Arun K. Sinha

Bulk separation of neurones and glia: A comparison of techniques

Abstract Three different techniques of preparation of neurones and neuropil (glia) from rat cortex, those of Rose, Norton and Poduslo, and Sellinger and Azcurra, were compared according to the proposed criteria of purity, integrity and yield. Morphologically the cells prepared by the Norton and Poduslo and Sellinger and Azcurra methods appeared better and the fractions were purer than Rose method I, though not Rose method II. Based on carbonic anhydrase ratios, the glial contamination of neurones was 11% in Rose cells, 24.8% in Norton and Poduslo cells and 8.3% in Sellinger and Azcurra cells. Lactate dehydrogenase activity was 25% in Sellinger and Azcurra cell suspension and 37% in Norton and Poduslo cell suspension, of that in the Rose suspension. Oxygen consumption and carbon dioxide production in Sellinger and Azcurra suspension was only 15% and in Norton and Poduslo cell suspension 50%, of that in the Rose technique. The metabolic damage observed in the Sellinger and Azcurra preparation could be partially averted by alteration of the disaggregation medium. The relative utility of the techniques is discussed.

Compartmentation of lysosomes in neurones and neuropil and a new neuronal marker

Abstract The activity of 9 acid hypdrolases and 2 alkaline hydrolases has been determined in cortical subcellular fractions, separated neuronal and neuropil cell fractions, and neuronal and neuropil subcellular fractions, from the rat. A modification of Rose Method I for the bulk separation of neuronal and neuropil fractions, designed to reduce possible contamination by liberated lysosomes, was introduced. In general, the acid hydrolases were concentrated in the crude ‘mitochondrial’ and ‘lysosomal’ subcellular fractions, though some showed a bimodal distribution. The alkaline hydrolases were more generally distributed. All enzymes were more concentrated in the neuronal than the neuropil cell fractions. The acid hydrolases could be divided into 3 groups. Group I contained β-glucosidase, acid phosphatase, β-glucuronidase and arabinosidase, group II aryl-sulphatase, cathepsin, acid DNAse and β-glucosaminidase, and group III β-galactosidase. The neuronal-neuropil activity ratio for group I was between 1.4 and 3.4, for group II between 5.8 and 6.8 and for group III, 9.9 Solubilisation of the enzymes with Triton X-100 slightly enhanced the activity, but did not affect the neuronal-neuropil activity ratios of the enzymes β-glucuronidase, β-glucosidase and β-galactosidase. After subcellular fractionation of the neuronal and neuropil fractions the neuronal-neuropil compared with neuropil lysosomal protein; (b) the cellular compartmentation of cerebral lysosomes. The significance of these observations in terms of the relationship between neuronal and neuropil metabolism is discussed and appropriate predictions made. The enzyme β-galactosidase is proposed as a new neuronal marker.

Synthesis of tubulin-enriched fraction in rat visual cortex is modulated by dark-rearing and light-exposure

Microtubules are known to have an important role in the brain, especially in relation to the transport of materials from the nerve cell body to synaptic sites [l] . The principal component of microtubules is the polymerisable protein, tubulin. In this paper, we report evidence that the rate of synthesis of a polymerisable tubulin-enriched protein fraction in the visual cortex of the rat brain is reduced during dark-rearing and is enhanced by exposing the dark-rearing animals to light. In previous experiments [2] we have shown that the rate of protein synthesis in the neurons of the visual cortex of dark-reared rats is lowered compared to that in their normally-reared littermates [3]. When rats reared for 50 days in the dark are exposed to light, there is an increased protein synthetic rate in the visual cortex, lateral geniculate and retina [4]. An hour after onset of illumination and injection of radioactivity, most of the increased protein radioactivity is bound to the ribosomes or in a small number of soluble fractions [5]. Because of the known importance of tubulin in the neuronal economy [6,7] it seemed sensible to see if any of the soluble protein fractions whose synthetic rate was modulated by dark-rearing and light-exposure could be related to tubulin.

Incorporation of 3H-lysine into a rapidly labelling neuronal protein fraction in visual cortex is suppressed in dark reared rats

Abstract In normal rat cortex, incorporation of 3 H-lysine into protein is higher in neurons than neuropil at periods up to 2 hrs, but is higher in neuropil than neurons at later times. This observation implies the presence of a rapidly labelling neuronal protein fraction which is subsequently absent from the neurons, possibly due to transport into other cell compartments. When rats are reared in the dark, incorporation into neuronal proteins is suppressed in the visual cortex but not in the motor cortex; this suppression appears to include the rapidly-labelling fraction.

Rapidly labelling and exported neuronal protein; [3H]fucose as a precursor, and effects of cycloheximide and colchicine.

