Psychopharmacologia (Berl.) 42, 27-32 (1975) 9 by Springer-Verlag 1975
Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures* KURT SCHLESINGER and BERNARD LIEFF Department of Psychology and Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado Received July 19, 1974; Final Version November 19, 1974
Abstract. The effects of pyridoxine deficiency and the administration of supplemental vitamin B 6 on audiogenic and electroconvulsive seizures were studied in two inbred strains of mice and their F 1 hybrids. Pyridoxine deficient diets increased seizure risk, whereas supplemental vitamin B 6 protected these animals against seizures. Penicillamine and thiosemicarbazide, at doses which lowered brain levels of
pyridoxine by only 10%, increased seizure risk. Diets deficient in zinc and copper did not alter susceptibility to either audiogenic or electroconvulsive seizures. DBA/2J mice, genetically susceptible to audiogenic seizures, have the same endogenous levels of pyridoxine in the brain as do C57B1/6J mice, which are resistant to audiogenic seizures.
Key words: Pyridoxine - Audiogenic Seizures - Electroconvulsive Seizures.
Introduction Coursin (1969) has reported on a certain class of patients, usually children, who suffer from convulsions which cannot be controlled by the usual types of anticonvulsant drug therapies. The clinical symptoms exhibited by these children show a marked and rapid improvement following the administration of pyridoxine (vitamin B6). These patients, when stimulated acoustically, show larger auditory evoked responses than do control subjects. Tower (1969) has summarized the data obtained from many of these cases and has termed the syndrome a "familial pyridoxine dependency". This name suggests that a heritable mechanism underlies the disorder, and, indeed, the occurrence of these symptoms tends to run in families. Tower has also suggested that the clinical symptoms associated with familial pyridoxine deficiencies can be controlled by supplemental vitamin B 6. Susceptibility to audiogenic seizures in mice is also determined genetically, as proven by strain comparison studies (e.g., Fuller and Sjursen, 1967; Coleman and Schlesinger, 1965) and by selective breeding experiments (Frings and Frings, 1953). In these animals, * The work reported in this paper was supported by grant number MH 13026 from the National Institute of Mental Health.
susceptibility to audiogenic seizures is dependent on the developmental stage at which the seizure test is given, maximal susceptibility varying with the genotype of the mouse. The period of maximal seizure risk can be extended if these animals are maintained on a dietary regimen deficient in pyridoxine (Coleman and Schlesinger, 1965). Schlesinger and Schreiber (1969) have shown that susceptibility to audiogenic seizures can be increased in animals which are not genetically at risk, if these animals are maintained on pyridoxine deficient diets. Susceptibility to electroconvulsive seizures is also increased in animals fed a vitamin B 6 deficient diet. The purpose of the experiments reported here was to (1) replicate previous work on the effects of pyridoxine deficient diets on susceptibility to audiogenic and electroconvulsive seizures, (2) study the effects of the anti-vitamin B 6 drugs thiosemicarbazide and penicillamine on seizure susceptibility, (3) study the effects of supplemental pyridoxine on susceptibility to audiogenic and electroconvulsive seizures, and (4) measure endogenous levels of vitamin B 6 in susceptible and resistant strains of mice. Methods Subjects. DBA/2J, C57B1/6J and DBA/2J • C57B1/6J F 1 hybrid mice were used as subjects in these experiments.
28 The origins and degree of inbreeding of animals of these genotypes have been reported elsewhere (Jay, 1963). The mice used in these studies were bred in our laboratory from breeding stocks obtained from The Jackson Laboratory, Bar Harbor, Maine. Prior to use, all mice were maintained under standard conditions of temperature (74~ _+ 3 ~ and lighting (12-hrs light cycle), with ad libitum access to Purina Mouse Breeder Chow and tap water. The number and ages of the animals used in the various experiments are given in the results section. Approximately equal numbers of male and female mice were used in each experiment.
Audiogenic Seizure Tests. Susceptibility to audiogenic seizures was determined as follows: Animals were removed from their home cages, placed into a large chromatography jar (height, 43.2 cm; diameter, 29.2 cm), and were given 30 sec to adapt. A 12.7 cm electric bell, generating approximately 117 db's of noise at the level of the mouse, was sounded for 60 sec. During this interval the mice were observed and records made of the incidence of wild running, clonic, tonic, and lethal seizures. For statistical analyses of these data each mouse was given a "seizure severity score" based on its response. The responses were scored as follows: No response = 0; wild running = 1; wild running plus clonic seizure = 2; wild running, clonic plus tonic seizures = 3; wild running, clonic, tonic and lethal seizures = 4. These various phases of the sound-induced convulsion can be characterized as follows: Wild running by frenzied, stifflegged running around the boundary of the container, clonic seizures by flexor contractions of the hind-limbs toward the chin, and tonic seizures by extensor flexion of all four limbs caudally. Death is due to respiratory failure and most animals can be revived by artificial respiration. The seizures may terminate at any stage, but characteristically follow each other in order. This distinct sequence of events makes scoring of the responses a relatively simple and unambiguous matter, and although a variety of different scoring procedures have been used, the method employed in these experiments is the most frequently used procedure in research on audiogenic seizures. EIectroconvulsive Seizure Tests. Susceptibility to electrically-induced seizures was determined as follows: Ear clips were attached to the mice, and the animals were stimulated with a 833.3 cps square wave of 0.2 sec duration, having a negative and positive pulse width of 0.3 msec and a delay of 0.6 msec between each cycle (total period = 1.2 msec). Three current intensities were used: in experiments with thiosemicarbazide and penicillamine animals were stimulated with 4.5 mA of current, in experiments with supplemental pyridoxine injections animals were stimulated with 5.0 mA of current, and in experiments with diets deficient in pyridoxine animals were stimulated with 11.0 mA of current. Incidence of clonic, tonic and lethal seizures were noted, and each mouse was given a "seizure severity score" based on its response, scored as for audiogenic seizure tests except wild running was not applicable. Dietary Regimens. All mice used in the dietary experiments were weaned at 21 days of age and placed on one of the following dietary regimens obtained from Nutritional Biochemical Corpration: (1) Control diet: Ad libitum access to a pelleted vitamin B complex test diet complete and ter-
Psychopharmacologia (Berl.), Vol. 42, Fasc. 1 (1975) ramycin-treated (10 g/gallon) tap water. (2)Pyridoxine deficient test diet: Ad libitum access to a pelleted vitamin B 6 deficient test diet and terramycin-treated tap water. (3) Zinc deficient test diet: Ad libitum access to a pelleted zinc deficient test diet and terramycin-treated tap water. (4) Copper deficient test diet: Adlibitum access to a pelleted copper deficient test diet and terramycin-treated tap water. All mice used in these dietary experiments were weighed every third day. Animals on the pyridoxine deficient diets, and their controls, were tested for seizure susceptibility when 72 days of age. Animals on the zinc and copper deficient diets, and their control, were tested for seizure susceptibility when 49 days of age.
