BIPHASIC MYELINATION AND THE FATTY ACID COMPOSITION OF CEREBROSIDES AND CHOLESTEROL ESTERS IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE DOMESTIC PIG

Abstract— The chemical composition of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) was determined in 107 pigs at 11 stages of fetal and postnatal development and also in 6 adults. In cerebrum, cerebellum and brain stem, but not in spinal cord, the rate of increase in weight and the rates of change in lipid content slowed down for a period of about 10 days before and after birth. Cholesterol esters and desmosterol were only found in progressively decreasing amounts during the fetal stages of development and together with DNA these were exceptions to the general increases in the tissue concentrations and total amounts of other components during the period studied.
The onset of myelination, as measured by calculated daily increases in tissue contents of cerebroside took place between 70 and 80 days conceptual age and there were two peaks of activity, the first occurring 2 weeks before and the second 3 weeks after birth. Unlike the rate curve for total spinal cord weight the biphasic accumulation of DNA was not synchronous with myelin lipid accretion and the earlier prenatal DNA peak probably denotes proliferation of oligodendrocytes. The two phases of myelination are discussed in relation to an observed generalized pause in development immediately before and after birth.
Fatty acid analysis of cerebrosides indicated that, in spinal cord, chain elongation and desaturation are associated with myelination and continue with increasing activity until maturity. Consequently there was a progressive decrease in the proportion of saturated fatty acids. The fatty acid components of cholesterol esters in the developing pig were shown to be similar to those found during development in the CNS of other species but different from those found in demyelinating conditions.     

CHANGES OCCURRING IN THE CHEMICAL COMPOSITION OF THE CENTRAL NERVOUS SYSTEM DURING FOETAL AND POST-NATAL DEVELOPMENT OF THE SHEEP

(1) The chemical composition of the CNS (separated into cerebrum, cerebellum, brain stem and spinal cord) was determined in sheep during foetal and post-natal development and in adults. (2) The spinal cord differed from the remainder of the CNS in growing more after the period studied (50-day-old foetuses to 5-week-old lambs) than before it. This was largely attributable to lipid accumulation. (3) Chemical growth (accumulation of DNA, protein and lipid) proceeded linearly in spinal cord, logarithmically in cerebrum and cerebellum while in brain stem growth was described by a sigmoid function. (4) Fat-free dry matter, protein, total lipid, cholesterol and phospholipid concentrations increased progressively in all parts of the CNS but DNA concentrations changed little. In the cerebrum alone there was an increase in DNA concentration during maturation suggesting an increased cell population. Cholesterol was present predominantly in the free form but esters were detected in foetal tissues from 70 up to 120 days gestation. (5) Cerebroside, the characteristic lipid of myelin, increased in concentration soon after 85 days of gestation, up to which point very low values were recorded, the rate varying according to the region of the CNS examined. Rates of increase in total regional cerebroside content were used to identify periods of myelination and the results suggest that there are two periods of peak activity, one about 20 days before birth and the other at 10-20 days after birth. (6) The composition of lipids added during the two phases of myelination and during maturation were characteristically different. In the spinal cord, lipid analyses best reflect changes in myelin composition.     

LIPID DEFICIENCY IN THE CENTRAL NERVOUS SYSTEM OF LANDRACE PIGLETS AFFECTED WITH CONGENITAL TREMOR AIII, A FORM OF CEREBROSPINAL HYPOMYELINOGENESIS

—Congenital tremor, type AIII, is characterized by partial agenesis of the white matter of the CNS affecting mainly the spinal cord. The percentage water content of the fresh cord is consistently higher than normal and other parts of the CNS are sporadically affected. The total lipid content (mg/g fresh tissue) is markedly decreased in the cord but brain stem and cerebellum are less severely deficient; the cerebrum is barely deficient. Total amounts of cholesterol, cerebroside and phospholipid (mmol/part) are significantly reduced predominantly in cerebellum, brain stem and cord. Total DNA and protein contents are decreased to a significant extent only in spinal cord. Broadly similar lipid changes are found in fixed tissues. The data are consistent with sub-normal myelination, associated with a deficiency of oligodendrocytes.     

