Psychopharmacology (1985) 86:359-364 Psychopharmacology �9 Springer-Verlag 1985

The modifying influence of aging on behavior in mice neonatally injected with monosodium glutamate Max Goldman and George E. Stowe Department of Biology, University of South Dakota, Vermillion, SD 57069, USA

Abstract. Newborn mice were injected with monosodium glutamate (MSG), and behavioral testing and physical de- velopment in neonatal, juvenile, adult, and aged mice were observed by means of a battery of appropriate tests. Early exposure to MSG resulted in altered behavioral perfor- mances postnatally, which were transitory and reflected the neurotoxicity of the chemical. Although behavioral deficits were not observed in mice neonatally treated with MSG at 1 month of age, a definite decline was manifested at 12 months and 34 months of age.

Key words: Postnatal evaluation and development - Adult and aged mice - Automutilation - Behavioral testing

Monosodium glutamate (MSG) is a chemical substance that has been used extensively as an additive in a variety of foods because of its efficacy as a meat tenderizer, preserva- tive, and flavor enhancer (Saunders 1966). Several years ago a number of related incidents occurred in which people having eaten in Chinese restaurants became violently ill, all individuals displaying symptoms such as headache, chest pains, burning sensations, and facial pain (Schaumberg et al. 1969). This aggregation of clinical manifestations of toxicity due to the ingestion of MSG, which had been liber- ally used in the preparation of the food, was subsequently termed the Chinese restaurant syndrome (Ho Man Kwok 1968; Schaumberg and Byck 1968; Schaumberg et al. 1969).

The consequent notoriety generated a series of investiga- tions of the effects of MSG on a variety of physiological activities. These include retinal damage (Lucas and Ne- whouse 1957; Potts et al. 1960; Cohen 1967), hypothalamic lesions (Olney 1971; Perez and Olney 1972; Holzwarth- McBride et al. 1976; Nemeroff et al. 1977a; Clemens et al. 1978; Dawson et al. 1982), growth and obesity (Bernardis and Patterson 1968; Olney 1969; Bernardis and Frohman 1970; Nagasawa etal. 1974; Holzwarth-McBride etal. 1976), disturbances in bone marrow development (Dhindsa etal. 1978), endocrinological disturbances (Olney 1969; Bernardis and Frohman 1970; Redding et al. 1971; Naga- sawa et al. 1974; Pizzi et al. 1977; Nemeroff et al. 1977a; Bakke et al. 1978; Clemens et al. 1978; Dhindsa et al, 1981 ; Nemeroff et al. 1981), and behavioral manifestations (O1-

Offprint requests to: M. Goldman

ney 1969; Prabhu and Oester 1971; Pradham and Lynch 1972; Nikoletseas 1977; Nemeroff etal. 1977b; Dawson and Lorden 1981).

This investigation was undertaken to determine the in- fluence of neonatal administration of MSG on behavioral effects in male and female mice at different ages, including neonatal, juvenile, adult, and old age. While previous inves- tigations have been limited to relatively short-term effects following neonatal administration of MSG, this study endeavored to consider the modifying influence of age on the MSG insult administered to newborn mice.

Materials and methods

Male and female CF 1 mice bred in our laboratory were used throughout the study. All breeding and experiments were carried out at an environmental temperature of 21 ~ + 1 ~ C under a controlled photoperiod (12-h light-dark cycle). The animals were maintained on Purina Laboratory Chow and had continuous access to tap water.

Drugs. Monosodium glutamate (MSG), (Sigma, St. Louis, MO) was made up as a 10% solution in 0.9% saline and injected subcutaneously soon after birth.

Neonatal Treatment. Forty-seven litters obtained at birth were randomly assigned to 2 groups; a control saline in- jected group and an MSG injected group. The newborn pups in the latter group were injected sc with the 10% aqueous solution of MSG on day 2 through day 10 postpar- tum according to the schedule ofPotts et al. (1960) as modi- fied by Cohen (1967) in a gradually increasing concentra- tion of 2.2, 2.5, 2.8, 3.2, 3.4, 3.6, 3.8, 4.0, and 4.2 mg/g body weight. The control group received injections of 0.9% saline in accordance with the same schedule as did the MSG-injected group. The day of birth was designated as day 0 (Cohen 1967).

Testing schedule. All mice were subjected to behavioral tests and observations prior to weaning. Thereafter, mice were randomly selected for testing at four different age intervals: neonatal, 1 month of age (juvenile), 12 months of age (adult) and 34 months of age (old). When testing at a specif- ic age interval was completed, the mice were not returned to the same cage but placed in different cages randomly to minimize litter effects.

