantial. Statistical significance was determined using Student’s The levels of lactate, the end-product of anaerobic t-test (STUDENT, 1907). glycolysis, should indicate any changes in metabolic demands imposed by the various agents. The changes in lactate following isoniazid are in contrast to the RESULTS large increases observed following MES (LUSTet a/., The control values for the metabolites and cyclic 1978; MCCANDLESS t t a[., 1979). In the cerebral cortinucleotides in the cerebellar and cerebral cortical cal layers, the lactate levels were either unchanged layers are given in Table 1. The calculated values on or depressed (Fig. 2Aj. The low lactate concentrations a lipid-free basis are given in parentheses. It has pre- in the presence of valproate might be attributed to viously been demonstrated that after MES glucose the fact that at the dose level used, the mice were is rapidly consumed to meet the additional energy somewhat sedated; a condition which could decrease demands resulting from the excitable state. The effects the metabolic flux. In the cerebellum, the lactate levels of isoniazid on glucose concentrations in the layers are not affected by isoniazid but are depressed in all of the cerebral cortex alone or in combination with layers by valproate, and in all but the Purkinje cellvalproate are shown in Fig. 1A. There were small rich layer after the combination of valproate and changes in most of the layers following the various isoniazid (Fig. 2B). Many of the seizure-induced metaDrug treatment. Valproate and isoniazid were dissolved
in isotonic saline at concentrations of 40 and 20mg/ml, respectively. Mice were given either 400 mg/kg ip. of sodium valproate, or 200mg/kg sc of isoniazid, or a comI
TABLEl. CONCENTRATIONS OF METABOLITES AND M
Cerebellar layers P G
CYCLIC
W
NUCLEOTIDES I N CEREBELLAR AND CEREBRAL LAYERS
Cerebral layers IL
0s
P
Po
W
nrnol/mg dry wt Glucose
5.87 k0.96 (10.8) 25.0 k2.0 (46.5) 5.98 k0.50 (10.5)
Lactate GABA
5.47 k0.68 (9.77) 24.0 k2.0 (42.9) 6.04 k0.74 (10.8)
5.83 (9.90) 23.4 k1.8 (39.7) 6.35 k0.50 (10.79)
3.92 k0.32 (11.2) 15.6 k0.81 (44.5) 7.16 k0.3 (20.5)
4.59 k0.57 (7.78) 2.02 k0.51 (3.42) 59
4.61 k0.17 (13.2) 0.88 k0.20 (2.51) 35
k1.11
6.48 k0.37 (10.1) 19.2 If- 1.4 (30.0) 7.53 k0.2 (11.7)
6.67 k0.44 (10.1) 24.3 k 2.4 (37.8) 7.35 0.2 (11.1)
6.29 k0.25 (9.83) 24.7 k2.6 (38.7) 7.16 kO.1 (11.2)
5.67 k0.75 (8.60) 19.9 k 1.8 (30.2) 5.91 k0.5 (8.97)
8.10 k0.62 (18.4) 25.9 k 1.87 (58.8) 5.36 k0.75 (12.2)
8.42 k0.77 (12.8) 0.195 k0.026 (0.295) 66
6.53 k1.02 (10.2) 0.187 k0.024 (0.292) 64
7.15 k0.92 (10.8) 0.196 k0.026 (0.297) 66
4.53 k0.33 (10.3) 0.236 k0.075 (0.536) 44
*
pmol/mg dry wt Cyclic AMP Cyclic GMP Protein
(x of dry
5.38 k0.46 (9.96) 2.24 k0.25 (4.15) 54
-
56
8.92 k0.72 (13.9) 0.24 1 k0.037 (0.376) 64
wt.)
The tissues were prepared and analyzed as described in Materials and Methods. Cerebellar lipid-free dry weight is from BERCER et ( I / . (1977). Cerebral cortical lipid-free dry wcight was determined by extracting the dried tissue samples with hexane and absolute ethanol. The cerebellar layers are M. molecular; P, Purkinje cell-rich; G, granular; and W,white matter. The cerebral cortical layers are P. plexiform; 0s. outer small; IL, inner large; Po, polyrnorphous and W, white as described in Materials and Methods. Values represent the mean S.E.M.for 9-15 determinations from 3 mice.
