Effects of experimentally induced hyperlysinemia on maze learning in mice

Physiology and Behavior, Vol 11, pp 4 2 9 - 4 3 3 . Brain Research Pubhcatmns Inc, 1973. Prmted m the U.S A.

Effects of Experimentally Induced Hyperlysinemia on Maze Learning in Mice HAROLD ZENICK

Department o f Psychology, New Mexico Highlands Umversity, Las Vegas, New Mexico 87701 R U S S E L L V. B R O W N

Sinclair Comparative Medicme Research Farm, Umverslty o f Missouri, Columbia, Missouri 65201 AND D E N N I S C. W R I G H T

Department o f Psychology, University o f Missouri, Columbia, Missouri 65201

( R e c e i v e d 21 July 1 9 7 2 )

ZENICK, H., R V BROWN AND D C. WRIGHT Effects of experimentally reduced hyperlysmemta on maze learning m mice PHYSIOL. BEHAV 11(4) 4 2 9 - 4 3 3 , 1973 - T h e experiment was designed to test effects of experimentally reduced hyperlyslnemla on maze learning ability In the mouse Six% L-lyslne was placed in the drinking water during gestation, and/or during nursing and/or during postweanmg in a factorial combination The young were tested at 40 days of age in a water-escape T maze. A week later all animals were retested for retention Findings revealed that overloads of lysme resulted m temporary impairment, but permanent impairment occurred only if excess lysme was administered during gestation. Bmchemlcal analysis revealed a sharp decrease In urea nitrogen in the urine, and a blood ammonia and lyslne increase. Hyperlyslnemla

Learning deficits

Biochemical correlates

METHOD

P H E N Y L K E T O N U R I A (or h y p e r p h e n y l a l a n e m l a ) has b e e n e x t e n s i v e l y s t u d i e d [ 11 ]. A n i m a l research o n a n o t h e r aminoaclduraa, h y p e r l y s m e m l a , has b e e n l i m i t e d . T h e label, h y p e r l y s i n e m i a , is applied t o a h e t e r o g e n e o u s g r o u p of disorders. G h a d i m l et al [6] have described t w o t y p e s o f h y p e r l y s m e m i a : (1) p e r i o d i c h y p e r l y s l n e m i a associated w i t h h y p e r a m m o n e m l a , and (2) p e r s i s t e n t h y p e r l y s l n e m l a . T h e f o r m e r c o n d i t i o n has b e e n d o c u m e n t e d b y C o l u m b o [3] while G h a d l m l et al [7, 8, 9, 1 0 ] , W o o d y et al [ 1 9 , 2 0 ] a n d Dancls et al [4] have r e p o r t e d o n p e r s i s t e n t h y p e r l y slnemla. The developmental periods during which exposure to high lysine levels can result in l e a r n i n g deficits are n o t k n o w n . In t h e p r e s e n t e x p e r i m e n t , a s e g m e n t of t h e h y p e r l y s i n e m i c s y n d r o m e was d u p l i c a t e d b y a d m i n i s t e r i n g h i g h dosages o f lyslne to mice d u r i n g t h r e e d e v e l o p m e n t a l p e n ods in a c o m p l e t e factorial design. T h e effects of excess lyslne d u r i n g various d e v e l o p m e n t a l periods o n l a t e r m a z e learning and some b i o c h e m i c a l correlates o f excess lyslne a d m i n i s t r a t i o n were investigated.

Ammals S e v e n t y - t w o o f f s p r i n g f r o m t h e m a t i n g of 12 H A / B w mice w i t h males o f t h e same strain were used. T h e H A / B w stock w a s t h e result o f crosses a m o n g C 5 7 B L / 6 J , C 5 7 B R / c d J , a n d C 3 H e B / F e J strains. The m o t h e r s were h o u s e d individually w i t h ad lib f o o d and water. A f t e r weaning at Day 21, each o f f s p n n g was caged individually.

