R. Balázs

Effect of thyroid hormone on the biochemical maturation of rat brain: Postnatal cell formation

(1) Treatment with thyroid hormone (triiodothyronine, T3) in early life reduced the rate of growth in the rat: at 50 days of age the weights of the body and the brain were 20–30% less than those of controls. During the period of relatively rapid growth of the brain its size was less reduced than was that of the body. However the size of the brain corresponding to body weights exceeding 40–50 g was less than in controls. (2) The growth of the brain is related not only to an increase in the size of cells existing at birth, but also to an increase in their number amounting in the cerebrum to 80% of the number at birth and in the cerebellum to a rise by a factor of 35. The rate in the deposition of DNA is most rapid during the period from 5 to 14 days when it is about 3 times faster in the cerebellum than in the cerebrum; the rapid rate also continues later in life in the cerebellum than in the cerebrum. These observations indicate that in respect to the final assembly of cells the cerebellum develops later than the cerebrum. (3) The reduced brain size in rats treated with T3 is caused by a permanent reduction in the final cell number. Evidence is summarized which indicates that treatment with T3 affects cell formation rather than cell destruction, resulting in a 30–40% reduction in the number of cells formed after birth. The effect develops at a considerable time after the treatment has been started, and becomes significant about a week later in the cerebellum (at the end of the second week of life) than in the cerebrum. These observations suggest that the sensitivity of the tissue to thyroid hormone depends on the developmental state of the cells. The findings are consistent with the view that treatment with T3 results in a premature termination of cell proliferation that is related to an acceleration of either the differentiation of the cells or their migration from germinating sites. Postnatal cell formation in the rat brain involves neurones as well as glial cells. Hence the reduction in postnatal cell formation may be involved in the impairment of the adaptive behaviour of rats treated with thyroid hormone. (4) By contrast to the marked effect on cell number treatment with T3 did not affect the growth of the cells in the brain. There was no alteration in the maturational changes related to cell size: the packing density of the cells, the concentration of cell constituents and the cellular contents of RNA and protein respectively were similar to those found in controls.

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post‐natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period.

Preparation of cell bodies from the developing cerebellum: Structural and metabolic integrity of the isolated ‘cells’

A method is described for the isolation from the developing rat cerebellum of cell bodies which display a high level of ultrastructural organization. The procedure, which utilizes isotonic conditions throughout, begins with a brief trypsinisation at low enzyme concentration (0.025%). Proteolysis is terminated by trypsin inhibitor and followed by short exposure to EDTA. The technique is effective with cerebella from rats up to 2 weeks after birth. Recoveries of cell bodies vary from 130-410 million/g wet weight of tissue, depending on age: this represents, in terms of recovered DNA, a mean value for yield of 33%. Suspensions contain little debris, free nuclei are rare and about 80% of the perikarya excludes trypan blue. Survey electron micrographs show that most cell bodies possess uninterrupted plasma membrane profiles and retain highly organised cytoplasmic and nuclear ultrastructure. Structural preservation is highlighted in the case of Purkinje cell bodies in which may characteristic features survive including, most notably, perisomatic spines. Metabolic integrity appears to parallel morphological preservation as judged by several functional criteria, including the ability to metabolise glucose, accumulate K+ ions and synthesize proteins. SDS-polyacrylamide gel electrophoresis shows that the tissue dossociation technique does not lead to major deletions of cell proteins and that the pattern of perikaryal protein synthesis in vitro closely resembles that in vivo. These perikaryal preparations therefore hold out great promise as a simplified system for metabolic studies and as a starting material for the derivation of purified sub-populations of cell bodies from developing cerebellum.