Abstract— The characteristics of a rapidly labelled and rapidly transported neuronal perikaryal protein fraction (Rose & Sinha. 1974a) were investigated in three experiments. (1) The kinetics of labelling of neuronal cell body and neuropil fractions from [3H]fucose were followed and shown to be similar to those from [3H]lysine, the label first appearing in the neuronal fraction and then being exported. The neuronal/neuropil incorporation ratio fell from 1.37 at 1 h to 0.77 at 4 h. (2) When cycloheximide (5 mg/kg) was injected intraperitoneally 15 min after [3H]lysine, incorporation into neuronal protein was inhibited to a greater extent (85%) than into neuropil (60%). (3) Colchicine was injected at a dose (40 μg/kg) sufficient to prevent accumulation of radioactively labelled protein into synaptosomes but insufficient to affect total incorporation of precursor into protein. [3H]Lysine was injected 1 h after colchicine and neurons and neuropil fractions made 1 h and 4 h later; colchicine inhibited the export of labelled protein from the neuronal perikaryon and its accumulation in the neuropil. We conclude that the rapidly labelled neuronal protein is partially glycoprotein in character and may be normally transported from the cell body by way of the axonal/(dendritic?) flow mechanism.

DARK REARING and VISUAL STIMULATION IN THE RAT: EFFECT ON BRAIN ENZYMES

Abstract— Enzyme activities in motor and visual cortex and cerebellum of rats reared for 50 days in the dark (D) were compared to levels in normally reared (N) and in dark‐reared littermates exposed to 3 h of visual stimulation (L). Amongst 6 acid hydrolases, two, acid phosphatase and galactosaminadase, showed no effect of dark rearing. In three of the others, glucuronidase, glucosaminidase and galactosidase, activity tended to be lower in D than L. In glucosaminidase, N was similar to D and L above both, while in (total) glucosidase, galactosidase and glucuronidase, N was higher than D and L approached N. There were fewer changes in cerebellum than in cortex.

Neuronal locus of enhanced incorporation of 3H-lysine into acid insoluble material in visual cortex on first exposure of rats to light

Abstract Incorporation of 3 H-lysine into acid insoluble material of cell fractions derived from the visual cortex of dark reared and light exposed rats was compared. There was an elevation of incorporation of 35% in the whole cell suspension and 33% in the neuronal fraction but only 3% in the neuropil fraction. The ratio of incorporation of precursor into protein in the neuronal fraction of the dark reared animals to that in the neuropil was 63%; after 60 min light exposure the ratio was 93%; in normal animals the ratio was 137%. These observations suggest two hypotheses; that the elevation of protein synthesis in the visual cortex on first exposure to light, is primarily a neuronal phenomenon; and that in the absence of functional stimulation neuronal protein synthesis is depressed and glial protein synthesis elevated compared with the normal controls.

SUB-CELLULAR LOCALISATION OF ENHANCED LYSINE INCORPORATION INTO CEREBRAL CORTEX PROTEINS IN DARK-REARED AND LIGHT-EXPOSED RATS

Incorporation of lysine into acid‐insoluble material from subcellular fractions of rat cerebral cortex has been studied using double and single‐labelling techniques, in littermates reared for 50 days in the dark and then dark‐maintained or light‐exposed for 1 h. When light‐exposed animals were compared to dark controls the only subcellular fraction from the whole cortex in which lysine incorporation shows a significant elevation (168%, P < 0.05) was located in the ribosomal pellet of the cerebral cortex. A similar comparison of subcellular fractions from visual and motor cortices showed that the elevation was again in the ribosomes and confined to visual cortex only. Motor cortex of light‐exposed animals showed a small depression of incorporation in ribosomes as compared to dark controls. Sub‐fractionation of nuclei from whole cortex preparations showed varying, but non‐significant elevations in light‐exposed animals in all but the histone fraction in which there was negligible incorporation of precursor. It is concluded that enhancement of incorporation of precursor into proteins of the cerebral cortex, which accompanies first exposure to light, is a complex response. At exposure for 1 h it involves a number of particular protein species located in the visual cortex, a major proportion of which are ribosomally bound.

NEURONAL AND NEUROPIL FRACTIONS FROM DEVELOPING RAT BRAIN

Abstract— A method is described for the preparation of enriched fractions containing isolated neuronal and glial cells from brains derived from 1 to 20‐day‐old rats. The method is based on mechanical disaggregation in a medium containing Ficoll‐PVP followed by centrifugation on a single‐stage two‐step gradient at 13,000g for 30min. The neuronal and neuropil (glial) fractions are approx 70–80% pure in cellular terms.