Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal chromaffin cells.

The specific activity of tyrosine hydroxylase (TH) in bovine adrenal chromaffin cells can be controlled by changing cell density. Chromaffin cells initially plated at low density (2-3 X 10(4) cells/cm2), and subsequently replated at a 10-fold higher density showed a sixfold increase in specific TH activity within 48 h, resulting from enhanced synthesis (increased number of TH molecules as demonstrated by immunotitration and blockade by cycloheximide) rather than activation. The density-mediated TH induction was blocked by inhibitors of both messenger RNA synthesis (alpha-amanitin) and processing (9-beta-arabinofuranosyladenine), indicating a transcriptional level of regulation. Medium conditioned by high density replated cells could not mimic the effect of high density plating itself, thus direct cell contact, rather than a diffusible factor, is responsible for the density-mediated TH induction. Since neither acetylcholinesterase nor lactate dehydrogenase specific activities were increased by high cell density, it can be concluded that the contact-mediated induction of TH is rather specific, and not the result of a general process of enzyme induction.

Compensatory increase in tyrosine hydroxylase activity in rat brain after intraventricular injections of 6-hydroxydopamine.

The neurotoxin 6-hydroxydopamine produced a permanent loss of endogenous norepinephrine and of 3H-labeled norepinephrine uptake sites in the hippocampus within 5 days. These losses were initially accompanied by parallel decreases in tyrosine hydroxylase activity and synaptosomal norepinephrine synthesis. Within 21 days, however, hippocampal tyrosine hydroxylase activity and norepinephrine synthesis rate increased three- to fivefold. These data suggest a novel form of plasticity in brain-damaged animals characterized by an increase in the capacity for transmitter biosynthesis in residual neurons.

Neurochemical compensation after nigrostriatal bundle injury in an animal model of preclinical parkinsonism.

Parkinson's disease usually involves a lengthy preclinical period during which few neurological symptoms are observed despite extensive damage to the dopaminergic nigrostriatal bundle. Injury to this projection in the rat also fails to produce major neurological dysfunctions. In our studies, damage to the nigrostriatal bundle of the rat, resulting in the loss of up to 95% of the dopaminergic terminals in striatum, was accompanied by apparent increases in the synthesis and release of dopamine (DA) from those dopaminergic terminals that remained. More specifically, both the activity of the rate-limiting biosynthetic enzyme, tyrosine hydroxylase, and the content of the principal DA metabolite, dihydroxyphenylacetic acid, were increased in striatum relative to DA levels. The increases were exponentially related to DA loss.

Subtotal destruction of central noradrenergic projections increases the firing rate of locus coeruleus cells.

The intraventricular administration of 6-hydroxydopamine was used to destroy 80-90% of the noradrenergic terminals in the forebrain of male rats with little apparent damage to the cells of origin in locus-coeruleus. Both 36 h and 21 days later the basal firing rate of these cells was elevated 4-fold above control levels. Moreover, microiontophoretic application of norepinephrine was significantly less effective in inhibiting the spontaneous activity of locus coeruleus cells in these rats relative to control animals. The increased firing may represent a compensatory response to the injury, leading to increased transmitter release release from terminals spared by the lesion.

Short and long term changes in tyrosine hydroxylase activity in rat brain after subtotal destruction of central noradrenergic neurons.

The administration of 6-hydroxydopamine into the cerebroventricles of the rat produced a rapid and permanent decrease of norepinephrine in hippocampus due to an apparent degeneration of central catecholaminergic nerve terminals. The decrease in norepinephrine levels was accompanied by a decrease in the activity of the rate-limiting biosynthetic enzyme, tyrosine hydroxylase. However, the decrease in enzyme activity was less pronounced than the decrease in norepinephrine levels, resulting in an increase in the ratio of tyrosine hydroxylase activity to norepinephrine content. This relative increase in enzyme activity was shown to result from two processes. Within 36 hr after the lesion, the apparent Vmax had decreased in parallel to the norepinephrine loss. However, there was an apparent activation of the remaining enzyme molecules. This activation was only detectable in the presence of subsaturating cofactor concentrations and at a pH above the pH optimum. The activation resembled that produced in control samples by in vitro adenosine 3':5'-monophosphate-dependent protein-phosphorylating conditions, and incubation under these conditions had no further effect on enzyme activity. The activation was followed by a gradual increase in the apparent Vmax of tyrosine hydroxylase toward control values. This increase was preceded by a 2-fold rise in the amount of enzyme present in the region of the locus coeruleus, an area rich in noradrenergic cell bodies. The time course of the increased Vmax in terminal fields appeared to be related to their proximity to the locus coeruleus, since it was more rapid for cerebellum (peak activity, 7 days) than for hippocampus (21 days) and probably represented a 3- to 4-fold increase in the amount of tyrosine hydroxylase per residual terminal. The increase in the Vmax was accompanied by a return to a basal activation state of the enzyme molecules and a restoration of the ability of in vitro protein-phosphorylating conditions to increase enzyme activity. These short and long term alterations in tyrosine hydroxylase activity after 6-hydroxydopamine treatment may represent adaptive responses to the lesion.