Effects of Anoxia on the Stimulated Release of Amino Acid Neurotransmitters in the Cerebellum In Vitro

Abstract: The effect of anoxia and ischemia on the release of amino acid transmitters from cerebellar slices induced by veratridine or high [K+] was studied. Synaptic specificity was tested by examining the tetradotoxin (TTX)‐sensitive and the Ca2+‐dependent components of stimulated release. Evoked release of endogenous amino acids was investigated in addition to more detailed studies on the stimulated efflux of preloaded [14C]GABA and d‐[3H]aspartate (a metabolically more stable anologue of acidic amino acids).[14C]GABA release evoked by either method of stimulation was unaffected by periods of up to 35 min of anoxia and declined moderately by 45 min. In contrast, induced release of d‐[3H]Asp increased markedly during anoxia to a peak at about 25 min, followed by a decline when anoxia was prolonged to 45 min. Evidence was obtained that the increased evoked efflux of d‐[3H]Asp from anoxic slices was not due to impaired reuptake of the released amino acid and that it was completely reversible by reoxygenation of the slices. Results of experiments examining the evoked release of endogenous amino acids in anoxia were consistent with those obtained with the exogenous amino acids. Only 4 of the 10 endogenous amino acids studied exhibited TTX‐sensitive veratridine‐induced release under aerobic conditions (glutamate, aspartate, GABA, and glycine). Anoxia for 25 min did not affect the stimulated efflux of these amino acids with the exception of glutamate, which showed a significant increase. Compared with anoxia, effects of ischemia on synaptic function appeared to be more severe. Veratridine‐evoked release of [14C]GABA was already depressed by 10 min and that of d‐[3H]Asp showed a modest elevation only at 5 min. Stimulated release of d‐Asp and labelled GABA declined progressively after 5 min. These findings were compared with changes in tissue ATP concentrations and histology. The latter studies indicated that in anoxia the earliest alterations are detectable in glia and that nerve terminals were the structures by far the most resistant to anoxic damage. The results thus indicated that evoked release of amino acid transmitters in the cerebellum is compromised only by prolonged anoxia in vitro. In addition, it would appear that the stimulated release of glutamate is selectively accentuated during anoxia. This effect may have a bearing on some hypoxic behavioral changes and, perhaps, also on the well‐known selective vulnerability of certain neurons during hypoxia.

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of 14C incorporation into brain DNA at 30 min after administration of [2‐14C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 106 and 18 × 106 cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.

Effect of thyroid deficiency on cell acquisition in the postnatal rat brain: A quantitative histological study

The mechanisms underlying transient reduction of cell number in the developing cerebellum of thyroid-deficient rats have been studied by quantitative histological methods. Thyroid deficiency has no significant effect on the generation cycle of dividing cells in either the subependymal layer of the lateral ventricular walls or the external granulay layer of the cerebellum: the length of the cell cycle and the duration of the different phases of the cycle, including the DNA synthesis time appears to be normal. However, the external granular layer of the cerebellum contains fewer cells than in control at 12 days. Pyknotic nuclei are prominent in the granule cell layer of the hypothyroid cerebellum at this age. These amount to an estimated loss of about 1% of the total cerebellar cell population in 24 h. It is suggested that death of granule cells is for the most part a consequence of reduced Purkinje cell dendritic arborization, with failure of parallel fibre/Purkinje cell synaptogenesis. In the second postnatal week, granule cell death and reduced numbers of cells in the germinal zone can account to a great extent for the observed shortfall in cerebellar DNA content. The eventual attainment of normal cell numbers in the cerebellum of hypothyroid rats is related to a persistent external granular layer in the forth and fifth postnatal weeks.

Effect of thyroid deficiency on postnatal cell formation in the rat brain: A biochemical investigation

In thyroid deficiency, interference with postnatal cell formation seems to be confined to those regions of the brain where postnatal neurogenesis is significant. In comparison with controls the increase in cell number in the cerebellum is retarded in the second week of life, but a normal number is reached by 35 days. In contrast the DNA content of the olfactory bulbs is apparently irreversibly depressed. Mitotic activity, in terms of incorporation of [2-14C]thymidine into DNA, is mainly affected in the cerebellum: in thyroid deficient rats, it is depressed below control levels at day 12, but it is about 4 times higher than in controls at day 21 when, under normal conditions, cell proliferation has virtually ceased. The time course (15-240 min) of [14C]thymidine metabolism at day 14 shows regional differences in control rats. The rate of conversion of [14C]thymidine to [14C]thymidine nucleotides, and of these in turn to [14C]DNA is slower in the forebrain - where cell proliferation occurs on a smaller scale - than in the cerebellum. Consequently, in the forebrain nearly linear DNA synthesis rate is maintained for a longer time than in the cerebellum (1 h vs. 0.5 h), and since less 14C is conserved in DNA a significant efflux of unconverted [14C]thymidine is evident during the experimental period. The effect of thyroid deficiency on [14C]thymidine metabolism in the brain is only slight, and is due to an abnormally large supply of [14C]thymidine consequent to depressed systemic utilization of this precursor.

