- Email: [email protected]
Effects of monosodium glutamate on somatic development, obesity and activity in the mouse
Pharmacology Biochemistry & Behavior, Vol. 5, pp. 551-557. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Effects of Monosodium Glutamate on Somatic Development, Obesity and Activity in the Mouse WILLIAM J. PIZZI AND JUNE E. BARNHART
Department o f Psychology, Northeastern Illinois University, Chicago, IL 60625 (Received 7 October 1975) PIZZI, W. J. AND J. E. BARNHART. Effects o f monosodium glutamate on somatic development, obesity and activity in the mouse. PHARMAC. BIOCHEM. BEHAV. 5(5) 551-557, 1976. - Neonatal mice 1 and 5 days of age and older mice 25 days of age were injected with an increasing dose of monosodium glutamate (MSG) for a ten-day period and observed for at least 150 days. Both male and female animals in the 1- and 5-day age group treated with MSG showed large increases in weight over controls along with a shortened body length. The MSG group also showed decreases in locomotor and exploratory behavior. The 25-day animals took much longer to show effects or failed to show any effects, indicating that the MSG-induced changes studied are age dependent. Possible methodological considerations accounting for conflicting reports in the MSG literature are discussed in light of the present findings. Monosodium glutamate
Obesity
Activity
Developmental neurobiology
RECENT reports have implicated monosodium glutamate (MSG) in neuronal damage of the central nervous system [12, 19, 20, 21, 22, 23, 24]. While these anatomical findings have not gone uncriticized [2, 4, 32], they have more often been supported, with neuronal damage most often being reported to occur in the arcuate nucleus of the hypothalamus [1, 7, 11, 12, 17, 19, 21, 24, 34]. The thorough study by Lemkey-Johnston and Reynolds [12] has demonstrated neuronal damage in other structures including the preoptic region, the rectum, the dorsolateral surface of the thalamus, the dentate gyrus of the hippocampus, the habenular nucleus, the subfornical organ, and the cortex. Along with these CNS structures, the retina has been shown to be susceptible to damage by MSG [8, 14, 20]. Concomitant with this CNS damage there have been reports of a number of developmental, physiological, and behavioral abnormalities including obesity [3, 16, 19], stunted skeletal growth [ 3, 16, 19 ], sterility in females [ 19, 30], disruption of the electroretinogram [26], decreased and increased activity levels [3,19], and learning deficits [5,27]. The present study was designed to examine several of the developmental, physiological, and behavioral c o m plications which have been reported following neonatal administration of MSG; namely, obesity, skeletal abnormalities, and changes in activity levels. There have been a number of contradictory reports regarding MSG-induced obesity and changes in activity levels. Several studies [3, 16, 19] have reported the development of obesity and stunted skeletal structure following administration of MSG to neonatal mice. Failure to find MSG-induced obesity has also been reported by a number of investigators [9, 13, 15,
25, 29, 32, 33]. It should be pointed out that the studies reporting negative results differ in method of administration, age of treatment, and length of the followup period from those yielding positive findings. One investigation [9] which failed to find significant weight gains in animals treated with MSG did report that the carcass fat content was increased to approximately twice that of control animals. A second study in that laboratory [10] found that MSG-treated animals expend significantly less energy than controls as measured by oxygen consumption and carbon dioxide production. This last finding seems consistent with Olney's [19] observation that animals treated with MSG as neonates were lethargic as adults, but inconsistent with Araujo and Mayer's [3] finding of increased activity. The experiments reported on in this paper will attempt to resolve the inconsistencies mentioned above by using animals at several stages of development and by the use of a battery of activity measures in the adult mouse. Further, these experiments will explore the possibility that MSGinduced abnormalities are an age-dependent phenonmenon. Based on earlier studies in this laboratory with oral administration of MSG in rats, an experimental obesity is predicted if the followup period is of sufficient length. METHOD
Animals Animals were 178 albino mouse pups from 19 litters. All animals were born in the laboratory and housed in standard polycarbonate cages with their dams. Weaning and sexing was carried out when the animals reached 29 days of age 551
552
PIZZI AND BARNHART
w h i c h is c o n s i d e r e d o p t i m a l for t h e h e a l t h of t h e m o u s e [ 3 5 ] . All a n i m a l s were fed a s t a n d a r d l a b o r a t o r y c h o w and given w a t e r ad lib. Lighting was r e g u a l t e d at 12 h r o n an 12 hr off t h r o u g h o u t t h e e x p e r i m e n t .
Drug Treatment Mouse pups 1, 5, a n d 25 days of age were i n j e c t e d s u b c u t a n e o u s l y for 10 days w i t h an a q u e o u s s o l u t i o n (10% w/v) o f m o n o s o d i u m g l u t a m a t e a c c o r d i n g to a m o d i f i e d dose s c h e d u l e described by P o t t s , Modrell, and K i n g s b u r y [ 2 6 ] . A n i m a l s i n j e c t e d at o n e day of age b e g a n at a dose o f 2.2 mg/g b o d y weight w i t h doses of 2.5, 2.8, a n d 3.2 m g / g b o d y weight o n Days 2, 3, a n d 4 respectively. The dose o n Days 5 t h r o u g h 17 s t a r t e d at 3.4 mg/g b o d y w e i g h t and increased b y 0.2 mg/g b o d y w e i g h t to 5.8 m g / g b o d y weight. T h e 25-day g r o u p received t e n c o n s e c u t i v e doses at 5.8 mg/g b o d y weight. L i t t e r m a t e c o n t r o l s were i n j e c t e d w i t h e q u a l v o l u m e s of b a c t e r i o s t a t i c water. F o l l o w i n g the i n j e c t i o n s animals were weighed every 5 days o n an e l e c t r o n i c balance. A n i m a l s were placed in a p a p e r cup to r e d u c e m o v e m e n t s , a n d weighed to a h u n d r e d t h of a gram. G r o w t h curves were f o l l o w e d for a m i n i m u m of 150 days a n d in some cases as long as 250 days.
Activity T h r e e m e t h o d s were used to r e c o r d activity: (1) the s t a n d a r d a c t i v i t y wheel; (2) the o p e n field m e t h o d ; (3) t h e Boissier a n d S i m o n Poke Test [ 6 ] . All activity m e a s u r e s were t a k e n b e t w e e n 150 a n d 200 days of age u n d e r ad lib f o o d a n d w a t e r c o n d i t i o n s . E a c h a n i m a l was placed in an activity w h e e l for a 2 4 - h r p e r i o d w i t h f o o d a n d w a t e r available. No later t h a n five days f o l l o w i n g t h i s first measure t h e same a n i m a l s were t e s t e d in t h e o p e n field s i t u a t i o n . T h i s m e a s u r e c o n s i s t e d of placing each m o u s e in the c e n t e r of a 76 x 76 cm b o x w h i c h was lined o f f t o m a k e u p 36 squares. T h e m e a s u r e o f activity was the t o t a l n u m b e r o f squares e n t e r e d in a 3-rain period. T h e c r i t e r i o n for e n t r a n c e i n t o a square c o n s i s t e d of t h e a n i m a l p u t t i n g its t w o f r o n t paws i n t o a p a r t i c u l a r square. T h e a p p a r a t u s for t h e p o k e test was a m o d i f i c a t i o n of t h a t described b y Boissier and S i m o n [ 6 ] . It consisted of a black p l y w o o d b o a r d of 37.5 × 37.5 × 1.3 cm in w h i c h were located 16 e q u i d i s t a n t holes w i t h a d i a m e t e r of 4.5 cm. T h e b o a r d was s u r r o u n d e d b y walls w h i c h s u s p e n d e d it 35.0 cm a b o v e the floor. T h e walls e x t e n d e d 20.5 cm a b o v e the p o k e b o a r d surface. E a c h a n i m a l was placed in t h e c e n t e r of t h e b o a r d and the n u m b e r of p o k e s r e c o r d e d over a 5-min period. A poke was d e f i n e d as t h e i n s e r t i o n of the a n i m a l ' s h e a d , past the level of its eyes, i n t o a n y hole. A t t h e c o n c l u s i o n o f t h e e x p e r i m e n t s t h e a n i m a l s were a n e s t h e t i z e d and b o d y l e n g t h was calculated as the d i s t a n c e f r o m the tip of the s n o u t t o the anal orifice.
