Journoi

OJ

Neerochrmisrry. 1976. Vol. 26. pp. 893-900. Pergamon Press. Printed in Great Britain.

NEONATAL ASPHYXIA IN THE RAT: GREATER VULNERABILITY OF MALES AND PERSISTENT EFFECTS ON BRAIN MONOAMINE SYNTHESIS NICOLE SIMON' and L. VOLICER Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118, U.S.A. (Received 17 June 1915. Accepted 10 October 1975)

Abstract-In the rat, neonatal asphyxia produced by suffocation did not leave permanent visible lesions in thc brain, nor did it result in permanent motor impairment, although a delay in the development of some reflexes was observed. A transient retardation of body and brain growth, which was more pronounced in males, was found. By 5-6 weeks of age, body and brain weights of asphyxiated rats were no longer significantly different from control animals. However, an increase in brain norepinephrine synthesis was found to persist after maturation. An alteration of serotonin metabolism was found after maturation only in asphyxiated males. The possibility that neonatal asphyxia in the rat is a model for abnormal development of monoamine metabolism, relevant to early childhood behavior disorders such as infantile autism or the syndrome of minimal brain dysfunction, is discussed.

RESEARCH on the effects of asphyxia in the perinatal

by studies of differential circulatory rate in the brain period has revealed that in several species, including (KETY,1962) and order of myelin development (YAKman, the resulting injury to the brain may be confined OVLEV & LECOURS, 1967). Thc focal lesions result from to subcortical structures without involvement of the rapid production of carbon dioxide and acidosis in cerebral hemispheres (CLARK& ANDERSON,1961; these areas of high metabolic activity (WINDLE,1966; ROZDILSKY & OLSZEWSKI, 1961; CHEN, 1964; JILEK CHENet al., 1971). Animals with this pattern of focal et a/., 1964; CHENet a/., 1965; BRAND& BIGNAMI, brainstem lesions do not become spastic or suffer per1969; WINDLE, 1969a; TOWBIN, 1970; MYERS,1972; manent impairment of motor functions (WINDLE, NORMAN, 1972; GRIFFITHS & LAURENCE,1974; GRUN- 1969~).The effects of a prolonged partial deficiency of oxygen (hypoxia) are entirely different. The basal NET et al., 1974). Research on monamines in the central nervous system indicates that their normal meta- ganglia and paracentral cortex are often the most bolism depends upon integrity of some of the brain- severely damaged after prolonged hypoxia. The instem areas that are most vulnerable to perinatal volvement of these two motor systems is the characet al., 1969; HARVEY teristic pattern underlying the affliction of spastic ccrasphyxia (JOUVET,1969; POIRIER & GAL, 1974). We chose to study neonatal asphyxia ebral palsy and prolonged partial asphyxia results in in the rat as a possible naturalistic model for abnor- clear neurological signs. Early childhood behavior disorders such as infanmal development of central monoaminergic neural tile autism are not neccessarily accompanied by any networks. It is now well established that, in the monkey, two hard neurological signs (motor manifestations): hence entirely different patterns of brain damage result from the focal lesions of brainstem centers seen after a brief oxygen deprivation around the time of birth, depend- episode of total asphyxia at birth may be a more ing upon whether the insult is a brief acute episode likely cause for this type of disorder. SIMON(1975) of total asphyxia or a prolonged partial disruption has discussed the possible involvement of brainstem of the oxygen supply (MYERS,1972). Ten-15 min of auditory nuclei in the language disorder of autistic total asphyxia produced by umbilical cord clamp children. WINDLE(1963, 19696); SECHZERet nl. (1973) before the neonate is allowed to breathe, results in believe that the focal lesions of the brainstem and a highly predictable pattern of small, focal lesions, thalamus seen after an acute asphyxic insult may unmost prominent in brainstem auditory structures, reti- derly the clinical syndrome of Minimal Brain Dyscular formation and the thalamus. The vulnerability function (MBD). Perinatal injuries have been found of these subcortical sites follow a rank order predicted to be an important etiological factor underlying both Predoctoral Trainee of the National Institute of Men- autism and MBD (SECHZERet al., 1973; LOBASCHER ef al., 1970; CHESS,1971) and disorders of monoamine tal Health. Present address: Behavioral Sciences Division, Bolt metabolism have been implicated in these behavioral Beranck and Newman, Inc., 50 Moulton Street, Cam- disorders of childhood (COLEMAN,1973; WENDER, 1972). bridge, MA 02138, U.S.A. 893

