result of the reserve of flexibility which 3. E. H. Bloch, Ainer. J. Aniat. 110, 125 (1962).

5 (1962). of treatment at every dose tested, in-

4. P-I. Branemark and J. Lindstrom. Biorheol-
the red cells have in these larger ves- ogv 1, 139 (1963). cluding 0.5 mg/g (Fig. la). Certain
sels rather than of functional physiolog- 5. P-I. Brdnemark, in Proceedings of the Fouirth structures located in a paramedian
Intertnationial Congress on Rheology (Inter-
ical significance. science, New York, 1965), p. 459. plane and bordering on the roof and
R. SKALAK 6. M. M. Guest, T. P. Bond, R. G. Cooper, floor of the third ventricle were prefer-
J. R. Derrick, Science 142, 1319 (1963).
Department of Civil Engineering andl 7. A. C. L. Barnard, L. Lopez, J. D. Hellums, entially affected. At the base of the
Engineering Mechanics., Microvascidar Res. 1, 21 (1968). brain, preoptic and arcuate nuclei of
8. R. M. Hochmuth and S. P. Suterat, Bibi.
Columinbia University, New York 10027 Anat., in press. the hypothalamus were selectively de-
P-I BRANEMARK 9. H. Wang and R. Skalak, Office of Nai al stroyed along with scattered neurons
Research Rep. NR 062-393-1, Coltumbia
Laboratory of Experimental Biology, University, New York (1967). within the median eminence (nuclei
Department of Anatomy, University of 10. P. B. Canham and A. C. Burton, Circ. Res. tuberales). No acute changes were
22, 405 (1968).
Gotheniburg, Gothenburg, Sweden 11. Y. C. Fung, ibid. 25, 1761 (1966). found in other hypothalamic areas or
12. - and P. Tong, J. Biophys. 8, 175 (1968). in the pituitary. Dorsally, the subcom-
References and Notes 13. P. A. G. Monro, Bibl. Anat., in press.
14. Supported by grants from NIH (HE 5724-08), missural and subfornical organs and
1. M. 1. Gregersen, C. A. Bryant. W. E. Ham-
merle, S. Usami, S. Chien, Science 157, 825 NSF, and Swedish Medical Research Coun- neuronal groups contiguous with them
cil. We thank Miss Y. Winsnes for research
(1967).
2. P-I. Branemark, "Intravascular Anatomy of assistance. were involved, including the medial
Blood Cells in Man," in preparation. 3(1 Jantuary 1969 habenular nuclei and neurons of the
rostral hippocampus (dentate gyrus).
Acute lesions were also found in brains
of adult mice given high doses (5 to
Brain Lesions, Obesity, and Other Disturbances 7 mg/g) of MSG subcutaneously (Fig.
in Mice Treated with Monosodium Glutamate Ib). Whether lower dosages than those
tested might induce neuronal pathology
Abstract. In ne wborn mfli(e subcutaneous injectionis of inionosodilium gluttamnate in either the immature or mature CNS
induced acuite neluronal necrosis in several regions of developing brain including requires further systematic investiga-
the hypothalamus. As adults, treated animals showed stunted skeletal developnment, tion. Brain lesions were also found in
nmarked obesity, and femlale sterility. Pathlological chianges were also found in the C57BL/6 strain of mice and in
several organs associated wvith endocritne function. Studies of food constimptionr albino rats after MSG treatment in the
failed to demtonstrate hyperphagia to explain the obesity. It is postulated that the neonatal period.
adult syndromtie represents a in uiltifaceted neuroendocrine distllrbance arising from To study the possibility of long-
the disruption of developing nielurail centers cotncernled in the m?iedliatiotn of enfdo- range effects accruing from glutamate
critne functioni. treatment of the neonate I followed
five litters of Swiss albino mice, con-
Parenterally administered monosodi- to 9 da,tys old, were killed from 1 to sisting of 38 healthy animals, from
um glutamate (MSG) produces an 48 hours after a single subcutaneous birth to 9 months of age. Twenty ani-
acute degenerative lesion in the inner injection of MSG (dosages varied from mals received subcutaneous injections
retina of normal neonatal mice (1). 0.5 to 4 mg/g), and brains were ex- of MSG daily from 1 to 10 days
Although the acute lesion has been de- amined by light microscopy for acute after birth, according to a dose sched-
scribed both light and electron micro- pathology. Brain lesions characterized ule described by Cohen (4); 18 con-
scopically (2) and several biochemical by intracellular edema and neuronal trols received no treatment. All animals
parameters have been studied (3), the necrosis developed within a few hours were weighed individually on a weeklyI
specific mechanisms underlying the
effect of MSG on retinal neurons have
not been definitively clarified. That
MSG treatment might have a similar
deleterious effect on neurons in other
regions of the central nervous system
(CNS) has apparently not been con-
sidered. A suspicion that hypothalamic
lesions might be associated with glu-
tamate treatment was aroused by the
observation that several months after
neonatal mice were treated with glu-
tamate, for purposes of inducing retinal a .