RESULTS
Obesity and Somatic Development Figure 1 shows the m e a n b o d y weights of the MSGt r e a t e d mice versus t h e c o n t r o l s p l o t t e d each 10 days over the test period. The M S G - t r e a t e d a n i m a l s s h o w e d a greater weight gain in every g r o u p w i t h the e x c e p t i o n of the 25-day females. While a n i m a l s were assigned r a n d o m l y to the MSG and c o n t r o l groups, it s h o u l d be n o t e d t h a t n o n e of t h e MSG groups weighed m o r e at 30 days o f age. The p a t t e r n
of weight gain was c h a r a c t e r i z e d b y being slow and steady, and s h o w e d n o signs of a b a t i n g at 2 0 0 days of age. An A N O V A was p e r f o r m e d on the last 80 days of each g r o u p in Fig. 1. The n e o n a t a l groups s h o w e d highly significant weight differences (1-day males, F(15,280)=11.692, p < 0 . 0 0 1 ; 1-day females, F ( 1 5 , 2 3 1 ) = 7 . 3 0 5 , p < 0 . 0 0 1 ; 5-day males, F(1 5,192)=5.190, p<0.001; 5-day females, F ( 1 5 , 1 5 1 ) = 9 . 3 3 7 , p < 0 . 0 0 0 1 ) , while t h e m a t u r e animals s h o w e d m i x e d effects (25-day males, F ( 1 5 , 1 2 8 ) = 1 . 8 5 4 , p < 0 . 0 5 ; 25-day females, F ( 1 5 , 1 2 0 ) = 1 . 5 7 5 , p = NS). F u r t h e r analyses utilized a priori c o n t r a s t s [ 1 8 ] . The dashed vertical lines in Fig. 1 i n d i c a t e the p o i n t at w h i c h t h e M S G - t r e a t e d animals b e c a m e significantly heavier. It s h o u l d be e m p h a s i z e d t h a t every pair of p o i n t s to the right of t h e dashed vertical lines show a significant difference as d e m o n s t r a t e d b y a priori c o n t r a s t analysis. No line is s h o w n for the 25-day males since only t w o pairs of p o i n t s were f o u n d to be significantly d i f f e r e n t - these were at 190 and 210 days of age. Thus, animals i n j e c t e d at an earlier age t e n d e d to show o b e s i t y s o o n e r t h a n t h o s e injected at an older age. The l - d a y g r o u p s t o o k an average of l l 0 days following the first i n j e c t i o n to display significant differences in weight, while the 5-day groups averaged 135 days, and the male 25-day g r o u p s h o w e d significance only a f t e r 190 days. The 25-day MSG-female group failed to r e a c h significance or even equal the c o n t r o l a n i m a l s ' weights a f t e r 2 5 0 days. Figure 2 shows a c o n t r o l female m o u s e f r o m the 1-day g r o u p a n d a r e p r e s e n t a t i v e obese female m o u s e t r e a t e d w i t h MSG from day one to day ten. Table 1 shows the m e a n b o d y lengths for M S G - t r e a t e d and c o n t r o l animals in the various age and sex groups. All 1and 5-day M S G - t r e a t e d groups, b o t h male and female, were significantly s h o r t e r in b o d y length. The 25-day groups failed to s h o w a significant decrease in b o d y length. M o r b i d i t y rates were calculated f r o m the first day of i n j e c t i o n to 250 days of age for each of the three age groups of MSG a n d c o n t r o l animals. Results s h o w e d t h a t animals i n j e c t e d with MSG at 1 day of age e x h i b i t e d a 25.5% m o r b i d i t y rate. MSG animals injected at 5 days of age s h o w e d a m o r b i d i t y rate of 30.0%, and those administered MSG at 25 days of age s h o w e d a 1 0 . 5 ~ m o r b i d i t y rate. The m o r b i d i t y rate of all three age groups of l i t t e r m a t e c o n t r o l animals was relatively h o m o g e n e o u s at a p p r o x i m a t e l y 10%.
Activity Visual i n s p e c t i o n of t h e M S G - t r e a t e d animals b o t h in t h e i r h o m e cages a n d o n a flat surface i n d i c a t e d t h a t t h e y were lethargic. Figure 3 s h o w s t h e results of t h r e e f o r m a l m e a s u r e s of activity and e x p l o r a t o r y behavior. T h e 1-day group t r e a t e d w i t h MSG s h o w e d decreases in activity on all t h r e e m e a s u r e s ; h o w e v e r , o n l y the M S G - t r e a t e d males s h o w e d a decrease o n the wheel r u n n i n g test. I n s p e c t i o n of Figure 3 shows t h e l- a n d 5-day g r o u p s t r e a t e d w i t h MSG always s h o w lower m e a n scores o n t h e wheel r u n n i n g measure, b u t due to t h e large v a r i a t i o n s w i t h i n groups, these differences were n o t significant. In t h e o p e n field test b o t h male a n d female animals t r e a t e d w i t h MSG starting at 1-day of age s h o w e d decreased a c t i v i t y and e x p l o r a t o r y b e h a v i o r ( p < 0 . 0 0 1 , two-tailed). No significant d i f f e r e n c e s were f o u n d in animals t r e a t e d w i t h MSG at 5 or 25 days of age. All 1-day a n d 5-day groups t r e a t e d w i t h MSG s h o w e d decreased e x p l o r a t o r y b e h a v i o r o n t h e Boissier Poke Test ( p < 0.001, two-tailed). T h e 25-day males t r e a t e d w i t h MSG
MSG, SOMATIC DEVELOPMENT, OBESITY AND ACTIVITY
1- D A Y
•
MSG
o
CONTROL
MALE
553
(N=18)
( N = 20 )
4S
1-DAY
FEMALE
f
•
MSG
0
CONTROL
(N=17) (N=11)
4~
25
20
15 A
10
* i 30 40
i
t 60
=
= i DO
, , 100
i , 120
, i 140
i 160
1C
3'0 4'0
'
60
'
. 100 . .
J'O
. 120 . .
. 140 .
160
•
MSG
Q
CONTROL
E 5-DAY
•© CONTRO" MsG ~N-~0) ( N - 16
45
MALE
v
5-DAY
( N = 11 ) ( N ~ 9 )
FEMALE
40
1"
35
30
m
W
Z
< m
10
i
,
,
30 40
1
i
60
i
|
80
J
,
100
|
!
120
I
I
140
|
•
25-
i
180
DAY
i
180
MSG
i
i 200
30 40
60
80
10G
120
140
160
100
200
(N=10) •
o CONTROL ( N= o >
"
25-
DAY
4O
FEMALE
MSG
( N=10
o CONTROL ( N = ~'~
35
X
25
2O
t5
15
10
, 30
,
, 50
,
, 70
•
| |0
•
| 110
|
, 130
=
t IN
i
AGE
, 170
i
, , 100
• |10
i
(days
, • , 230 250
I0
from
* 30
•
• 50
t
birth
* 70
|
|
,
90
t t i 110 130
)
FIG. 1. Mean weights of MSG-treated and control groups plotted in 10-day intervals.
|
• t 190
• 1"0
*
. NO
*
• 210
•
| 230
i--
~o
554
PIZZI AND B A R N H A R T
FIG. 2. A. Randomly selected female mice from the one-day age group. The control female mouse (right) was treated with the control vehicle from Days 1-10, while the obese mouse (left) was treated with MSG from Days l 10. This particular pair of mice do not show the reduced body length referred to in this paper. B. Male mice from the 1-day age group following the treatment described above. The MSG-treated animal (left) shows an extreme accumulation of adipose tissue and reduced body length.
