Life
Sciences Vol . 17, pP . Printed in the U.S .A .
1369-1372
Pergamon Preas
THE EFFECT OF VITAMIN dfi SUPPLEMENTS ON DEVELOPMENT OF SEI2URE5 fN THE t:ER91L, MERIOIIES UNGUIÇULATUS Harrlett Kaplan and Alan Kluger N . y . S . Instituts for Basic Research in Mental Mtardatton 1050 Forest Hî11 Road, Staten Island, New York 1031k (Received in final form September 22, 1975) SUMMARY To see if a Vttawin B6 deficiency is responsibls for seizureproneness to tM gerbil, Meriones care uiculatus, a diet supplewented with 0 .5wg of pyri x M per grave o urine Lab Cha+ was fed to B expsriawntal (E) weted pair : and their sitters while 8 control (C) pairs and tMir litters were fed Purina Lab which oontalns .OO~wg per grew of pyridoxine . All litters wan tee cad for seizures awry 2 weeks fraw 2 through 7 woe . of age ants a worth in a standard (SS) test tags and 2 woks latsr in a clean bows tags (CL) - and again at i yr In tM SS cage . TMre wen no dlffsrenas on any of tM wsasures between E and C litters . Stgnifluntly wore seizures wen elicited in tM SS than In tM CC tests, and tM SS fast beewwr increasingly eon effeetl w with aye . It was concluded that gerbil seizures were not due to a Vitawin D6 deficiency and that soave age-related process Wight M a products w area for future investigation . TMre has been considerable interest to tM wechanisws responsible for seizure activity in tM Mongolian gsrbll, Merianss care uitulatus . TMss seizures, which appw r to bs reflex (1) as well as ap ept orw n character (2), are triggered by external stiwul l that haw been terwsd "naves" (2) and 'stressful" Nothing is a: yet known about eM internal physiological oonditlons (9, y) . undsriying seizure proneness, although processes related to water balance (2) and to nutritional defttits (5) haw been suggested as possible causes . Sires relati wly itttls is known of tM dietary requireswnts of tM gerbil (6), this latter possibility saewsd worthy of investigation . Yitawin 06 (pyridoxine) was chosen as tM first dlstary constituent to bs studied, since It Is well established that a deficiency of this vitawin preeipttatss seizures in both aniswls In addition certain strains of seizure-susceptible wins (7, B) and wen (!) . appear to be pyridoxine dependent (10) . 06 supplewents, on tM otMr hand, haw provided protection against eieetroconvulsiw and audiogsnic ssisures in wip (li) . Msthods Several preliwinary studies were run to dsterwins tM wost effeetl w wathod of adwinlstsring tM vitawin supplewent . Adwinistering pyridoxine via intraperitonsal In)ettion was tried and found unsatisfactory, slots tM handling netassitated by tM dally inJsctlons sowtiwss triggered seizure:, tfws confounding tM recalls . Ws also tried powdered !6 dlesolwd in tap water but found that tM awount of water ingested was highly variable and sows of our subJetts drank only wtniswl awounts . TM awount of food Ingested wes found to be wore s tab le and appared to be p~s 1 t i w 1 y wrre 1 ated wi th 1369
1370
Vitamin B6 and Seizures in Gerbils
Vol. 17, No . 9
body weight . lie thsrefo» obtained an experlwntal Purina lab chw diet supplewanted with pyridoxine (in a dextrose vehicle) and a control dial with the dextrose vehicle only frow 91o-Sere, In~ ., Frenchta+n, N .J . Tha control pellet contained approxtrtely O .OOhwg of pyridoxine per grave whils the experiwental pellet had about 0 .5wg of pyridoxine per grew or over 100 firs as wuch . This was confirrd in an independent assay (Loo, psrsanal coww .) To be sun the diets had a rxi rl effect, they were gi wn to the 16 pairs of gerbils (obtsiead Prow Tuwblebroalc Farr) which were to be the parents of our sxperiwental (E) and control (C) litters . These ant rls wen r ud