Effect of depolarization on the maturation of cerebellar granule cells in culture

The effect of depolarization on the maturation of granule cells derived from cerebella of 8-day-old rats can be studied in cultures in chemically defined media because their survival is not dependent on elevated K+ as it is when they are grown in serum-containing media. As an index of maturation, stimulus-coupled transmitter release was examined. This was chosen because it is closely associated with the neuronal phenotype and, in contrast to granule cells grown under depolarizing conditions in serum-containing media, it has not been known whether this property is expressed during the development of serum-free cells in culture. Veratrine-induced release of preloaded D-[3H]aspartate (Asp), an analogue of glutamate (Glu; the transmitter of the granule cells), was not detectable in the serum-free cells at a time (8-12 days in vitro) when this property was fully developed in cells grown in a medium containing serum and 25 mM K+ (reference cultures). This finding may be related to the failure of the expression of voltage-sensitive calcium channels in the serum-free granule cells. However, in comparison with reference cultures, voltage-sensitive 45Ca2+ entry was only transiently retarded in the serum-free cells. Furthermore, in contrast to the exogenous D-[3H]Asp, stimulated release of endogenous Glu was detectable, although it was substantially lower than in the reference cultures. Autoradiographic studies indicated that the failure to elicit evoked release of the exogenous amino acid was due to a severe retardation of the expression of the acidic amino acid carrier in the serum-free granule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular fractionation of rat cerebellum: an electron microscopic and biochemical investigation. I. preservation of large fragments of the cerebellar glomeruli

Abstract By a combination of differential and sucrose density gradient (both discontinuous and linear) centrifugation, large fragments of the cerebellar glomeruli were isolated in high purity from hand homogenised tissue. The final preparation contained only about 1% of the tissue protein, but over 90% of its volume was accounted for by the glomerulus particles. The ultrastructure of the glomerulus particles was well preserved. The enzyme profile was characteristic: the glomerulus particles were enriched in glutamate decarboxylase (GAD) activity (relative specific activity (RSA), 2.54), but the RSA of choline acetyltransferase (ChAc) was only 1.05. These findings are consistent with the view that GAD activity is very high in the inhibitory Golgi terminals, which occupy only a small fraction of the total volume of the particles, and acetylcholine may be a transmitter only in a relatively small fraction of the mossy fibre terminals. The glomerulus particles also contained a high concentration of succinate dehydrogenase (SDH) activity (RSA, 1.91), whereas the RSA of glutamate dehydrogenase (GDH) was only 1.15. The great asset of this preparation for future investigations is that it is composed almost exclusively from pre- and postsynaptic neuronal structures. Fractions containing neuropil fragments of non-glomerular origin were also obtained, but the profile of the estimated enzymes did not indicate unique characteristics.

Regulation of the expression of NMDA receptor subunits in rat cerebellar granule cells: effect of chronic K(+)-induced depolarization and NMDA exposure.

Abstract: The influence of K+‐induced membrane depolarization and NMDA treatment on the regulation of NMDA receptor subunit (NR) expression was investigated during the development of granule cells in culture, as a follow‐up of previous work on NMDA receptor activity. In spite of the increase in NMDA receptor activity elicited by these treatments (K25 or K10 + NMDA cultures), the main developmental changes in receptor mRNA levels were similar to those in untreated cells (K10) (a threefold increase in total NMDA receptor mRNA, quantitative dominance of NR1 mRNA, late expression of NR2C, and virtual absence of NR2D). However, high K+ and NMDA treatment resulted in a greater increase of NR2A mRNA levels and a retardation in the developmental changes in the relative amounts of NR2B and NR2C mRNAs. The correspondence between NMDA receptor activity and the amount of NR1 and NR2A subunit proteins was excellent, the rank order being K25 > K10 + NMDA > K10 at 9 days in vitro. Because the increase in subunit mRNA was not always paralleled by an increase in subunit protein, the control of NMDA receptor expression involves critically, in addition to gene transcription, regulation of translational and/or posttranslational events.