894

NICOLE SIMONand L. VOLICER

any maternal rejection of the asphyxiated pups. All animals were weaned between 21 and 24 days of age. Experimental procedures. Reflex development was tested in the pre-weanling pups according to the schedule of SMART& DOBBING(1971~).A few rats were sacrificed 20 or 40 days after the asphyxic insult and their brains k e d and stained according to the method of NAUTA(1957). Most of the animals were allowed to mature and were used for biochemical studies between 5 and 6 weeks of age. At the time of sacrifice, the maximum age difference between litters was 4 days. Six-nine litters were used lor each study of monoamine turnover and paired experimental-control littermates were selected from each litter for every time-point measurement. Radioactive L-tyrosine or L-tryptophan was injected into the tail vein. The amount injected was about 100 pCi/rat, MATERIALS AND METHODS contained in 0.5 ml of saline/100 g wt of the animal. Each Materials. Radioactive-~-[3,5-'H]tyrosine (30-50 Ci/ animal was killed by decapitation exactly 90, 120 or mmol) and ~-[~H]tryptophan,generally labelled (1-5 Ci/ 150min after injection. The brain was quickly removcd, mmol) were purchased from New England Nuclear COT. blotted and the lower brainstem trimmed off by a cut di(Boston). Ion-exchange resin (AG 50-X4, 2 W 0 0 mesh) rectly behind the cerebellum; the olfactory lobes were was purchased from Biorad Labs (Richmond, CA). Alu- trimmed off by a cut directly in front of the frontal lobes. minum oxide (alumina) was purchased from the British The brain was wrapped in aluminum foil and immediately Drug Houses, LTD. (obtained through Gallard-Schlesinger frozen on dry ice. The frozen brains were kept for up to Chemical Mfg. Corp., Carle Place, NY). Amines and amino 1 week in a freezer at -4°C. acids used for standards were purchased from Calbiochem The brains were homogenized in 4 vol of ice cold (LaJolla, CA), Nutritional Biochemicals (Cleveland, OH) 0.4 M-perchloric acid containing 0.050/, sodium metabisuland Sigma Chemical Co. (St. Louis, MO). Tris, hydroxyfile. Monoamines in the supernatants of the brain homomethyl amino methane, buffer was purchascd from Sigma genates were separated from their precursor amino acids Chemical Co. (St. Louis). Otherwise, analytical grade re- by ion exchange columns as described by NEFFet al. (1971), agents purchased from Fisher Scientific Co. (Fair Lawn, with some modifications. Norepinephrine was eluted with NJ) were used throughout all procedures. Pregnant rats 1.0~ rather than O ~ M - H C IBetter . recovery of serotonin were obtained from the Charles River Breeding Labs. was achieved by elution with saturated trisodium phos(Wilmington, MA). phate than with the 0 . 5 ~solution used by NE=F et a/. Animal treatment. The pregnant rats were shipped on (1971). The norepinephrine and dopamine eluates were the fourteenth to sixteenth day of gestation and placed, further purified on washed alumina according to the upon arrival, in individual breeding cages. Within 24 h method of CHANG(1964). Both catecholamines were after a natural birth, the mother was removed from the measured fluorometrically in the final effluent from alumina nest cage. The infant rats were weighed and individually (1968). by the method of LAWRTY& TAYLOR marked by cutting off one