and housed in ~8x27xigcw clear polyurbanste cages and rintained as dsscribed In an eariisr ri r~t (3) . Eight rsndawly ssiectid poi n wen fed the pyridoxins-snrichsd (E dlst and the other 8, the control (C) diet . Every 2 wasks the rtsd gerbils wen wsighed to see if the diets we» egwily acceptable . Yhen litters wars born they wen culled to 6, sar of the extra pups being distributed to swallsr litters within the saw expsriwental group. For statisti ul purposes, however, data w» based on weans eowputed fraw the original Inters . Pups wen warned at 28 days of age, at which ti w they wen rrked and housed in stwilar cages, 2 to i likesexsd gerbils per cage and rtntainad on the save diet that had been fed to their Mrents . Frow 2 to 7 woe . of age, all subjects were tasted for sals~res under ~ canditlans . Once a worth they wen gt wn the standard test for seizures (SS test), after which they were placed in a clean how tags . In our standard test, the subJect is lifted by its tail and placed in tM ewnter of a ~8x;8x20cw white plastic cage for ; win or until a seizure is cosgleted . After this the subject is placed in a clean haw cage . Since cages were cleaned awry Z wks ., we also tested the gerbils for sstsure activity in a clean hgwe cage (CC test) 2 wks . after the standard fast . The clean hor cage was lined with 2 paper towels, covered with San-l-Cell bedding and divided le half by a plexiglass partition . A gerbil was placed in one cagartwent for ; win or until cowpletton of a set :un . Its cagewate was then tested in the second cowpartswnt . if won than 2 gerbils were housed togetMr, the second one tested was rewowd after Its test and placed in the coapartwent with tM first gerbil . Thus sn awpty ooarpartwant was again available . Each subJeet was given 6 SS tests and 6 CC tests . Measures recorded Included seizure latency and duration . Resylt : 1.
Acceptability of diets
Both experirntal (E) and control (C) diets wen agwlly acceptable to the original rtad pairs and to their young, all of whow showed nom dewlopewnt and weight gains . At the five the diets wen intr+odueed the ran weight of wted E ferles was ~6g and of wted C ferles, kSg . Two wks . later the E fawles shawad a wean weight gain of 5g and 8 wks . later a ran weight gain of 16g . During the corresponding period the C ferles gained 7g and 17g respects w ly, a surprising concordance stop sow ferles to each group had becar pregnant and given birth 'during this ti w . A stwilar picture (s obtained frow the wted rles . 9oth diets also rtntained equal rates of growth in the C and E litters . Malysis of Variance :bawd that then was no significant diffsrence in weights of E and C litters at weaning, at 7 wos, and at 1 yr . of age (F < 1 .0 ; df I,I~) . Z.
Main offsets
Diet : TMre wen no significant difhrences in any of the wasuns, which ineluâeâ-percent seisures, latency and duration of sat :are (Table 1) at aMr ago between the pyridoxine-enriched and control groups . Thw Interaction bet~wen effects of age and diet was also eat significant . (In all gees F < I .O ; df I,lk) .
Vol. 17, No . 9
Vitamin H6 and Seiznree in Gerbils
1371
TABLE 1 Percent satsuns in sxparlrntal (E) and control (C) litters during the standard seizure (SS) and clean cage (CC) tests . Parant Seiwrss SS Tast
CC T~st
8-Tb
ô-~ 12 3 6 6 3 5 3 5 ...._
(Mo.)
2y 11 21 19 30 33
3 ti 5 6 7 1 yr .
Mean latency to ssize In the C litters ; in the CC fast it Moan ssizura duration in the while In the CC fasts it was
15 6 1 I1 19 y9
6 SS casts was 53 sac for E litters and ~1 sac for was ~5 sec for the E litters and 58 sac for C litters . SS fasts was 127 sac for E and 1~0 sac for C litters, 122 sae for E litters and 12~.sae for C litters.