TABLE1
DISCUSSION
MEAN BODY LENGTHS (IN MM) OF MSG-TREATED AND CONTROL ANIMALS MEASURED FROM THE TIP OF THE SNOUT TO THE ANUS (ALL MEASURES TAKEN BETWEEN 259 AND 269 DAYS OF AGE)
1-Day 5-Day 25-Day
Sex
N
M F M F M F
19 11 7 7 8 8
Control 106.7 108.6 103.1 98.4 110.4 111.3
(_ 1.05) (_+ 0.63) (+ 1.40) (_+ 1.13) (_+ 1.84) (_+ 1.09)
N
MSG-treated
17 14 7 10 10 7
100.8(_+ 1.60)? 102.0(_+ 1.31)$ 98.1 (_+ 1.51)* 92.9 (_+ 1.89)* 109.2(_+ 0.94) 108.0(_+ 2.57)
*p <0.05. tp<0.01. :~p<0.001. Number in parenthesis = SEM. All probability values are two-tailed.
also s h o w e d a significant decrease on this task ( p < 0 . 0 1 , two-tailed). The p a t t e r n o f e f f e c t s in this e x p e r i m e n t are similar to t h o s e f o u n d in the first e x p e r i m e n t , w i t h t h e animals t r e a t e d at an earlier age m o r e readily s h o w i n g an effect.
The data g a t h e r e d in this s t u d y s u p p o r t the findings of those investigators w h o have r e p o r t e d increased weight gain and decreased b o d y length following n e o n a t a l administration o f MSG. All animals t r e a t e d at 1 or 5 days o f age w h e t h e r male or female s h o w e d the effect. Those animals t r e a t e d later in the course of d e v e l o p m e n t s h o w e d the weight gains m u c h later or n o t at all. In t h o s e studies failing to r e p o r t increased weight gains [25, 31, 35] or t h o s e in which depressed weights are seen [13, 15, 2 5 ] , the length o f t i m e for which the animals are f o l l o w e d b e c o m e s the i m p o r t a n t factor. During the initial a d m i n i s t r a t i o n of MSG and for a period o f time t h e r e a f t e r , the animals lose weight; h o w e v e r , the p h e n o m e n o n is transient and is f o l l o w e d by a slow steady weight gain leading to obesity. The g r o w t h curves m u s t be f o l l o w e d for several m o n t h s to see this effect. F u r t h e r m o r e , if the p h e n o m e n o n is age d e p e n d e n t , as these e x p e r i m e n t s suggest, then m e t h o d and age o f MSG t r e a t m e n t are critical factors. In t h o s e studies using rat c h o w with various a m o u n t s o f MSG a d d e d [15, 31, 3 3 ] , one would n o t e x p e c t to see an e f f e c t since adult animals are b e y o n d the critical age and m o u s e and rat pups do n o t begin to eat solid f o o d s until after t h e y have passed the critical age. A n u m b e r o f studies [15, 28, 29] in w h i c h physiological and e n d o c r i n e factors were of p r i m a r y concern have r e p o r t e d the initial weight loss but t h e n failed to follow the g r o w t h curves for a sufficient length o f time for
MSG, SOMATIC DEVELOPMENT,
OBESITY AND ACTIVITY
555
WHEEL RUNNING
WHEEL RUNNING 2400 3200 3000
_o
~
CONTROL B
2800
MSG
2600 O. 2400 rr 2200
uJ D.
NS
O "Mr i 1400
1200
GO Z n-
I000
I-
400
I-DAYMALES
OPEN FIELD
_o iT LU (~
160 18o 140 130
pc.001
O m
CC
.i et
I'--] c O N T R O L
l~
~
MSG
NS ~3 I~1 nILl I-
~ UI I]~
1-DAYFEMALES S-DAYFEMALES 25-DAYFEMALES
5"DAYMALES 25-DAYMALES
Q ILl rr
6o ~o
~
CONTROL
so 6o
eo
(/) UJ
80 4o 30
n-
30
P<.OOl
I00
7
U~
6o
IBo 140 130 120
NS
.o Ioo i)o
OPEN FIELD
16o
2o
10
Io
I-~Y II~LES
5-~AYMALES
25-~Y BALES
I-DA'ffEMALES S-OA'fFEMALES 25-DAYFEMALES
POKE TEST
POKE TEST
{~ CONTROL
r-"l CONTROL IIIm MSG
14o 1. i
m MSG
~4o
p<.m
6o I It)
p-.0z
6o 80
p<.O01
I'J'l ~
Z
.o
m v
eo I10
m
p<.NI
p~,O01
P.
Io
Io
I-O~r MALES
5"NY BALES
25"0MMALES
I-OAYf I N . l ~
5-m NIULES ~ - ~ ' NMAL[$
FIG. 3. Mean scores on three measures of activity for male and female mice treated with MSG and a control vehicle at 1, 5, and 25 days o f age. The N ' s are the same as those in Fig. 1, excepting t h e 1-day control male group which was reduced by one for all three tests.
556
PIZZI AND B A R N H A R T
the o b e s i t y to m a n i f e s t itself. The l e n g t h o f time for w h i c h the animals m u s t be f o l l o w e d will p r o b a b l y vary f r o m s t u d y to s t u d y due to d i f f e r e n c e s in the strain o f animal and general l a b o r a t o r y p r o c e d u r e s such as l o c a t i o n and t y p e of f o o d , lighting schedules, and h o u s i n g c o n d i t i o n s . However, the f o l l o w u p p e r i o d for investigations o f M S G - i n d u c e d o b e s i t y s h o u l d p r o b a b l y be set at a m i n i m u m o f 120 days. The data r e p o r t e d in this s t u d y c o n f i r m the findings b y o t h e r s [3, 16, 19] o f s h o r t e n e d b o d y l e n g t h in the M S G - t r e a t e d animals. A cursory i n s p e c t i o n o f several x-rays of m a t c h e d e x p e r i m e n t a l and c o n t r o l animals failed to s h o w any severe skeletal d e f o r m i t i e s . The increase in activity r e p o r t e d b y Araujo and Mayer [3] was not c o n f i r m e d in this study. Several investigators have r e p o r t e d M S G - t r e a t e d animals to be lethargic based on i n f o r m a l o b s e r v a t i o n s . This o b s e r v a t i o n was i n d i r e c t l y s u p p o r t e d by the o b s e r v a t i o n s o f Djazayery and Miller [ 1 0 ] , w h o f o u n d t h a t M S G - t r e a t e d animals e x p e n d e d significantly less energy as m e a s u r e d by o x y g e n cons u m p t i o n and c a r b o n d i o x i d e p r o d u c t i o n . The p r e s e n t s t u d y utilized a b a t t e r y o f activity measures w h i c h s h o w e d the M S G - t r e a t e d animals to have l o w e r levels of activity and e x p l o r a t o r y behavior. Again the age at w h i c h the animals are t r e a t e d with MSG seems to be a factor. The animals t r e a t e d at 1 day o f age s h o w e d an e f f e c t o n all t h r e e measures o f activity w i t h t h e e x c e p t i o n o f the females on the w h e e l r u n n i n g test. The p o k e test best d e m o n s t r a t e d the d i f f e r e n c e b e t w e e n MSG-treated animals and controls. The animals t r e a t e d with MSG at 1 and 5 days o f age s h o w e d highly significant decreases in activity on this test. The 25-day males t r e a t e d w i t h MSG s h o w e d a decrease, but the 25-day females did not. The results o f these tests
c o n f i r m the i n f o r m a l o b s e r v a t i o n s of o t h e r investigators that M S G - t r e a t e d animals are lethargic and h y p o a c t i v e . Several i n t e r e s t i n g q u e s t i o n s are raised by the MSG literature and the data r e p o r t e d above. The m o s t i m m e d i a t e q u e s t i o n s c o n c e r n o t h e r physiological and b e h a v i o r i m p a i r m e n t s w h i c h m a y be i n d u c e d by MSG a d m i n i s t e r e d during the n e o n a t a l period. T h e r e have been brief r e p o r t s of e n d o c r i n e d y s f u n c t i o n and sterility in female organisms treated w i t h MSG [19,30] along w i t h several r e p o r t s o f impaired learning [ 5 , 2 7 ] . The data indicating t h a t MSG disrupts the e l e c t r o r e t i n o g r a m e x e m p l i f i e s the need for behavioral studies at various dose levels and stages of d e v e l o p m e n t . Each o f t h e s e topics deserves e x p a n d e d s t u d y and q u a n t i f i c a t i o n . Finally, t h e r e arises the q u e s t i o n as to w h e t h e r MSGi n d u c e d o b e s i t y r e p r e s e n t s an e x a m p l e o f r e g u l a t o r y or m e t a b o l i c o b e s i t y . The slow steady p a t t e r n o f weight gain along w i t h a n u m b e r of r e p o r t s w h i c h failed to show any evidence of h y p e r p h a g i a lend s u p p o r t for a m e t a b o l i c i n t e r p r e t a t i o n . However, Araujo and Mayer [3] c a u t i o n that o n e - q u a r t e r o f a gram o f c h o w per day could a c c o u n t for a l-g per week weight increase, and t h a t this might be missed by the f o o d - m o n i t o r i n g s y s t e m s currently emp l o y e d . This along w i t h the fact t h a t MSG-induced brain lesions can be f o u n d in the a r c u a t e - v e n t r o m e d i a l h y p o thalamic region requires that a careful investigation be m a d e o f these alternative e x p l a n a t i o n s . ACKNOWLEDGEMENT This work was supported in part by a grant from the Committee on Organized Research, Northeastern Illinois University.
