Frances B. Nesbett

In Vitro Retina as an Experimental Model of the Central Nervous System

Abstract Methods are described for isolating adult rabbit retina and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, and electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue

INTRACELLULAR C1−, Na+, K+, Ca2+, Mg2+, AND P IN NERVOUS TISSUE; RESPONSE TO GLUTAMATE AND TO CHANGES IN EXTRACELLULAR CALCIUM*

THE AIMS of this study were to determine the normal pattern of intracellular electrolytes in a sample of mammalian central nervous tissue and to observe how this pattern is affected by changes in Ca2+ or glutamate in the extracellular fluid. Particular attention has been paid to the intracellular Ca2+ and Mg2+ because of the importance of these ions to neuronal function and because relatively little is known about their levels within the cells and about how these levels are maintained. The experiments were performed on an in uitro preparation of nervous tissue, the isolated retina of the rabbit. This made it possible to control and to modify the composition of the fluid bathing the tissue and to use an elution technique (AWES and NESBETT, 1966) for partitioning the water and electrolytes between the intracellular and extracellular phases. Measurements of the electrolytes were made by semi-micro chemical techniques, rather than with isotopes, so that it was possible to determine the amounts of all five of the major inorganic ions as well as P on a single specimen and to observe how they changed with relation to each other. Though about 15 per cent of the retina is specialized for photoreception, the rest appears to be representative of CNS grey matter in general, on the basis of its embryology, morphology, chemistry and function. As will be shown from the present study, it is possible to isolate and incubate the tissue under control conditions with little or no change in the concentrations of the intracellular electrolytes. It was shown previously that the electron microscopic appearance (WEBSTER and AMES, 1965) and the electrophysiological function (AMES and GURIAN, 1960) of the isolated tissue also remain quite normal during incubations lasting up to 2 hr.

Improved Fixation for Histological Demonstration of Glycogen and Comparison with Chemical Determination in Liver.

Summary Glycogen of rat liver was well preserved throughout histological blocks and was not displaced in the cells when pieces of liver were fixed in ice-cold picro-alcohol-formalin. After fixation, liver sections were stained by the Bauer-Feulgen method and the optical density of each section was measured on a Photovolt electronic photometer. Chemical determinations were made of the glycogen contained in other pieces of the same livers. The livers of rats undergoing progressive starvation from 6 to 48 hours showed good correlation between the chemically and histologically determined glycogen concentrations.

Protein synthesis in central nervous tissue: studies on retina in vitro.

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000μM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100‐1000μM, leucine was incorporated into protein at 2.03 ± 0.04 (s.E.M.) μmol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 μM with other amino acids constant, 14C‐leucine incorporation fell 70%; without significant change in 3H‐threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 μM. It fell markedly when medium leucine was reduced to 2 μM or increased to 1000 μM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 μM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.

A method for multiple electrolyte analyses on small samples of nervous tissue.

THIS paper describes a system of analysis designed to measure all of the major inorganic electrolytes-Na, K, Ca, Mg, C1, and P-in a single small sample of nervous tissue. Because of the interrelationships between the movements of the different electrolytes in living tissue, it is important to be able easily to measure a number of them in the same specimen. At the same time, the multiplicity of the determinations must not increase unduly the size of the sample required. The advantage of limiting the size of the specimen becomes evident in the analysis of small tissues such as retinas and sympathetic ganglia and in the discrete sampling of a heterogeneous organ such as brain. In the latter instance, each significant reduction in sample size opens new opportunities for correlations between chemistry and the function of successively smaller structural units. The potentialities of this approach, as applied particularly to organic compounds in brain, have been remarkably demonstrated by the work of LOWRY and his associates (e.g. LOWRY, ROBERTS and CHANG, 1956). Methods have been described for the analysis, in biological materials, of small amounts of Na and K (KEYNES and LEWIS, 1951; SOLOMON and CATON, 1955) and of C1 and P (LOWRY, ROBERTS, LEINER, Wu and FARR, 1954a). Equally satisfactory microanalyses have not been available for Mg and Ca, though GLICK, FREIER and OCHS (1957) have recently published a method for measuring Mg in mpmole amounts using a microscope colorimeter, and a fluorescent indicator for Ca of considerable promise has been synthesized (WALLACH, to be published). A number of systems for the simultaneous analysis of four or more electrolytes are described in the literature (CULLEN, WILKINS and HARRISON, 1933; YANNET and DARROW, 1938; EICHELBERGER and RICHTER, 1944; LOWRY, HASTINGS, MCCAY and BROWN, 1946; L I L L I E N T H A L ~ ~ ~ ~ . , 1950; Conom, HOLLIDAY, S C H W A R T Z ~ ~ ~ WALLACE, 1951). The total size of the specimen required for these analyses varies between 0.3 and 19 g. Different methods of extracting, digesting or ashing the tissue are used prior to the h a 1 analyses for the different typ:s of electrolytes; and, since these preparatoxy steps are not interchangeable, the several analyses require either partitioning of the tissue in its original state or after it has been minced, dried and ground, or homogenized. Partitioning the fresh tissue creates difficulties in interpreting the results if the specimen is not entirely homogeneous. Mincing, grinding, or homogenizing are difficult to perform quantitatively on a microscale and may increase the

Analysis for potassium, sodium, chloride, and water in a 2-μl sample of extracellular fluid

Abstract Methods are described for analyzing Na, K, Cl, and water on a single 2-μl sample of extracellular fluid, with an accuracy and reproducibility of better than 2%. Conventional glassware was used throughout. Cl was determined colorimetrically; Na and K were measured by a flame photometer of special design (Baird-Atomic Inc. of Cambridge, Mass.) with a modified system for atomization. Considerable further reduction in sample size is probably possible with slight modifications of these methods.