Supplemental Pyridoxine Injections. Supplemental pyridoxine was administered to animals maintained on normal diets by daily intraperitoneal injections of pyridoxine-HCl dissolved in phosphate buffer, final pH = 7.0. Control mice were given daily injections of vehicle. DBA/2J mice were injected with doses of 0.0, 0.5 and 1.0 mg/g body weight; F 1 hybrid mice were injected with 0.0, 0.5, 1.0 and 1.5 mg/g body weight. All mice were tested for seizures 4 - 5 hrs after the last injection. For audiogenic seizure tests DBA/2J mice were given the first injection when 14 days of age and the last when 28 days of age; F 1 hybrid mice were given the first injection when 14 days of age and the last when 21 days of age. For electroconvulsive seizure tests, DBA/2J and F 1hybrid mice were given the first injection when 7 days of age and the last when 21 days of age. Volumes of all injections were 0.02 ml/g body weight. Drug Regimes. Both thiosemicarbazide and penicillamine were injected intraperitoneally to animals which had been maintained on normal diets. The drugs were dissolved in distilled water and control animals were given injections of vehicle. Only F~ hybrid mice were used in these experiments, and all animals were 28 days of age. Dose of the drugs were 0.0, 0.3, and 0.6 mg/g for penicillamine, and 0.0, 0.03, 0.045 and 0.06 mg/g for thiosemicarbazide. Volumes of all injections were 0.02 ml/g body weight. Tests for audiogenic and electroconvulsive seizures were conducted 15 min after penicillamine injection and 30 min after thiosemicarbazide injections. Pyridoxine Determinations. Pyridoxine determinations were performed by the method described by Fujita et al. (1955), with the exception that the pyridoxine was batch eluted from the permutit resin, instead of the column elution procedure described in the original assay. From 3 to 7 brains were pooled for each determination. Samples were read in an Aminco-Bowman spectrophotofluorometer, exciting wavelength = 350 m~t and emitting wavelength = 450 m~t. Blanks, extracted and unextracted standards were measured with each experiment. Results The effects of diets deficient in pyridoxine on susceptibility to audiogenic seizures are s u m m a r i z e d in Table 1. D a t a are given as average seizure severity scores, and the probabilities listed in the table are those o b t a i n e d from t-tests b e t w e e n each treated g r o u p and its appropriate control group. I n the two
29
K. Schlesinger and B. Lieff: Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures Table 1. Effects of diets deficient in pyridoxine on susceptibility to audiogenic and electroconvulsive seizures Genotype
Treatment
N
2 Seizure severity score • 1 S.D.
P
C57B1/6J C57B1/6J
Control Deficient
10 10
0.20 + 0.43 1.10 • 0.88
P < 0.01
DBA/2J DBA/2J
Control Deficient
10 10
0.00 • 0.00 0.10 • 0.32
N.S.
F1 F1
Control Deficient
10 10
0.20 • 0.63 2.30 + 1.16
P < 0.01
C57B1/6J C57B1/6J
Control Deficient
10 10
0.00 + 0.00 1.80 +_ 1.93
P < 0.01
DBA/2J DBA/2J
Control Deficient
10 10
1.70 • 1.83 2.80 • 1.03
N.S.
F1 F1
Control Deficient
10 10
1.00 • 1.37 2.00 • 1.41
N.S.
Audiogenic seizures
Electroconvulsive seizures
cases in which the control animals exhibited no seizure activity whatever, i.e., where there was zero variability, X2 tests were used to evaluate the statistical significance of the data; a clonic seizure was used as the criterion to distinguish between seizure and nonseizure activity. These data indicate that diets deficient in vitamin B 6 increase susceptibility to both audiogenic and eleetroconvulsive seizures. However, the magnitude of the effect varies in mice of different genotypes, being statistically significant in animals of certain strains and not significant in others. Fig. 1 summarizes the effects of penicillamine and thiosemicarbazide on susceptibility to audiogenic and electroconvulsive seizure in F 1 hybrid mice. The data are given as average seizure severity scores, _+ 1 standard error. Both thiosemicarbazide and penicillamine tended to increase susceptibility to audiogenic and electroconvulsive seizures. With respect to audiogenic seizures, seizure severity increased with increasing drug doses, and at the higher doses the results were statistically significant. For electroconvulsive seizures, the effects of penicillamine were statistically significant, whereas those obtained with thiosemicarbazide were not. Ten mice each from strains D B A / 2 J and C57B1/ 6J and 10 F 1 hybrids, were maintained on control diets, and on diets deficient in either zinc or copper, starting at 21 days of age. After 4 weeks on these dietary regimens, all animals were tested for susceptibility to audiogenic seizures. No statistically significant effects were noted for either of these metal deficient
4.0THIOSEM[CARBAZIDE
PENICILLAMINE n=lO
3,5audiogenic electroconvulsive
3.0-
n=lO n=t5
g~
n=15
2.5-
n=10
-r_
I
2.0-
1.5
ii
n=10
ix
n=I~ n=15
1.0-
iii
0.5 ~
0
.03
.06
0
.30
.60
Dose [mglg)
Fig. 1. The effects of thiosemicarbazide and penicillamine on audiogenic and electroconvulsive seizures. Data are given average seizure severity scores; number of animals and standard errors of the mean are given on top of each bar test diets with respect to susceptibility to audiogenic seizures. The effects of supplemental pyridoxine administration on audiogenic and electroconvulsive seizures in D B A / 2 J and F 1 hybrid mice are summarized in Fig. 2. The data are summarized as average seizure severity scores, _+ 1 standard error. Supplemental vitamin B 6 tended to protect these animals against both audiogenic and electroconvulsive seizures, the degree of protection increasing with the amount of supplemental vitamin B 6 which was administered. The
Psychopharmaeologia (Berl.), Vol. 42, Fasc. 1 (1975)
30 4.0-
DBAI 2J
n=lO
F, (DBAI 2d x C57BL/6d)
3.5-
3.0-
n=lO
Discussion
[ Z ~ audiogenic
2.5-
electro-
n=_Jo
convulsive
n:~ n=lO
o3 2.0-
.= g~
i~
1.5A_
Ix
~ill n=15 j
0.5-
l i
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:
!::::
.