Behavioral A nalysls Apparatus. A water-escape, T m a z e , similar t o t h o s e emp l o y e d in previous studies [5, 13, 1 4 ] , was c o n s t r u c t e d o f galvamzed i r o n and p a i n t e d w i t h a flat b l a c k e n a m e l p a i n t . The s t e m was 76.2 cm long a n d 7.62 cm wide. T h e alleyways were 15.24 cm long a n d 7.62 cm wide. T h e w a t e r in t h e maze was 17 78 cm deep and was m a i n t a i n e d at 25.5 ° C. Groups and conditions. L-lyslne was a d m i n i s t e r e d in the d r i n k i n g w a t e r at a dose of 6 g / 1 0 0 ml Pilot w o r k i n d i c a t e d

1Supported in part by N.I.H. Grants AM-12793 and RR00285 429

430

ZENICK, BROWN AND WRIGHT

t h a t higher dosages p r e c l u d e d successful p r e g n a n c y T h r e e d e v e l o p m e n t a l periods were d e f i n e d , w i t h lysine a d m r n i s t e r ed. (a) to t h e m o t h e r s , d u n n g g e s t a t i o n , f r o m t h e onset of p r e g n a n c y (defined b y the a p p e a r a n c e of vaginal plugs) to p a r t u r i t i o n , (b) t o t h e m o t h e r s d u r i n g the 21 days o f nursing; and, (c) directly to t h e offspring f r o m age 21 days to 4 0 days. The t r e a t m e n t p r o t o c o l was such t h a t lysine was, or was n o t , a d m i n i s t e r e d d u r i n g these t h r e e d e v e l o p m e n t a l periods, yielding t h e 2 x 2 x 2 design o u t l i n e d in Table 1 R e f e r e n c e to specific groups in t h e t e x t e m p l o y s a yes (Y), n o (N) n o t a t i o n for t h e a d m i n i s t r a t i o n of lysine d u r i n g each d e v e l o p m e n t a l period. F o r e x a m p l e , g r o u p YYN was composed o f mice e x p o s e d to lysine d u r i n g g e s t a t i o n and nursing b u t n o t d u r i n g p o s t w e a n i n g ( n o t e n o m e n c l a t u r e in Table 1) Water b o t t l e s were s i t u a t e d so t h a t only t h e m o t h er could d r i n k d u r i n g g e s t a t m n and nursing. A t weaning, h t t e r s were s p h t a n d r a n d o m l y assigned to groups, w i t h t h e sexes equally d i s t r i b u t e d across g r o u p s Procedure Water c o n s u m p t i o n a n d weight gains were r e c o r d e d weekly for m o t h e r s and o f f s p r i n g t h r o u g h o u t the e x p e r i m e n t . Testing began for all animals at 4 0 days o f age. On e a c h test trial, t h e animals were placed in t h e start p o s i t i o n a n d allowed to swim to an escape r a m p placed at t h e end o f an arm o f t h e T maze A 30 m i n u t e inter-trial interval was used t h r o u g h o u t testing T h e animals were placed u n d e r a w a r m a:r fan d u r i n g t h e inter-trial interval to facilitate drying. The animals were lowered i n t o and r e m o v e d f r o m t h e maze w i t h a h a r d w a r e c l o t h ladder. To e h m i n a t e d i f f e r e n c e s in t h e t i m e required to disengage t h e a n i m a l f r o m t h e ladder, escape l a t e n c y was m e a s u r e d f r o m t h e time b o t h o f the a n i m a l ' s forelegs passed a m a r k 12 7 cm f r o m the start of t h e maze u n t i l h e t o u c h e d t h e escape r a m p Day 1 o f testmg consisted o f t h r e e p r e t r a l n l n g trials m w h i c h escape r a m p s were available at t h e ends of b o t h maze arms so t h a t exit was possible f r o m e i t h e r arm During acquisition, o n Days 2, 3, 4, and 5, a single r a m p was always placed on t h e side o p p o s i t e the p r e f e r r e d side of exit for each animal. Preferred side of exit was defined as t h a t side c h o s e n b y t h e a n i m a l o n at least t w o of t h e t h r e e p r e t r a l n l n g trials. Five trials were r u n o n each of t h e f o u r a c q m s i t i o n days. The a n i m a l ' s m o v e m e n t s t h r o u g h the maze, escape l a t e n c y , a n d n u m b e r o f w r o n g t u r n s were recorded A w r o n g t u r n was scored w h e n t h e a n i m a l t u r n e d in a d I r e c t m n i n c o n s i s t e n t w i t h escape. Thus, ff t h e a n i m a l