,,U.
pathology (4), they became quite
obese. Data establishing that glutamate Fig. 1. (a) Section throuigh hypothalalmMUs of 5-day-old Swiss albino mouse showing
lesion formation 3 hours after a subcutaneous dose of MSG (I mg/g). Scattered
treatment does induce brain lesions are neurons in the median eminence (ME) are necrotic wvith bloated cytoplasm and
now presented, and a preliminary char- pyknotic nuclei. The majority of neurons in the arcuate nuclei (AR) are necrotic, but
acterization is given of a syndrome re- those of the ventromedial nuclei (VM) are unaffected (X 100). (b) Section through
sulting from glutamate treatment hypothalamus of adult C57BL/6 moLuse 3 hours after a stubcutaneous dose of MSG
which features obesity as its most (6 mg/g). The arcuate nuclei (AR) are completely destroyed along with neuronal
constituents in the median eminence (ME). Capillairy lumina are empty and widely
striking characteristic. dilated because this animal was killed by perfusion of glutaraldehyde throLugh the
Ten litters of Swiss albino mice, 2 ascending aorta (x 115).
9 MAY 1969 719
basis frorn the 1 st to 5th months, and for controls throughout the period from
data on Ifood consumption were gath- 30 to 150 days. Treated animals sur-
ered weelkly on all males for the same passed controls in weight at a mean
period. ALnimals were given free access age of approximately 45 days, and at
to Purina mouse breeder chow in con- 150 days the range of weights for
taineis dt:signed to avoid food loss by treated animals showed no overlap with
spillage or soiling of food by urine those of controls. Experimental females
and fecess. in this series gained more weight by
Treate J animals appeared stunted at comparison to controls of their own
terminati4on of treatment on the 10th sex than did treated males.
day after birth and remained smaller Data on food consumption compiled
than conttrols on the 30th day. Data on for all males of the five litters studied
growth piresented separately for females are included in Fig. 2b for direct com-
(Fig. 2a)) and males (Fig. 2b) illus- parison with their growth data for
trate that the rate of weight gain for comparable periods. Contrary to ex-
experimernrtal animals was greater than pectation, treated animals consumed
slightly less food than controls in every
period for which data were collected.
Food consumption was also measured
60r
(a) (52-70) on all animals, both male and female.
from the same five litters for 4 hours
of unrestricted eating after the 24
E hours during which food was withheld.
.-L
Mean per capita consumption over the
4 hours was 2.5 g for controls and 1.7
a:)0 g for experimental animals.
3 (34-42) Treated animals continued to gain
>1
-o weight on unrestricted diets beyond the
m age of 5 months (Fig. 3). Despite
/ Eweight excesses, however, treated ani-
*91 E
rntal mals were approximately 10 percent
o°10 Controls shorter in mean body length than con-
trols. These differences were reflected
30 90 150 in measurements of the long bones and
Days spines of animals x-rayed at 9 months.
Treated animals were quite lethargic
('5a70s aS adults, and they lacked the sleekness
(b) of body coat seen in controls (Fig. 3).
The reproductive capacity of treated
females was also affected in that thex
I4 consistently failed to conceive in spite
(,40-492 of adequate exposure, from 5 to 9
months of age, to both treated and nor-
mal males. Mating of treated males
*f Experimental with untreated females resulted in preg-
*I
o 8 Contro/s nancies and normal offspring.
Autopsies performed at 9 months of
age revealed massive accumulations of
4& adipose tissue in experimental animals
140i
- compared with small to moderate
amounts in controls. The livers of
80 i tieated animals showed fatty changes,
1 and the ovaries contained approxi-
20 2 mately twice as many atretic follicles
(S were found in controls. The uteri of
30 9C 150
DAYS treated animals were easily distin-
guished from controls by their slender,
Fig. 2. ( ) Composite growth recordls for attenuated appearance. The endome-
experimelntal and control females from
five litter:s of mice covering the 1st to 5th trium was thinner and contained only
months o If life. Weight ranges on the 150th small secretion-poor glands. The testes
day are in parentheses. (b) Composite of treated males were indistinguishable
growth rrecords and data on food con- from controls. Findings suggestive of
sumption L for experimental and control
males fr4 om five litters of mice covering amild adrenocortical hypertrophy re-
the 1st t,o 5th months of life. Ranges of main under study at this time. The pars
weight O0 n the 150th day are in parentheses. nervosa and pars intermedia of pituli-
72 0
Fig. 3. A 9-month-old Swiss albino mouse
(left) which was treated, as a newborn,
with MSG is shown beside the heaviest
tuntreated male (right) from the same lit-
ter. The experimental animal weighed 84 g
compared to 44 g for the control. In
addition, the treated animal is shorter
than the control, and his body coat is not
as sleek as thait of the control.