!::::::
[ 0
0.5
1.0
n=lO
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[
semicarbazide by approximately 14%; the former difference was not statistically significant, whereas the latter difference was statistically significant at the 0.05 level of confidence.
i:ii 0
0.5
1.0
! !
.....
i !
.....
1.5
Dose (mg/g/day)
Fig. 2. Effects of supplemental vitamin B 6 o n susceptibility to audiogenic and electroconvulsive seizures. Data are given as average seizure severity scores; number of animals and standard errors of the mean are given on top of each bar
magnitude of protection against these types of seizures afforded by pyridoxine varied in animals of different genotypes. For example, supplemental vitamin B 6 protected both DBA/2J and F 1 hybrid mice against audiogenic seizures, but this effect was statistically significant only in F 1 hybrid mice and not in DBA/2J animals. Susceptibility to electroconvulsive seizures also tended to decrease in animals injected with supplemental vitamin B6; in this case these effects were statistically significant in DBA/2J, but not in F1 hybrid mice. Endogenous levels of pyridoxine in brain tissue of these animals were as follows: DBA/2J, 21 day old, 7 experiments, 2 = 0.65 m~tg/mg tissue, S.D. = 0.51. DBA/2J, 42 day old, 6 experiments, = 0.89 m~tg/mg tissue, S.D. = 0.62. C57B1/6J, 21 day old, 7 experiments, s = 0.68 m~tg/mg tissue, S.D. = 0.53. C57B1/6J, 42 day old, 6 experiments, 2 = 1.02 m~tg/mg tissue, S.D. = 0.66. These levels were not significantly different either across strains or age. The effects of thiosemicarbazide and penicillamine on levels of pyridoxine in brain were also measured. Twenty-eight day old F 1 hybrid mice were injected with either vehicle, 0.6 mg/g penicillamine, or 0.06 mg/g thiosemicarbazide and brain pyridoxine determinations were made 15 and 30 rain after penicillamine and thiosemicarbazide administrations, respectively. At these doses, penicillamine lowered pyridoxine levels by approximately 10 % and thio-
Vitamin B 6 deficiencies are known to cause a wide spectrum of central nervous system effects, including hyperirritability, hyperexcitability and convulsions. Such symptoms are not surprising since pyridoxine is important in the metabolism of amino acids, gammaaminobutyric acid (GABA), the biogenic amines, nucleic acids, proteins, and lipids. In this paper, we have reported on the effects of manipulating levels of pyridoxine on susceptibility to audiogenic and electroconvulsive seizures in mice. Deficiencies in levels of pyridoxine, produced by feeding mice diets lacking this vitamin, tended to lower thresholds for sound-induced and electrically induced seizures. In general, these results are in good agreement with data previously reported (see, e.g., Coleman and Schlesinger, 1965; Schlesinger and Schreibet, 1969). In no instance did we observe spontaneous seizures in animals maintained on the pyridoxine deficient test diets, suggesting that under these conditions deficiencies in vitamin B 6 lower the thresholds at which stimulation, in this case by sound and an electric current, results in behavioral convulsions. The magnitude of this lowering of the convulsive threshold by pyridoxine deficiency varied as a function of the genotype of the animals being tested. For example, DBA/2J mice maintained on diets lacking pyridoxine were not appreciably more susceptible to audiogenic seizures than were mice maintained on control diets. Nevertheless, DBA/2J mice maintained on pyridoxine deficient diets could be demonstrated to have lower convulsive thresholds as indicated by the data on electroconvulsive seizures. This apparently contradictory result is probably due to the fact that DBA/2J mice do not normally exhibit sound-induced convulsions at 72 days of age, the age at which the animals used in these experiments were tested to ensure that they were truly pyridoxine deficient. The fact that older DBA/2J mice do not show audiogenic seizures at these ages can be explained on the basis of data reported by Rails (1967); this investigator demonstrated that DBA/2J mice of approximately this age have very poor hearing, especially high frequency hearing as shown by auditory evoked potential studies. Pyridoxine deficient test diets did significantly enhance audiogenic seizure susceptibility in C57B1/6J and F 1 hybrid mice. In a previous preliminary experiment, Schlesinger (1968) tested the effects of supplemental vitamin B 6
K. Schlesinger and B. Lieff: Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures on susceptibility to audiogenic seizures in DBA/2J mice. We noted that supplemental pyridoxine ameliorated the effects of sound stimulation, protecting the animals against the often fatal effects of such treatment. In the present experiments, we have extended these observations to F 1hybrid mice and to electrically induced seizures. In addition, the effects of varying amounts of supplemental vitamin B 6were studied. The results indicate that supplemental pyridoxine protects both DBA/2J and Fj hybrid mice against audiogenic and electroconvulsive seizures. The magnitude of protection afforded by the administration of supplemental pyridoxine varied with the amount of the supplemental dose and also with the genotype of the animal being treated. In animals of some strains the degree of protection was statistically significant, whereas in other groups, the treatment effect failed to reach statistical significance. We had predicted that since pyridoxine deficient diets increase seizure susceptibility, penicillamine and thiosemicarbazide