CONGENITAL HYPOMYELINOGENESIS (BORDER DISEASE) OF LAMBS: POSTNATAL NEUROCHEMICAL RECOVERY IN THE CENTRAL NERVOUS SYSTEM

Abstract— In a neurochemical study of experimental Border Disease in lambs it was found that the fresh weights of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) from clinically affected lambs were significantly smaller than those of controls at birth but by 20 weeks of age the cerebrum, cerebellum and brain stem had reached near normal weights. The spinal cord was still considerably smaller, however. Clinical symptoms of the disease (muscular spasms and'hairy'birthcoat) had disappeared during this period, accompanied by a regression in the neurochemical abnormalities seen at birth. Thus the deficiency of myelin lipids was partially made up by the rapid deposition of cerebrosides and by 20 weeks differences in the fatty acid composition of this lipid fraction were no longer apparent. Myelin degeneration as indicated by the presence of elevated levels of esterified cholesterol was apparently absent at 20 weeks of age and this was parallelled by a fall in the level of'anti-myelin'antibodies in the sera of affected lambs. The altered distribution of copper in spinal cord myelin seen at birth had also become nearly normal at the end of the period.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Vulnerability of myelin development of the chick to the herbicide 2,4-dichlorophenoxyacetic butyl ester

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b. e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.     

SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.     

ACTIVITY OF 2',3'-CYCLIC-NUCLEOTIDE 3'-PHOSPHODIESTERASE IN REGIONS OF RAT BRAIN DURING DEVELOPMENT: QUANTITATIVE RELATIONSHIP TO MYELIN BASIC PROTEIN

2′,3′-Cyclic-nucleotide-3′-phosphodiesterase activity was examined in several regions of rat brain during development, namely optic nerve, olfactory bulb, cerebrum, cerebellum, midbrain, brain stem, and spinal cord. From 4 to 120 days the total activity increased in all regions, although the specific activity approached a constant value in adults. The developmental profile of the enzyme appeared to correlate with the onset of myelination and with the levels of myelin basic protein as well as the appearance of galactocerebroside sulfotransferase. A correlation coefficient of 0.91 was found between total basic protein, expressed as the per cent of the adult (120 day) value, and total enzyme activity over 12–42 days of age (P < 0.001) from six different brain regions as well as for whole brain. By increasing the sensitivity of the assay with the use of [³H-8]adenosine 2′,3′-cyclic monophosphate, we were able to detect activity at birth in both whole brain and spinal cord.     

DISTRIBUTION OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE TRANSAMINASE IN SEVERAL AREAS OF THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The regional distributions of serine hydroxymethyltransferase (SHMT) and glycine transaminase (GT) have been determined in five areas of the CNS of the rat. The SHMT activity per mg protein varied in these areas in the following order: medulia-pons and spinal cord > cerebellum > midbrain > telencephalon. The GT activity per mg protein was essentially the same in the four brain areas, whereas, in the spinal cord it was lower. The activity of GT did not correlate with the glycine content (r=?0.45. P > 0.05). However, SHMT activity per mg protein was correlated with the glycine content in four regions (the telencephalon, midbrain, medulla-pons and spinal cord; r= 0.997, P < 0.05). When the activity of SHMT was expressed per relative number of mitochondria, the enzyme levels were correlated with the glycine content in all five areas (r= 0.952, P < 0.05). The distribution of SHMT was determined in the primary subcellular fractions of the CNS. The SHMT activity in these areas of the CNS appeared to be located predominately in paniculate structures, while only 1 to 4 per cent was found in the soluble fraction. The crude nuclear (P₁) and the crude mitochondrial (P₂) fractions contained 90–97 per cent of the activity. Subfractionation of P₂ pellets obtained from the telencephalon, medulla-pons and spinal cord indicated the SHMT activity was localized in both ‘free’ and occluded mitochondria.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

NEUROTOXICITY OF A NON-METABOLIZABLE AMINO ACID, 1-AMINOCYCLOPENTANE-l-CARBOXYLIC ACID: REGIONAL PROTEIN LEVELS AND LIPID COMPOSITION OF NERVOUS TISSUE