360

Postnatal evaluation by behavior testing and physical devel- opment. These tests and observations were initiated soon after birth and continued 1 h after injection of MSG or saline and continued until termination of the testing period.

Pinna detachment. Observations were made from day 1 until both pinnae were detached in all pups (Irwin 1968).

Incisor eruption. Observations were continued from day 1 until both the upper and lower incisors had erupted in all pups (Irwin 1968).

Eye opening. Pups were observed daily from day 1 until both eyes were open (Irwin 1968).

Surface righting. A pup was placed on its back on a smooth surface and allowed to right itself. When all four feet were in contact with the surface, the time it took to complete the test was recorded. Each pup was given two trials daily and a maximum time limit o f 30 s for performance of each test was allowed (Brunner et al. 1978). Observations were made from day 1 until day 10 of life.

Cliff avoidance. A pup was placed near the edge of a raised box (raised platform) with its forepaws and face positioned near the edge o f the "cliff." An animal that drew away from edge of the cliff was given a positive score. The maxi- mum time allotted for retreating from the edge was 30 s, and a pup that either remained in this position without any attempt to withdraw or that fell off the platform was given a negative score (Fox 1965). Observations were made from day 1 until day 10 of life.

Swimming test. Observations were made from day 6 until all pups had demonstrated the ability to swim in a straight line. The test animal was placed in a small tank containing water at a temperature of 27~ for no more than 5-15 s and a positive score was recorded if the pup swam directly to the opposite tank wall without showing any circling be- havior (Schapiro et al. 1970).

At 21 days of age the mice in each group were weaned, the young separated on the basis of sex and four to five pups were housed together in translucent plastic cages.

Behavioral studies in young, adult and old mice. Older male and female mice 1, 12, and 34 months of age were randomly selected for testing from each of the control and MSG in- jected groups.

Acceleration righting reflex. The righting reflex test is uti- lized to test equilibrium and neuromuscular coordination (Fox 1965). A mouse was held by the tail and gently swung in a circular manner to disrupt its equilibrium and then, in a face-up position, dropped from a height of not more than 30 cm into a cage containing 5 cm or more of wood shavings to cushion its fall. This test was performed on each mouse, once per day, for 7 consecutive days. A positive score was assigned if the mouse was able to land in the normal upright position (on all four feet) and a negative score was recorded if the animal failed to land in an upright manner.

Roto-rod test. The roto-rod test devised by Dunham and Miya (1957) was used to test for neurological deficiency.

The rolling rod apparatus was powered by a 115 V, 60 cycle AC Bond Electric Kymograph with the motor shaft connected to one end of a rod 3 cm in diameter with the other end supported in a free rotating fashion. To prevent injury to an animal falling off the bar, a cage containing a large quantity of wood shavings was placed below the rotating rod. A mouse was placed on the roto-rod appara- tus with all four paws in contact with the rotating bar, and the length of time that the mouse maintained itself on the bar was determined with a stopwatch. A mouse that maintained its balance on the rotating rod for a period of 3 min was considered as having successfully completed the test. Each mouse was tested once a day for 7 consecutive days and the data expressed as the mean percentage for the 7-day test period for those mice capable o f maintaining their equilibrium for the 3-min testing period.

Grip strength test. This test is a measure of muscular endur- ance (Prabhu and t e s t e r 1971). A string 1 mm in diameter was stretched between two supporting rods 40 cm above a cage containing wood shavings. A mouse was placed on the tightly strung string and allowed to grasp the string with its forelimbs only. After the mouse was released, it should have been able to bring its hind legs up to grasp the cord. The duration of time that the mouse could main- tain itself on the string was measured with a stopwatch. Each animal was tested once each day for 3 consecutive days and the results averaged to find a mean "hang t ime" for each animal.

String test. The string test measures muscular coordination and vigor (Miquel and Blasco 1978). A string 1 mm in di- ameter was strung between two support rods 60 cm apart and 40 cm above a cage containing wood shavings. A mouse was held by the tail and suspended in a head down position for 30 s. The mouse was allowed to grasp the string with its forelimbs and the tail held until the mouse obtained a solid grasp on the string with its forepaws, at which time the mouse was released. Mice able to pull themselves up with all 4 legs in contact with the string and pull themselves along the string to one of the supporting rods in 1 min were given a positive score; those mice unable to pull them- selves up onto the string and so unable to cross to one of the support rods were given a negative score. The results were then reported as the percentage of mice able to com- plete the test in the ascribed manner.