e n t e r e d t h e w r o n g arm {entry past forelegs) or t u r n e d in t h e stem and h e a d e d b a c k t o w a r d the start, an error was scored In o r d e r t o assess t h e d u r a t i o n of t r e a t m e n t effects, animals were r e t e s t e d o n e week later (52 days of age) o n the same task, w i t h five trials a day for four days U n u s u a l b e h a v i o r o u t s i d e t h e l e a r n i n g task a n d gross physical a b n o r m a h t i e s were also r e c o r d e d t h r o u g h o u t t h e e x p e r i m e n t . The m e a n p e r f o r m a n c e / d a y / a n i m a l was c o m p u t e d The data c o n f o r m e d to a 2 x 2 x 2 ( t r e a t m e n t g r o u p s ) × 4 (days) r e p e a t e d m e a s u r e s A N O V A [ 18 ]

Biochemical Analysts T h i r t y H A / B w mice, 4 0 days o f age, were r a n d o m l y placed in six m e t a b o l i s m cages (five/cage), w i t h half recewing L-lysine in t h e i r d r i n k i n g w a t e r (6 g / 1 0 0 ml). Urine was collected a n d m e a s u r e d daily for o n e week. Daily f o o d a n d w a t e r c o n s u m p t i o n were n o t e d for each g r o u p . Urea conc e n t r a t i o n was d e t e r m i n e d a c c o r d i n g to a p r o c e d u r e o f F a w c e t t a n d Scott as m o d i f i e d b y P r e s t o n [ 1 6 ] . A t-test was e m p l o y e d to assess t h e statistical significance of differences in g r o u p m e a n urea c o n c e n t r a t i o n s . F i f t e e n a d d i t i o n a l , 4 0 day old, H A / B w mice were bled from t h e tail (0 3 m l / m o u s e ) a n d the samples p o o l e d at t h r e e periods before e x p o s u r e to lysine, a f t e r 96 h r exposure to lysIne in d r i n k i n g w a t e r (6 g / 1 0 0 ml), and a f t e r 144 h r e x p o s u r e . F o u r h r a f t e r each bleeding, serum was s e p a r a t e d b y c e n t r l f u g a t i o n at 1150 g. T h e samples were frozen at 10°C u n t i l analyzed. Analyses of lysine and a m m o n i a levels were p e r f o r m e d b y c o l u m n c h r o m a t o g r a p h y o n a B e c k m a n 121 A u t o m a t e d A m i n o Acid Analyzer following t h e m e t h o d o f Moore a n d S t e m [ 17 ]. RESULTS

Water c o n s u m p t i o n a n d weight gains did n o t differ a m o n g m o t h e r s or a m o n g t h e i r offspring, suggesting n o d e b i h t a t i o n a t t r i b u t a b l e to decreased w a t e r i n t a k e or anorexia F u r t h e r m o r e , t h e r e were n o significant d i f f e r e n c e s in escape latencies a m o n g groups d u r i n g p r e t r a i n i n g , indicating t h a t t h e r e were no initial d i f f e r e n c e s in s w i m m i n g a b l h t y a m o n g groups. Since e r r o r and latency data analyses yielded identical results, only error scores are p r e s e n t e d . Mean e r r o r scores m a c q u i s i t i o n and retest are p l o t t e d in Fig. 1 a n d t h e A N O V A