Abstract— The non-metabolizable amino acid, 1-aminocyclopentane-l-carboxylic acid (ACPC), when administered to mice, induces primary degeneration of axons in the cerebellum, rostral spinal cord and peripheral nerves. One to 4 weeks after a single intraperitoneal injection of ACPC (0.5–2 mg/g body wt) in adult mice, the fresh and dry weights of brain, cerebellum and spinal cord were reduced compared with those of normal and pair-fed controls. The protein content of all CNS regions, but particularly that of the cerebellum and cervical spinal cord, was lowered in ACPC-treated mice relative to that of normal controls. Sciatic nerve protein was also decreased in mice following 2 mg/g of ACPC. Pair-fed controls exhibited protein deficits in the cerebellum and cervical spinal cord but to a significantly smaller degree. In ACPC-treated mice, the sulfatide content of spinal cord and peripheral nerve was reduced but that of brain was normal. Sphingomyelin levels in these three regions increased except in the brains of mice given 0.5 mg/g of ACPC where the levels fell.
The protein and sulfatide deficits were greatest in the regions which are known to exhibit the highest proportion of degenerating nerve fibers. The correlation of ACPC treatment with protein and sulfatide loss is consistent with the reported disruptive effects of ACPC on protein metabolism and with the involvement of proteins in sulfatide. metabolism. The protein deficits in pair-fed mice are considered in relation to the exacerbating effect of reduced dietary protein intake on ACPC neurotoxicity.     

Myelin Lipids in the Developing Cerebrum, Cerebellum, and Brain Stem of Normal and Undernourished Children

Abstract: The developmental lipid profiles in the human cerebrum, cerebellum and brain stem are presented, with special reference to galactolipids as myelin markers to trace myelination in the three main parts of the human CNS. A group of undernourished children were also studied to test the vulnerability of myelinogenesis in the different regions of the human brain. Myelination was well advanced in the brain stem with regard to the other brain regions, a fact reflected in the much higher concentration of myelin lipids in the brain stem of the human foetus of 26 weeks of gestational age. The cerebrum, on the other hand, had the lowest galactolipid concentration during the prenatal period, galactolipid levels in the cerebellum being four times higher. From just before the end of gestation the accretion of galactolipids accelerated enormously in the cerebrum, whereas it slowed down considerably in the cerebellum. Consequently, in relation to prenatal levels galactolipids increased most rapidly in the cerebrum, followed by the cerebellum and finally by the brain stem. These regional differences were in clear contrast to data from the rat, as was the finding that only the cerebrum of undernourished children had a galactolipid concentration significantly decreased with respect to normal values. A relationship between the different myelination patterns in the human and the rat and the distinct vulnerability of myelinogenesis in the two species is suggested.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.     

Pathway of cholesterol biosynthesis in the brain of the neonatal rat

Suckling rats were killed at various intervals after intraperitoneal injection of acetate-1-(14)C and their brain sterols were analyzed by column, thin-layer, paper, and gas-liquid chromatography. The crude sterol (to which carrier zymosterol was added) was separated by column chromatography into cholesterol, desmosterol, and zymosterol fractions, and the specific activities of the recovered digitonides were determined. The zymosterol fraction, mainly carrier, was not uniformly labeled, in that the trailing half of the peak had a higher specific activity than the leading half. Evidence obtained suggests that this carbon activity was present in one or more sterols resembling zymosterol (Delta(8,24)-cholestadienol), Delta(7,24)-cholestadienol, and Delta(7,5.24)-cholestatrienol. The desmosterol and cholesterol were also carbon-labeled. The time course of the distribution of carbon activity among the above fractions indicated that the zymosterol fraction is a precursor of the desmosterol and that the desmosterol is, in turn, a precursor of the cholesterol. The data suggest that, in the developing brain of the rat, the course of the transformation of cholesterol precursors into cholesterol is influenced by the presence of at least two slow steps, one involving the conversion of Delta(7)- and Delta(8)-compounds to Delta(5)-compounds and the other, the reduction of the Delta(24)-unsaturation.     

Accumulation of zymosterol in yeast grown in the presence of ethionine.

