The acetylcholine release enhancer linopirdine induces Fos in neocortex of aged rats.

Centrally acting cholinergic agents induce the immediate early gene c-fos in the rat brain resulting in transient increases of Fos protein, most notably in the cerebral cortex. In this study we have monitored by Fos immunohistochemistry the effect of the acetylcholine release enhancer linopirdine (DUP996) on the immediate early gene c-fos in brains of 3 months and 30 months old rats. In young rats linopirdine had only a marginal effect on Fos expression. In contrast, in aged rats linopirdine caused widespread expression of Fos throughout neocortex. In somatosensory cortex, the induction of the c-fos gene by linopirdine was nearly completely blocked by atropine and scopolamine and strongly attenuated by the NMDA receptor blockers CPP and MK-801. The results suggest that the age-related decline in acetylcholine release in rodents can be partially compensated for by administration of linopirdine.

Positive-negative epitope-tagging of beta amyloid precursor protein to identify inhibitors of A beta processing.

In this report, a novel positive-negative epitope tagging approach was developed to study the cellular processing of beta amyloid precursor protein (beta APP). Amino acids centered around the alpha-secretase cleavage site within the A beta sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant beta APP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled beta APP by beta- and gamma-secretase(s) results in the release of an epitope-tagged A beta peptide, whereas cleavage by alpha-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled beta APP via the amyloidogenic pathway. Secretion of epitope-tagged A beta was prevented by MDL 28170, a previously described gamma-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged beta APP was not different from wild-type beta APP. These results suggest that positive-negative epitope-tagged beta APP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer's disease.

Intrastriatal and intraventricular injections of oligodeoxynucleotides in the rat brain: tissue penetration, intracellular distribution and c-fos antisense effects.

We have determined the time course, the spatial spread in brain tissue, and the intracellular distribution of biotin- and fluorescein-labeled phosphorothioate oligodeoxynucleotides (ODNs) following single injections into the rat striatum or the lateral ventricle. These time and space parameters were correlated with the ability of c-fos phosphorothioate antisense ODNs to suppress the induction of Fos protein by cocaine. A rapid and dose-dependent tissue penetration of labeled ODNs was observed following either intrastriatal or intraventricular injections of a constant sample volume. Inspection of tissue sections by confocal microscopy uncovered a distinct change in the intracellular disposition of labeled ODNs during the 24 h post-injection period. At 1, 6 and 12 h, the vast majority of the fluorescent signal was confined to the interstitial spaces throughout the zone penetrated by ODNs. Neuronal nuclei displayed faint labeling along the outer portion of the nucleus at 1 and 6 h post-injection. At these time-points, ODNs were not detected in the cytoplasm. By 16 h, ODNs were barely detectable in the extracellular space and absent from neuronal nuclei. Instead, ODNs were seen in large cytoplasmic granules of neurons throughout the tissue zone penetrated by the ODNs. Experiments with intrastriatal injections of antisense ODNs to c-fos mRNA revealed Fos suppression between 3 and 12 h, but not at 16 and 24 h. This combined analysis has revealed that (1) restricted tissue penetration by ODNs limits their antisense effects on protein expression, and (2) depletion of extracellular ODNs and sequestration of c-fos antisense ODNs into large intracellular granules coincides with the loss of their biological activity.

Characterization of [125I]sauvagine binding to CRH2 receptors: membrane homogenate and autoradiographic studies.