digit of either the left or right Calculations. The mean efficiency in counting of radioac(1971~) tivity was found to be the same (30%) for all of the final forepaw; this was described by SMART& DOBBING as a non-traumatic procedure. Littermate pairs were then aqueous eluates, hence this factor was not carried through selected according to sex and birthweight for long-term the calculations. To compute the specific activity (SA) for comparisons. One member of each pair was subjected to each amine and amino acid, the counts per minute (c.p.m.) asphyxia and the other kept as a control. We sometimes were multiplied by the molecular weight (MW x 10 3, transferred a pair from one litter to another to equalize and three factors to correct for the fraction of the total litter size; in general, 6 - 8rats were reared in each cage. tissue sample used for counts (F), the recovery (R), and The control littermates were placed in the corners of the fraction of tritium ions remaining after conversion to the nest cage during the asphyxiation of the experimental amine (T): pups; this kept the pups isolated from each other, but the c.p.m. x MW x 1 1 1 control animals were not subjected to any additional stress. x-x-x--. SA = The experimental animals were asphyxiated in 12 cm' L F R T capacity. air-tight vials. The duration of asphyxia was For serotonin counts, corrections were made for the between 45 min and 2 h at room temperature (24-28" C). Animals were removed from the vials when gasping could leakage of free tritium released from the generally labelled no longer be elicited by tapping the side of the vial, the tryptophan during the separation procedure. This leakage mouth had closed and the pup appeared pale and flaccid. was estimated by carrying standards of the tritium labelled The animals were placed, supine, on a paper towel covertryptophan through all the steps of the procedures for isoing a heating pad, which maintained a temperature of 34°C lation of serotonin and the fraction (f) of tritium counts and thcy were dried off with absorbant tissue. Resusci- in the serotonin eluate was measured. Then, for serotonin, tation was accomplished by intermittently massaging or c.p.m. = (c.p.m.5_HI- f x c.p.m.Tr,p). NEFF et al. (1971) softly tapping the chest and applying, at intervals, a slow estimated that of the twelve hydrogen atoms in the tryptostream of air to the nose and mouth. phan molecule, two would be lost in conversion to seroThe mother was returned to the nest cage only after tonin and thus 1/6 of the 'tritium radioactivity should be the resuscitated pups had been replaced. There was never assumed to be lost. We therefore set T = 5/6 for serotonin. Metabolic anomalies in children are generally thought t o be hereditary. O u r study was designed t o investigate whether long term biochemical dysfunctions could result from an exogenous insult such as perinatal asphyxia. We chose the rat for o u r studies because, in the neonatal rat, acute or chronic anoxia affects only brainstem areas and the injury appears to be transient (JILEK et ul., 1964). With maturation, however, there is a residual defect in acquisition and cxtinction of conditioned reflexes, which JILEK et al. (1964) suggested may be d u e to a long-term alteration of sub-microscopic structure or metabolism in the nervous system.

