Chapter 20 - Selective effects of DSP-4 on locus coeruleus axons: are there pharmacologically different types of noradrenergic axons in the central nervous system?

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There is considerable evidence from biochemical studies that the transmitter-depleting action of drugs and neurotoxins which act upon central noradrenergic (NA) axon terminals is not uniform in different brain regions. Among NA axons, those originating in the locus coeruleus (LC) have been proposed to be most susceptible to the action of NA neurotoxins such as N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). The studies described here were conducted to determine whether this differential susceptibility to DSP-4 reflects a pharmacological heterogeneity between different populations of NA axons. To determine whether DSP-4 acts selectively upon LC axons, we have characterized the effects of this drug on NA axons in different brain regions, by using noradrenaline and dopamine-β-hydroxylase (DβH) immunohistochemistry. Following systemic administration of DSP-4, there was an almost complete loss of noradrenaline and DβH staining in brain regions innervated by LC axons. No effects of the drug treatment were detected in brain regions innervated primarily by non-coerulean NA axons. These results demonstrate that both the transmitter-depleting and the neurodegenerative action of DSP-4 are restricted to NA axons originating in the LC. To explore the basis for this selectivity, noradrenaline uptake studies were conducted using synaptosomes from brain regions in which NA axons differ in their response to DSP-4. The results reveal a significant difference in the affinity of DSP-4 for the noradrenaline uptake carrier in cortical and hypothalamic synaptosomes. This finding is compatible with the hypothesis that the noradrenaline uptake carrier is pharmacologically distinct in LC and non-coerulean NA axons. This heterogeneity in noradrenaline uptake raises the question whether other drugs may also have differential actions on LC and non-coerulean NA neurons.

Introduction

Many widely prescribed drugs exert their therapeutic effects by interaction with noradrenergic (NA) neurons. Observations that central NA neurons may differ in their response to pharmacological manipulation date back to the pioneering studies by Dahlström and Fuxe and their colleagues (1964; Carlsson et al., 1966; Anden, 1967). Using drugs which influence noradrenaline synthesis, storage and release, they observed nonuniform effects on NA axons in different brain regions. For example, Carlsson et al. (1966) documented that the noradrenaline-releasing effects of amphetamine are more pronounced in cerebral cortex than in hypothalamus and brainstem. Despite the considerable implications of these observations for understanding the functional organization of central NA neurons, these studies did not stimulate further efforts to characterize the differential responses of NA neurons to therapeutic drugs.

Pronounced regional differences in the response of NA axons were also observed with catecholamine neurotoxins (Iversen and Uretsky, 1970; Sachs and Jonsson, 1972; Jonsson and Sachs, 1976; Pappas et al., 1976). After systemic injections of 6-hydroxydopa, Jacobowitz and Kostrzewa (1971) observed long-lasting reductions in noradrenaline levels in cerebral cortex, hippocampus and cerebellum, but found few effects in the pons, medulla, hypothalamus, preoptic area and septum. In their comprehensive review on the pharmacology of 6-hydroxydopamine, Kostrzewa and Jacobowitz (1974) emphasized, among the NA axons, that LC axons are most susceptible to the effects of this drug. A striking example for distinct responses of LC and non-coerulean NA axons to 6-hydroxydopamine was reported by McBride et al. (1985). These authors demonstrated that systemic administration of this neurotoxin to neonatal rats results in selective elimination of the coeruleo-spinal projection, whereas descending axons of A5 and A7 NA neurons were spared. Further evidence that the action of neurotoxins may be more pronounced on LC axons has been reported for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; Hallman et al., 1985; Forno et al., 1986) and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; Jonsson et al., 1981; Jonsson, 1983; Logue et al., 1985). For instance, these studies provided evidence that the noradrenaline-depleting effects of DSP-4 are more pronounced in cerebral cortex, cerebellum and spinal cord than in hypothalamus and brainstem.

Commenting on the correlation between the regional effects of neurotoxins on noradrenaline levels and the distribution of LC axons, several authors stressed that these drugs do not have an absolute specificity for LC axons, and that non-coerulean NA fibers are also affected, but to a lesser degree (Jonsson and Sachs, 1973; Jonsson et al., 1981; Kostrzewa, 1988). These investigators argued that the pharmacological properties of NA axons are identical in all brain regions, and that structural features account for the differential response of NA axons to neurotoxins. Most investigators have employed biochemical assays to evaluate the effects of NA neurotoxins in various brain areas. However, in regions where a drug causes only partial depletion of noradrenaline, it is not possible to determine with biochemical assays whether a partial decrease in transmitter levels reflects an incomplete loss of noradrenaline from all NA axons or a complete loss from a subpopulation of NA axons. Thus a potential selectivity of neurotoxins for different populations of NA axons may have escaped detection. Unlike biochemical assays, immunohistochemistry permits direct assessment of the effects of drugs on individual NA axons. The recent introduction of specific antibodies to noradrenaline (Geffard et al., 1986) makes it possible to visualize NA axons directly based upon their neurotransmitter content.