Protein Turnover in Retina

Abstract: Rabbit retinas were exposed in vitro to 0.5‐h pulses of [3H]leucine or [14C]Ieucine. Some retinas were harvested promptly after labeling to measure synthesis. These were combined, in double‐labeling experiments, with retinas that had been returned to unlabeled medium for a subsequent 1 h or 3.75 h to measure degradation. All of the proteins were solubilized, and separated according to size by gel electrophoresis. The gels were cut into 95 slices, and each slice was differentially counted. The amount of protein in the slice was estimated from the Coomassie blue staining, and its molecular weight from the distribution of molecular weight (MW) standards. Turnover rates of the various sizes of proteins were calculated from these data using certain well‐defined assumptions. Retinal protein contained about 32 ± 103 nmol of polypeptide per g, with a median MW of 27,000. Total synthesis was at the rate of 103 nmol/g of protein/h, with the most rapid synthesis in the 33,000–43,000 MW range, at 2 nmol/g/h for every 1000 increment in MW. Protein renewal averaged 0.52%/h, but varied directly (p < 0.0001) with MW, so that proteins of 10,000 MW were being renewed at about 0.1%/h and proteins of 140,000 MW at about 1.4%/h. Taken together, the measurements of fractional renewal and the measurements of degradation of the newly synthesized proteins demonstrated that each slice contained proteins with markedly different breakdown coefficients, and provided enough information to characterize the proteins in the slice in terms of a fast and a slow subgroup. This analysis indicated that: breakdown coefficients varied much more than rates of synthesis and were therefore the prime determinant of the amount of each protein that was present; as MW increased, breakdown coefficients of the long‐lived proteins increased (p < 0.0001), accounting in major part for the correlation between size and turnover; most staining bands were due to proteins with peculiarly long lifespans; the proteins with the slowest turnover of all appeared to be histones: there was an unusually rapid synthesis of a 138,000 MW polypeptide with a moderately short half‐life (about 3 h).

Transport of leucine and sodium in central nervous tissue: studies on retina in vitro.

Unidirectional leucine fluxes were measured in isolated rabbit retina maintained under steady state conditions in medium resembling CSF but with leucine varied from 2 to 20,000 μM. At physiological leucine concentration (11 μM), 1/2 time for outward transport was 88 s and intracellular fluid was cleared of Isotopically labelled leucine at 2.3ml/g dry wt./min; 1/2 time for inward transport was 16 s and interstitial fluid was cleared at 7.5 ml/g dry wt./min. The rate of leucine influx corresponded quite well with its rate of disappearance from the intracellular fluid, over a wide range of concentrations. Exchange diffusion was demonstrated for transport in both directions. There was competition by other amino acids, but no interaction between Na+ and leucine transport could be demonstrated. Kinetic analysis indicated the presence of more than one transport system for leucine. There was an unexpected fall in the efflux coefficient, with reduction in leucine concentration at the lower end of the concentration range, for which an explanation is proposed. Under control conditions, 1/2 time for efflux of intracellular 24Na+ was about 0.9 min. With intracellular Na+ increased 4 fold, l/2 time for efflux was slightly reduced. Problems encountered in measuring fluxes in organized tissue are discussed.

Effect of Bile Acids on Activity of GIucose-6-Phosphatase.

Summary (1) Bile acids have been found to influence, in vitro, the activity of rat liver glucose-6-phosphatase, concentrations of 5 × 10-4 M inactivating and 1 × 10-3 M activating the enzyme. (2) Glucose-6-phosphatase from both normal and diabetic rats responds similarly. The increased glucose-6-phospha-tase activity found in liver of alloxan diabetic rats therefore does not appear to be due to any such activation mechanism, but rather due to an actual increase in the amount of enzyme present.

SYNTHESIS AND BREAKDOWN OF DIFFERENT SIZED RETINAL PROTEINS IN DARKNESS AND DURING PHOTIC STIMULATION

Rabbit retinas were maintained in vitro in medium resembling cerebrospinal fluid and were exposed to (3)H-leucine or (14)C-leucine in double-labeling experiments designed to measure rates of protein turnover and to determine the effects of photic stimulation on protein synthesis and degradation. The retinas were solubilized, and the proteins separated according to size by polyacrylamide gel electrophoresis. The gels were cut into 95 slices and each slice differentially counted. Protein content of the slices was estimated from Coomassie Blue staining, and molecular weight (MW) from distribution along the gel of MW standards. The retinas appeared to function nearly normally in vitro for many hours. Protein renewal was rapid and reproducible at 0.55 +/- 0.01 (S.E.M.) %/h and remained quite constant for at least 7 h. Synthesis and degradation were approximately equal. Two retinas were maintained in vitro at 37 degrees C for 52 h, and showed good preservation of morphology, electrophysiological response to light, and protein synthesis. Total synthesis of new polypeptides was at the rate of 103 nmole per g of protein, per h; there was a sharp peak in the 33,000 to 43,000 MW range. Proteins in every size group were very heterogeneous with respect to breakdown coefficients (i.e. longevity) which were the prime determinants of the amount of each protein present. Fractional renewal showed a highly significance (p<0.0001) correlation with MW, due apparently to a reduction in maximal longevity as size increased. Neither synthesis nor degradation was significantly affected by intense continuous light or flashing light, though the latter increased uptake of 2-deoxyglucose by 38%.