which are known to be pyridoxine antagonists, should yield the same results. We used these drugs as a convenient and efficient way of lowering levels of this vitamin in tissue, particularly in brain. The results supported our hypothesis, but measurements of pyridoxine in brain following treatment with these drugs indicated that vitamin B 6 levels were lowered by only 10% by these compounds. It seems doubtful that such a small decrease in levels of pyridoxine could account for the behavioral effects of these drugs. Neither penicillamine nor thiosemicarbazide is a specific inhibitor of pyridoxine; they have many other pharmacological actions, including alteration of zinc and copper (McCall et al., 1967; Foreman, H., 1960). To determine whether the effects of these drugs on zinc and copper concentrations might mediate the observed increases in seizure susceptibility, animals were placed on diets lacking these metals. No effects on seizure susceptibility were observed. We must conclude that the increased seizure susceptibility observed after treatment with penicillamine and thiosemicarbazide is not due to the effects of these drugs on either pyridoxine or zinc and copper content in the brain. The mechanisms underlying increased susceptibility to audiogenic seizures in pyridoxine deficient animals are not fully understood. Since vitamin B 6 is a co-factor in the synthesis of the biogenic amines and of GABA, it is possible to speculate that animals deficient in this vitamin are also deficient in levels of serotonin (5-HT), norepinephrine (NE), and GABA, and that this is the reason for the increased incidence of convulsions observed in these animals. Mice of these genotypes, maintained on pyridoxine deficient diets for approximately the periods of time used in the experiments reported above, however, are not
31
deficient in levels of 5-HT and NE (Schlesinger and Schreiber, 1969). Tews and Lovell (1967) have shown that these treatments lower levels of G A B A in brain, and it seems possible to attribute the increased central nervous system excitability observed in pyridoxine deficient animals to this effect. DBA/2J mice, susceptible to audiogenic seizures, do not have lower levels of pyridoxine in brain than C57B1/6J mice which are resistant to these seizures. This result rules out the possibility of attributing heritable differences in seizure susceptibility to endogenous differences in vitamin B 6 levels in brain tissue. Nevertheless, we have found that the administration of supplemental vitamin B 6 tends to protect both DBA/2J and F 1 hybrid mice against sound induced and electrically induced seizures. As already indicated, Coursin (1969) has reported that certain patients, especially children, suffer from convulsions which are not controlled by the usual anticonvulsants. These children have a greater auditory evoked response to acoustic stimulation than do control subjects. The clinical symptoms exhibited by these patients show marked and immediate improvement following the administration of pyridoxine. These two phenomena, i.e., patients with "familial pyridoxine deficiencies" and mice susceptible to audiogenic seizures, are analogous in the sense that both types of seizures are controlled by the administration of vitamin B 6. Whether the mechanism underlying these two types of seizures is the same remains to be determined, as does the mechanism whereby supplemental vitamin B 6 protects animals against convulsions.
References Coleman, D. L., Schlesinger, K.: Effects of pyridoxine deficiency on audiogenic seizure susceptibility in inbred mice. Proc. Soc. exp. Biol. (N.Y.) 119, 264-266 (1965) Coursin, D. B.: Vitamin B 6 and brain function in animals and man. Ann. N.Y. Acad. Sci. 166, 7-15 (1969) Foreman, H.: The pharmacology of some useful chelating agents. In: Metal binding in medicine, M. J. Seven, ed., pp. 82-94. Philadelphia: Lippincott 1960 Frings, H., Frings, M.: Development of strains of albino mice with predictable susceptibilities to audiogenic seizures. Science 117, 283-284 (1953) Fujita, A., Matsuura, K., Fujino, K.: Fluorometric determination of vitamin B 6. I. Determination of pyridoxine. J. Vitaminol. (Osaka) 1, 267-274 (1955) Fuller, J. L., Sjursen, F. H., Jr.: Audiogenic seizures in eleven mouse strains. J. Hered. 58, 135-140 (1967) Jay, G. E., Jr.: Genetic strains and stocks. In: Methodology in mammalian genetics, W.J. Burdette, ed., pp. 83-123. San Francisco: Holden-Day 1963 McCall, J.T., Goldstein, N.P., Randall, R.V., Gross, J.B.: Comparative metabolism of copper and zinc in patients with Wilsons disease (Lepatolenticular degeneration). Amer. J. reed. Sci. 254, 13-23 (1967)
32 Ralls, K.: Auditory sensitivity in mice: Peromyscus and mus musculus. Anim. Behav. 15, 123-128 (1967) Schlesinger, K.: Experimentally induced seizures in mice. In: Mind as a tissue. C. Rupp, ed., pp. 271-302. New York: Harper and Row 1968 Schlesinger, K., Schreiber, R. A.: Interaction of drugs and pyridoxine deficiency on central nervous system excitability. Ann. N.Y. Acad. Sci. 166, 281-287 (1969)
Psychopharmacologia (Berl.), Vol. 42, Fasc. 1 (1975) Tews, J.K., Lovell, R. A.: The effect of a nutritional pyridoxine deficiency on free amino acids and related substances in mouse brain. J. Neurochem. 14, 1-7 (1967) Tower, D. B.: Neurochemical mechanisms. In: Basic mechanisms of the epilepsies. H. H. Jasper, A. A. Ward and A. Pope, eds., pp. 611-638. Boston: Little, Brown 1969
Dr. Bernard Lieff, Department of Psychology and Institute for Behavioral Genetics University of Colorado, Boulder, Col. 80302, U.S.A.
Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures* KURT SCHLESINGER and BERNARD LIEFF Department of Psychology and Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado Received July 19, 1974; Final Version November 19, 1974
Abstract. The effects of pyridoxine deficiency and the administration of supplemental vitamin B 6 on audiogenic and electroconvulsive seizures were studied in two inbred strains of mice and their F 1 hybrids. Pyridoxine deficient diets increased seizure risk, whereas supplemental vitamin B 6 protected these animals against seizures. Penicillamine and thiosemicarbazide, at doses which lowered brain levels of
pyridoxine by only 10%, increased seizure risk. Diets deficient in zinc and copper did not alter susceptibility to either audiogenic or electroconvulsive seizures. DBA/2J mice, genetically susceptible to audiogenic seizures, have the same endogenous levels of pyridoxine in the brain as do C57B1/6J mice, which are resistant to audiogenic seizures.
Key words: Pyridoxine - Audiogenic Seizures - Electroconvulsive Seizures.
Introduction Coursin (1969) has reported on a certain class of patients, usually children, who suffer from convulsions which cannot be controlled by the usual types of anticonvulsant drug therapies. The clinical symptoms exhibited by these children show a marked and rapid improvement following the administration of pyridoxine (vitamin B6). These patients, when stimulated acoustically, show larger auditory evoked responses than do control subjects. Tower (1969) has summarized the data obtained from many of these cases and has termed the syndrome a "familial pyridoxine dependency". This name suggests that a heritable mechanism underlies the disorder, and, indeed, the occurrence of these symptoms tends to run in families. Tower has also suggested that the clinical symptoms associated with familial pyridoxine deficiencies can be controlled by supplemental vitamin B 6. Susceptibility to audiogenic seizures in mice is also determined genetically, as proven by strain comparison studies (e.g., Fuller and Sjursen, 1967; Coleman and Schlesinger, 1965) and by selective breeding experiments (Frings and Frings, 1953). In these animals, * The work reported in this paper was supported by grant number MH 13026 from the National Institute of Mental Health.
susceptibility to audiogenic seizures is dependent on the developmental stage at which the seizure test is given, maximal susceptibility varying with the genotype of the mouse. The period of maximal seizure risk can be extended if these animals are maintained on a dietary regimen deficient in pyridoxine (Coleman and Schlesinger, 1965). Schlesinger and Schreiber (1969) have shown that susceptibility to audiogenic seizures can be increased in animals which are not genetically at risk, if these animals are maintained on pyridoxine deficient diets. Susceptibility to electroconvulsive seizures is also increased in animals fed a vitamin B 6 deficient diet. The purpose of the experiments reported here was to (1) replicate previous work on the effects of pyridoxine deficient diets on susceptibility to audiogenic and electroconvulsive seizures, (2) study the effects of the anti-vitamin B 6 drugs thiosemicarbazide and penicillamine on seizure susceptibility, (3) study the effects of supplemental pyridoxine on susceptibility to audiogenic and electroconvulsive seizures, and (4) measure endogenous levels of vitamin B 6 in susceptible and resistant strains of mice. Methods Subjects. DBA/2J, C57B1/6J and DBA/2J • C57B1/6J F 1 hybrid mice were used as subjects in these experiments.
28 The origins and degree of inbreeding of animals of these genotypes have been reported elsewhere (Jay, 1963). The mice used in these studies were bred in our laboratory from breeding stocks obtained from The Jackson Laboratory, Bar Harbor, Maine. Prior to use, all mice were maintained under standard conditions of temperature (74~ _+ 3 ~ and lighting (12-hrs light cycle), with ad libitum access to Purina Mouse Breeder Chow and tap water. The number and ages of the animals used in the various experiments are given in the results section. Approximately equal numbers of male and female mice were used in each experiment.
Audiogenic Seizure Tests. Susceptibility to audiogenic seizures was determined as follows: Animals were removed from their home cages, placed into a large chromatography jar (height, 43.2 cm; diameter, 29.2 cm), and were given 30 sec to adapt. A 12.7 cm electric bell, generating approximately 117 db's of noise at the level of the mouse, was sounded for 60 sec. During this interval the mice were observed and records made of the incidence of wild running, clonic, tonic, and lethal seizures. For statistical analyses of these data each mouse was given a "seizure severity score" based on its response. The responses were scored as follows: No response = 0; wild running = 1; wild running plus clonic seizure = 2; wild running, clonic plus tonic seizures = 3; wild running, clonic, tonic and lethal seizures = 4. These various phases of the sound-induced convulsion can be characterized as follows: Wild running by frenzied, stifflegged running around the boundary of the container, clonic seizures by flexor contractions of the hind-limbs toward the chin, and tonic seizures by extensor flexion of all four limbs caudally. Death is due to respiratory failure and most animals can be revived by artificial respiration. The seizures may terminate at any stage, but characteristically follow each other in order. This distinct sequence of events makes scoring of the responses a relatively simple and unambiguous matter, and although a variety of different scoring procedures have been used, the method employed in these experiments is the most frequently used procedure in research on audiogenic seizures. EIectroconvulsive Seizure Tests. Susceptibility to electrically-induced seizures was determined as follows: Ear clips were attached to the mice, and the animals were stimulated with a 833.3 cps square wave of 0.2 sec duration, having a negative and positive pulse width of 0.3 msec and a delay of 0.6 msec between each cycle (total period = 1.2 msec). Three current intensities were used: in experiments with thiosemicarbazide and penicillamine animals were stimulated with 4.5 mA of current, in experiments with supplemental pyridoxine injections animals were stimulated with 5.0 mA of current, and in experiments with diets deficient in pyridoxine animals were stimulated with 11.0 mA of current. Incidence of clonic, tonic and lethal seizures were noted, and each mouse was given a "seizure severity score" based on its response, scored as for audiogenic seizure tests except wild running was not applicable. Dietary Regimens. All mice used in the dietary experiments were weaned at 21 days of age and placed on one of the following dietary regimens obtained from Nutritional Biochemical Corpration: (1) Control diet: Ad libitum access to a pelleted vitamin B complex test diet complete and ter-
Psychopharmacologia (Berl.), Vol. 42, Fasc. 1 (1975) ramycin-treated (10 g/gallon) tap water. (2)Pyridoxine deficient test diet: Ad libitum access to a pelleted vitamin B 6 deficient test diet and terramycin-treated tap water. (3) Zinc deficient test diet: Ad libitum access to a pelleted zinc deficient test diet and terramycin-treated tap water. (4) Copper deficient test diet: Adlibitum access to a pelleted copper deficient test diet and terramycin-treated tap water. All mice used in these dietary experiments were weighed every third day. Animals on the pyridoxine deficient diets, and their controls, were tested for seizure susceptibility when 72 days of age. Animals on the zinc and copper deficient diets, and their control, were tested for seizure susceptibility when 49 days of age.
