Ian S. McLennan

Characterization of a cardiac muscle factor required for the survival of cultured parasympathetic neurones

A macromolecule from ox cardiac muscle which supports the survival of dissociated embryonic chick ciliary ganglionic neurones in culture has been partially purified and characterized. Gel filtration after ammonium sulphate fractionation separated two peaks of activity, a high molecular weight form (greater than 50,000) which dissociated to a lower molecular weight component (approximately 20,000). The high molecular weight form was retained by DEAE-cellulose at neutral pH and was eluted by 0.11-0.16 M NaCl. The isoelectric point of the factor was 6.2.

Parasympathetic neuronal survival induced by factors from muscle

Abstract A factor has been identified in extracts of rat cardiac and skeletal muscle which increases the activity of choline acetyltransferase in cultures of avian ciliary ganglia. It is likely that this factor differs from the previously described factor present in conditioned medium that stimulates the outgrowth of neurites in these ganglia.

Influence of cardiac extracts on cultured ciliary ganglia.

Inclusion of extracts of rat hearts in culture medium resulted in both a fibre outgrowth and maintenance of choline acetyltransferase (CAT) activity in explained chicken embryo ciliary ganglia. CAT activity was maintained in ganglia from 7- to 19-day-old embryos whereas fibre outgrowth was maximal in ganglia from 8- to 11-day-old embryos and as the embryo matured ganglia became less responsive, showing virtually no fibre response by 16 days. The fibre outgrowth response had an absolute dependence on serum in the medium and a pH optimum of 7.1--7.3. Maintenance of CAT was stimulated by, but not absolutely dependent on, the presence of serum in the medium and was insensitive to changes in the pH of the culture medium between 7.2 and 8.0 but was sensitive to decreases in pH below 7.2. Horse and fetal calf serum were equally effective in stimulating the cardiac extract medium-induced maintenance of CAT activity and induction of fibre outgrowth.

Inhibition of prostaglandin synthesis produces a muscular dystrophy-like myopathy

Administration of inhibitors of prostaglandin synthetase to chicken embryos produced myopathies in their skeletal muscles which were characterized by ringbinden, hypercontraction, loss of Z disks, M bands, and thick and thin filaments. The effects of the inhibitors were reversed by administration of PGE1. The pathology of the myopathy induced in the chicken embryos was identical to that observed in human muscular dystrophy and had some features in common with other congenital myopathies. Altered prostaglandin function may therefore be directly or indirectly involved in the pathogenesis of congenital myopathies.

Effects of haemin on neurones derived from the neural crest.

Haemin at high concentrations promoted the survival and neuritic outgrowth in vitro of dissociated sensory and parasympathetic neurones from 8-day-old chick embryos. Similar concentrations of haemin increased the activities of the neurotransmitter biosynthetic enzymes, tyrosine hydroxylase and choline acetyltransferase, in explant cultures of neonatal rat sympathetic ganglia, and in explant and dissociated 14-day-old embryonic chick parasympathethic ganglia, respectively. Enzyme activities of both dissociated neonatal rat sympathetic ganglia and explant and dissociated 8-day-old embryonic chick parasympathetic ganglia were not affected by haemin. Since the neurotrophic effects of haemin were small compared to those of tissue extracts, and were seen mainly at concentrations approaching the limits of its solubility, it is concluded that haemin resulting from the breakdown of haem proteins in tissue extracts is unlikely to contribute significantly to their effects.

Development of cholinergic neurones in cultures of rat superior cervical ganglia. Role of calcium and macromolecules.

Abstract Superior cervical ganglia from 3-day-old rats were grown in vitro for 14 days. Addition of an extract of rat heart to the culture medium led to no change in the activity of tyrosine hydroxylase but a doubling of the activity of choline acetyltransferase in the ganglia. To see whether this effect was due to a change in the concentration of unbound Ca 2+ , the calcium-binding capacity of extracts of adult rat heart was determined in a dialysis-binding study. Both cardiac extracts and serum lowered the levels of free calcium in the medium, but by relatively small amounts. When ganglia were grown in media containing 0, 2.5 and 6 mm Ca 2+ , there was no change in the ratio of choline acetyltransferase activity to tyrosine hydroxylase activity, although the activities of both enzymes increased at lower Ca 2+ concentrations. When superior cervical ganglia were grown in Rose chambers under cellophane strips, the choline acetyltransferase/tyrosine hydroxylase ratio was higher than when superior cervical ganglia were loose in the medium. When a larger number of ganglia was grown under cellophane this further increased the ratio, while positioning of the ganglia so that substances could diffuse out from under the cellophane decreased the choline acetyltransferase/tyrosine hydroxylase ratio. When ganglia from 3-day-old rats were grown side by side with ganglia from 3-week-old rats, there was an even greater effect on the ratio of the two enzyme activities. We conclude that the ability of cardiac extracts to increase the ratio of choline acetyltransferase activity to tyrposine hydroxylase activity in ganglia is not due to changes in the Ca 2+ concentration in the medium, but to the presence of a non-dialysable macromolecule. A similar factor also seems to be secreted by the ganglion itself.

RU38486 blocks the steroid regulation of transmitter choice in cultured rat sympathetic ganglia

Sympathetic neurones grown in tissue culture change phenotype from adrenergic to cholinergic due to a factor released by non-neuronal cells. Glucocorticosteroids prevent the production of this factor by non-neuronal cells and hence prevent the change in phenotype. Conventional steroid antagonists, however, fail to block this effect. We report here that the steroid antagonist RU38486 is effective in preventing the action of corticosterone on cultured sympathetic ganglia and hence may be a useful in vivo tool to study the steroid regulation of development.