We describe the binding of [125I]tyr(o)sauvagine to membranes of corticotropin-releasing hormone (CRH2) receptor expressing HEK293/EBNA (293ECRH2 alpha) cells. The binding of [125I]tyr(o)sauvagine to CRH2 receptors was temperature, time and tissue dependent. Equilibrium was reached in 2 hr at 23 degrees C. Saturation data best fit a two-site model with affinity constants of 44 pM and 4.1 nM for high and low affinity states, respectively. The high affinity [125I]tyr(o)sauvagine binding sites were eliminated with 200 microM Gpp (NH) p, indicating coupling to G proteins. The rank order potency of peptide analogs of CRH to inhibit [125I]tyr(o)sauvagine binding to CRH2 alpha receptors was: urotensin > sauvagine = urocortin > alpha-helical CRH9-41 > rh-CRH >> o-CRH. This was in contrast to the rank order potency of the peptides at inhibiting [125I]tyr(o)oCRH binding to CRH, receptors: urotensin > urocortin > r/h-CRH > o-CRH = sauvagine > alpha-helical CRH9-41. We show that two recently identified small molecule CRH antagonists with nanomolar potency at the CRH1 receptor, have little or no affinity for CRH2 alpha receptors as labeled by [125I]tyr(o)sauvagine. Two selective CRH1 receptor antagonists enabled us to examine comparative densities of CRH1 and CRH2 receptors in several brain areas. We also used [125I]tyr(o)sauvagine in combination with a specific CRH1 antagonist to examine the anatomic distribution of CRH2 receptors using receptor autoradiography. With a few notable exceptions the CRH2 receptors demonstrated autoradiographically in this study match the data obtained by in situ hybridization studies on the localization of CRH2 mRNA. The anatomic overlap of the autoradiographic and in situ hybridization data suggest that CRH2 receptors are postsynaptic. This study demonstrates the utility of using [125I]tyr(o)sauvagine to study cloned CRH2 receptors expressed in cell lines. In addition, [125I]tyr(o)sauvagine used in conjunction with saturating concentrations of a specific CRH1 receptor antagonist allows the study of CRH2 receptors in brain tissues using both in vitro homogenate binding and receptor autoradiography techniques.

Restoration of ascending noradrenergic projections by residual locus coeruleus neurons: compensatory response to neurotoxin-induced cell death in the adult rat brain.

There is clinical and experimental evidence that monoamine neurons respond to lesions with a wide range of compensatory adaptations aimed at preserving their functional integrity. Neurotoxin-induced lesions are followed by increased synthesis and release of transmitter from residual monoamine fibers and by axonal sprouting. However, the fate of lesioned neurons after long survival periods remains largely unknown. Whether regenerative sprouting may contribute significantly to recovery of function following lesions which induce cell loss has been questioned. We have previously analyzed the response of locus coeruleus (LC) neurons to systemic administration of the noradrenergic (NE) neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to adult rats. This drug causes ablation of nearly all LC axon terminals within 2 weeks after administration, followed by a profound loss of LC cell bodies 6 months later. The present study was conducted to determine the fate of surviving LC neurons and to characterize their potential for regenerative sprouting during a 16 month period after DSP-4 treatment. The time-course and extent of LC neuron degeneration were analyzed quantitatively in Nissl-stained sections, and the regenerative response of residual neurons was characterized by dopamine-beta-hydroxylase immunohistochemistry. The results document that LC neurons degenerate gradually after DSP-4 treatment, cell loss reaching on average 57% after 1 year. LC neurons which survive the lesion exhibit a vigorous regenerative response, even in those animals in which cell loss exceeds 60-70%. This regenerative process leads progressively to restoration of the NE innervation pattern in the forebrain, with some regions becoming markedly hyperinnervated. In stark contrast to the forebrain, very little reinnervation takes place in the brainstem, cerebellum and spinal cord. These findings suggest that regenerative sprouting of residual neurons is an important compensatory mechanism by which the LC may regain much of its functional integrity in the presence of extensive cell loss. Furthermore, regeneration of LC axons after DSP-4 treatment is region-specific, suggesting that the pattern of reinnervation is controlled by target areas. Elucidation of the factors underlying recovery of LC neurons after DSP-4 treatment may provide insights into the compensatory mechanisms of central neurons after injury and in disease states.

The neurotoxic effects of p-chloroamphetamine in rat brain are blocked by prior depletion of serotonin.