TABLE 1 SCHEDULES OF LYSINE ADMINISTRATION

Developmental Period

Treatment

Gestation

Y

I

Nursing

Postweamng

Y

Y

N

N

Y

Y

N

Y

N

N

Y

N

EXPERIMENTALLY INDUCED HYPERLYSINEMIA

431

4 Y : Received Lysine N = No Lysine .°. YYN = Received Lysine During Gestation, Nursing, But Not Post-Weaning

YNN ) YNY i nO

n.-

:5

0::

LIJ e YYY.

XYNNE,. OYYN~ A Y N

2.5

2 NYNG.%

NNY

\

NNY

\

,%

eNYY

%

1.5

"-Ak

NNNI~-

-

- "6,,,, %

~

YNN YNY

/

:%

Y

'% •~ % % % %

" ~ ; - - =" ~" '"

yy ~"~YNY

NYN 0... " ~ , ~ , . NNN e.., " ",~

....eNYN - -eNNN

-~NYN

- -

"e~lN

Test

I

I

, I

I

!

I

!

2

3

4

I

2

5

4

Retest

Days

Days

FIG. 1. Dally Acquisition and Retest Performance.

F ratios are presented m Table 2. Sigmficantly poorer performances (p<0.01) m acquisition were shown by groups receiving lysme during the gestation and/or postweaning intervals. The group that received lysine only during nursing (NYN) did not differ from the controls (NNN). There was a significant interaction ( p < 0 . 0 5 ) b e t w e e n gestational and postweaning acquisition errors; so group means were also collapsed across gestation and postweanlng to derive the following acqmsitlon scores: 2.82 for groups YYY and YNY (lyslne during gestatxon and postweanmg), 2.69 for groups YYN and YNN (lyslne during gestation but not postweaning), 2.12 for groups NYY and NNY (postweanmg but not gestation), and 1 33 for groups NYN and NNN (neither gestation nor postweaning). A least squares difference comparison [18] of these means revealed that all groups receiving lyslne d u n n g gestation (YYY, YNY, YYN, YNN) showed more errors (p<0.01) than the postweaning groups (NNY and NYY) who were in turn, inferior (pC 0.01) to the groups that did not receive during gestation or postweanmg (NYN, NNN). There was significant days effect ( p < 0 . 0 0 0 t ) for both

acquisition and retest error scores. The groups x days interaction was not significant, indicating similar rates of learnlng by all groups. Day by day error scores for acquisition and retest, collapsed across groups, are presented m Table 3. Least squares difference analyses revealed a significant improvement (p<0.05) in performance across all days in both acqmsitlon and retest. Analysis of retest error scores (Table 2) indicated that only those animals exposed to lysme during gestation (YYY, YYN, YNN, YNY) continued to show inflated error scores (p< 0.01 ). Since the absence of a groups x days interaction m acquisition and retest indicated similar learning rates across groups, it was possible that the inabdity to overcome initial learning deficits, accounted for the poor performance of the postweanlng groups in acquisition. A group comparison on acqmsltion Day 1 revealed significantly poorer performance (p<0.01) by gestation-exposed groups (YYY, YYN, YNY, YNN) compared to the postweanlng groups (NNY, NYY), which differed (p<0.01) from the nursing only (NYN) and control (NNN) groups, which did not differ

432

Z E N I C K , BROWN A N D WRIGH]TABLE 2

SUMMARY OF

1~ R A T I O S F O R E R R O R S TION AND RETEST

Treatment

Acqmsltlon F

dJ

Gestation (G) Nursmg (N) Postweanmg (W) G\ N G xW NxW GxNxW Between Error

1 1 1 1 1 1 1 64

Days (D) DxG DIN DxW DxGxN DxGxW DxNxW DxGxNxW

3 3 3 3 3 3 3 3

Within krror

192

~p~<0 05

DURING ACQUISI-

Retest F

74 70+ 0 31 14 71+ 0 09 7 13 ~ 017 0196

112 68? 2.23 1 22 0 0001 (I.95 056 (131

(MS = 1 1)