Developmental potential of quail dorsal root ganglion cells analyzed in vitro and in vivo.

The cell types present in quail dorsal root ganglia during early development were identified using markers for neurons, glial cells, and fibroblasts (Rohrer et al., 1985). Using the quail-chick transplantation technique, the potential of quail dorsal root ganglion cells to differentiate to adrenergic chromaffin cells, as identified by tyrosine hydroxylase immunostaining, was analyzed. A population of undifferentiated cells, which is present in quail dorsal root ganglia at embryonic day 5, was separated from neurons and glial cells. We show that this population contains cells that differentiate to adrenergic chromaffin cells after back-transplantation into 2-d-old chick embryos. A large proportion of these undifferentiated cells also differentiates to neurons in vitro. Precursors for adrenal chromaffin cells and for neurons are present in dorsal root ganglia in significant numbers only during early development.

Nerve growth factor-mediated enzyme induction in primary cultures of bovine adrenal chromaffin cells: specificity and level of regulation.

Primary cultures of bovine adrenal chromaffin cells provide large quantities of a homogeneous population of target cells for nerve growth factor (NGF) and, thus, are a suitable system for studying the molecular mechanism of action of NGF. In this study, we have shown that NGF mediates the specific induction of the key enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). Acetylcholinesterase (AChE), an enzyme which catalyzes the breakdown of acetylcholine, is also induced by NGF. We have compared NGF-mediated TH and AChE induction and have provided pharmacological evidence that TH induction involves a post-transcriptional, polyadenylation-dependent event (blockable by 9-beta-arabinofuranosyladenine but not by alpha-amanitin), whereas AChE induction requires transcription (blockable by alpha-amanitin). DBH and PNMT appear to be regulated via the same mechanism as TH. The time course of TH induction is such that NGF must be continuously present for at least the first 36 hr (during which time TH levels remain unchanged), and then the entire increase takes place during the subsequent 12 hr. In contrast, AChE induction proceeds linearly with time of NGF exposure. These data suggest that there may be multiple mechanisms by which NGF regulates enzyme induction. We have also compared the effects of cAMP with those of NGF. As compared to NGF, cAMP produces a different pattern of enzyme induction (in addition to TH, DBH, PNMT, and AChE, dopa decarboxylase (DDC) is also induced), it acts rapidly (a 12-hr exposure produces the full effect), and it acts only at the transcriptional level (its effects are blocked by alpha-amanitin). These data provide evidence that cAMP does not act as a second messenger for NGF with regard to enzyme induction.

Anterograde transport of brain-derived neurotrophic factor and its role in the brain.

The role of neurotrophins as target-derived proteins that promote neuron survival following their retrograde transport from the terminals to the cell bodies of neurons has been firmly established in the developing peripheral nervous system. However, neurotrophins appear to have more diverse functions, particularly in the adult central nervous system. Brain-derived neurotrophic factor (BDNF), for example, produces a variety of neuromodulatory effects in the brain that are more consistent with local actions than with long-distance retrograde signalling. Here we show that BDNF is widely distributed in nerve terminals, even in brain areas such as the striatum that lack BDNF messenger RNA, and that inhibition of axonal transport or deafferentation depletes BDNF. The number of striatal neurons that contain the calcium-binding protein parvalbumin was decreased in BDNF+/- and BDNF-/- mice in direct proportion to the loss of BDNF protein, which is consistent with anterogradely supplied BDNF having a functional role in development or maintenance. Thus the anterograde transport of BDNF from neuron cell bodies to their terminals may be important for the trafficking of BDNF in the brain.