REFERENCES 1. Abraham, R., W. Doughtery, L. Goldberg and F. Coulston. The response of the hypothalamus to high doses of monosodium glutamate in mice and monkeys. Cytochemistry and ultrastructural study of lysosomal changes. Expl. Molec. Path. 15: 43 - 60, 1971. 2. Adamo, N. J. and A. Ratner. Monosodium glutamate: Lack of effects on brain and reproductive function in rats. Science 169: 673 - 674, 1970. 3. Araujo, P. E. and J. Mayer. Activity increase associated with obesity induced by monosodium glutamate in mice. Am. J. PhysioL 225:764 765, 1973. 4. Arees, E. and J. Mayer. Monosodium glutamate-induced brain lesions: Electron microscope examination. Science 170:549 - 5 5 0 , 1970. 5. Berry, H. K., R. E. Butcher, L. A. Elliot and R. L. Brunner. The effect of monosodium glutamate on the early biochemical and behavioral development of the rat. Devl Pwchobiol. 1: 165 - 173, 1974. 6. Boissier, J. R. and P. Simon. Dissociation a deux composantes le comportment d'investigation de la souris. Archs int. Pharmacodyn. ThOr. 147:372 - 387, 1964. 7. Burde, R. M., B. Schainker and J. Kayes. Monosodium glutamate: Acute effect of oral and subcutaneous administration on the arcuate nucleus of the hypothalamus in mice and rats. Nature, (Lond.), 233:58 - 60, 1971. 8. Cohen, A. I. An electron microscopic study of the modification by monosodium glutamate of the retinas of normal and rodless mice. Am. J. Anat. 120: 319, 1967. 9. Djazayery, A. and D. S. Miller. The use of gold-thioglucose and monosodium glutamate to induce obesity in mice. Proc. Nutr. Soc. 32: 30A, 1973. 10. Djazayery, A., D. S. Miller and M. J. Stock. Energy balances of mice treated with gold-thioglucose and monosodium glutamate. Proc. Nutr. Soc. 32: 31A, 1973.
11. Everly, J. L. Light microscopic examination of MSG-induced lesions in brain of fetal and neonatal rats. Anat. Rec. 169:312, 1971. 12. Lemkey-Johnston, N. and W. A. Reynolds. Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate: A light and electron microscope study. J. Neuropath. exp. Neurol. 33:74 97, 1974. 13. Lewis, L. M., J. F. Lynch and J. S. Adkins. Effect of protein level and source on the growth of rats fed excess monosodium L-glutamate. Fedn Proc. 29:567 abs, 1970. 14. Lucas, D. R. and J. P. Newhouse. The toxic effect of sodium L-glutamate on the inner layers of the retina. Archs Ophthal. 58:193 - 201, 1957. 15. Lynch, J. F., L. M. Lewis, E. L. Hove and J. S. Adkins. Effect of monosodium L-glutamate on development and reproduction in rats. FedrProc. 29:567 abs, 1970. 16. Matsuyan, S. Studies on experimental obesity in mice treated with monosodium glutamate. ,lap. J. Vet. Sci. 32: 206, 1970. 17. Murakami, U. and M. Inouye. Brain lesions in the mouse fetus caused by maternal administration of monosodium glutamate. CongenitalAnomalies, 11:171 - 177, 1971. 18. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner and D. H. Bent. SPSS. Statistical Package for the Social Sciences. New York: McGraw-Hill, 1975, p. 425. 19. Olney, J. W. Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate. Science 164:719 721, 1969. 20. Olney, J. W. Glutamate-induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion. J. Neuropath. exp. NeuroL 28:455 - 474, 1969. 21. Olney, J. W. and O. L. Ho. Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature (Lond.), 227:609 - 610, 1970.
MSG, SOMATIC D E V E L O P M E N T , OBESITY AND A C T I V I T Y 22. Olney, J. W. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus: An electron microscopic study. J. Neuropath. exp. NeuroL 30:75 - 90, 1971. 23. Olney, J. W., O. L. Ho and V. Rhee. Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system. Expl Brain Res. 14:61 - 76,1971. 24. Olney, J. W., L. G. Sharpe and R. D. Feigin. Glutamateinduced brain damage in infant primates. J. Neuropath. exp. Neurol. 31:464 - 488, 1972. 25. Oser, B. L., S. Carson, E. E. Vogin and G. E. Cox. Oral and subcutaneous administration of monosodium glutamate on infant rodents and dogs. Nature 229:411 - 413, 1971. 26. Ports, A. M., K. W. Modrell and C. Kingsbury. Permanent fractionation of the ERG by sodium glutamate. Am. J. Ophthal. 50:900 - 907, 1960. 27. Pradhan, S. N. and J. F. Lynch Jr. Behavioral changes in adult rats treated with monosodium glutamate in the neonatal stage. Archs int. Pharmacodyn. Thbr. 197:301 - 304, 1972. 28. Redding, T. W. and A. V. Schally. Effect of monosodium glutamate on the endocrine axis in rats. Fedr Proc. 2 9 : 3 7 8 abs. 1970. 29. Redding, T. W., A. V. Schally, A. Arimura and I. Wakaboyashi. Effects of monosodium glutamate on some endocrine functions. Neuroendocrinology 8 : 2 4 5 - 2 5 6 , 1 9 7 1 .
557
30. Reynolds, W. A., A. S. Bingel and N. Lemkey-Johnston. Female sterility in the MSG-induced hypothalamic lesion of the mouse. Society for Gynecological Investigation. 19th Meeting, Abstracts, p. 46, 1972. 31. Semprini, M. E., M. A. Frasca and A. Mariani. Effects of monosodium glutamate administration on rats during the intrauterine and neonatal period. Quat. Nutr. 3 1 : 8 5 - 100, 1971. 32. Semprini, M. E., L. Conti, A. Ciofi-Luzzatto and A. Mariani. Effect of oral administration of monosodium glutamate (MSG) on the hypothalamic arcuate region of rat and mouse: A histological assay. Biomedicine 21:398 - 403, 1974. 33. Semprini, M. E., A. D'Amicis and A. Mariani. Effect of monosodium glutamate on fetus and newborn mouse. Nutr. Metabol. 16:276 - 284, 1974. 34. Shimizu, K., A. Mizutani and M. Inouye. Electron microscopic studies on the hypothalamic lesions in the mouse fetus caused by monosodium glutamate. Teratology 8: 105, 1973. 35. Strong, L. C. The care of experimental mice. In: The Care and Breeding of Laboratory Animals edited by E. J. Farris. New York: John Wiley and Sons, 1950, pp. 79 96.