Systemic administration of p-chloroamphetamine (PCA) causes degeneration of serotonergic (5-HT) axons, but recent data indicate that this drug itself is not neurotoxic when applied directly to 5-HT axons. The present study was designed to test whether the toxic effects of PCA in the brain are dependent on release of endogenous 5-HT and to identify which stores of 5-HT are involved. The long-term effects of PCA on brain levels of 5-HT and on central 5-HT axons were determined in rats that had been initially depleted of 5-HT by administration of p-chlorophenylalanine and reserpine. The results show that transient depletion of 5-HT provides substantial protection against subsequent PCA-induced degeneration of 5-HT axon terminals; the neurotoxicity induced by PCA thus appears to be dependent on the presence of endogenous stores of 5-HT. In addition, the protective effect of 5-HT depletion is found only after pretreatment regimens that deplete peripheral as well as central stores of 5-HT. We interpret this finding as evidence that release of 5-HT from peripheral storage sites may be necessary for the expression of PCA-induced toxicity. Based on these results, we propose that central neurotoxicity is not induced by a direct action of PCA alone but may require or be augmented by a toxic metabolite of 5-HT.

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry.

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimentally-induced neuron loss in the locus coeruleus of adult rats.

Systemic administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to adult rats causes widespread degeneration of locus coeruleus (LC) axon terminals. The present study was conducted to determine the effects of DSP-4-induced LC axon lesions on LC cell bodies. Six months after DSP-4 treatment, quantitative analysis of Nissl-stained sections revealed a profound loss of LC perikarya, ranging from 20 to 73% of control. The remaining LC neurons appeared shrunken, but stained strongly with dopamine beta-hydroxylase immunohistochemistry. These findings support the conclusion that DSP-4-induced LC axon lesions cause retrograde degeneration of LC neurons. DSP-4 may serve as a useful tool in studies of the mechanisms of LC neuron degeneration.

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

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-beta-hydroxylase (D beta H) immunohistochemistry. Following systemic administration of DSP-4, there was an almost complete loss of noradrenaline and D beta H 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.

Efferent projections of different subpopulations of central noradrenaline neurons.

Early anatomical studies of the projections of central noradrenergic (NA) neurons led to the widely accepted view of NA cells as a class of diffusely projecting neurons. This view greatly influenced the formulation of numerous hypotheses about the functional role of these neurons in the central nervous system (CNS). With the introduction of transmitter-specific retrograde and anterograde transport methods, two powerful tools became available to rigorously re-examine whether the projections of NA neurons are diffuse or topographically organized. This article summarizes some of the results of these studies in which retrograde transport of fluorescent tracers and anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L), respectively, were combined with immunohistochemical identification of NA neurons and their projections. The results of these studies revealed a remarkable degree of specificity in the projections of different subgroups of NA neurons. In the rat CNS, the differential distribution of NA axons of the locus coeruleus (LC) and non-coerulean NA cells is particularly striking in the spinal cord and brainstem. In these regions, NA axons of the LC are primarily distributed to sensory nuclei while NA axons of non-coerulean NA neurons are distributed to motor nuclei. The results support the proposition that NA neurons can be divided into subgroups which differ in their connections and hence represent separated anatomical entities with different functional capacities.

Unlike systemic administration of p-chloroamphetamine, direct intracerebral injection does not produce degeneration of 5-HT axons.

Systemic administration of the amphetamine derivative p-chloroamphetamine (PCA) causes degeneration of 5-HT axon terminals in rat brain. The present study was designed to determine whether PCA induces neurotoxic effects by a direct action on 5-HT axon terminals. PCA was administered by microinjection directly into the cerebral cortex of rats. Continuous intracerebral infusions were made over extended time periods (10 min-48 h) to explore whether the induction of neurotoxicity requires a prolonged exposure of axon terminals to the drug. Two weeks after drug administration, brain sections that passed through the injection site were processed for 5-HT immunohistochemistry. The 5-HT innervation of cerebral cortex in PCA-injected animals was compared with that after intracortical injection of saline or of 5,7-dihydroxytryptamine. The results demonstrate that, in the concentrations used, direct application of PCA into the neocortex does not elicit axonal degeneration, even after a continuous infusion for 2 days. This finding suggests that PCA itself is not directly toxic to 5-HT axons.