(MS = 0 04)

41 42+ 2.15 144 0 39 1 04 0.32 032 1 89 (MS = 0 35)

[] Before Exposure • 96 Hours Exposure 171144 Hours Exposure

48 56? 1 25 124 1.38 082 021 1 997 083

o

E o o

2

AMMONIA

LYSINE

FIG 2 Lyslne and Ammonia Levels in Mouse Serum

(MS = 0 161

?p<0 0l

TABLE 3 D A Y T O D A Y M E A N P E R b O R M A N C E I~OR A C Q U I S I T I O N A N D RETEST*

Day

Acquisition

Retest

1

2 81

2 17

2

2 37?

1 835

3

1 96?

1 555

4

1.76?

1 425

~Means computed across groups per day ?Required difference of 0 19 = p<0.05 (method least squares difference ) :[:Reqmrcd difference 0 13 = p<0.05 f r o m each o t h e r . On retest Day 1, o n l y t h e g e s t a t m n groups s h o w e d significantly i m p a i r e d p e r f o r m a n c e (p~<0.01 ). Differences in l o n g - t e r m m e m o r y storage a n d retrieval could also have c o n t r i b u t e d to t h e o b s e r v e d l e a r n i n g deftcits This d i f f e r e n c e s h o u l d have b e e n reflected best b y retest Day 1 savings of Day 4 a c q u i s i t i o n p e r f o r m a n c e . A 2 x 2 x 2 ( t r e a t m e n t groups) x 2 (days) r e p e a t e d m e a s u r e s A N O V A was used to c o m p a r e errors o n a c q u i s i t i o n Day 4

and retest Day 1 The g e s t a t i o n groups (YYY, YYN, YNY, Y N N ) s h o w e d i n f e r i o r p e r f o r m a n c e (p<~ 0 01 ) on b o t h days while t h e p o s t w e a n i n g groups (NYY, NNY) had elevated e r r o r scores o n l y o n a c q u i s i t i o n Day 4 ( p ~ 0 01 ) T h e r e was a significant days effect (F = 11 62, d j 1,64, p<-0 0 5 ) w i t h all groups e x c e p t NNY s h o w i n g negative retest savings scores (Table 4) The a b s e n c e of a g r o u p s x days I n t e r a c t i o n (F = 1 48, d)' 1,64) i n d i c a t e s similarity a m o n g the groups in forgettang, and n o difference a m o n g t h e groups in l o n g - t e r m m e m o r y capabilities Data f r o m t h e m e t a b o h s m s t u d y i n d i c a t e d t h a t t h e lysine g r o u p s e x c r e t e d significantly less urea t h a n t h e c o n t r o l s (t-test for t h e d i f f e r e n c e b e t w e e n m e a n s , df 28, t = 14.58, p < 0 . 0 0 1 ) F u r t h e r m o r e , analyses of t h e serum revealed sign i f i c a n t l y elevated c o n c e n t r a t i o n s (p<~O0 1 ) of lyslne and a m m o n i a o n t h e days t h a t t h e animals were o n lysme treatm e n t (Fig 2) The t r a i n i n g a n d retest data i n d i c a t e t h a t e x p o s u r e to high lysme levels d u r i n g g e s t a t m n p r o d u c e s a p e r m a n e n t learning i m p a i r m e n t High lysane d u r i n g t h e p o s t w e a n a n g period p r o d u c e d Impaired p e r f o r m a n c e , b u t t h e retest scores i n d i c a t e t h a t this effect was t e m p o r a r y and n o longer d e m o n s t r a b l e 11 days a f t e r lysme a d m i n i s t r a t i o n ceased. In h u m a n s and a variety o f o t h e r species, excess lysIne clears t h e b o d y w i t h i n 72 h r [q] Since the mice did n o t receive lysine a f t e r t h e c o m p l e t i o n of p r e t r a i n i n g , t h e 2 6 4 h r interval b e t w e e n the start of a c q u i s i t i o n and retestang allowed for t h e d i s s i p a t m n of excess a m o u n t s of lysine a n d t h e r e t u r n of t h e p o s t w e a n i n g mice to n o r m a l p e r f o r m a n c e levels ( G r o u p s NYY, N N Y ) These findings i n d i c a t e t h a t p o s t p a r t u r a t t o n a l , excess lysme causes only t e m p o r a r y I m p a i r m e n t in maze l e a r m n g ability If excess lysme led to p e r m a n e n t d a m a g e w h e n e x p o s u r e o c c u r r e d d u r i n g g e s t a t i o n , t h e n t h e d i s s i p a t i o n o f lyslne b e t w e e n a c q u i s i t i o n and retest s h o u l d n o t have a f f e c t e d