Effects of Monosodium Glutamate on Somatic Development, Obesity and Activity in the Mouse WILLIAM J. PIZZI AND JUNE E. BARNHART
Department o f Psychology, Northeastern Illinois University, Chicago, IL 60625 (Received 7 October 1975) PIZZI, W. J. AND J. E. BARNHART. Effects o f monosodium glutamate on somatic development, obesity and activity in the mouse. PHARMAC. BIOCHEM. BEHAV. 5(5) 551-557, 1976. - Neonatal mice 1 and 5 days of age and older mice 25 days of age were injected with an increasing dose of monosodium glutamate (MSG) for a ten-day period and observed for at least 150 days. Both male and female animals in the 1- and 5-day age group treated with MSG showed large increases in weight over controls along with a shortened body length. The MSG group also showed decreases in locomotor and exploratory behavior. The 25-day animals took much longer to show effects or failed to show any effects, indicating that the MSG-induced changes studied are age dependent. Possible methodological considerations accounting for conflicting reports in the MSG literature are discussed in light of the present findings. Monosodium glutamate
Obesity
Activity
Developmental neurobiology
RECENT reports have implicated monosodium glutamate (MSG) in neuronal damage of the central nervous system [12, 19, 20, 21, 22, 23, 24]. While these anatomical findings have not gone uncriticized [2, 4, 32], they have more often been supported, with neuronal damage most often being reported to occur in the arcuate nucleus of the hypothalamus [1, 7, 11, 12, 17, 19, 21, 24, 34]. The thorough study by Lemkey-Johnston and Reynolds [12] has demonstrated neuronal damage in other structures including the preoptic region, the rectum, the dorsolateral surface of the thalamus, the dentate gyrus of the hippocampus, the habenular nucleus, the subfornical organ, and the cortex. Along with these CNS structures, the retina has been shown to be susceptible to damage by MSG [8, 14, 20]. Concomitant with this CNS damage there have been reports of a number of developmental, physiological, and behavioral abnormalities including obesity [3, 16, 19], stunted skeletal growth [ 3, 16, 19 ], sterility in females [ 19, 30], disruption of the electroretinogram [26], decreased and increased activity levels [3,19], and learning deficits [5,27]. The present study was designed to examine several of the developmental, physiological, and behavioral c o m plications which have been reported following neonatal administration of MSG; namely, obesity, skeletal abnormalities, and changes in activity levels. There have been a number of contradictory reports regarding MSG-induced obesity and changes in activity levels. Several studies [3, 16, 19] have reported the development of obesity and stunted skeletal structure following administration of MSG to neonatal mice. Failure to find MSG-induced obesity has also been reported by a number of investigators [9, 13, 15,
25, 29, 32, 33]. It should be pointed out that the studies reporting negative results differ in method of administration, age of treatment, and length of the followup period from those yielding positive findings. One investigation [9] which failed to find significant weight gains in animals treated with MSG did report that the carcass fat content was increased to approximately twice that of control animals. A second study in that laboratory [10] found that MSG-treated animals expend significantly less energy than controls as measured by oxygen consumption and carbon dioxide production. This last finding seems consistent with Olney's [19] observation that animals treated with MSG as neonates were lethargic as adults, but inconsistent with Araujo and Mayer's [3] finding of increased activity. The experiments reported on in this paper will attempt to resolve the inconsistencies mentioned above by using animals at several stages of development and by the use of a battery of activity measures in the adult mouse. Further, these experiments will explore the possibility that MSGinduced abnormalities are an age-dependent phenonmenon. Based on earlier studies in this laboratory with oral administration of MSG in rats, an experimental obesity is predicted if the followup period is of sufficient length. METHOD
Animals Animals were 178 albino mouse pups from 19 litters. All animals were born in the laboratory and housed in standard polycarbonate cages with their dams. Weaning and sexing was carried out when the animals reached 29 days of age 551
552
PIZZI AND BARNHART
w h i c h is c o n s i d e r e d o p t i m a l for t h e h e a l t h of t h e m o u s e [ 3 5 ] . All a n i m a l s were fed a s t a n d a r d l a b o r a t o r y c h o w and given w a t e r ad lib. Lighting was r e g u a l t e d at 12 h r o n an 12 hr off t h r o u g h o u t t h e e x p e r i m e n t .
Drug Treatment Mouse pups 1, 5, a n d 25 days of age were i n j e c t e d s u b c u t a n e o u s l y for 10 days w i t h an a q u e o u s s o l u t i o n (10% w/v) o f m o n o s o d i u m g l u t a m a t e a c c o r d i n g to a m o d i f i e d dose s c h e d u l e described by P o t t s , Modrell, and K i n g s b u r y [ 2 6 ] . A n i m a l s i n j e c t e d at o n e day of age b e g a n at a dose o f 2.2 mg/g b o d y weight w i t h doses of 2.5, 2.8, a n d 3.2 m g / g b o d y weight o n Days 2, 3, a n d 4 respectively. The dose o n Days 5 t h r o u g h 17 s t a r t e d at 3.4 mg/g b o d y w e i g h t and increased b y 0.2 mg/g b o d y w e i g h t to 5.8 m g / g b o d y weight. T h e 25-day g r o u p received t e n c o n s e c u t i v e doses at 5.8 mg/g b o d y weight. L i t t e r m a t e c o n t r o l s were i n j e c t e d w i t h e q u a l v o l u m e s of b a c t e r i o s t a t i c water. F o l l o w i n g the i n j e c t i o n s animals were weighed every 5 days o n an e l e c t r o n i c balance. A n i m a l s were placed in a p a p e r cup to r e d u c e m o v e m e n t s , a n d weighed to a h u n d r e d t h of a gram. G r o w t h curves were f o l l o w e d for a m i n i m u m of 150 days a n d in some cases as long as 250 days.
Activity T h r e e m e t h o d s were used to r e c o r d activity: (1) the s t a n d a r d a c t i v i t y wheel; (2) the o p e n field m e t h o d ; (3) t h e Boissier a n d S i m o n Poke Test [ 6 ] . All activity m e a s u r e s were t a k e n b e t w e e n 150 a n d 200 days of age u n d e r ad lib f o o d a n d w a t e r c o n d i t i o n s . E a c h a n i m a l was placed in an activity w h e e l for a 2 4 - h r p e r i o d w i t h f o o d a n d w a t e r available. No later t h a n five days f o l l o w i n g t h i s first measure t h e same a n i m a l s were t e s t e d in t h e o p e n field s i t u a t i o n . T h i s m e a s u r e c o n s i s t e d of placing each m o u s e in the c e n t e r of a 76 x 76 cm b o x w h i c h was lined o f f t o m a k e u p 36 squares. T h e m e a s u r e o f activity was the t o t a l n u m b e r o f squares e n t e r e d in a 3-rain period. T h e c r i t e r i o n for e n t r a n c e i n t o a square c o n s i s t e d of t h e a n i m a l p u t t i n g its t w o f r o n t paws i n t o a p a r t i c u l a r square. T h e a p p a r a t u s for t h e p o k e test was a m o d i f i c a t i o n of t h a t described b y Boissier and S i m o n [ 6 ] . It consisted of a black p l y w o o d b o a r d of 37.5 × 37.5 × 1.3 cm in w h i c h were located 16 e q u i d i s t a n t holes w i t h a d i a m e t e r of 4.5 cm. T h e b o a r d was s u r r o u n d e d b y walls w h i c h s u s p e n d e d it 35.0 cm a b o v e the floor. T h e walls e x t e n d e d 20.5 cm a b o v e the p o k e b o a r d surface. E a c h a n i m a l was placed in t h e c e n t e r of t h e b o a r d and the n u m b e r of p o k e s r e c o r d e d over a 5-min period. A poke was d e f i n e d as t h e i n s e r t i o n of the a n i m a l ' s h e a d , past the level of its eyes, i n t o a n y hole. A t t h e c o n c l u s i o n o f t h e e x p e r i m e n t s t h e a n i m a l s were a n e s t h e t i z e d and b o d y l e n g t h was calculated as the d i s t a n c e f r o m the tip of the s n o u t t o the anal orifice.