The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunohistochemical study using antibodies to noradrenaline and dopamine-beta-hydroxylase.

The response of noradrenaline (NA) axons to the effects of systemic injections of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) was studied in the rat brain. Antibodies to NA and to dopamine-beta-hydroxylase (DBH) were employed to assess by immunohistochemistry the effects of DSP-4 on NA axons between 6 h and 2 weeks after drug administration. The changes in NA and DBH staining after DSP-4 treatment were restricted to brain regions innervated by the locus coeruleus. In these areas, DSP-4 induced profound loss of both NA and DBH from NA axons, but with a distinctly different time-course. While NA disappeared within hours after drug treatment, DBH staining of NA axons remained unchanged during the first 4 days after DSP-4 treatment. Thereafter, there was an abrupt loss of DBH staining which coincided with the appearance of numerous brightly stained, thick and swollen NA axons. The distribution of these fibres suggests that they represent the distal ends of preterminal NA axons. Two weeks after drug treatment, NA axons could no longer be visualized by either NA or DBH immunohistochemistry in regions affected by DSP-4. During this 2-week time-period, the staining of cell bodies in the locus coeruleus and of ascending NA axons in the dorsal bundle was unaffected. The results suggest two phases in the response of NA axons to DSP-4: an acute phase, marked by loss of transmitter, and a neurodegenerative phase, characterized by loss of DBH and structural disintegration of NA axons.

Differential effects of DSP-4 on noradrenaline axons in cerebral cortex and hypothalamus may reflect heterogeneity of noradrenaline uptake sites.

The effect of the noradrenergic neurotoxin DSP-4 on high affinity transport of noradrenaline (NAT) was studied using rat brain synaptosomes. DSP-4 decreased NAT with the characteristics of a competitive inhibitor. The neurotoxin was more potent in inhibiting NAT into cortical synaptosomes (Ki = 179 +/- 39 nM) than into hypothalamic synaptosomes (Ki = 460 +/- 35 nM). Differences in NAT into cortical and hypothalamic synaptosomes were also observed with noradrenaline itself (Km = 39.5 +/- 7.5 nM and 100 +/- 12.1 nM, respectively) and with the catecholamine uptake blocker mazindol (Ki = 0.55 +/- 0.05 nM and 0.30 +/- 0.08 nM, respectively). The differences in the pharmacological properties of the noradrenaline uptake carrier in cerebral cortex and hypothalamus may account for the differential effects of DSP-4 on noradrenergic axons in these two brain regions.

Distribution of locus coeruleus axons within the rat brainstem demonstrated by Phaseolus vulgaris leucoagglutinin anterograde tracing in combination with dopamine-beta-hydroxylase immunofluorescence.

Projections of the locus coeruleus (LC) to the midbrain and hindbrain were analyzed by anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). Following iontophoretic application of PHA-L into the LC, the distribution of labeled axons was analyzed in sections processed for the immunoperoxidase method and in sections processed for double-immunofluorescence staining using antibodies to PHA-L and to dopamine-beta-hydroxylase. This combined staining approach proved to be necessary for the unequivocal identification of LC axons in the brainstem since all injections labeled many non-noradrenergic axons whose distribution was different from that of LC fibers. The major new finding of the present study was the observation that large territories of the brainstem that receive a dense noradrenergic input are very sparsely innervated by the LC. Numerous labeled LC axons were observed in somatic afferent nuclei, tectum, pontine nuclei, interpenduncular nucleus, and inferior olivary complex. In contrast, very few labeled fibers were observed in autonomic and motor nuclei, and throughout the brainstem reticular formation, including raphe nuclei. Our data show that the distribution of LC axons in the brainstem is far less prominent than the projections of this nucleus to the forebrain and spinal cord. Our findings suggest that the dense NA projections to the core of the brainstem originate principally in non-LC NA neurons. On the basis of the present anatomical findings, a prominent role of the LC in motor and integrative functions of the brainstem appears unlikely.

