E. De Robertis

Gangliosides and acetylcholinesterase in isolated membranes of the rat-brain cortex

Abstract The subcellular distribution of gangliosides was studied in the rat-brain cortex. In the primary fractions most of the gangliosides are in the microsomal and crude mitochondrial fractions, with the highest concentration in the light microsomes which contain mainly membranous components. After osmotic disruption of the synaptic complex, gradient techniques were used to purify the synaptic vesicles and to isolate the synaptic membranes. Two layers of synaptic membranes were found to contain the highest concentrations of gangliosides and acetylcholinesterase. Another layer of synaptic membranes poor in acetylcholinesterase had a low content of gangliosides. The lack of gangliosides and acetylcholinesterase in synaptic vesicles is emphasized, and the possible physiological significance of these complex acidic glycolipids in some neuronal membranes is discussed.

Lipids and proteolipids in isolated subcellular membranes of rat brain cortex.

The cerebral cortex of the rat was submitted to an extensive cell fractionation schedule and in the various fractions, protein, proteolipid protein, total phospholipids, cholesterol, galactolipids, plasmalogens, and gangliosides were determined. With increasing purification the different isolated membranous structures: i.e. myelin, nerve ending membranes, synaptic vesicles, mitochondria, and microsomes, show a definite biochemical specialization reflected in their lipid composition. The presence of gangliosides in some nerve ending membranes is confirmed, and the possible functional role of these acid glyco‐lipids is discussed.

Benzodiazepine receptors in rat cerebral cortex and hippocampus undergo rapid and reversible changes after acute stress

Rats were submitted to forced swimming and were killed 15 min after initiation of the stress and at 1 h, 1 day and 4 days thereafter. Immediately after the stress there was a decrease of 30% in the density of [3H]flunitrazepam binding sites in the cerebral cortex and of 27% in the hippocampal formation, with no changes in all the other brain areas studied. These changes in the number of benzodiazepine receptors were also corroborated by the binding of [3H]ethyl-beta-carboline carboxylate. For both ligands there were no changes in affinity. These effects were selective for the benzodiazepine receptors and no changes in alpha 1, alpha 2 and beta adrenoceptors and in dopaminergic receptors were observed. One hour after the stress, the number of benzodiazepine receptors had recovered in the cerebral cortex (8% above the control) and had increased greatly in the hippocampal formation (+53%). One day after the stress, the [3H]flunitrazepam binding in the cerebral cortex reached the normal level but it was still slightly elevated (+16%) in the hippocampus. These results are discussed in relation to some contradictory findings in the literature and to the fact that the hippocampal formation is related to neural mechanisms underlying behavior and neuroendocrine regulation.

Triton X-100 as a channel-forming substance in artificial lipid bilayer membranes

The nonionic detergent Triton X-100, introduced into artificial membranes of various lipid compositions, induced current fluctuations that may correspond to the formation of channels across the bilayer. Independently of the lipid used, these fluctuations vary in amplitude between 1 and 4·10−10, Ω−1, show a strong dependence on the applied voltage, and are selective for cations in the sequence Rb+ > K+ > Na+ > Li+. These results are discussed in relation to the chemical structure of the Triton X-100 molecule.

NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium‐3 (HC‐3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [32P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [l4C]HC‐3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC‐3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.

Binding ofdimethyl-C14-d-tubocurarine, methyl-C14-hexamethonium, and H3-alloferine by isolated synaptic membranes of brain cortex

Abstract The binding capacities for dimethyl-C 14 -d-tubocurarine , methyl-C 14 -hexamethonium and H 3 -alloferine were studied in subcellular fractions of the rat brain cortex and compared with the acetylcholinesterase activity of the same fractions. An in vitro technique for the uptake of the radioactive agent was developed and the best conditions regarding incubation medium, time, washing, particle and blocking agent concentration were determined. The uptake was studied in the primary particulate fractions (nuclear, mitochondrial and microsomal), in the particulate fractions after the osmotic shock of the crude mitochondrial fractions (subfractions M 1 and M 2 ) and in the subfractions of M 1 on a sucrose density gradient. The most interesting results were found in subfractions M 1 0·9 and M 1 1·0 separated at 0·9 and 1·0 M sucrose, which showed the highest binding capacity and also the highest cholinesterase activity. Under the electron microscope these fractions showed typical synaptic membranes. Subfraction M 1 1·2, also of synaptic membranes, showed tittle binding and acetylcholinesterase activity. These and other results indicate that synaptic membranes having cholinergic and non-cholinergic properties may be isolated from the brain cortex. Preliminary experiments were made to study the interferebce of atropine, eserine and acetylcholine with the uptake. Others are planned to establish if this drug-membrane interaction is truly specific for the cholinergic receptor.

Conductance Changes Produced by Acetylcholine in Lipidic Membranes Containing a Proteolipid from Electrophorus

Ultrathin lipidic membranes containing one ten-thousandth of a special proteolipid from electric organ of Electrophorus reacted to the addition of acetylcholine by a rapid and transient increase in conductance. Such a change was not induced by choline and is greatly reduced by a previous application of d-tubocurarine. These properties, resembling those from chemically excitable membranes, were not observed with another proteolipid from the same tissue.

Isolation of hydrophobic proteins binding amino acids. Stereoselectivity of the binding of L-[14C]glutamic acid in cerebral cortex.

From the total lipid extract of ncrve‐ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L‐[14C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [14C]GABA‐binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd1= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L‐enantiomer. The binding of L‐[14C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL‐a‐methylglutamic acid, L‐nuciferine, L‐aspartic acid and L‐glutamic acid diethyl ester. These results are compared with those previously obtained with the L‐glutamic acid‐binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.

Catechol-o-methyltransferase in nerve endings of rat brain

Abstract The distribution of COMT was studied in subcellular fractions of rat brain. About 50 percent of the enzyme is particulate and concentrated in the nerve ending fractions. The solubility of the enzyme after the osmotic rupture of the nerve ending makes impossible a finer localization of the enzyme within the synaptic complex. The difference in localization with MAO and the probable physiological action of the two enzymes on brain catecholamines are discussed.

Early effects of antiserum against the nerve growth factor on fine structure of sympathetic neurons

Abstract The early changes produced by the antiserum (AS) against the nerve growth factor (NGF) on sympathetic ganglia of newborn mice were studied under the electron microscope. The AS was injected at birth and the ganglia observed between few hours and 15 days. Four- to six-hour in vitro experiments were also done on pieces of newborn ganglia by adding AS to Morgans TC 199 fluid. Within a few hours after the AS injection there is considerable damage of most neurons while neurilemma and endothelial cells remain intact. The early alterations consist of folding of the nuclear membrane and condensation of chromatin. This is accompanied by nuclear swelling, dilatation of the vacuolar system, mitochondrial alteration and final lysis of both the nucleus and cytoplasm. In vitro the cytolytic effects are similar but more marked. These findings are interpreted as indicative of a cytotoxic antigen-antibody reaction taking place in most sympathetic neurons. In this reaction the presence of complement seems indispensable.