Metabolite distribution after isoniazid and/or valproate bolic events, particularly in MES, are associated with hypoxia which results in elevated lactate levels. The low brain lactate in this study indicates that there is no hypoxic component to be considered. Fluctuations in GABA concentrations in the brain are thought to play a role in the regulation of seizure activity; GABA has been shown to be increased by some anticonvulsants, and decreased by some convulsants. In the present study we have examined GABA concentrations in the cerebral cortical layers as well as in the layers of the cerebellum. Following the administration of valproate, GABA was significantly elevated in all regions ofthe cortex except the polymorphous layer (Po) (Fig. 3A). When valproate and isoniazid were both given, the GABA levels were unchanged from those of control animals. Isoniazid given alone had no effect 30 min after injection; however at the onset of seizures at 40min, GABA was significantly decreased in the outer small cell layer (OS), in the inner large cell layer (IL) and in the subjacent white matter (W). As previously seen in measurements in the whole cerebellum, GABA increased significantly following administration of valproate; the effect was seen in all 4 layers. While this effect was partially antagonized
1-
A.
CEREBRAL CORTEX
P I
0s
Po
IL
I
I
I
w I
8.
CEREBELLUM M
P
G
W
I
I
1
I
FIG. 1. Glucose concentrations in 5 layers of the cerebral cortex (A) and 4 layers of the cerebellum (B) 30min after treatment with (400 mg/kg, IP) valproate, (0);treatment with (200 mg/kg, SC), isoniazid (A); treatment with both drugs, ( 0 )or approximately 40 min after treatment with isoniazid at the onset of seizure activity (0).The layers of each brain region are described in Materials and Methods. The shaded area indicates control values s.E.M., the filled symbols indicate differences significant from control values (P < 0.05). There were 9 determinations for each treatment from 3 animals (a total of at least 15 mice)
I57
~
(LACTATEI
A. 4
2'
c
e
'0
P
E -m E
I
\4
CEREBRAL CORTEX IL Po
0s I
I
I
w I
8.
0
2
M
CEREBELLUM P G
w
I
cortex (A) and 4 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are the same as for Fig. 1. There were 9-15 determinations for each treatment from at least 3 animals. by isoniazid in the molecular (M), Purkinje (P) and granular layers (G), the depression of GABA was most dramatic in the white matter (W) (Fig. 3B). Isoniazid alone significantly depressed GABA levels in all cerebellar layers except the molecular (M) and a further decrease in GABA was evident at the onset of seizures. The two GABAergic agents do affect the GABA levels in the layers as would be predicted by studies on the whole cerebellum and cerebral cortex; however, the results d o not reflect a specific locus of action and in fact it would appear that there is generalized diffuse effect on GABA at multiple sites. Cyclic AMP levels in the cerebral cortex in mice treated with valproate alone or in combination with isoniazid were not significantly different from control values (Fig. 4A). When isoniazid was given, there were significant increases in cyclic A M P in the outer small cell layer (OS), inner large cell layer (IL) and the white matter (W). At the onset of seizures, there was an additional increase in cyclic AMP in all layers of the cerebral cortex. As in the cerebral cortex, cerebellar cyclic A M P was unaffected by valproate (Fig. 4B). Isoniazid alone caused a significant increase in the granular layer (G) and the increase was not prevented by valproate. There were dramatic increases in cyclic A M P (2.5-fold) at the onset of seizures in all the layers of both the cerebellum and cerebral cortex. A similar cyclic A M P effect in the molecular (M) and granular (G) layers of the cerebellum has been reported by RUBIN& FERRENDELLI (1977) at the onset of a pentylenetetrazol-induced seizure.
D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU
758
' . CEREBRAL CORTEX v E
P
QS
IL
Po
w
D
I
I
I
I
I
10 '
5 CEREBELLUM M
P
G
W
I
FIG. 3. GABA'concentrations in 5 layers of the cerebral cortex (A) and 4 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are as for Fig. 1. There were 9-15 determinations for each treatment from at least 3 animals.
Cyclic GMP was significantly elevated in the middle 3 layers of the cerebral cortex following the administration of valproate (Fig. 5A). Treatment with isoniazid significantly increased cyclic GMP in all but the white layer (W). Surprisingly, valproate and isoniazid given in tandem resulted in no significant changes in cyclic GMP from control values. Furthermore, the isoniazid-induced increase in cortical cyclic GMP disappeared with the onset of convulsions. The effects of valproate and isoniazid in the cerebellar layers mimicked those in an earlier report on the whole cerebellum (LUST et al., 1976). Valproate significantly decreased cyclic GMP in all layers examined, and isoniazid produced an increase. Valproate prevented the increase in cyclic GMP when given with isoniazid. In contrast to the cerebral cortex, the elevated cyclic GMP in the cerebellum persisted after the onset of seizures. Unlike the other metabolites measured after treatment, the cyclic GMP response in the cerebral cortex is markedly different in several respects from that in the cerebellum. These differences between the two regions may offer some clue as to the physiological interrelationships of the cerebral cortex and cerebellum during seizures.