RESULTS
Obesity and Somatic Development Figure 1 shows the m e a n b o d y weights of the MSGt r e a t e d mice versus t h e c o n t r o l s p l o t t e d each 10 days over the test period. The M S G - t r e a t e d a n i m a l s s h o w e d a greater weight gain in every g r o u p w i t h the e x c e p t i o n of the 25-day females. While a n i m a l s were assigned r a n d o m l y to the MSG and c o n t r o l groups, it s h o u l d be n o t e d t h a t n o n e of t h e MSG groups weighed m o r e at 30 days o f age. The p a t t e r n
of weight gain was c h a r a c t e r i z e d b y being slow and steady, and s h o w e d n o signs of a b a t i n g at 2 0 0 days of age. An A N O V A was p e r f o r m e d on the last 80 days of each g r o u p in Fig. 1. The n e o n a t a l groups s h o w e d highly significant weight differences (1-day males, F(15,280)=11.692, p < 0 . 0 0 1 ; 1-day females, F ( 1 5 , 2 3 1 ) = 7 . 3 0 5 , p < 0 . 0 0 1 ; 5-day males, F(1 5,192)=5.190, p<0.001; 5-day females, F ( 1 5 , 1 5 1 ) = 9 . 3 3 7 , p < 0 . 0 0 0 1 ) , while t h e m a t u r e animals s h o w e d m i x e d effects (25-day males, F ( 1 5 , 1 2 8 ) = 1 . 8 5 4 , p < 0 . 0 5 ; 25-day females, F ( 1 5 , 1 2 0 ) = 1 . 5 7 5 , p = NS). F u r t h e r analyses utilized a priori c o n t r a s t s [ 1 8 ] . The dashed vertical lines in Fig. 1 i n d i c a t e the p o i n t at w h i c h t h e M S G - t r e a t e d animals b e c a m e significantly heavier. It s h o u l d be e m p h a s i z e d t h a t every pair of p o i n t s to the right of t h e dashed vertical lines show a significant difference as d e m o n s t r a t e d b y a priori c o n t r a s t analysis. No line is s h o w n for the 25-day males since only t w o pairs of p o i n t s were f o u n d to be significantly d i f f e r e n t - these were at 190 and 210 days of age. Thus, animals i n j e c t e d at an earlier age t e n d e d to show o b e s i t y s o o n e r t h a n t h o s e injected at an older age. The l - d a y g r o u p s t o o k an average of l l 0 days following the first i n j e c t i o n to display significant differences in weight, while the 5-day groups averaged 135 days, and the male 25-day g r o u p s h o w e d significance only a f t e r 190 days. The 25-day MSG-female group failed to r e a c h significance or even equal the c o n t r o l a n i m a l s ' weights a f t e r 2 5 0 days. Figure 2 shows a c o n t r o l female m o u s e f r o m the 1-day g r o u p a n d a r e p r e s e n t a t i v e obese female m o u s e t r e a t e d w i t h MSG from day one to day ten. Table 1 shows the m e a n b o d y lengths for M S G - t r e a t e d and c o n t r o l animals in the various age and sex groups. All 1and 5-day M S G - t r e a t e d groups, b o t h male and female, were significantly s h o r t e r in b o d y length. The 25-day groups failed to s h o w a significant decrease in b o d y length. M o r b i d i t y rates were calculated f r o m the first day of i n j e c t i o n to 250 days of age for each of the three age groups of MSG a n d c o n t r o l animals. Results s h o w e d t h a t animals i n j e c t e d with MSG at 1 day of age e x h i b i t e d a 25.5% m o r b i d i t y rate. MSG animals injected at 5 days of age s h o w e d a m o r b i d i t y rate of 30.0%, and those administered MSG at 25 days of age s h o w e d a 1 0 . 5 ~ m o r b i d i t y rate. The m o r b i d i t y rate of all three age groups of l i t t e r m a t e c o n t r o l animals was relatively h o m o g e n e o u s at a p p r o x i m a t e l y 10%.
Activity Visual i n s p e c t i o n of t h e M S G - t r e a t e d animals b o t h in t h e i r h o m e cages a n d o n a flat surface i n d i c a t e d t h a t t h e y were lethargic. Figure 3 s h o w s t h e results of t h r e e f o r m a l m e a s u r e s of activity and e x p l o r a t o r y behavior. T h e 1-day group t r e a t e d w i t h MSG s h o w e d decreases in activity on all t h r e e m e a s u r e s ; h o w e v e r , o n l y the M S G - t r e a t e d males s h o w e d a decrease o n the wheel r u n n i n g test. I n s p e c t i o n of Figure 3 shows t h e l- a n d 5-day g r o u p s t r e a t e d w i t h MSG always s h o w lower m e a n scores o n t h e wheel r u n n i n g measure, b u t due to t h e large v a r i a t i o n s w i t h i n groups, these differences were n o t significant. In t h e o p e n field test b o t h male a n d female animals t r e a t e d w i t h MSG starting at 1-day of age s h o w e d decreased a c t i v i t y and e x p l o r a t o r y b e h a v i o r ( p < 0 . 0 0 1 , two-tailed). No significant d i f f e r e n c e s were f o u n d in animals t r e a t e d w i t h MSG at 5 or 25 days of age. All 1-day a n d 5-day groups t r e a t e d w i t h MSG s h o w e d decreased e x p l o r a t o r y b e h a v i o r o n t h e Boissier Poke Test ( p < 0.001, two-tailed). T h e 25-day males t r e a t e d w i t h MSG
MSG, SOMATIC DEVELOPMENT, OBESITY AND ACTIVITY
1- D A Y
•
MSG
o
CONTROL
MALE
553
(N=18)
( N = 20 )
4S
1-DAY
FEMALE
f
•
MSG
0
CONTROL
(N=17) (N=11)
4~
25
20
15 A
10
* i 30 40
i
t 60
=
= i DO
, , 100
i , 120
, i 140
i 160
1C
3'0 4'0
'
60
'
. 100 . .
J'O
. 120 . .
. 140 .
160
•
MSG
Q
CONTROL
E 5-DAY
•© CONTRO" MsG ~N-~0) ( N - 16
45
MALE
v
5-DAY
( N = 11 ) ( N ~ 9 )
FEMALE
40
1"
35
30
m
W
Z
< m
10
i
,
,
30 40
1
i
60
i
|
80
J
,
100
|
!
120
I
I
140
|
•
25-
i
180
DAY
i
180
MSG
i
i 200
30 40
60
80
10G
120
140
160
100
200
(N=10) •
o CONTROL ( N= o >
"
25-
DAY
4O
FEMALE
MSG
( N=10
o CONTROL ( N = ~'~
35
X
25
2O
t5
15
10
, 30
,
, 50
,
, 70
•
| |0
•
| 110
|
, 130
=
t IN
i
AGE
, 170
i
, , 100
• |10
i
(days
, • , 230 250
I0
from
* 30
•
• 50
t
birth
* 70
|
|
,
90
t t i 110 130
)
FIG. 1. Mean weights of MSG-treated and control groups plotted in 10-day intervals.
|
• t 190
• 1"0
*
. NO
*
• 210
•
| 230
i--
~o
554
PIZZI AND B A R N H A R T
FIG. 2. A. Randomly selected female mice from the one-day age group. The control female mouse (right) was treated with the control vehicle from Days 1-10, while the obese mouse (left) was treated with MSG from Days l 10. This particular pair of mice do not show the reduced body length referred to in this paper. B. Male mice from the 1-day age group following the treatment described above. The MSG-treated animal (left) shows an extreme accumulation of adipose tissue and reduced body length.
