Gerard M. Lehrer

Cerebroside antibody inhibits sulfatide synthesis and myelination and demyelinates in cord tissue cultures.

Antiserum to cerebroside was prepared in rabbits by injection of cerebroside together with bovine serum albumin in complete Freunds adjuvant. When applied to cultures of embryo mouse spinal cord at explantation, this antiserum inhibited sulfatide synthesis and myelination; when applied to myelinated cultures it inhibited sulfatide synthesis and produced demyelination. Complement fixation assays also show antibody to cerebroside in serums from rabbits with experimental allergic encephalomyelitis induced by injection of whole white matter. Absorption of such serum with cerebroside abolishes the inhibiting and demyelinating activities.

Enzymes of carbohydrate metabolism in the rat cerebellum developing in situ and in vitro

Abstract Seven enzymes of the glycolytic, citric acid cycle, and pentose phosphate shunt pathways have been studied by quantitative micro-methods in areas corresponding to the internal granular layer from both frozen-dried cultures and sections of cerebella of rats at various ages. Characteristic increases with age were observed for hexokinase, lactic dehydrogenase and malic dehydrogenase during development in situ and in culture. However, the increases in the cultures occurred about 2 days earlier than in situ. Glucose-6-phosphate dehydrogenase showed a sharp rise with a peak at the eighth day followed by a gradual decline in situ, whereas its activity continued high in the cultures, even to the twenty-fourth day. NADP+-dependent isocitric dehydrogenase paralleled glucose-6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase showed no particular trend. We concluded that the enzymatic spectrum during development in culture, with some modifications, closely reproduces that observed during in situ development. These results genrate further confidence in the use of the rat cerebellum cultures as a biological model for studies of central nervous system development and function. Lactic dehydrogenase may be inducible in culture. Glucose-6-phosphate dehydrogenase and NADP+-dependent isocitric dehydrogenase may serve in pathways which supply NADPH required for synthetic processes.

Sulfatide Synthesis: Inhibition by Experimental Allergic Encephalomyelitis Serum

The rate of 35S incorporation into cerebroside sulfate in cultures of embryo mouse spinal cord shows a rapid acceleration at the time of myelin formation. Exposure of cultures to dilute serum from rabbits with experimental allergic encephalomyelitis results in almost complete inhibition of sulfatide synthesis. Within 24 hours after replacement of inhibiting medium with normal medium there is an increase in sulfatide synthesis followed by myelination.

Enzymatic maturation of mouse cerebral neocortex in vitro and in situ

Abstract Enzymes active in various metabolic pathways have been studied by quantitative micromethods in histologically defined areas corresponding to layers III–V of the superior frontal neocortex from frozen-dried long-term tissue cultures and from frozen sections of the forebrains of mice at ages between birth and 24 days. Characteristic, parallel increases were seen for hexokinase, lactic dehydrogenase, and malic dehydrogenase during in vitro and in situ development. NADP + -dependent isocitric dehydrogenase showed parallel decreases. The greatest rates of change under both conditions occurred between Days 8 and 15. Only β-glucuronidase and glucose-6-phosphate dehydrogenase showed differences between in situ and in vitro findings. These results generate further confidence in the use of the mouse cerebral cortex culture as a biological model for studies of neocortical development and function. Evidence is offered that lactic dehydrogenase may be inducible in nervous tissue.

Changes in LDH isoenzymes of brain developing in situ and in vitro

Postnatal changes in lactate dehydrogenase activity and isoenzyme pattern studied in mouse cerebrum and rat cerebellum by quantitative microassay and acrylamide ‘disc’ gel electrophoresis were compared to those occurring in the same tissues growing in tissue culture. Both in vivo and in vitro there is a relative and absolute increase in those isoenzymes which migrate more rapidly toward the anode. These are associated with function at high relative oxygen concentrations and are characteristic of adult brain. Furthermore, increase of the slower moving isoenzymes was induced in the cultures, related to the oxygen gradient in the tissue. In vivo and in vitro the enzyme changes accompany structural and physiological developments such as neuropil growth, synapse formation and myelination, which have large energy requirements.

Regional changes in cerebellar creatine phosphate metabolism during late maturation

Abstract The activity of creatine kinase, the steady-state level of creatine phosphate, and the rate of change of creatine phosphate during a brief period of ischemia have been studied by quantitative micromethods in the cerebellar molecular and granular layers and white matter of prepubescent and adult mice. In the neuropil-rich molecular layer there is a large increase with maturation in creatine kinase activity, creatine phosphate level, and the rate of disappearance of creatine phosphate during ischemia. In the granular layer there is a decline in creatine kinase activity with maturation but no change in creatine phosphate level or its ischemic flux. Neither creatine kinase activity nor the creatine phosphate level changes in the white matter with maturation. The differences could not be attributed to differences in lipid or cell density. The findings suggest a significant change in energy metabolism of synapse-rich areas after the apparent morphological maturation of the cerebellar cortex. This change would be obscured if histologically more complex regions are studied.

The Tissue Culture as a Model for The Biochemistry of Brain Development

Publisher Summary The purpose of this chapter is to demonstrate the concomitant development of biochemical specializations and to offer some examples of the past and current experiments that could not easily have been performed in other systems with equal reliability. The great advantages of the culture system are that it is free from the homeostatic mechanisms of the organism, susceptible to controlled manipulations of its environment, and available for constant, direct microscopic observation of structure and function. Glucose-6-phosphate dehydrogenase is a NADP-dependent enzyme of the pentose phosphate shunt, which serves to reduce NADP, a pyridine nucleotide not so much concerned with cell respiration as it is with synthetic processes in the cell, particularly the final steps in lipid synthesis (long-chain fatty acids and cholesterol) where it serves as the principal reducing agent. NADP-dependent isocitrate dehydrogenase decreases during cerebellar development in vivo, whereas in the tissue culture, it starts high and does not decrease, a difference between the culture and the in vivo system.

CIRCULATORY FACTORS IN THE CARBOHYDRATE METABOLISM OF AN EXPERIMENTAL GLIAL NEOPLASM

Lowry and coworkers ( 1964) have shown that decapitation essentially converts the cranial contents into a closed biological system, and that valid conclusions can be drawn about steady-state, in vivo modes of brain carbohydrate metabolism from observed changes in the levels of high energy and glycolytic intermediates during the period following decapitation. lt is likely that such an approach can yield more valid information about in vivo metabolism of tissue than can be obtained by metabolic studies in tissue slices or homogenates. We SUCcessfully applied this method to a previous study of a transplanted mouse ependymoblastoma growing intracerebrally (Maker et al., 1966). These studies have now been extended to the same neoplasm, transplanted subcutaneously. Marked differences in the nietabolic modes of this tumor at the two different sites have become apparent. Results of studies at the two sites will be compared, and implications regarding relative blood supply of the tumors will be discussed.

Microchemical assay of galactose containing cerebrosides

Abstract A method for the estimation of galactose containing lipids based on acid hydrolysis and fluorometric assay of galactose with galactose dehydrogenase has been developed. The characteristics of cerebroside hydrolysis under several different conditions are described. Under the conditions of hydrolysis sphingosine and galactose are released in parallel. A microchemical modification of the procedure has been used to assay galactose cerebroside-sulfatide in fragments of mouse cerebellar layers weighing 1–3 μg dry wt dissected from freeze-dried tissue sections.