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The objective of this study was to identify cellular proteins that are associated with foetal brain transplants effective in reinstating memory function in adult rats with brain lesions. Quantitative memory deficits can be created in rats by lesioning the cholinergic projection system, using ibotenic acid. Previous work suggested that injection of cell suspensions prepared from presumptive cholinergic cells of foetal basal forebrain into adult brain, after such lesions, are most effective in restoring cognitive function. It was not clear, however, whether it was the cholinergic nature of the transplants that was critical for their success or whether other factors were involved. In this study, the proteins present in transplanted tissues and control brains were analysed by two-dimensional polyacrylamide gel electrophoresis to identify markers for the cells that were specifically correlated with restoration of cognitive function. On each gel, the relative optical densities of the same 33 selected proteins were measured on an interactive computerised image analyser. The amount of each protein was compared between treatment groups and correlated with four behavioural measurements. Seven of the proteins analysed had levels of expression that were either related to transplantation or correlated with behavioural performance. The proteins of interest were divided into the following three groups: (1) transplant-related proteins, (2) cholinergic transplant-specific proteins, and (3) behaviour-related proteins. Notable among the proteins of interest was one of the cholinergic transplant-specific proteins that was positively correlated with three of the four behavioural measurements and was also the only protein among those analysed that was significantly correlated with choline acetyltransferase (ChAT) levels. This has been identified, by immunoblotting, as glial fibrillary acidic protein, an astrocytic cell marker. These results suggest, therefore, that at least two cell types, astrocytes and ChAT(+)-staining cells, play an important role in the successful recovery of cognitive function. This study also identified possible protein markers for cognitive performance. The level of expression of two of the proteins analysed was not affected by lesioning or transplantation, but was significantly correlated with behaviour. One of these proteins, whose amounts correlated negatively with behavioural measurements, has been identified as neurone-specific enolase, a brain-specific neuronal cell marker.

3-Bromopyruvate inhibits pyruvate decarboxylase in brain homogenates and causes a 90% drop in acetylcholine tissue content at a concentration of 2 mM. Stereotaxic injection of 3-bromopyruvate into the basal forebrain causes after 7 days a 40% drop of acetylcholine concentration and pyruvate decarboxylase activity in the cortex and hippocampus, and greater decreases at the site of injection. However, values return to normal 18 days after injection. Choline acetyltransferase is partially inhibited only at the site of injection after 7 days. Choline transport and choline concentration are not affected at either 7 or 18 days after injection. Impairments in spontaneous alternation and in retention of passive avoidance were seen only 7 days after the injection. The results suggest that stereotaxic injection of bromopyruvate can induce discrete reversible cholinergic lesions on a time scale useful for behavioural experiments and for comparison with neurodegeneration.

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.

The coupling of choline transport to acetylcholine synthesis has been investigated by measurement of the isotopic dilution of a pulse of [3H]choline during its incorporation into the recently synthesised acetylcholine of cerebral cortex synaptosomes. Recently synthesised acetylcholine was identified as that containing 14C-labelled precursors introduced by a preincubation before the pulse. When [14C]glucose was used to label acetyl-CoA coupling ratios (calculated as the inverse of the dilution of extracellular [3H]choline during its incorporation into [3H]acetylcholine) of about 0.05-0.2 were found at a choline concentration of 1 microM, rising to 0.5 at choline concentrations of 10-50 microM. Experiments using [14C]choline as a precursor gave similar results, and it was shown that the isotopic dilution did not occur extrasynaptosomally and was not affected by low glucose concentrations. Coupling ratios were always less than unity and rose as the choline concentration increased. It is concluded that choline transported into the nerve terminal has no privileged access to choline acetyltransferase. The results can be explained by a rate-controlling transport of choline into the terminal followed by its rapid acetylation rather than any linkage or coupling of the two processes.

Stores of methionine-enkephalin were labelled on the N-terminal by incubation of whole brain slices with [3H]tyrosine (10 microCi/ml). The 3H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD2 and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37 degrees C and was inhibited in the presence of 10 microM cycloheximide. Isolated nerve terminals failed to incorporate any [3H]tyrosine. The labelled compound had opiate-like activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N-terminus, and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [14C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N-terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 mM) elicited a Ca2+-dependent release of the synthesized [3H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [3H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release, and its effect was partially reversed by atropine.

The release of lactate dehydrogenase and K+ when synaptosomes are exposed to resuspension in media of various osmolarity has been investigated in order to measure their disruption. Even when resuspended in distilled water a significant percentage (10-20%) of lactate dehydrogenase and K+ remains unreleased. The particles containing these substances sediment to the same density as synaptosomes. Synaptosomes retaining their internal organelles after hypoosmotic treatment can be seen in electron micrographs. Resealing of disrupted synaptosomes was measured by the inclusion of [14C]sucrose. The resealing is spontaneous, essentially complete (80-90%) within 20 min and not noticeably affected by temperature, pH, or the addition of fusogen. The synaptosome preparation after hypoosmotic disruption will therefore contain some undisrupted synaptosomes with some or all of their complement of cytoplasmic constituents, as well as resealed synaptosomes. The retention of the ability of the hypoosmotically treated preparation to convert [14C]choline to [14C]acetylcholine is demonstrated as an example of the disproportionate effect these undisrupted particles have on its properties.

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.

As in intact tissues, choline influx into synaptosomes is enhanced after a period of depolarization induced release of acetylcholine. The activation of uptake is dependent on the presence of Ca2+ and inhibited by high Mg2+ concentrations in the medium during depolarization. Choline transport in erythrocytes was not activated by prior treatment with potassium. The permeability constant of the synaptosome membrane to choline was found to be 2.7 x 10(-8) cm . s-1 and to acetylcholine 1.8 x 10(-8) cm . s-1. Choline influx has been studied after pre-loading synaptosomes with choline. Different radiolabels were used to measure efflux of preloaded choline and influx simultaneously. Isotopic dilution in flux studies was estimated and corrected for. Influx was stimulated by high internal concentrations of choline, and efflux similarly stimulated by high outside concentrations of choline. The maximal influx and efflux at saturating opposite concentrations of choline were equal with a value of about 500 pmol . min-1 per mg synaptosomal protein. A reciprocating carrier would explain the equality of the maximal influx and efflux. Acetylcholine competes with choline for binding to the carrier but is itself hardly transported. Increased acetylcholine concentrations were shown to inhibit both choline influx and efflux from the trans position. Raising intrasynaptosomal acetylcholine concentrations by pre-loading abolished the stimulation of influx by prior depolarization. It is proposed that high concentrations of acetylcholine immobilize the carrier on the inside of the synaptic membrane. The stimulation of choline influx consequent upon depolarization is caused by release of ACh which results in relief of this immobilisation. The enhanced supply of choline achieved by this mechanism is likely to be important in maintaining stores of the acetylcholine in vivo.