Supplemental Pyridoxine Injections. Supplemental pyridoxine was administered to animals maintained on normal diets by daily intraperitoneal injections of pyridoxine-HCl dissolved in phosphate buffer, final pH = 7.0. Control mice were given daily injections of vehicle. DBA/2J mice were injected with doses of 0.0, 0.5 and 1.0 mg/g body weight; F 1 hybrid mice were injected with 0.0, 0.5, 1.0 and 1.5 mg/g body weight. All mice were tested for seizures 4 - 5 hrs after the last injection. For audiogenic seizure tests DBA/2J mice were given the first injection when 14 days of age and the last when 28 days of age; F 1 hybrid mice were given the first injection when 14 days of age and the last when 21 days of age. For electroconvulsive seizure tests, DBA/2J and F 1hybrid mice were given the first injection when 7 days of age and the last when 21 days of age. Volumes of all injections were 0.02 ml/g body weight. Drug Regimes. Both thiosemicarbazide and penicillamine were injected intraperitoneally to animals which had been maintained on normal diets. The drugs were dissolved in distilled water and control animals were given injections of vehicle. Only F~ hybrid mice were used in these experiments, and all animals were 28 days of age. Dose of the drugs were 0.0, 0.3, and 0.6 mg/g for penicillamine, and 0.0, 0.03, 0.045 and 0.06 mg/g for thiosemicarbazide. Volumes of all injections were 0.02 ml/g body weight. Tests for audiogenic and electroconvulsive seizures were conducted 15 min after penicillamine injection and 30 min after thiosemicarbazide injections. Pyridoxine Determinations. Pyridoxine determinations were performed by the method described by Fujita et al. (1955), with the exception that the pyridoxine was batch eluted from the permutit resin, instead of the column elution procedure described in the original assay. From 3 to 7 brains were pooled for each determination. Samples were read in an Aminco-Bowman spectrophotofluorometer, exciting wavelength = 350 m~t and emitting wavelength = 450 m~t. Blanks, extracted and unextracted standards were measured with each experiment. Results The effects of diets deficient in pyridoxine on susceptibility to audiogenic seizures are s u m m a r i z e d in Table 1. D a t a are given as average seizure severity scores, and the probabilities listed in the table are those o b t a i n e d from t-tests b e t w e e n each treated g r o u p and its appropriate control group. I n the two
29
K. Schlesinger and B. Lieff: Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures Table 1. Effects of diets deficient in pyridoxine on susceptibility to audiogenic and electroconvulsive seizures Genotype
Treatment
N
2 Seizure severity score • 1 S.D.
P
C57B1/6J C57B1/6J
Control Deficient
10 10
0.20 + 0.43 1.10 • 0.88
P < 0.01
DBA/2J DBA/2J
Control Deficient
10 10
0.00 • 0.00 0.10 • 0.32
N.S.
F1 F1
Control Deficient
10 10
0.20 • 0.63 2.30 + 1.16
P < 0.01
C57B1/6J C57B1/6J
Control Deficient
10 10
0.00 + 0.00 1.80 +_ 1.93
P < 0.01
DBA/2J DBA/2J
Control Deficient
10 10
1.70 • 1.83 2.80 • 1.03
N.S.
F1 F1
Control Deficient
10 10
1.00 • 1.37 2.00 • 1.41
N.S.
Audiogenic seizures
Electroconvulsive seizures
cases in which the control animals exhibited no seizure activity whatever, i.e., where there was zero variability, X2 tests were used to evaluate the statistical significance of the data; a clonic seizure was used as the criterion to distinguish between seizure and nonseizure activity. These data indicate that diets deficient in vitamin B 6 increase susceptibility to both audiogenic and eleetroconvulsive seizures. However, the magnitude of the effect varies in mice of different genotypes, being statistically significant in animals of certain strains and not significant in others. Fig. 1 summarizes the effects of penicillamine and thiosemicarbazide on susceptibility to audiogenic and electroconvulsive seizure in F 1 hybrid mice. The data are given as average seizure severity scores, _+ 1 standard error. Both thiosemicarbazide and penicillamine tended to increase susceptibility to audiogenic and electroconvulsive seizures. With respect to audiogenic seizures, seizure severity increased with increasing drug doses, and at the higher doses the results were statistically significant. For electroconvulsive seizures, the effects of penicillamine were statistically significant, whereas those obtained with thiosemicarbazide were not. Ten mice each from strains D B A / 2 J and C57B1/ 6J and 10 F 1 hybrids, were maintained on control diets, and on diets deficient in either zinc or copper, starting at 21 days of age. After 4 weeks on these dietary regimens, all animals were tested for susceptibility to audiogenic seizures. No statistically significant effects were noted for either of these metal deficient
4.0THIOSEM[CARBAZIDE
PENICILLAMINE n=lO
3,5audiogenic electroconvulsive
3.0-
n=lO n=t5
g~
n=15
2.5-
n=10
-r_
I
2.0-
1.5
ii
n=10
ix
n=I~ n=15
1.0-
iii
0.5 ~
0
.03
.06
0
.30
.60
Dose [mglg)
Fig. 1. The effects of thiosemicarbazide and penicillamine on audiogenic and electroconvulsive seizures. Data are given average seizure severity scores; number of animals and standard errors of the mean are given on top of each bar test diets with respect to susceptibility to audiogenic seizures. The effects of supplemental pyridoxine administration on audiogenic and electroconvulsive seizures in D B A / 2 J and F 1 hybrid mice are summarized in Fig. 2. The data are summarized as average seizure severity scores, _+ 1 standard error. Supplemental vitamin B 6 tended to protect these animals against both audiogenic and electroconvulsive seizures, the degree of protection increasing with the amount of supplemental vitamin B 6 which was administered. The
Psychopharmaeologia (Berl.), Vol. 42, Fasc. 1 (1975)
30 4.0-
DBAI 2J
n=lO
F, (DBAI 2d x C57BL/6d)
3.5-
3.0-
n=lO
Discussion
[ Z ~ audiogenic
2.5-
electro-
n=_Jo
convulsive
n:~ n=lO
o3 2.0-
.= g~
i~
1.5A_
Ix
~ill n=15 j
0.5-
l i
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:
!::::
.