Neonatal asphyxia in the rat

895

It was assumed that 1/2 of the tritium ions would be displaced during hydroxylation of tyrosine, which was labelled in the 3,5 positions of the phenyl ring. We therefore set T = 1/2 for dopamine and norepinephrine. The logarithm of the specific activities, for each animal, were used t o compute linear regression curves with respect to time. Values of specific activity for calculation of the synthesis rate constant wcrc calculated from the slope and intercept of the regression lines. at time points 90, 100, 110, 120, 130, 140 and 150. The synthesis rate constant (K) was computed for five 20-min intervals, using the formula derived by NEFFet al. (1971): dMjdi A-M where dM/dt is the rate of decline of monoamine activity computed between time points tl and t 2 as:

K=-

dM/dt =

Mf2 - M,, t2 -

tl

and A - M is the average difference between amino acid activity and monoamine activity in the interval, computed by the approximation:

A-M=

(A

-

MI,> + (A

-

M)t2

2

RESULTS

Survival and vulnerability to asphyxia Only about half of the experimental animals survived a severe asphyxic insult. Mean death rate in eight experiments (n = 186) was 49 & 7% for males and 47 8% for females. The survivors appeared to be quite depressed during the first 24 h after asphyxia. Most did not gain a normal amount of weight during this period and many lost up to 0.5 g in the first 24 h after asphyxia. Those animals with large weight loss (0.5-1 g) or those who appeared jaundiced (with yellow discoloration of the skin), often died within the first 24-48 h after asphyxiation. Control animals (n = 145) gained 1.03 & 0.06g in the first 24 h after birth. Only one control animal lost weight in the first 24 h, a female pup who lost 0-75g. In contrast to this the average weight gain of 122 experimental animals 24h after asphyxia was only 0.21 +_ 0.06g; this included 37 animals (30.3%) who lost weight during the first 24 h. It was thought that vulnerability to asphyxia might be related to the metabolic rcquirements of the animal and that this might be related to birth weight and sex. Male animals were significantly heavier at n = 90) than females birth (7.21 & 0.06g (667 k 0.07 g, n = 91j and 35.9% of the males, as opposed to 24.6% of the females, lost weight during the first 24 h after asphyxia. Computation of correlation coefficient for initial weight loss/gain with respect to birthweight however was not significant for either males (r = -0-12,n = 82) or females (r = 0.02, n = 57). There was a significant correlation of initial weight gain/loss with respect to duration of asphyxia, at room temperature (24-28"C), for female pups ( P = -056, n = 46, P < 0001); this was not significant for male pups ( r = -0.16, n = 55).

L

:

.

0

2

4

6 days

8

1012

FIG.1. Weight gain in normal males ( 0 - 0 ) , n = 8, nor(,).n = 19, asphyxiated males mal females + (o----o), n = 10 and asphyxiated females (@---a), n = 18.

Short-term growth retardation Growth retardation persisted in the first 2 weeks; this is illustrated in Fig. 1, for the data from one experiment. This pattern of early growth retardation was strikingly similar in all experiments. A three-way analysis of variance of growth rate indicates that the differences between asphyxiated and control animals are highly significant ( P < 0.001),while those between males and females are not; the effect of asphyxiation is however significantly greater for males than females in retarding growth ( P < 0.001). The weight differences for paired male and paired female littermates from another experiment are shown in Fig. 2; these differences are significantly larger in males (P < 0.0016). Brain growth was also retarded in asphyxiated animals. The average brain weight (& s.E.M.) of 25 male pups 1 week after asphyxia was 721.5 i 10.2mg as compared with an average 755.9 & 8.5 mg for 27 control males. In asphyxialed females, the brain weights averaged 703.1 f 9.3 mg (n = 30) in contrast to 725.2 k 8.0mg (n = 28) in control females. A 2-way

t i m e , days

FIG. 2. Weight differences between control and asphyxiated male (+a) and female (0---0) pairs during the first week aftcr asphyxia. Pairs were selccted a priori on the basis of similar birth weight. Numbers beside each point represent the number of pairs weighed on each day.

896

NICOLE SMON and L. VOLICER

analysis of variance on data from three experiments with 7-day old rats indicated that brain weight was significantly lower ( P < 0.01) 1 week after neonatal asphyxia, that the female brain was significantly smaller than the male brain at this age ( P < @01), but that brain growth was affected to the same degree in both sexes by neonatal asphyxia. The ratio of brain/body wt was 00378 f 0-008 and 00385 f 0.007 for 7-day old control males and females respectively; this ratio was 0.0413 f 0008 for both asphyxiated males and females; this difference was significant for both males (P < 0-001)and females ( P < 0.01). At 5-6 weeks of age the trend for lower brain and body 1 0 *' 90 120 I50 weights was still evident in male animals, but the difminuter ferences with respect to the control groups were no FIG.3. Decline in specific activity of tritium-labelled tryplonger statistically significant. tophan (0)and serotonin (0)in male rats. The points are Reflex development and histology

mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and asphyxiated (broken lines) animals.