Test Condition : TMn was a signtficant difference in the nusber of salsuns trigqai+a~ ur~lnq~ and CC fast: (F ~ ly .k22 ; df 1,1~ ; p < .O1) . At aw ry aqs son satzurss wars elicited during the SS than in the CC fasts (Fig . 1) . Sixtytwo percent of the 16 subjects hwinq saisures in the CC fast had only one saizurs each . Tha rawalninq sub)acts had either 2 or 3 saizuns . Of the ~7 subjects hwinq satsuma in the SS condition only 28t had a sirgla satsun ; 31i had 2 salsvras, 28t had 3 satsuns and the rarininq 1~ had either k or 5 salaams. df ~
s
TMre was a signtficant ineraase in saizuras'as aqe Ineraasad (F~2 .379, ; p < .05) . Ths partant saizuras eitcitad In tha CC tests nr lnad ao-
ss-
0
s s AsstrssMsl
Fr a
Fiq. 1 Percent selzuns (based on eowblnad axparirntal and control litter sans) during clean uqa (CC) and standard saisuns (SS) fasts frow 2 to 7 wtonths of aga and a final SS fast at 1 year .
1372
Vitamin H6 and Sauures in Gerbils
Vol. 17, No .
6
about tM saws fraw 2 to 7 ~ . of age, while the pergnt seizures during ehs SS test won than doubled during tM sar period (Fig . 1) . When the subjects were rs-tested at i yr of age, then wss a significant increase in setsures In both the E and C litters . Diseusstan Since than wen no difhrenees in weights between the E and C litters it can be assuwed that tM E litters ingested about 100 tl rs as wuch pyridoxine as the C litters did . This large supplernt, however, had no sff~ct on the de w lopwant of seizures, seizure latency or duration . The two variables that did haw an effect on seisuns were age and test condition . As reported earlier (3), seizures Increase with increasing sge . In this experiwent, however, the increase wes found In one test condition only . In the CC test, tM percentage of seizures mined about the save during the entire testing period, while in the SS test It rose considerably . Ores mason for this is that subjects habitwte fairly rapidly to the CC test as evidenced by the fact that 62t of those that :sized in this condition had only ores seizure . Obviously a clean her cage is less howl aced less stressful thaw the standard seisun-test cage which the subJset expertsnoes only once a worth . Ws can conclude that seizun "proneness in gerbils is probably not the rssult of a Yltaw(n 06 deficiency, it fs unlikely that larger supplewsnts would be any won effects w than the awoa~ts already adwinistered . Sing seizure-proneness exhibits consistent increases with age, it w(ght ba product) w to tnwstigate next sor process (horwonaf, revere) or othen+tse) that also shows terporal changes . Acknaw le~ts 11a wish to thank 0r . y . H . Loo, Dr . Y, Silwrrn, C . MiezsJeskl and 0 . Sktnitis for their hip In various aspects of this study . Refereaas 1. 2. 3. ~. 5. 6. 7. 8. 9. 10 . 11 .
W . J . Loskota, P . Larx i S . T . Rieh, E tl ia ~ 109-119 (19I0 . D . 0 . Thtsssen, C . Ltndzey s H . C . Friand, . Scl . 11 227-228 (1968) . H . Kaplan s C . Mte :eJeski, J . Coso . Pht slor l 36y-273 (1972) H . Kaplan, Life Sei . In Press D . C . Robt wonT, Sci . Hews . ~ 16-18 (1968) . L . R . Arr(ngton, C . (I, wÂ~ssrwsn and D . E . Franke, Lab . Miw . Scl . 851-85~ (1968) . R . A . Patton, H . W. Karn s H . E . Longenecker, J . 9to1 . Chew . 1~ 181-191 (19 ;6) . H . Chick, M . M . EI Sadr s A . H . Worden, 6lochew . J . ~ 595-600 (19+a) . 0 . tl . Counln, Mn . H .y . Atad . Sci . 16 6 - , K. Schwinger i L . p uss van`as In tilocMwicel Psyehopharwscolo~r k 105-1ti0 Rawn Press, H .y . ~ltj K. Schissinger s B . Lisff, Psychopharwsoologia (Serl .) . y2 27-32 (1975) .