tary glands from experimental animals
atppeared normal but an overall reduc-
tion in mass and in the number of cells
was evident in the pars distalis (adeno-
hypophysis).
Three additional litters of Swiss al-
bino mice (10 experimental and 13
control animals) were used to test the
obesity-inducing potential of a single
subcutaneous injection of MSG (3
mg/g) 2 days after birth. Treated ani-
mnals in this series were on the average
16.9 g heavier than littermate controls
at 9 months of age. However, this rep-
resented a more slowly developing and
less severe syndrome than was created
by daily treatments for the first 10 days
of life.
These observtations, linking MSG
treatment of the neonatal mouse with
a syndrome of manifestations, including
skeletal stunting, marked adiposity, and
sterility of the female, coupled with
histopathological findings in several
organs associated with endocrine func-
tion, suggest a complex endocrine dis-
turbance. In view of the additional
finding of lesions in regions of the brain
thought to function as neuroendocrine
regulatory centers (5), a unitary hy-
pothesis might be constructed relating
all or most of the findings to the neo-
natal disruption of neuronal develop-
ment in these centers. Since acute
degenerative changes were not found
in neonatal pituitary glands, the small
size of adult pituitary glands from
treated animals suggests an interference
in some extrapituitary influence (per-
haps hypothalamic) on the develop-
ment of this gland.
SCIENCE, VOL. 164
 Obesity, the most striking clinical doses of phenylalanine were given to a incorporation of radioactive methyl
manifestation of MSG treatment, has pregnant rhesus monkey, the ratio of groups from 3H-methyl-S-adenosyl-L-
been produced experimentally in mice mother to fetus for this amino acid re- methionine into material which sur-
treated with two other chemical com- mained unchanged so that exceedingly vives deproteinization by the Marmur
pounds, gold thioglucose (GTG) (6) high fetal blood levels resulted (9). procedure (5), is precipitable in cold
and bipiperidyl mustard (7). In each The possibility that brain lesions could 5 percent perchloric acid, and is not
case, however, animals were treated in occur in the developing primate em- degraded by 0.5M NaOH at 60°C.
adulthood, lesions were reported in the bryo in response to increased glutamic This assay removes protein and RNA
ventromedial nucleus ("satiety center") acid concentrations in the maternal cir- that is methylated by other enzymes
of the hypothalamus, and treated ani- culation, therefore, warrants investiga- present in the crude nuclear extracts.
mals were considered hyperphagic. In tion. In plants deproteinization has the ad-
that hypothalamic lesions in MSG- JOHN W. OLNEY ditional virtue o _removing some green
treated animals routinely spared ventro- Department of Psychiatry, Washington material that interferes with scintilla-
medial nuclei and these animals were University School of Medicine, tion counting of the product (Table 1).
consistently hypophagic by comparison St. Louis, Missouri Most of the radioactivity remaining in
with littermate controls, a mechanism acid-precipitable material after this
other than appetite disturbance must References and Notes procedure appeared to be in DNA, as
be considered. Whether a regulatory 1. D. R. Lucas and J. P. Newhouse, Amer. Med. it was rendered acid-soluble by treat-
mechanism affecting fat metabolism in 2. J.Ass.W.Arch. Ophthalmol. 58, 193 (1957).
Olney, in press. ment with pancreatic deoxyribonuclease
the mouse can be localized to the arcu- 3. J. K. Freedman and A. M. Potts, Invest.
Ophthalmol. 1, 118 (1962); ibid. 2, 252
(Table 1). The product of the reaction
ate nucleus, or other brain areas selec- (1965). was further characterized by hydrolysis
tively destroyed by MSG treatment, 4. A. I. Cohen, Amer. J. Anat. 120, 319 (1967). in 90 percent formic acid at 180°C for
5. E. Scharrer and B. Scharrer, Neuroendocrinol-
requires further study. ogy (Columbia Univ. Press, New York, 1963). 30 minutes to generate the free bases,
The assumption that MSG is an en- 6. G. Brecher and S. Waxler, Proc. Soc. Exp.