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[
semicarbazide by approximately 14%; the former difference was not statistically significant, whereas the latter difference was statistically significant at the 0.05 level of confidence.
i:ii 0
0.5
1.0
! !
.....
i !
.....
1.5
Dose (mg/g/day)
Fig. 2. Effects of supplemental vitamin B 6 o n susceptibility to audiogenic and electroconvulsive seizures. Data are given as average seizure severity scores; number of animals and standard errors of the mean are given on top of each bar
magnitude of protection against these types of seizures afforded by pyridoxine varied in animals of different genotypes. For example, supplemental vitamin B 6 protected both DBA/2J and F 1 hybrid mice against audiogenic seizures, but this effect was statistically significant only in F 1 hybrid mice and not in DBA/2J animals. Susceptibility to electroconvulsive seizures also tended to decrease in animals injected with supplemental vitamin B6; in this case these effects were statistically significant in DBA/2J, but not in F1 hybrid mice. Endogenous levels of pyridoxine in brain tissue of these animals were as follows: DBA/2J, 21 day old, 7 experiments, 2 = 0.65 m~tg/mg tissue, S.D. = 0.51. DBA/2J, 42 day old, 6 experiments, = 0.89 m~tg/mg tissue, S.D. = 0.62. C57B1/6J, 21 day old, 7 experiments, s = 0.68 m~tg/mg tissue, S.D. = 0.53. C57B1/6J, 42 day old, 6 experiments, 2 = 1.02 m~tg/mg tissue, S.D. = 0.66. These levels were not significantly different either across strains or age. The effects of thiosemicarbazide and penicillamine on levels of pyridoxine in brain were also measured. Twenty-eight day old F 1 hybrid mice were injected with either vehicle, 0.6 mg/g penicillamine, or 0.06 mg/g thiosemicarbazide and brain pyridoxine determinations were made 15 and 30 rain after penicillamine and thiosemicarbazide administrations, respectively. At these doses, penicillamine lowered pyridoxine levels by approximately 10 % and thio-
Vitamin B 6 deficiencies are known to cause a wide spectrum of central nervous system effects, including hyperirritability, hyperexcitability and convulsions. Such symptoms are not surprising since pyridoxine is important in the metabolism of amino acids, gammaaminobutyric acid (GABA), the biogenic amines, nucleic acids, proteins, and lipids. In this paper, we have reported on the effects of manipulating levels of pyridoxine on susceptibility to audiogenic and electroconvulsive seizures in mice. Deficiencies in levels of pyridoxine, produced by feeding mice diets lacking this vitamin, tended to lower thresholds for sound-induced and electrically induced seizures. In general, these results are in good agreement with data previously reported (see, e.g., Coleman and Schlesinger, 1965; Schlesinger and Schreibet, 1969). In no instance did we observe spontaneous seizures in animals maintained on the pyridoxine deficient test diets, suggesting that under these conditions deficiencies in vitamin B 6 lower the thresholds at which stimulation, in this case by sound and an electric current, results in behavioral convulsions. The magnitude of this lowering of the convulsive threshold by pyridoxine deficiency varied as a function of the genotype of the animals being tested. For example, DBA/2J mice maintained on diets lacking pyridoxine were not appreciably more susceptible to audiogenic seizures than were mice maintained on control diets. Nevertheless, DBA/2J mice maintained on pyridoxine deficient diets could be demonstrated to have lower convulsive thresholds as indicated by the data on electroconvulsive seizures. This apparently contradictory result is probably due to the fact that DBA/2J mice do not normally exhibit sound-induced convulsions at 72 days of age, the age at which the animals used in these experiments were tested to ensure that they were truly pyridoxine deficient. The fact that older DBA/2J mice do not show audiogenic seizures at these ages can be explained on the basis of data reported by Rails (1967); this investigator demonstrated that DBA/2J mice of approximately this age have very poor hearing, especially high frequency hearing as shown by auditory evoked potential studies. Pyridoxine deficient test diets did significantly enhance audiogenic seizure susceptibility in C57B1/6J and F 1 hybrid mice. In a previous preliminary experiment, Schlesinger (1968) tested the effects of supplemental vitamin B 6
K. Schlesinger and B. Lieff: Levels of Pyridoxine and Susceptibility to Electroconvulsive and Audiogenic Seizures on susceptibility to audiogenic seizures in DBA/2J mice. We noted that supplemental pyridoxine ameliorated the effects of sound stimulation, protecting the animals against the often fatal effects of such treatment. In the present experiments, we have extended these observations to F 1hybrid mice and to electrically induced seizures. In addition, the effects of varying amounts of supplemental vitamin B 6were studied. The results indicate that supplemental pyridoxine protects both DBA/2J and Fj hybrid mice against audiogenic and electroconvulsive seizures. The magnitude of protection afforded by the administration of supplemental pyridoxine varied with the amount of the supplemental dose and also with the genotype of the animal being treated. In animals of some strains the degree of protection was statistically significant, whereas in other groups, the treatment effect failed to reach statistical significance. We had predicted that since pyridoxine deficient diets increase seizure susceptibility, penicillamine and thiosemicarbazide