The asphyxiated animals did not become spastic or noticeably incapacitated in any way. To determine if there were any developmental reflex or motor deficonstant for serotonin was significantly increased ciencies, a total of nine experimental and nine control (Table 1). litters, in three separate experiments, were examined Although the rate of decline in tryptophan activity according to the method of SMT & DOBBING was steeper in the asphyxiated females, and the speci(1971a). The differenax between control and asphyxfic activity in the serotonin fraction was somewhat iated animals were significant (P < 0.05) in two .of higher at all time points (Fig. 4), the rate of decline these tests (grasp and negative geotaxis). in serotonin activity was the same in control and The forelimb grasp reflex usually develops between asphyxiated animals. There was no significant differthe second and third day after birth (Fox, 1965) and ence between asphyxiated and control females in the wanes between the sixth and seventh days of life. After computed rate constant, or in serotonin synthesis rate neonatal asphyxia, the development of the forelimb in nmol/h per g of tissue. The serotonin synthesis grasp was delayed by a full day in male pups; the rate in the female brain was higher than in the male delay was somewhat smaller in females but an analybrain, control or asphyxiated, (Table 1). sis of variance indicated that the males were not significantly more delayed than females. Tyrosine and catecholamine metabolism By the fourth or fifth day of life, the rat pup placed The decline in specific activity of tyrosine, dopahead-down on a 20-degree inclined plane, instinctively turns to a head-up position; this is called nega- mine and norepinephrine in the brain, 9CL150 min after injection of tritium labelled tyrosine into the tailtive geotaxis. Partial turning (to a horizontal posvein, is shown in Fig. 5. Tyrosine activity was higher ition) begins around the third day of life. In asphyxat all three time points in asphyxiated animals and iated male pups the turning was again delayed by a full day; the female pups were somewhat less delayed, but the male-female difference was not significant. A histological study, using NAUTA'S (1957) procedure for degenerating fibers, 3 and 6 weeks after asphyxia revealed no focal lesions or visible disruption of neural pathways. Tryptophan to serotonin metabolism The decline of specific activity of tryptophan and serotonin id the brain, 9@150 min after injection of tritium-labelled tryptophan into the tail-vein of 5-6-week old rats is shown in Fig. 3 for males and Fig. 4 for females. In male rats, the specific activity of tryptophan was slightly higher at all time points and steeper decline in the specific activity of serotonin was found in asphyxiated males compared with the male control group (Fig. 3); and the computed rate

1

0

$,

90

minuter

120

150

FIG.4. Decline in specific activity of tritium-labelled tryptophan (0)and serotonin @ ) in female rats. The points are mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and asphyxiated (broken lines) animals.

Neonatal asphyxia in the rat

I 0

//

SB

"

minuter

rio

rbo

FIG.5. Decline in specific activity of tritium-labelled tyrosine (U),dopamine (0),and norepinephrine (A). The points are mean values and the bars indicate S.E.M. The lines are regression curves computed for control (solid lines) and asphyxiated (broken lines) groups of animals.

its decline was somewhat steeper. Norepinephrine activity was also higher at all time points, with a steeper decline during the 9G-150 min interval after injection of its precursor amino acid. Synthesis rate for norepinephrine computed from this data indicates that the rate of norepinephrine synthesis in the asphyxiated animals was nearly double that of t6e control group (Table 1). There was no difference in the decline of dopamine activity, as seen in Fig. 5, and the rate constant for dopamine synthesis was not significantly different in asphyxiated compared with control animals (Table 1).There were no male-female differences evident in the catecholamine data. DISCUSSION

Growth and development Newborn rats subjected to asphyxia by suffocation did not become spastic or display any signs of sub-

TA~LE 1. EFFECTOF

897

sequent gross motor impairment. Only with careful, day by day assessement was a delay in the acquisition of some motor responses revealed. Permanent necrotic lesions were not seen in the brains of asphyxiated animals. The most striking result of neonatal asphyxia in the rat was growth retardation, with asphyxiated male animals showing a greater lag in weight-gain than asphyxiated females. Brain growth was also retarded in the asphyxiated animals, but to a lesser degree than body growth. By 5-6 weeks of age, the body and brain weight differences of the asphyxiated and control animals were no longer statistically significant. These results stand in contrast to the studies of perinatal malnutrition (SHOEMAKER & WUKTMAN, 1971; ShlART & DOBBING, 1971a), in which weight differences between nutritionally deprived and control rats did not become significant until 5 days after birth (SMART& DOBBMG, 1971b), but the degree of growth retardation increased with maturation. 'Ile effects on the rat of asphyxia and malnutrition in the perinatal period then can be clearly differentiated; they are not equivalent insults. Growth retardation was also observed by FRANCESCONI & MAGER(1969) in infant rats reared under hypoxic conditions, with normal growth rate seen after return to normal atmospheric environment. Prolonged hypoxia and brief total asphyxia thus appear to be related, with rcspect to growth impairment, in the rat. Measurement of monoamine synthesis

