Culture of rat locus coeruleus explants: a model to study transneuronal regulation of tyrosine hydroxylase in central noradrenergic neurons

Abstract

A culture method is described to maintain explants of rat locus coeruleus in vitro for several days. Both anatomical and biochemical methods were used to control the evolution of the cultures with time. Explants were first examined 7 days after culture by immunohistochemistry with specific antisera directed against enzymes specific for non-neuronal or neuronal cells. Non-neuronal enolase immunoreactive cells were observed throughout the explants except at their edge where outgrowth zones were observed. Neuronal cells which were separately stained by three markers (neuron-specific enolase, tyrosine hydroxylase and dopamine-β-hydroxylase antisera) share common morphological features: they are grouped in clusters; their soma have similar shape and size and the staining was always seen inside the cytoplasm of the cell bodies and their proximal processes. These results show that noradrenergic neurons are still present in the explants 7 days after culture. Biochemical characteristics of the explants were followed throughout culture. Non neuronal enolase activity doubled during the first week in vitro. In contrast neuron specific enolase activity decreased to about half its initial value. Similarly the amount of 1-[ 3H]noradrenaline taken up in the explants decreased abruptly during the two first days in culture to reach a stable value. Tyrosine hydroxylase activity decreased significantly to about a third its initial value. All biochemical markers became stable after 7 days in culture. The cholinergic control of tyrosine hydroxylase in the noradrenergic cells has been investigated using 1-week old cultures. Tyrosine hydroxylase activity in locus coeruleus explants incubated in the presence of oxotremorine (10 −5 M, during 24 h) is significantly increased (+108%) as compared to non treated tissues. This increase was abolished if atropine sulfate (10 −5 M) was present together with oxotremorine. Immunotitrations of tyrosine hydroxylase in homogenates of the explants treated with the cholinergic agonist revealed that the increased enzyme activity is due to a change in its catalytic properties without variation in the number of enzyme molecules (delayed activation). This experiment indicates that an activation of tyrosine hydroxylase can result from the stimulation of cholinergic receptors located in the locus coeruleus region. The present investigation shows that central noradrenergic neurons of the locus coeruleus region can survive in culture conditions. These neurons retain most of their in situ morphological and biochemical characteristics. This in vitro approach appears to be an interesting means to study tyrosine hydroxylase regulation in central noradrenergic neurons.