The data are also reported as the biomass transported in order to minimize the favorable influence of a light body weight. Biomass transported was calculated by adding the body weight of all the mice that received a positive score in each group and dividing the result by the total number of mice tested, which is the sum total of all of the mice receiving positive and negative scores (Miquel and Blasco 1978).

The data were subjected to one-way analysis of variance, and the significance between means was determined by Duncan 's multiple range. A P value <0.05 was considered statistically significant.

Results

Neonatal evaluation

No disparities with respect to age when eye opening, incisor eruption, or pinna detachment occurred in control and MSG-injected animals.

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Table 1. Effect of neonatal injection of monosodium glutamate on roto-rod, grip strength, and string test in I month, 12 month, and 34 month old male mice

Group No. of Body weight Lee index Roto-rod Grip strength String test animals (g) (%) (s) (%)

1 Month Control 13 20.4 _ 0.8 2.9 • 0.03 96.7 • 1.7 a MSG 14 18.4 • 1.2 2.9 • 0.04 95.9 • 2.4

12 Months Control 18 39.2 -+ 0.8 3.2 • 0.02 95.2 • 2.3 MSG 24 44.3+0.8 c 3.4_+0.02 c 92.2_+3.5

34 Months Control 8 40.0_+ 0.7 3.2 • 0.03 7.5 • 1.5 MSG 8 42.6 • 1.1 3.4 • 0.04 c 0.0 _+ 0.0 ~

100.2• b 91.8• 98.5-+2.3 92.3-+7.7

81.2-+4.8 58.7-+9.8 49.3-+3.9 c 11.9• c

12.5• 2.9• c

" Results expressed as mean percent positive score • SEM b Results expressed as mean seconds _+ SEM

Differ from control group, P<0.05

Table 2. Effect of neonatal injection of monosodium glutamate on roto-rod, grip strength, and string test in 1 month, 12 month, and 34 month old female mice

Group No. of Body weight Lee index Roto-rod Grip strength String test animals (g) (%) (s) (%)

1 Month Control 19 16.1 • 0.6 2.8 • 0.02 93.9 _+ 2.6" MSG 15 17.7 __. 0.6 c 2.9 + 0.03 d 92.3 • 3.7

12 Months Control 16 33.6 -+ 0.8 3.1 • 0.02 91.9 _+ 4.1 MSG 16 38.9 _ 1.7 c 3.4 + 0.03 c 90.1 • 5.7

34 Months Control 7 32.6 • 1.8 3.1 • 0.03 45.7 _+ 5.3 MSG 10 38.5_+ 1.5 ~ 3.4_+0.03 ~ 0.0_+ 0.0 ~

87.0 • 4.2 b 82.7 • 7.7 a 86.1 -I-4.0 88.6___7.4

75.5-+3.3 42.0_+9.2 63.8+4.2 c 25.9_+8.7 c

31.4_+13.7 17.7--+ 6.2

a Results expressed as mean percent positive score + SEM b Results expressed as mean seconds + SEM c Differ from control group, P<0.05

Surface righting reflex. The mean age at which all MSG- injected pups had completed the test successfully (100% completion) was on day 7 after birth while 100% of the control pups completed the test on day 5 (P<0.05). The mean time for completion of the test was 1.6_+0.2 s for 25 control pups and 2.8_+ 1.0 s for 25 MSG-injected pups on day 7 and 1.6+0.3 for control pups and 1.9-t-0.3 s for MSG-injected pups on day 10.

Cliff avoidance. In the control group, 60%, 80%, and 100% completion of the test was observed, respectively, on days 4, 5, and 6 while 40%, 56%, 80%, and 100% completion occurred on Days 4-7 in the MSG-injected group. The mean time for completion was 6.2___0.8 s for 25 control pups and 8.6___1.0 s for 25 MSG-injected pups on day 6 when the control group showed 100% completion and 4.8 _+ 0.7 s for control pups and 6.5 + 0.8 s for MSG-injected pups on day 7 when the MSG group displayed 100% com- pletion. The mean time for completion of the test on day 10 was 4.9+0.1 for the control group and 5.0_+0.7 for the MSG-injected group. Despite the differences in percent completion and mean time for completion of this test, none of the data were statistically significant.