Biol. Med. 70, 498 (1949); J. Mayer, Physiol.
tirely innocuous substance for human Rev. 33, 472 (1953); A. H. Perry and R.
A. Liebelt, Proc. Soc. Exp. Biol. Med. 106,
consumption has been questioned re- 55 (1961); R. L. Deter and R. A. Liebelt, c.-p
cently in view of its role in the Chinese Tex. Rep. Biol. Med. 22, 229 (1964).
7. R. J. Rutman, F. S. Lewis, W. D. Bloomer,
restaurant syndrome (8). The finding Science 153, 1000 (1966).
that neuronal necrosis can be induced 8. H. H. Schaumburg and R. Byck, N. Engl. J.
Med. 279, 105 (1968); M. Ambos, N.
in the immature mouse brain by 0.5 Leavitt, L. Mormotek, S. Wolsilrina, ibid.,
mg/g of MSG raises the more specific p. 105; H. H. Schaumburg, R. Byck, R.
Gerstl, J. H. Mashman, Science 163 826
question whether there is any risk to (1969).
the developing human nervous system 9. G. R. Kerr and H. A. Waisman, in Amino
Acid Metabolism and Genetic Variation, W.
by maternal use of MSG during preg- L. Nyan, Ed. (McGraw-Hill, New York,
nancy. The primate placenta maintains 1967), p. 429.
10. Supported in part by PHS grants NB-04816,
amino acids in consistently higher con- MH-07081, MH-13002, and MH-38894. I
thank Drs. E. Robins, A. I. Cohen, M.
centrations in the fetal circulation than Constant, and D. Kipnis for advice, and
are found in the maternal circulation, Miss S. Freeman for the original observa-
tion that glutamate-treated mice appeared
the ratio for glutamic acid being great- abnormally fat.
er than 2:1 (9). In fact, when high 11 March 1969 a

Fig. 1. Chromatography of the meth-

ylated product. The reaction mixture and
conditions of incubation were the same as
Deoxyribonucleic Acid Methylase Activity in Pea Seedlings those described under Table 1. Product
measured by the standard assay procedure
Abstract. Deoxyribonucleic acid methylase activity has been detected in a described in the text. (A) The product was
preparation of disrupted nuclei prepared from pea seedlings. S-Adenosyl-L- hydrolyzed at 180'C for 45 minutes with
0.3 ml of 90 percent formic acid and
methionine acted as a donor of methyl groups, and the product of the reaction chromatographed in butanol: H,O: NHs for
was identified as 5-methylcytosine. The reaction had a sharp temperature opti- 16 hours. (B) A second sample of the
mum at about 30°C and was unusual in that the DNA methylase was able to formic acid hydrolyzate was chromato-
methylate DNA in the crude extract. graphed in isopropanol:HCI for 20 hours.
(C) A third sample of the formic acid
hydrolyzate was treated with HNO2 for
5-Methylcytosine is a minor com- from S-adenosyl-L-methionine to spe- 4 hours at 20'C and chromatographed
ponent of the DNA of many organisms cific sites in DNA of high molecular in isopropanol: HCI for 20 hours. (D)
(1), but it is a major component of the weight (3). Despite the superabund- The final alkali stabile sediment was
DNA's of higher plants (2). In bacterial washed with ether, dried, and then hydro-
ance of 5-methylcytosine in plant lyzed with 20 ,ug of pancreatic deoxyribo-
DNA's only about 1 percent and in DNA's, nothing has been known about nuclease (Worthington) in 2.5 ml of tris
animal DNA's only about 5 percent of its biosynthesis. To our knowledge this (pH 7.5) 0.O1M containing 0.005M
the cytosines are methylated (1), but is the first report of DNA-methylase MgCl2 for 3 hours at 37'C. Then 0.3 ml
in plant DNA's between 20 and 30 of 0.SM tris (pH 8.5) was added with 210
activity in a higher plant, pea seedlings. ,Ag of venom phosphodiesterase (Crotalus
percent of the cytosine is methylated. The assay for DNA-methylase activ- adamanteus) (Worthington), and the in-
In bacteria and in animal tissues, 5- ity of pea seedlings was identical with cubation was continued for 3 more hours.
methylcytosine is the product of highly that developed for detection of DNA- The material was dried, resuspended in
specific enzymes, called DNA methy- methylase activity in extracts of mam- H20, applied to Whatman No. 3 paper,
lases, which transfer methyl groups and chromatographed for 16 hours in
malian tissues (4). This assay measures borate buffer.
9 MAY 1969
721