be derived from using chemical convulsants like isoniazid. Certainly, the investigation of the metabolic events in the preconvulsive state is fundamental to understanding the biochemical perturbations that lower seizure threshold. For this type of study, MES is not useful. MES decreased glucose significantly in et al., 1979) and has all cerebral layers (MCCANDLESS been shown to decrease glucose in cerebral cortical tissue as well (KINGet al., 1973; FERRENDELLI & MCDOUGAL,1971). Following the administration of isoniazid even at the onset of seizures, glucose was depressed only in white matter of cortex. There were no changes in glucose elicited in the cerebellum by isoniazid. Lactate was significantly elevated by MES in the cerebellar layers, and this response was not modified by phenytoin. Other reports have shown that lactate is increased in the cortex by electroconvulsive shock (LUST et al., 1978; KINGet al., 1973; FERRENDELLI & MCDOUGAL,1971). In the cerebral cortical layers, lactate was less than control values in all instances. While valproate appeared to have some sedative effect which might account for the decrease in lactate, it was surprising that even in animals which had begun to convulse, the lactate levels were not elevated. In the cerebellum, lactate was also decreased in the presence of valproate while isoniazid had no significant effect. It appears that metabolism of these two brain regions is not compromised to any
pmmq
A.
P
0s
M
CEREBRAL CORTEX IL Po
CEREBELLUM G
W
W
DISCUSSION It is of interest to compare the effects of MES and isoniazid on the metabolic events in brain. Although the convulsive behavior after isoniazid is less predictable than that for MES, certain advantages are to
FIG.4. Cyclic AMP concentrations in 5 layers of the cerebral cortex (A) and 3 layers of the cerebellum (B) from the same experiment as Fig. 1. The abbreviations and symbols are as for Fig. i. There were 9-15 determinations of each treatment from at least 3 animals.
Metabolite distribution after isoniazid and/or valproate
759
Valproate caused significant increases in GABA in all layers of the cerebral cortex except that containing polymorphous cells. However, GABA concentrations were not decreased in the cortical layers by isoniazid as they were in the cerebellum. Only in animals at the onset of seizures were GABA levels significantly decreased in 3 of the 5 cortical layers examined. The difference in response to isoniazid between the two O 0 23 I brain regions may be significant in relation to seizures. The triggering event could be in part the reducCEREBRAL CORTEX tion of GABA in the cerebellar layers, with conse0: :1 9" y quent changes in the cerebellar influence on cortical activity. Using these GABAergic agents, the initiation of, as well as the prevention of seizures is associated with derangements in the steady state levels of GABA. However, other seizures both chemically and electrically-induced apparently occur independent of GABA levels. Thus, it would appear that perturbation of GABA metabolism is only one of several vulnerable CEREBELLUM sites capable of eliciting seizures. The changes in cyclic nucleotide concentrations may play a key role in modulating neuronal activity. Valproate had no effect on cyclic AMP concentrations in either the cortex or the cerebellum, nor FIG.5. Cyclic GMP concentrations in 5 layers of the cere- did it have much effect on the isoniazid induced bral cortex (A) and 3 layers of the cerebellum (B) from changes. Isoniazid alone caused some increases in the same experiment as Fig. 1. The abbreviations and sym- both brain regions, and at the onset of seizures, the bols are as for Fig. 1. There were 9-15 determinations increases were magnified. The increase in cyclic AMP contrasts with our previous studies in whole cerebelof each treatment from at least 3 animals. lum and cerebral cortex; increases in cyclic AMP great extent by isoniazid or the resultant limited seiz- were never observed during seizures with the excepures. The glucose-lactate changes are in general mir- tion of MES (LUST et al., 1976; LUST et al., 1978). ror images of one another; the agents which elicited Cyclic AMP has been shown to have an inhibitory increases in glucose caused decreases in lactate con- effect on pyramidal tract neurons as well as on Purcentrations. The contrast to the effect of MES is kinje cells (BLOOM,1975). The elevation in cyclic AMP sharp; the metabolic events concerned with energy in both brain