which are known to be pyridoxine antagonists, should yield the same results. We used these drugs as a convenient and efficient way of lowering levels of this vitamin in tissue, particularly in brain. The results supported our hypothesis, but measurements of pyridoxine in brain following treatment with these drugs indicated that vitamin B 6 levels were lowered by only 10% by these compounds. It seems doubtful that such a small decrease in levels of pyridoxine could account for the behavioral effects of these drugs. Neither penicillamine nor thiosemicarbazide is a specific inhibitor of pyridoxine; they have many other pharmacological actions, including alteration of zinc and copper (McCall et al., 1967; Foreman, H., 1960). To determine whether the effects of these drugs on zinc and copper concentrations might mediate the observed increases in seizure susceptibility, animals were placed on diets lacking these metals. No effects on seizure susceptibility were observed. We must conclude that the increased seizure susceptibility observed after treatment with penicillamine and thiosemicarbazide is not due to the effects of these drugs on either pyridoxine or zinc and copper content in the brain. The mechanisms underlying increased susceptibility to audiogenic seizures in pyridoxine deficient animals are not fully understood. Since vitamin B 6 is a co-factor in the synthesis of the biogenic amines and of GABA, it is possible to speculate that animals deficient in this vitamin are also deficient in levels of serotonin (5-HT), norepinephrine (NE), and GABA, and that this is the reason for the increased incidence of convulsions observed in these animals. Mice of these genotypes, maintained on pyridoxine deficient diets for approximately the periods of time used in the experiments reported above, however, are not
31
deficient in levels of 5-HT and NE (Schlesinger and Schreiber, 1969). Tews and Lovell (1967) have shown that these treatments lower levels of G A B A in brain, and it seems possible to attribute the increased central nervous system excitability observed in pyridoxine deficient animals to this effect. DBA/2J mice, susceptible to audiogenic seizures, do not have lower levels of pyridoxine in brain than C57B1/6J mice which are resistant to these seizures. This result rules out the possibility of attributing heritable differences in seizure susceptibility to endogenous differences in vitamin B 6 levels in brain tissue. Nevertheless, we have found that the administration of supplemental vitamin B 6 tends to protect both DBA/2J and F 1 hybrid mice against sound induced and electrically induced seizures. As already indicated, Coursin (1969) has reported that certain patients, especially children, suffer from convulsions which are not controlled by the usual anticonvulsants. These children have a greater auditory evoked response to acoustic stimulation than do control subjects. The clinical symptoms exhibited by these patients show marked and immediate improvement following the administration of pyridoxine. These two phenomena, i.e., patients with "familial pyridoxine deficiencies" and mice susceptible to audiogenic seizures, are analogous in the sense that both types of seizures are controlled by the administration of vitamin B 6. Whether the mechanism underlying these two types of seizures is the same remains to be determined, as does the mechanism whereby supplemental vitamin B 6 protects animals against convulsions.
References Coleman, D. L., Schlesinger, K.: Effects of pyridoxine deficiency on audiogenic seizure susceptibility in inbred mice. Proc. Soc. exp. Biol. (N.Y.) 119, 264-266 (1965) Coursin, D. B.: Vitamin B 6 and brain function in animals and man. Ann. N.Y. Acad. Sci. 166, 7-15 (1969) Foreman, H.: The pharmacology of some useful chelating agents. In: Metal binding in medicine, M. J. Seven, ed., pp. 82-94. Philadelphia: Lippincott 1960 Frings, H., Frings, M.: Development of strains of albino mice with predictable susceptibilities to audiogenic seizures. Science 117, 283-284 (1953) Fujita, A., Matsuura, K., Fujino, K.: Fluorometric determination of vitamin B 6. I. Determination of pyridoxine. J. Vitaminol. (Osaka) 1, 267-274 (1955) Fuller, J. L., Sjursen, F. H., Jr.: Audiogenic seizures in eleven mouse strains. J. Hered. 58, 135-140 (1967) Jay, G. E., Jr.: Genetic strains and stocks. In: Methodology in mammalian genetics, W.J. Burdette, ed., pp. 83-123. San Francisco: Holden-Day 1963 McCall, J.T., Goldstein, N.P., Randall, R.V., Gross, J.B.: Comparative metabolism of copper and zinc in patients with Wilsons disease (Lepatolenticular degeneration). Amer. J. reed. Sci. 254, 13-23 (1967)
32 Ralls, K.: Auditory sensitivity in mice: Peromyscus and mus musculus. Anim. Behav. 15, 123-128 (1967) Schlesinger, K.: Experimentally induced seizures in mice. In: Mind as a tissue. C. Rupp, ed., pp. 271-302. New York: Harper and Row 1968 Schlesinger, K., Schreiber, R. A.: Interaction of drugs and pyridoxine deficiency on central nervous system excitability. Ann. N.Y. Acad. Sci. 166, 281-287 (1969)
Psychopharmacologia (Berl.), Vol. 42, Fasc. 1 (1975) Tews, J.K., Lovell, R. A.: The effect of a nutritional pyridoxine deficiency on free amino acids and related substances in mouse brain. J. Neurochem. 14, 1-7 (1967) Tower, D. B.: Neurochemical mechanisms. In: Basic mechanisms of the epilepsies. H. H. Jasper, A. A. Ward and A. Pope, eds., pp. 611-638. Boston: Little, Brown 1969
Dr. Bernard Lieff, Department of Psychology and Institute for Behavioral Genetics University of Colorado, Boulder, Col. 80302, U.S.A.