EXPERIMENTALLY INDUCED HYPERLYSINEMIA

433

TABLE 4 GROUP MEAN ERRORS AND SAVINGS

Group

Acqmsmon Day 4

-

Retest Day 1

=

Savings

YYY

2 18

-

2.67

=

-0.49

YYN

1 89

-

2.64

=

-0.75

YNY

2.22

-

2 58

=

-0.36

YNN

2 30

-

2 66

=

-0.33

NYY

1 63

-

1.87

=

- 0 24

NNY

1.92

-

1 80

=

+0.12

NYN

1.22

-

1 60

=

-0.38

NNN

1.07

-

1 50

=

- 0 43

s u b s e q u e n t p e r f o r m a n c e . R e t e s t d a t a s u p p o r t s this p o s i t i o n , since mice e x p o s e d to excess lysine d u r i n g g e s t a t i o n ( Y Y Y , YYN, YNY, Y N N ) c o n t i n u e d to e x h i b i t i m p a i r e d perform a n c e d u r i n g retest. T h e a d m l m s t r a t i o n o f lyslne d u r i n g n u r s i n g failed to have an effect. While it is n o t k n o w n w h a t effect excess lysine m a y have o n t h e a m i n o acid c o n t e n t o f milk, d i e t a r y deficiencies f r e q u e n t l y d o n o t q u a l i t a t i v e l y a f f e c t m o t h e r ' s m i l k [1, 2, 12, 1 5 ] . A m i n o acid c o n t e n t o f m i l k b e t w e e n strains m a y differ s o m e w h a t , b u t are q m t e c o n s i s t e n t w i t h in strains [ 2 ] . T h u s it m i g h t b e e x p e c t e d t h a t increased d i e t a r y lyslne w o u l d n o t a p p r e c m b l y a l t e r t h e a m i n o acid c o m p o s i h o n o f t h e m d k . F u r t h e r m o r e , mice e x p o s e d t o lysine d u r i n g n u r s i n g h a d a 19 day interval b e t w e e n weaning a n d a c q u i s i t i o n testing. This interval s h o u l d have all o w e d s u f f i c i e n t t i m e for t h e clearance of a n y excess lysine from the body. T h e b i o c h e m i c a l data suggest a slower urea f o r m a t i o n and decompensated ammonia toxicity. Whether the observed l e a r n i n g deficits r e s u l t e d f r o m a m m o m a t o x i c i t y is equivocal. H o w e v e r , since t h e p l a c e n t a is p e r m e a b l e t o lysine a n d a m m o n i a , t h e e m b r y o m a y have b e e n e x p o s e d t o t h e t o x i c i t y m its o w n s y s t e m as well as its m o t h e r ' s . Postp a r t u n t l o n a l d i s s i p a t i o n o f t h e t o x i c s u b s t a n c e s w o u l d have h a d n o c o n s e q u e n c e s since p e r m a n e n t d a m a g e h a d already occurred

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2. 3 4 5 6.

7 8. 9.

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