The studies described below were conducted to evaluate, with an anatomical method, the responses of NA axons to DSP-4. This drug was chosen because of its unique ability to cross the blood-brain barrier and to induce selective loss of NA neuron markers without affecting dopaminergic neurons (Ross, 1976; Ross and Renyi, 1976). To determine whether DSP-4 acts selectively upon NA axons of the LC, we have characterized the transmitter-depleting and the neurodegenerative actions of this neurotoxin on NA axons in different brain regions. Both noradrenaline and dopamine-β-hydroxylase (DβH) immunohistochemistry were used at the light microscopy level to visualize the effects of DSP-4 on these two markers of NA axons over a two-week period. The rapid loss of noradrenaline induced by DSP-4 is likely to reflect an interaction between the drug and NA axon terminals (Landa et al., 1984). In contrast, DβH is a membrane-bound protein and may serve as an index of the structural integrity of NA axons. By characterizing the sequence of events that follows exposure to DSP-4, we sought to determine whether the selectivity of this drug for LC axons can be observed in relation to its transmitter-releasing effects as well as its neurodegenerative action. Furthermore, by using an in vitro uptake assay, we explored whether the preferential action of DSP-4 on LC axons reflects a pharmacological difference between LC and non-coerulean NA axons.

Rats were treated with a single systemic injection of DSP-4 (50 mg/kg). Noradrenaline and DβH were visualized in brain sections using specific antibodies in combination with the avidin-biotin peroxidase method of Hsu et al. (1981) (see Fritschy and Grzanna, 1989, and Fritschy et al., 1990 for details). Antibodies to noradrenaline were kindly provided by Dr. M. Geffard, Bordeaux, France (Geffard et al., 1986); antibodies to rat DβH were prepared and characterized in our laboratory (Grzanna and Coyle, 1976).

Section snippets

Acute effects of DSP-4: evidence for a selective loss of noradrenaline from locus coeruleus axon terminals

Changes in noradrenaline levels were measured by high performance liquid chromatography in four brain regions at 6 h, 24 h and 2 weeks after DSP-4 treatment (Grzanna et al., 1989). This biochemical analysis confirmed that DSP-4 induces rapid and profound loss of noradrenaline in brain regions innervated by the LC (e.g., cerebral cortex and cerebellum), but has only moderate effects in brain regions innervated by non-coerulean NA axons (ventral forebrain and hypothalamus).

Processing of brain

Long-term effects of DSP-4: degeneration of LC axon terminals

In contrast to the profound loss of noradrenaline immunoreactivity induced within hours by DSP-4, the structural integrity of NA axons, as visualized by DβH immunohistochemistry, was unchanged until day 4 after drug treatment (Fritschy et al., 1990). During this time span, no effect of the drug treatment could be observed on NA axons by DβH staining. Thereafter, loss of DβH immunoreactivity was abrupt and exhibited the same regional pattern as the loss of noradrenaline described above (see

What is the basis for the selectivity of DSP-4 for locus coeruleus axons?

A striking property of DSP-4, revealed by noradrenaline as well as DβH immunohistochemistry, is the regional specificity of its effects on NA axons. At all levels of the neuraxis, there is a strong correlation between the pattern of DSP-4-induced NA axon degeneration and the distribution of LC fibers (Fritschy and Grzanna, 1990a,b; see Foote et al., 1983; Björklund and Lindvall, 1986, for reviews). This correlation strongly suggests that the effects of this drug are restricted to LC axons.

As

Evidence that the differential effects of DSP-4 may be due to differences in the pharmacological properties of locus coeruleus and non-coerulean noradrenergic axons

An interaction of DSP-4 with the noradrenaline uptake carrier is required for the drug to exert its neurotoxic effects (Hallman and Jonsson, 1984; Ross, 1987). Several studies have related the toxicity of DSP-4 to its alkylating properties and suggested that the key target of this drug is the noradrenaline uptake carrier itself (Ross, 1976; Lee et al., 1982). It is unknown whether the destruction of the noradrenaline transporter by DSP-4 is sufficient to induce NA axon degeneration. Such a

Conclusions

By demonstrating that DSP-4 acts specifically upon LC axons, the results suggest the existence of subsets of central NA neurons with different pharmacological properties. The evidence summarized here, that NA axons are pharmacologically heterogeneous, implies that other drugs may also have differential actions on LC and non-coerulean NA neurons. A reinvestigation of the effects of neurotoxins such as 6-hydroxydopa or MPTP on NA axons using immunohistochemical methods may confirm the present

Acknowledgements

This research was supported by USPHS Grant NIMH MH-41977 and by a Biomedical Research Support Grant #RR5378. We are grateful to Dr. S.B. Ross, Astra Lakemedel AB, Sweden for a generous gift of DSP-4 and to Dr. M. Geffard, Bordeaux for providing antibodies to noradrenaline. J.M.F. is a recipient of a fellowship from the Swiss National Fund for Scientific Research.

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