Sciences Vol . 17, pP . Printed in the U.S .A .
1369-1372
Pergamon Preas
THE EFFECT OF VITAMIN dfi SUPPLEMENTS ON DEVELOPMENT OF SEI2URE5 fN THE t:ER91L, MERIOIIES UNGUIÇULATUS Harrlett Kaplan and Alan Kluger N . y . S . Instituts for Basic Research in Mental Mtardatton 1050 Forest Hî11 Road, Staten Island, New York 1031k (Received in final form September 22, 1975) SUMMARY To see if a Vttawin B6 deficiency is responsibls for seizureproneness to tM gerbil, Meriones care uiculatus, a diet supplewented with 0 .5wg of pyri x M per grave o urine Lab Cha+ was fed to B expsriawntal (E) weted pair : and their sitters while 8 control (C) pairs and tMir litters were fed Purina Lab which oontalns .OO~wg per grew of pyridoxine . All litters wan tee cad for seizures awry 2 weeks fraw 2 through 7 woe . of age ants a worth in a standard (SS) test tags and 2 woks latsr in a clean bows tags (CL) - and again at i yr In tM SS cage . TMre wen no dlffsrenas on any of tM wsasures between E and C litters . Stgnifluntly wore seizures wen elicited in tM SS than In tM CC tests, and tM SS fast beewwr increasingly eon effeetl w with aye . It was concluded that gerbil seizures were not due to a Vitawin D6 deficiency and that soave age-related process Wight M a products w area for future investigation . TMre has been considerable interest to tM wechanisws responsible for seizure activity in tM Mongolian gsrbll, Merianss care uitulatus . TMss seizures, which appw r to bs reflex (1) as well as ap ept orw n character (2), are triggered by external stiwul l that haw been terwsd "naves" (2) and 'stressful" Nothing is a: yet known about eM internal physiological oonditlons (9, y) . undsriying seizure proneness, although processes related to water balance (2) and to nutritional defttits (5) haw been suggested as possible causes . Sires relati wly itttls is known of tM dietary requireswnts of tM gerbil (6), this latter possibility saewsd worthy of investigation . Yitawin 06 (pyridoxine) was chosen as tM first dlstary constituent to bs studied, since It Is well established that a deficiency of this vitawin preeipttatss seizures in both aniswls In addition certain strains of seizure-susceptible wins (7, B) and wen (!) . appear to be pyridoxine dependent (10) . 06 supplewents, on tM otMr hand, haw provided protection against eieetroconvulsiw and audiogsnic ssisures in wip (li) . Msthods Several preliwinary studies were run to dsterwins tM wost effeetl w wathod of adwinlstsring tM vitawin supplewent . Adwinistering pyridoxine via intraperitonsal In)ettion was tried and found unsatisfactory, slots tM handling netassitated by tM dally inJsctlons sowtiwss triggered seizure:, tfws confounding tM recalls . Ws also tried powdered !6 dlesolwd in tap water but found that tM awount of water ingested was highly variable and sows of our subJetts drank only wtniswl awounts . TM awount of food Ingested wes found to be wore s tab le and appared to be p~s 1 t i w 1 y wrre 1 ated wi th 1369
1370
Vitamin B6 and Seizures in Gerbils
Vol. 17, No . 9
body weight . lie thsrefo» obtained an experlwntal Purina lab chw diet supplewanted with pyridoxine (in a dextrose vehicle) and a control dial with the dextrose vehicle only frow 91o-Sere, In~ ., Frenchta+n, N .J . Tha control pellet contained approxtrtely O .OOhwg of pyridoxine per grave whils the experiwental pellet had about 0 .5wg of pyridoxine per grew or over 100 firs as wuch . This was confirrd in an independent assay (Loo, psrsanal coww .) To be sun the diets had a rxi rl effect, they were gi wn to the 16 pairs of gerbils (obtsiead Prow Tuwblebroalc Farr) which were to be the parents of our sxperiwental (E) and control (C) litters . These ant rls wen r ud