TABLE1
DISCUSSION
MEAN BODY LENGTHS (IN MM) OF MSG-TREATED AND CONTROL ANIMALS MEASURED FROM THE TIP OF THE SNOUT TO THE ANUS (ALL MEASURES TAKEN BETWEEN 259 AND 269 DAYS OF AGE)
1-Day 5-Day 25-Day
Sex
N
M F M F M F
19 11 7 7 8 8
Control 106.7 108.6 103.1 98.4 110.4 111.3
(_ 1.05) (_+ 0.63) (+ 1.40) (_+ 1.13) (_+ 1.84) (_+ 1.09)
N
MSG-treated
17 14 7 10 10 7
100.8(_+ 1.60)? 102.0(_+ 1.31)$ 98.1 (_+ 1.51)* 92.9 (_+ 1.89)* 109.2(_+ 0.94) 108.0(_+ 2.57)
*p <0.05. tp<0.01. :~p<0.001. Number in parenthesis = SEM. All probability values are two-tailed.
also s h o w e d a significant decrease on this task ( p < 0 . 0 1 , two-tailed). The p a t t e r n o f e f f e c t s in this e x p e r i m e n t are similar to t h o s e f o u n d in the first e x p e r i m e n t , w i t h t h e animals t r e a t e d at an earlier age m o r e readily s h o w i n g an effect.
The data g a t h e r e d in this s t u d y s u p p o r t the findings of those investigators w h o have r e p o r t e d increased weight gain and decreased b o d y length following n e o n a t a l administration o f MSG. All animals t r e a t e d at 1 or 5 days o f age w h e t h e r male or female s h o w e d the effect. Those animals t r e a t e d later in the course of d e v e l o p m e n t s h o w e d the weight gains m u c h later or n o t at all. In t h o s e studies failing to r e p o r t increased weight gains [25, 31, 35] or t h o s e in which depressed weights are seen [13, 15, 2 5 ] , the length o f t i m e for which the animals are f o l l o w e d b e c o m e s the i m p o r t a n t factor. During the initial a d m i n i s t r a t i o n of MSG and for a period o f time t h e r e a f t e r , the animals lose weight; h o w e v e r , the p h e n o m e n o n is transient and is f o l l o w e d by a slow steady weight gain leading to obesity. The g r o w t h curves m u s t be f o l l o w e d for several m o n t h s to see this effect. F u r t h e r m o r e , if the p h e n o m e n o n is age d e p e n d e n t , as these e x p e r i m e n t s suggest, then m e t h o d and age o f MSG t r e a t m e n t are critical factors. In t h o s e studies using rat c h o w with various a m o u n t s o f MSG a d d e d [15, 31, 3 3 ] , one would n o t e x p e c t to see an e f f e c t since adult animals are b e y o n d the critical age and m o u s e and rat pups do n o t begin to eat solid f o o d s until after t h e y have passed the critical age. A n u m b e r o f studies [15, 28, 29] in w h i c h physiological and e n d o c r i n e factors were of p r i m a r y concern have r e p o r t e d the initial weight loss but t h e n failed to follow the g r o w t h curves for a sufficient length o f time for
MSG, SOMATIC DEVELOPMENT,
OBESITY AND ACTIVITY
555
WHEEL RUNNING
WHEEL RUNNING 2400 3200 3000
_o
~
CONTROL B
2800
MSG
2600 O. 2400 rr 2200
uJ D.
NS
O "Mr i 1400
1200
GO Z n-
I000
I-
400
I-DAYMALES
OPEN FIELD
_o iT LU (~
160 18o 140 130
pc.001
O m
CC
.i et
I'--] c O N T R O L
l~
~
MSG
NS ~3 I~1 nILl I-
~ UI I]~
1-DAYFEMALES S-DAYFEMALES 25-DAYFEMALES
5"DAYMALES 25-DAYMALES
Q ILl rr
6o ~o
~
CONTROL
so 6o
eo
(/) UJ
80 4o 30
n-
30
P<.OOl
I00
7
U~
6o
IBo 140 130 120
NS
.o Ioo i)o
OPEN FIELD
16o
2o
10
Io
I-~Y II~LES
5-~AYMALES
25-~Y BALES
I-DA'ffEMALES S-OA'fFEMALES 25-DAYFEMALES
POKE TEST
POKE TEST
{~ CONTROL
r-"l CONTROL IIIm MSG
14o 1. i
m MSG
~4o
p<.m
6o I It)
p-.0z
6o 80
p<.O01
I'J'l ~
Z
.o
m v
eo I10
m
p<.NI
p~,O01
P.
Io
Io
I-O~r MALES
5"NY BALES
25"0MMALES
I-OAYf I N . l ~
5-m NIULES ~ - ~ ' NMAL[$
FIG. 3. Mean scores on three measures of activity for male and female mice treated with MSG and a control vehicle at 1, 5, and 25 days o f age. The N ' s are the same as those in Fig. 1, excepting t h e 1-day control male group which was reduced by one for all three tests.
556
PIZZI AND B A R N H A R T
the o b e s i t y to m a n i f e s t itself. The l e n g t h o f time for w h i c h the animals m u s t be f o l l o w e d will p r o b a b l y vary f r o m s t u d y to s t u d y due to d i f f e r e n c e s in the strain o f animal and general l a b o r a t o r y p r o c e d u r e s such as l o c a t i o n and t y p e of f o o d , lighting schedules, and h o u s i n g c o n d i t i o n s . However, the f o l l o w u p p e r i o d for investigations o f M S G - i n d u c e d o b e s i t y s h o u l d p r o b a b l y be set at a m i n i m u m o f 120 days. The data r e p o r t e d in this s t u d y c o n f i r m the findings b y o t h e r s [3, 16, 19] o f s h o r t e n e d b o d y l e n g t h in the M S G - t r e a t e d animals. A cursory i n s p e c t i o n o f several x-rays of m a t c h e d e x p e r i m e n t a l and c o n t r o l animals failed to s h o w any severe skeletal d e f o r m i t i e s . The increase in activity r e p o r t e d b y Araujo and Mayer [3] was not c o n f i r m e d in this study. Several investigators have r e p o r t e d M S G - t r e a t e d animals to be lethargic based on i n f o r m a l o b s e r v a t i o n s . This o b s e r v a t i o n was i n d i r e c t l y s u p p o r t e d by the o b s e r v a t i o n s o f Djazayery and Miller [ 1 0 ] , w h o f o u n d t h a t M S G - t r e a t e d animals e x p e n d e d significantly less energy as m e a s u r e d by o x y g e n cons u m p t i o n and c a r b o n d i o x i d e p r o d u c t i o n . The p r e s e n t s t u d y utilized a b a t t e r y o f activity measures w h i c h s h o w e d the M S G - t r e a t e d animals to have l o w e r levels of activity and e x p l o r a t o r y behavior. Again the age at w h i c h the animals are t r e a t e d with MSG seems to be a factor. The animals t r e a t e d at 1 day o f age s h o w e d an e f f e c t o n all t h r e e measures o f activity w i t h t h e e x c e p t i o n o f the females on the w h e e l r u n n i n g test. The p o k e test best d e m o n s t r a t e d the d i f f e r e n c e b e t w e e n MSG-treated animals and controls. The animals t r e a t e d with MSG at 1 and 5 days o f age s h o w e d highly significant decreases in activity on this test. The 25-day males t r e a t e d w i t h MSG s h o w e d a decrease, but the 25-day females did not. The results o f these tests
c o n f i r m the i n f o r m a l o b s e r v a t i o n s of o t h e r investigators that M S G - t r e a t e d animals are lethargic and h y p o a c t i v e . Several i n t e r e s t i n g q u e s t i o n s are raised by the MSG literature and the data r e p o r t e d above. The m o s t i m m e d i a t e q u e s t i o n s c o n c e r n o t h e r physiological and b e h a v i o r i m p a i r m e n t s w h i c h m a y be i n d u c e d by MSG a d m i n i s t e r e d during the n e o n a t a l period. T h e r e have been brief r e p o r t s of e n d o c r i n e d y s f u n c t i o n and sterility in female organisms treated w i t h MSG [19,30] along w i t h several r e p o r t s o f impaired learning [ 5 , 2 7 ] . The data indicating t h a t MSG disrupts the e l e c t r o r e t i n o g r a m e x e m p l i f i e s the need for behavioral studies at various dose levels and stages of d e v e l o p m e n t . Each o f t h e s e topics deserves e x p a n d e d s t u d y and q u a n t i f i c a t i o n . Finally, t h e r e arises the q u e s t i o n as to w h e t h e r MSGi n d u c e d o b e s i t y r e p r e s e n t s an e x a m p l e o f r e g u l a t o r y or m e t a b o l i c o b e s i t y . The slow steady p a t t e r n o f weight gain along w i t h a n u m b e r of r e p o r t s w h i c h failed to show any evidence of h y p e r p h a g i a lend s u p p o r t for a m e t a b o l i c i n t e r p r e t a t i o n . However, Araujo and Mayer [3] c a u t i o n that o n e - q u a r t e r o f a gram o f c h o w per day could a c c o u n t for a l-g per week weight increase, and t h a t this might be missed by the f o o d - m o n i t o r i n g s y s t e m s currently emp l o y e d . This along w i t h the fact t h a t MSG-induced brain lesions can be f o u n d in the a r c u a t e - v e n t r o m e d i a l h y p o thalamic region requires that a careful investigation be m a d e o f these alternative e x p l a n a t i o n s . ACKNOWLEDGEMENT This work was supported in part by a grant from the Committee on Organized Research, Northeastern Illinois University.