Swimming response. Both control and MSG-injected pups swam in circles until day 5 at which time they began to swim in a straight line towards the opposite side of the tank. In the control group 24%, 80%, 96%, and 100% completion occurred on days 5-8, respectively, while 12%, 40%, 56%, 64%, 72%, and 100% completion was observed on days 5-10 in the MSG-injected group. On those days where differences in swimming response were noted be- tween control and MSG-injected groups, the data were stat- istically significant (P < 0.05).

Post-weaning evaluation

Aggressive behavior. Male mice in the control group con- sistently displayed aggressive behavior toward each other, which resulted in varying degrees of muti lat ion and castra- tion when they were housed in the same cage. This behavior is consistently observed in our colony in male CF 1 mice, which are maintained in the same cage. However, such ag- gressive behavior was conspicuously absent in adult male mice that had been injected neonatally with MSG.

Automutilation. Neonatal injection of MSG did not result in a single case of automuti la t ion (i.e., tail biting) in any of the male or female mice.

362

Acceleration righting reflex. There was no significant differ- ence between MSG-injected and control male and female mice when these tests were performed at either 1 or 12 months of age.

Roto-rod test. No significant impairment in performance of this test was observed in either 1-month-old or in 12- month-old male and female mice that had been injected with MSG neonatally (Tables 1 and 2). While both male and female control mice 34 months of age displayed a signif- icant reduction in performance of the roto-rod test, an even greater deficit in performance was observed in similar aged male (P<0.05) and female mice (P<0.05) that had been injected with MSG at birth (Tables 1 and 2).

Grip strength. No significant difference in muscular strength was observed in male or female MSG-injected or nonin- jected animals when tested at 1 month of age (Tables 1 and 2). However, a significant reduction in muscle strength (P<0.05) was observed in both male and female MSG in- jected mice at 12 months of age (Tables 1 and 2).

Comparing the performance of 12-month-old male mice having body weights from 38 to 42 g showed that nine con- trol mice (mean body weight 38.2-t-3.0 g) had a score of 80.3_+3.1 s, while nine MSG-injected mice (mean body weight 40.2_2.1) had a score of 44.1 ___6.8 s. Similarly, 12- month-old female mice having body weights from 26 to 38 g showed that nine control mice (mean body weight 33.6_+2.0 g) had a score of 74.1-t-2.1 s, while nine MSG- injected mice (mean body weight 34.9 +_ 1.4 g) had a score of 57.3_+ 3.3 s. The above data for the grip strength test were statistically significant.

xm oJ 4~ m co

w u

%

c

m E co 5-

0J

o

m

4~ x =

o c o

4~ o

25

20

15

lO -

5-

1

I

1

Months

Male

Controls

MSG

3 4

Fig. 1. Neuromuscular activity based upon transported biomass in male mice neonatally injected with monosodium glutamate. Values represent the mean • SEM of eight or more mice. Single asterisk indicates P<0.05 when compared with their controls; double asterisk, P < 0.05 when compared with their controls

String test. There was no significant difference in perfor- ;- mance of this test in either male and female control or MSG-injected mice at 1 month of age (Tables 1 and 2). However, a significant reduction in the ability to perform this test was observed in both male (P<0.05) and female (P<0.05) 12-month-old mice that had been injected with MSG neonatally (Tables 1 and 2). Impairment in perfor- mance of this test as a consequence of age was again ob- served in control male and female mice, 34 months of age

c~

(Tables 1 and 2). Although a marked decline in test perfor- mance was observed at this age in both male and female 7_ mice that had been injected with MSG neonatally, only the deficit in the 34 month old males was statistically signifi- .~ cant (P<0.05) (Tables 1 and 2).

Since a reduction in neuromuscular ability could be as- o

cribed to the increased body weight observed in obese MSG-injected mice, the data were also calculated on the 4~

basis of transported biomass in order to compensate for the enhanced body weight. The data derived in this way revealed that the neuromuscular decline observed in 12- and 34-month-old mice that had been injected with MSG at birth were statistically significant in both male and female obese mice (Figs. 1, 2).