and housed in ~8x27xigcw clear polyurbanste cages and rintained as dsscribed In an eariisr ri r~t (3) . Eight rsndawly ssiectid poi n wen fed the pyridoxins-snrichsd (E dlst and the other 8, the control (C) diet . Every 2 wasks the rtsd gerbils wen wsighed to see if the diets we» egwily acceptable . Yhen litters wars born they wen culled to 6, sar of the extra pups being distributed to swallsr litters within the saw expsriwental group. For statisti ul purposes, however, data w» based on weans eowputed fraw the original Inters . Pups wen warned at 28 days of age, at which ti w they wen rrked and housed in stwilar cages, 2 to i likesexsd gerbils per cage and rtntainad on the save diet that had been fed to their Mrents . Frow 2 to 7 woe . of age, all subjects were tasted for sals~res under ~ canditlans . Once a worth they wen gt wn the standard test for seizures (SS test), after which they were placed in a clean how tags . In our standard test, the subJect is lifted by its tail and placed in tM ewnter of a ~8x;8x20cw white plastic cage for ; win or until a seizure is cosgleted . After this the subject is placed in a clean haw cage . Since cages were cleaned awry Z wks ., we also tested the gerbils for sstsure activity in a clean hgwe cage (CC test) 2 wks . after the standard fast . The clean hor cage was lined with 2 paper towels, covered with San-l-Cell bedding and divided le half by a plexiglass partition . A gerbil was placed in one cagartwent for ; win or until cowpletton of a set :un . Its cagewate was then tested in the second cowpartswnt . if won than 2 gerbils were housed togetMr, the second one tested was rewowd after Its test and placed in the coapartwent with tM first gerbil . Thus sn awpty ooarpartwant was again available . Each subJeet was given 6 SS tests and 6 CC tests . Measures recorded Included seizure latency and duration . Resylt : 1.
Acceptability of diets
Both experirntal (E) and control (C) diets wen agwlly acceptable to the original rtad pairs and to their young, all of whow showed nom dewlopewnt and weight gains . At the five the diets wen intr+odueed the ran weight of wted E ferles was ~6g and of wted C ferles, kSg . Two wks . later the E fawles shawad a wean weight gain of 5g and 8 wks . later a ran weight gain of 16g . During the corresponding period the C ferles gained 7g and 17g respects w ly, a surprising concordance stop sow ferles to each group had becar pregnant and given birth 'during this ti w . A stwilar picture (s obtained frow the wted rles . 9oth diets also rtntained equal rates of growth in the C and E litters . Malysis of Variance :bawd that then was no significant diffsrence in weights of E and C litters at weaning, at 7 wos, and at 1 yr . of age (F < 1 .0 ; df I,I~) . Z.
Main offsets
Diet : TMre wen no significant difhrences in any of the wasuns, which ineluâeâ-percent seisures, latency and duration of sat :are (Table 1) at aMr ago between the pyridoxine-enriched and control groups . Thw Interaction bet~wen effects of age and diet was also eat significant . (In all gees F < I .O ; df I,lk) .
Vol. 17, No . 9
Vitamin H6 and Seiznree in Gerbils
1371
TABLE 1 Percent satsuns in sxparlrntal (E) and control (C) litters during the standard seizure (SS) and clean cage (CC) tests . Parant Seiwrss SS Tast
CC T~st
8-Tb
ô-~ 12 3 6 6 3 5 3 5 ...._
(Mo.)
2y 11 21 19 30 33
3 ti 5 6 7 1 yr .
Mean latency to ssize In the C litters ; in the CC fast it Moan ssizura duration in the while In the CC fasts it was
15 6 1 I1 19 y9
6 SS casts was 53 sac for E litters and ~1 sac for was ~5 sec for the E litters and 58 sac for C litters . SS fasts was 127 sac for E and 1~0 sac for C litters, 122 sae for E litters and 12~.sae for C litters.