REFERENCES 1. Abraham, R., W. Doughtery, L. Goldberg and F. Coulston. The response of the hypothalamus to high doses of monosodium glutamate in mice and monkeys. Cytochemistry and ultrastructural study of lysosomal changes. Expl. Molec. Path. 15: 43 - 60, 1971. 2. Adamo, N. J. and A. Ratner. Monosodium glutamate: Lack of effects on brain and reproductive function in rats. Science 169: 673 - 674, 1970. 3. Araujo, P. E. and J. Mayer. Activity increase associated with obesity induced by monosodium glutamate in mice. Am. J. PhysioL 225:764 765, 1973. 4. Arees, E. and J. Mayer. Monosodium glutamate-induced brain lesions: Electron microscope examination. Science 170:549 - 5 5 0 , 1970. 5. Berry, H. K., R. E. Butcher, L. A. Elliot and R. L. Brunner. The effect of monosodium glutamate on the early biochemical and behavioral development of the rat. Devl Pwchobiol. 1: 165 - 173, 1974. 6. Boissier, J. R. and P. Simon. Dissociation a deux composantes le comportment d'investigation de la souris. Archs int. Pharmacodyn. ThOr. 147:372 - 387, 1964. 7. Burde, R. M., B. Schainker and J. Kayes. Monosodium glutamate: Acute effect of oral and subcutaneous administration on the arcuate nucleus of the hypothalamus in mice and rats. Nature, (Lond.), 233:58 - 60, 1971. 8. Cohen, A. I. An electron microscopic study of the modification by monosodium glutamate of the retinas of normal and rodless mice. Am. J. Anat. 120: 319, 1967. 9. Djazayery, A. and D. S. Miller. The use of gold-thioglucose and monosodium glutamate to induce obesity in mice. Proc. Nutr. Soc. 32: 30A, 1973. 10. Djazayery, A., D. S. Miller and M. J. Stock. Energy balances of mice treated with gold-thioglucose and monosodium glutamate. Proc. Nutr. Soc. 32: 31A, 1973.
11. Everly, J. L. Light microscopic examination of MSG-induced lesions in brain of fetal and neonatal rats. Anat. Rec. 169:312, 1971. 12. Lemkey-Johnston, N. and W. A. Reynolds. Nature and extent of brain lesions in mice related to ingestion of monosodium glutamate: A light and electron microscope study. J. Neuropath. exp. Neurol. 33:74 97, 1974. 13. Lewis, L. M., J. F. Lynch and J. S. Adkins. Effect of protein level and source on the growth of rats fed excess monosodium L-glutamate. Fedn Proc. 29:567 abs, 1970. 14. Lucas, D. R. and J. P. Newhouse. The toxic effect of sodium L-glutamate on the inner layers of the retina. Archs Ophthal. 58:193 - 201, 1957. 15. Lynch, J. F., L. M. Lewis, E. L. Hove and J. S. Adkins. Effect of monosodium L-glutamate on development and reproduction in rats. FedrProc. 29:567 abs, 1970. 16. Matsuyan, S. Studies on experimental obesity in mice treated with monosodium glutamate. ,lap. J. Vet. Sci. 32: 206, 1970. 17. Murakami, U. and M. Inouye. Brain lesions in the mouse fetus caused by maternal administration of monosodium glutamate. CongenitalAnomalies, 11:171 - 177, 1971. 18. Nie, N. H., C. H. Hull, J. G. Jenkins, K. Steinbrenner and D. H. Bent. SPSS. Statistical Package for the Social Sciences. New York: McGraw-Hill, 1975, p. 425. 19. Olney, J. W. Brain lesions, obesity and other disturbances in mice treated with monosodium glutamate. Science 164:719 721, 1969. 20. Olney, J. W. Glutamate-induced retinal degeneration in neonatal mice. Electron microscopy of the acutely evolving lesion. J. Neuropath. exp. NeuroL 28:455 - 474, 1969. 21. Olney, J. W. and O. L. Ho. Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine. Nature (Lond.), 227:609 - 610, 1970.
MSG, SOMATIC D E V E L O P M E N T , OBESITY AND A C T I V I T Y 22. Olney, J. W. Glutamate-induced neuronal necrosis in the infant mouse hypothalamus: An electron microscopic study. J. Neuropath. exp. NeuroL 30:75 - 90, 1971. 23. Olney, J. W., O. L. Ho and V. Rhee. Cytotoxic effects of acidic and sulphur containing amino acids on the infant mouse central nervous system. Expl Brain Res. 14:61 - 76,1971. 24. Olney, J. W., L. G. Sharpe and R. D. Feigin. Glutamateinduced brain damage in infant primates. J. Neuropath. exp. Neurol. 31:464 - 488, 1972. 25. Oser, B. L., S. Carson, E. E. Vogin and G. E. Cox. Oral and subcutaneous administration of monosodium glutamate on infant rodents and dogs. Nature 229:411 - 413, 1971. 26. Ports, A. M., K. W. Modrell and C. Kingsbury. Permanent fractionation of the ERG by sodium glutamate. Am. J. Ophthal. 50:900 - 907, 1960. 27. Pradhan, S. N. and J. F. Lynch Jr. Behavioral changes in adult rats treated with monosodium glutamate in the neonatal stage. Archs int. Pharmacodyn. Thbr. 197:301 - 304, 1972. 28. Redding, T. W. and A. V. Schally. Effect of monosodium glutamate on the endocrine axis in rats. Fedr Proc. 2 9 : 3 7 8 abs. 1970. 29. Redding, T. W., A. V. Schally, A. Arimura and I. Wakaboyashi. Effects of monosodium glutamate on some endocrine functions. Neuroendocrinology 8 : 2 4 5 - 2 5 6 , 1 9 7 1 .
557
30. Reynolds, W. A., A. S. Bingel and N. Lemkey-Johnston. Female sterility in the MSG-induced hypothalamic lesion of the mouse. Society for Gynecological Investigation. 19th Meeting, Abstracts, p. 46, 1972. 31. Semprini, M. E., M. A. Frasca and A. Mariani. Effects of monosodium glutamate administration on rats during the intrauterine and neonatal period. Quat. Nutr. 3 1 : 8 5 - 100, 1971. 32. Semprini, M. E., L. Conti, A. Ciofi-Luzzatto and A. Mariani. Effect of oral administration of monosodium glutamate (MSG) on the hypothalamic arcuate region of rat and mouse: A histological assay. Biomedicine 21:398 - 403, 1974. 33. Semprini, M. E., A. D'Amicis and A. Mariani. Effect of monosodium glutamate on fetus and newborn mouse. Nutr. Metabol. 16:276 - 284, 1974. 34. Shimizu, K., A. Mizutani and M. Inouye. Electron microscopic studies on the hypothalamic lesions in the mouse fetus caused by monosodium glutamate. Teratology 8: 105, 1973. 35. Strong, L. C. The care of experimental mice. In: The Care and Breeding of Laboratory Animals edited by E. J. Farris. New York: John Wiley and Sons, 1950, pp. 79 96.