A completely reliable method for the true rate of neurotransmitter formation has still to be develet al., 1974). We chose the oped (MOKOT-GAUDRY method of NEFF et al., (1971) because we felt that it was suitable for the measurement of differences between our control and asphyxiated groups. We found no indication of severe alteration of neuronal development after neonatal asphyxia in the rat that would make the asphyxiated animals incomparable

NEONATAL ASPHYXIA ON SYNTHESIS

RATE OF BRAIN MONOAMINES IN &DAY

OLD RATS

(C = Control, A = Asphyxiated Within 24 Hours After Birth) Dopamine

Norepinephrine

Serotonin Females A

C

A

C

A

C

0.354

0.380

0.261

0.497

0.853

Males C

A

0.573

0.740

Mean Rate

Constant/hr

0.852

Steady-State Concentration iig/g

Tissue

(mean+ S . E . M . )

Synthesis Rate rnpmole/g/hr (mean? s . E . M . )

Number of Animals Used

0.665

0.727

0.138

20.03S

20.041

20.009

0.202 3.007

0.46 +o.O2

0 49

0.48

0.41

+O.OZ

z0.03

fp.03

2.36

1.55

1.54

1.80

0.305

0.593

2.32

20.10

+o.z8

20,014

20 020

9.20

+a.lo

20.14

-0.13

1.72

19

16

19

18

23

17

19

21

898

NICOLE SIMONand L. VOLICER

to the controls. JILEK et al. (1964) noted microscopic changes in the brains of neonatal rats within 24h after anoxic insult, but then found no evidence of lesions after maturation ; it was their suggestion that behavioral deficits might be due to a long-term alteration of metabolic systems that prompted our research. Because we were injecting the precursor amino acids into the general circulation via the tailvein, we investigated the rate of transport of tyrosine and tryptophan into the brain (SIMON,1974). We thought this might explain the increased activity of tyrosine and tryptophan in the brain 9Ck-120min after injection; but there was no evidence of increased penetration of the precursor amino acids into the brain (SIMON,1974). We cannot relate the biochemical differences we have found to any neurobehavioral deficits, but we can conclude that an asphyxic insult in the neonatal period can affect metabolic systems of the brain without producing permanent, gross brain damage. Retardation of body growth and the greater growth lag in male animals suggest, in particular, a possible interference with development of neuroendocrine systems.

in male animals by altering the normal interaction

of testosterone with serotonin in the central nervous system. Growth retardation after neonatal asphyxia may result directly from monoamine depletion in the brain. Secretion of growth hormone from the pituitary appears to be initiated by serotonin or (more likely) a serotonin metabolite (SASSIN et al., 1969; COLLUet al., 1972); growth hormone release is also impaired after depletion of monoamine stores by reserpine administration (SACHAR et al., 1972). The apparently greater vulnerability of males to neonatal asphyxia suggests that the asphyxic insult had a greater effect on the testosterone mediated stages of postnatal development of the male brain and that the asphyxic insult had a lesser effect on estrogen mediated stages of brain development in female animals. Perhaps estrogens protect the female and prevent, or minimize the results of, asphyxic damage.