Test Condition : TMn was a signtficant difference in the nusber of salsuns trigqai+a~ ur~lnq~ and CC fast: (F ~ ly .k22 ; df 1,1~ ; p < .O1) . At aw ry aqs son satzurss wars elicited during the SS than in the CC fasts (Fig . 1) . Sixtytwo percent of the 16 subjects hwinq saisures in the CC fast had only one saizurs each . Tha rawalninq sub)acts had either 2 or 3 saizuns . Of the ~7 subjects hwinq satsuma in the SS condition only 28t had a sirgla satsun ; 31i had 2 salsvras, 28t had 3 satsuns and the rarininq 1~ had either k or 5 salaams. df ~
s
TMre was a signtficant ineraase in saizuras'as aqe Ineraasad (F~2 .379, ; p < .05) . Ths partant saizuras eitcitad In tha CC tests nr lnad ao-
ss-
0
s s AsstrssMsl
Fr a
Fiq. 1 Percent selzuns (based on eowblnad axparirntal and control litter sans) during clean uqa (CC) and standard saisuns (SS) fasts frow 2 to 7 wtonths of aga and a final SS fast at 1 year .
1372
Vitamin H6 and Sauures in Gerbils
Vol. 17, No .
6
about tM saws fraw 2 to 7 ~ . of age, while the pergnt seizures during ehs SS test won than doubled during tM sar period (Fig . 1) . When the subjects were rs-tested at i yr of age, then wss a significant increase in setsures In both the E and C litters . Diseusstan Since than wen no difhrenees in weights between the E and C litters it can be assuwed that tM E litters ingested about 100 tl rs as wuch pyridoxine as the C litters did . This large supplernt, however, had no sff~ct on the de w lopwant of seizures, seizure latency or duration . The two variables that did haw an effect on seisuns were age and test condition . As reported earlier (3), seizures Increase with increasing sge . In this experiwent, however, the increase wes found In one test condition only . In the CC test, tM percentage of seizures mined about the save during the entire testing period, while in the SS test It rose considerably . Ores mason for this is that subjects habitwte fairly rapidly to the CC test as evidenced by the fact that 62t of those that :sized in this condition had only ores seizure . Obviously a clean her cage is less howl aced less stressful thaw the standard seisun-test cage which the subJset expertsnoes only once a worth . Ws can conclude that seizun "proneness in gerbils is probably not the rssult of a Yltaw(n 06 deficiency, it fs unlikely that larger supplewsnts would be any won effects w than the awoa~ts already adwinistered . Sing seizure-proneness exhibits consistent increases with age, it w(ght ba product) w to tnwstigate next sor process (horwonaf, revere) or othen+tse) that also shows terporal changes . Acknaw le~ts 11a wish to thank 0r . y . H . Loo, Dr . Y, Silwrrn, C . MiezsJeskl and 0 . Sktnitis for their hip In various aspects of this study . Refereaas 1. 2. 3. ~. 5. 6. 7. 8. 9. 10 . 11 .
W . J . Loskota, P . Larx i S . T . Rieh, E tl ia ~ 109-119 (19I0 . D . 0 . Thtsssen, C . Ltndzey s H . C . Friand, . Scl . 11 227-228 (1968) . H . Kaplan s C . Mte :eJeski, J . Coso . Pht slor l 36y-273 (1972) H . Kaplan, Life Sei . In Press D . C . Robt wonT, Sci . Hews . ~ 16-18 (1968) . L . R . Arr(ngton, C . (I, wÂ~ssrwsn and D . E . Franke, Lab . Miw . Scl . 851-85~ (1968) . R . A . Patton, H . W. Karn s H . E . Longenecker, J . 9to1 . Chew . 1~ 181-191 (19 ;6) . H . Chick, M . M . EI Sadr s A . H . Worden, 6lochew . J . ~ 595-600 (19+a) . 0 . tl . Counln, Mn . H .y . Atad . Sci . 16 6 - , K. Schwinger i L . p uss van`as In tilocMwicel Psyehopharwscolo~r k 105-1ti0 Rawn Press, H .y . ~ltj K. Schissinger s B . Lisff, Psychopharwsoologia (Serl .) . y2 27-32 (1975) .