Moreover, comparing the performance of 12-month-old male mice having body weights from 37 to 42 g showed that ten control mice (mean body weight 38.8 + 0.4 g) had a score of 64.8% _-t-10.0% completion, while ten MSG-in- jected mice (mean body weight 39.7+0.4) had a score of 25.1% _+ 10.1% completion of the test. Similarly, 12-month- old female mice having body weights from 26 to 40 g showed that seven control mice (mean body weight

25

20

15

I0

5 -

1 2

Months

Female

F " l c o n t r o l s

~ - ~ MSG

7-

3 4

Fig. 2. Neuromuscular activity based upon transported biomass in female mice neonatally injected with monosodium glutamate. Values represent the mean _+ SEM of seven or more mice. Asterisk indicates P < 0.05 when compared with their controls

33.6 • 1.0 g) had a score of 42.0% • 8.0% completion, while seven MSG-injected mice (mean body weight 35.6-t-1.5 g) had a score of 14.3%_ 9.2% completion. In 34-month-old male mice having body weights from 38 to 40 g, five control mice (mean body weight 39.2 + 2.0 g) scored 14.3 % +_ 4.0 % completion while five MSG-injected mice (mean body

363

weight 39.4 + 2.1 g) scored 2.8 % complet ion of the test. The above da ta for score performance of the string test were statistically significant.

Discussion

The present study emphasizes the modifying influence of age on neuromuscular endurance and muscular coordina- tion as a consequence of pr ior neonatal t reatment with M S G in mice and suggests that the behavioral discrepancies reported in the l i terature (Wincze and Vogel 1969; P radham and Lynch 1972; Araujo and Mayer 1973; Trentini et al. 1973) may be a reflection of observations limited to relative- ly short- term effects of M S G treatment. Indeed, Prabhu and Oester (1971), while report ing a lack of neurological deficiency in mice tested at 50 days of age that had been treated with M S G neonatally, did suggest that the degree of neurological deficit observed at a specific age might be a reflection of the length of t ime after t reatment with M S G such that the chemical insult would lead to greater exacer- bat ion with aging. Early exposure to M S G resulted in al- tered performances in behavioral tests that were used in this study to evaluate the progression of development dur- ing preweaning. These disparit ies were most likely a conse- quence o f the neurotoxic characteristics o f early treatment with MSG. While hypothalamic damage is associated with early M S G treatment (Olney 1971; Holzwar th-McBride et al. 1976; Nemerof f et al. 1977a; Clemens et al. 1978; Dawson et al. 1982), compensatory mechanisms during fur- ther development of the nervous system may mask observa- t ion of short- term neuromuscular and behavioral deficien- cies until such time as the aging process results in an attenu- at ion of this compensatory mechanism (Frolkis et al. 1972; Timiras 1972; Robinson 1975; Everett and Porter 1976; Di lman 1976).

Al though neonatal mice injected with M S G demon- strated no discernabTe neuromuscular changes at 1 month of age, a definite decline in activity was observed at 12 months and 34 months of age (Tables 1 and 2). This loss in neuromuscular vitality was mainta ined even after consid- erat ion of the body weight da ta on the basis of t ranspor ted biomass (Figs. 1, 2), which el iminated the body weight dif- ferential between obese and lean mice as a variable. Indeed, this was confirmed by compar ing the scores obtained in the performance of this test by control and MSG-injected mice having similar body weights.

Al though there have been some reports that M S G treat- ment in mice soon after birth results in some degree of hyperirr i tabi l i ty (Nemeroff e t a l . 1977b), older mice, 9 months of age, appeared to be hypoactive (Olney 1969). The MSG-injec ted mice in our study demonstra ted both behavioral pat terns; hyperirr i tabi l i ty between 20-30 days of age and lethargy and very little activity after 5 months of age.

The phenomenon of tail automut i la t ion associated with neonatal adminis t ra t ion of M S G in rats, which had been reported by Nemerof f et al. (1977b) and Clemens e ta l . (1978), was completely absent in any of the more than 300 mice injected neonata l ly with MSG. However, newborn Sprague-Dawley rats from our colony when injected with M S G displayed automut i la t ion at the time of weaning. This dispari ty in behavior may represent a species difference.

Disrupt ion and destruction of neuronal areas in the hy- po tha lamus by early t reatment with M S G in newborn mice

leads to disrupt ion in neuromuscular vitality, equilibrium, muscular coordinat ion, and other behavioral activities (O1- ney 1969; Olney 1971; Pizzi et al. 1977; Nemerof f et al. 1977a, 1981). The da ta in this investigation indicates that the influence of neonatal t reatment with M S G on behavior is significantly modif ied by the aging process.

Acknowledgements. This study was supported by a University of South Dakota General Research Committee grant and is gratefully acknowledged.

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Received September 12, 1983; Final version October 29, 1984