Maturation and stress The possibility exists that increased norepinephrine synthesis after neonatal asphyxia, at least in some regions of the brain, may be due to maturational delay. PORCHER & HELLEX(1972) in an investigation Sex dgerences in serotonin metabolism of regional development of catecholamine synthesis in Sex differences in rat brain serotonin levels and rat brain found that in this species, between 7 and metabolism have been noted in several studies (KATO, 45 days of age, the tyrosine hydroxylase activity in 1960: SKILLEN et al., 1961; LADOSKY& GAZ~RI, 1970; several brainstem areas equalled or exceeded the GIULIAN et al., 1973; HARDIN,1973). A higher sero- adult rate. The activity of tyrosine hydroxylase in foretonin synthesis rate in females had been suggested by brain areas of immature animals is lower than in SKILLEN et al. (1961) and HARDIN (1973) on the basis adult rats however and approaches the adult level of in vitro studies of 5-hydroxytryptophan decarboxy- of enzyme activity slowly, reaching only 75% of the lase and monoamine oxidase; our finding of greater normal adult rate by 45 days of age. Since only whole in viuo conversion of tryptophan to serotonin in brain was assayed in the experiments after neonatal female rat brain confirms this. Administration of tes- asphyxia presented here, it is not possible to say tosterone, estrogens and castration have been found whether the increased synthesis rate found was due to affect the development of brain serotonin primarily to increased biosynthetic activity in subcor(LADOSKY& GAZIRI,1970; GIULIANet al., 1973). tical or cortical regions. LADOSKY& GAZIRI (I 9-70), studying the androgenizHUTTUNEN (1971) observed an increase.in forebrain ing effect of testosterone administration to female rats norepinephrine turnover in W 5 - d a y old rats after in the neonatal period, found a normal increase of prenatal stress (maternal footshock). ADAMSONS et al. forebrain and midbrain serotonin levels in females at (1971) noted that maternal stress causes release of 12 days of age; this increase was prevented by a single catecholamines, which interferes with uterine bloodinjection of testosterone propionate (0.1 mg.) on the flow and produces, in monkey offspring the same patfirst day of life. A comparable (though somewhat tern of focal brainstem lesions found after a brief smaller) increase in brain serotonin levels at 12 days period of total asphyxia at birth. The stress produced of age was found in castrated males. by maternal footshock during pregnancy, then, may Although biosynthesis and storage of monoamines compromise the offspring in a way comparable to is limited in the neonatal rat, probably the acute effect neonatal asphyxia. HUTTUNEN’S (1971) finding of inof anoxia or hypoxia is to deplete the monoamine creased norepinephrine turnover in the forebrain of stores of the brain (HURWITZ et al., 1971; CVMERMAN prenatally stressed rats suggests that the effect is due et al., 1972). Reserpine is known to deplete m o n e less to immaturity than to premature induction of amine stores (BRODIEet al., 1959), and if reserpine enzyme transcription and synthesis. This would correis injected at the time of neonatal testosterone spond to the premature development of hepatic trypadministration to female rats, the androgenizing tophan oxygenase in rats reared in an hypoxic eneffects on later development are prevented (LAWSKY vironment (FRANCESCONI & MAGER,1969), or early & GAZIRI,1970). A depletion of monoamines by induction of other hepatic enzymes found after preasphyxic stress in the neonatal perod may selectively natal or neonatal hormone injections (GREENGARD, alter the development of serotonin metabolism only 1969).

Neonatal asphyxia in the rat In conclusion, we cannot be sure that neonatal asphyxia in the rat provides a useful model for any human affliction. It is of possible significance that male animals were more vulnerable to neonatal asphyxia, as there is about a 4 : l greater preponderance of males over females in the human population who develop early schizophrenic illness, or infantile autism (WING,1966). In one pilot experiment (SIMON, 1974) we found a decreased serotonin binding capacity of blood platelets from asphyxiated rats compared with control animals. A similar deficiency in scrotonin binding capacity has been reported for autistic children (BOUILLINet al., 1970). The use of paired littermates in our experiments excludes the possibility of genetic causes underlying the differences we have found. This suggests that similar metabolic anomalies in human congenital disorders may also be due t o factors other than genetic abnormalities. Acknowledgements-This work was supported by a grant from the Benevolent Foundation of Scottish Rite Freemasonry, Northern Jurisdiction, U.S.A. (the Scottish Rite Schizophrenia Research Program). We are also grateful for support, for NICOLESIMON: from the American Association of University Women, for the Connie M. Guion Fellowship for Medical Research in 1971-1972 and from the National Institute of Mental Health, for predoctoral fellowship number l F01 MH51868-01 for 1972-1974. The authors would like to thank Mr. B. HURTEX and Mr. R. WICKE(who was supported, in part, by U.R.O.P. Workstudy Program of the Massachusetts Institute of Technology) for their valuable technical assistance.

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