In order to identify the methyl acceptor for the methylation of sterol side-chains in ergosterol biosynthesis, Saccharomyces cerevisiae (wild type) was grown in the presence and absence of ethionine which was expected to be an inhibitor of the methylation. Gas-liquid chromatographic analyses of the sterols in the cells grown in the absence of ethionine showed that ergosterol was the most abundant sterol. On the other hand, a sterol, named sterol Z, accounted for more than 50% of the total sterols in the cells grown in the presence of ethionine. As a result of experiments to raise the yield of sterol Z, the best concentration of DL-ethionine for the production was found to be 1.0 mM. The use of the methionine-less mutant was less effective for the production of sterol Z. Sterol Z was isolated by repeated TLC and was identified as zymosterol from its melting point, GLC and mass spectrometry. The role of zymosterol and other sterols as the methyl-acceptor sterol in ergosterol biosynthesis is also discussed.     

Cerebral, cerebellar, and brain stem gangliosides in mice susceptible to audiogenic seizures

Abstract— The quantitative and qualitative distribution of gangliosides was investigated in the cerebrum, cerebellum and brain stem of audiogenic seizure resistant (C57BL/6J) and susceptible (DBA/2J) mice at 21 days of age. The concentration of gangliosides (μg/unit weight) was higher in the DBA cerebrum and brain stem, but lower in the DBA cerebellum compared to the concentration in C57 mice. In general, the brain water content was lower in DBA mice than in C57 mice. The distributions of a number of gangliosides were found to be different between the two strains and the differences were often in the same direction across the three brain regions. The most consistant and significant difference in ganglioside pattern observed between the strains was the higher concentration of G_M1 in all three regions of the DBA brain. These results suggest that DBA mice have a more heavily myelinated CNS than C57 mice. The relationship of these observations to inherent audiogenic seizure susceptibility is discussed.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF HOMOCARNOSINE–CARNOSINE SYNTHETASE IN THE CENTRAL NERVOUS SYSTEM OF RATS

Homocarnosine–carnosine synthetase and carnosinase were assayed in homogenates, 100,000 g supernatants, and ammonium sulfate fractions of the supernatants from nine regions of the central nervous system (CNS), as well as subcellular fractions of whole brains. The enzymes were detected in all CNS regions tested, with olfactory bulbs having the highest activities of both enzymes. In the subcellular fractions, the synthetase was found mainly in the cell-sap; carnosinase was detected in all fractions, the highest activity being in the mitochondria. The synthetases from olfactory bulbs, cerebellum and spinal cord have similar K_m's for β-alanine and GABA.     

Seasonal and geographical variations of sterol composition in snow crab hepatopancreas and pelagic fish viscera from Eastern Quebec

Sterol composition was determined in snow crab hepatopancreas and mackerel and herring viscera for various locations and collection periods. A simple and valuable method, using direct saponification and extraction with water-cyclohexane has been optimized to recover total sterol. They were identified and quantified as trimethylsilyl ether derivatives by GC-MS analysis. Method validation indicated excellent sensitivity (limit of quantification: 1.25 mg/100 g wet basis for cholesterol and desmosterol; 0.03-0.05 mg/100 g for other sterols), good reproducibility (CV%: 1.5-6.8) and accuracy (recovery%: 94-107). In crab hepatopancreas, cholesterol was the main sterol (67-76%), followed by desmosterol (19-24%). Phytosterols and molluscan sterols were also present in low quantity. A lower total sterol content with different composition was found in crabs from Magdalen Islands compared to those from Gaspé Peninsula or North Shore of the St-Lawrence Gulf. No seasonal variation was observed between collection periods, which were probably too close. Mackerel and herring viscera contained the same sterols as crab except for campesterol and sitosterol, but the cholesterol proportion was higher (93-98%). The higher abundance of sterols in herring caught in September vs. May would be related to an increase of the body lipid content during the summer.     

Developmental Changes of N-Acetyl-L-Aspartic Acid, N-Acetyl-α-Aspartylglutamic Acid and β-Citryl-L-Glutamic Acid in Different Brain Regions and Spinal Cords of Rat and Guinea Pig

Abstract The developmental changes of N -acetylaspartic acid (NA-Asp), N -acetyl-α-aspartylglutamic acid (NA-Asp-Glu), and β -citryl-L-glutamic acid ( β -CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β -CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β -CG content decreased to an adult level at birth.