regions could result in opposing effects; use may, in part, be dissociated from a direct role a decrease in the inhibitory output from the cerebelin seizure activity. The pronounced imbalance of lum with a consequent effect on the cerebral cortex energy production and utilization which occurs dur- to sustain seizures; and a suppression of the seizure ing MES is certainly not a factor in the present study. activity in the cortex by the local elevated concenBy limiting the development of the seizures, the dele- tration of cyclic AMP. terious effects of energy depletion and anoxia which Cyclic G M P changes in the cerebellar layers were are central to the electroshock studies can be disre- like those seen in the whole cerebellum and have been garded. discussed in the preceding paper (MCCANDLESS et a/., Both isoniazid and valproate have previously been 1979). The net effect is that the anticonvulsant valshown to affect GABA metabolism and it is through proate decreases cyclic G M P and permits increased this GABAergic component that these agents are output of the Purkinje cells, while the convulsant thought to exert their effects (MELDRUM 1975; LUST isoniazid increases cyclic AMP and thus counteracts et a/., 1976). GABA levels in the cerebellar layers were this effect. Cyclic G M P has been shown to stimulate increased following valproate and decreased following pyramidal tract neurons. Surprisingly, the anticonvulisoniazid; these changes confirmed previous observa- sant valproate increased cyclic G M P in the layers of tions in the whole cerebellum (LUSTet a)., 1978). Val- the cerebral cortex containing neurons. Isoniazid also proate has been shown to prevent decreases in GABA elevated cyclic G M P in cerebral cortical layers, which due to isoniazid in the whole cercbellum; in the cere- might be expected. However, the two agents given bellar layers there was a similar antagonism in all simultaneously had no effect on cyclic G M P concenlayers except the white matter. Since valproate was trations. Such disparate results are hard to reconcile. effcctive in preventing seizures, the lack of effect in The decreased cyclic G M P in the cerebral cortical the white layer is of no consequence, as would be layers once seizures have begun may play a role in expected if the primary action of GABA is at the the modulation of seizure activity. The predicted effect of decreased cyclic G M P would be a decrease synapses.
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D. W. MCCANDLESS, G. K. FEUSSNER, W. D. LUSTand J. V. PASSONNEAU
in the stimulus t o the cortex; a t the same time there is a n increase in cyclic AMP, with a predicted inhibitory effect. The combination of events might influence the course of seizure activity and ultimately result in its cessation. REFERENCES BLOOM F. E. (1975) The role of cyclic nucleotides in central synaptic function. Rev. Physiol. Biochem. Pharmac. 14, 1-81. FERRENDELLI J. A. & MCDOUGALD. B., JR. (1971) The effect of electroshock on regional CNS energy reserves in mice. J. Neurochem. 18, 1197-1205. KINGL. J., CARLJ. L. & LAO L. (1973) Carbohydrate metabolism in brain during convulsions and its rnodification by phenobarbitone. J . Neurochem. 20, 477485. LARSELL 0. (1952) The morphogenesis and adult pattern of the lobules and fissues of the cerebellum of the white rat. J . Comp. Neurol. 97, 281-356. H. J., PASSONNEAU J. V. & LUST W. D., KUPFERBERG 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 Yafproate (Epipilim) in the Treatment ofEpilepsy (LEGGN. J., ed.), pp. 123129. MCS Consultants, Tunbridge Wells, England. Hr J., YONEKAWA W. D., PEmv LUSTW. D., KUPFERBERG J. K., PASSONNEAU J. V. & WHEATONA. D. (1978) Changes in brain metabolites induced by convulsants or electroshock: effects of anticonvulsant agents. Mol. Pharmac. 14, 347-356. G. K., LUSTW. D. & PASMCCANDLES D. W., FEUSSNER SONNEAU J. V. (1979) Metabolite levels in brain following experimental seizures: the effects of maximal electroshock and phenytoin in cerebellar layers. J. Neurochem. 32, 743-753. MELDRUM B. S. (1975) Epilepsy and gamma-aminobutyric acid-mediated inhibition. In International Review of Neurobiology (PFEIFFERC. C . & SMYTHIES J. R., eds.), pp. 1-36. Academic Press, New York. RUBIN E. H. & FERRENDELLI J. A. (1977) Distribution and regulation of cyclic nucleotide levels in cerebellum in vivo. J . Neurochem. 29, 4351. STUDENT (GOSSETW. S.) (1907) O n the error of counting with a hemocytometer. Biometrika 5, 351-360.