Demonstration of two separate descending noradrenergic pathways to the rat spinal cord: evidence for an intragriseal trajectory of locus coeruleus axons in the superficial layers of the dorsal horn.

The rat spinal cord receives noradrenergic (NA) projections from the locus coeruleus (LC) and the A5 and A7 groups. In contradiction to previous statements about the distribution of descending NA axons, we have recently proposed that in the rat LC neurons project primarily to the dorsal horn and intermediate zone, whereas A5 and A7 neurons project to somatic motoneurons and the intermediolateral cell column. The aim of the present study was to determine the funicular course and terminal distribution of descending NA axons from the LC and from the A5 and A7 groups. The organization of the coeruleospinal projection was analyzed by using the anterograde tracer Phaseolus vulgaris leucoagglutinin in combination with dopamine-beta-hydroxylase immunohistochemistry. The trajectory of A5 and A7 axons was studied in spinal cord sections of rats following ablation of the coeruleospinal projection with the neurotoxin DSP-4. To assess the relative contribution of the LC and the A5 and A7 groups to the NA innervation of the spinal cord, unilateral injections of the retrograde tracer True Blue were made at cervical, thoracic, and lumbar levels, and retrogradely labeled NA neurons were identified by dopamine-beta-hydroxylase immunofluorescence. The results of the anterograde tracing experiments confirm our previous findings that LC neurons project most heavily to the dorsal horn and intermediate zone. Analysis of horizontal sections revealed that LC axons descend the length of the spinal cord within layers I and II. In contrast to the intragriseal course of LC fibers, A5 and A7 axons travel in the ventral and dorsolateral funiculi and terminate in the ventral horn and the intermediolateral cell column. Retrograde transport studies indicate that the contribution of the A5 and A7 groups to the NA projection to the spinal cord is greater than that of the LC. We conclude that descending axons of the LC and A5 and A7 groups differ in their course and distribution within the spinal cord. The documentation of a definite topographic order in the bulbospinal NA projections suggests that the LC and the A5 and A7 groups have different functional capacities. The LC is in a position to influence the processing of sensory inputs, in particular nociceptive inputs, whereas A5 and A7 neurons are likely to influence motoneurons.

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects.

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.

The noradrenergic neurotoxin DSP-4 eliminates the coeruleospinal projection but spares projections of the A5 and A7 groups to the ventral horn of the rat spinal cord.

Systemic administration of the noradrenergic neurotoxin DSP-4 results in a complete loss of staining of noradrenergic (NA) axons in the dorsal horn and intermediate zone of the rat spinal cord. NA axon staining in the ventral horn and in the intermediolateral cell column is only slightly decreased by the drug treatment. We have taken advantage of this differential effect of DSP-4 on NA axons to determine the location and number of cells that give rise to NA axons in the ventral horn and the intermediolateral cell column. Retrograde transport of the fluorescent tracer True blue was combined with dopamine-beta-hydroxylase immunohistochemistry 2 weeks after treatment of rats with 50 mg/kg of DSP-4. Compared with controls, the drug treatment resulted in a more than 90% decrease in the number of retrogradely labeled NA neurons in the locus coeruleus and an only 30-50% reduction in the number of retrogradely labeled NA cells in the A5 and A7 groups. The results reveal different sites of termination in the spinal cord of NA axons originating in the LC and in NA cells of the A5 and A7 groups: the LC distributes fibers mainly to the dorsal horn and the intermediate zone, while NA cells of the A5 and A7 groups project to motoneurons of the ventral horn and the intermediolateral cell column.

Depletion of serotonin using p-chlorophenylalanine (PCPA) and reserpine protects against the neurotoxic effects of p-chloroamphetamine (PCA) in the brain.

The present study attempts to determine whether the neurotoxicity of p-chloroamphetamine (PCA) is dependent on a releasable pool of serotonin (5-HT). Rats treated with PCA alone or with reserpine and PCA exhibit a profound loss of 5-HT innervation in cerebral cortex after a 2-week survival period. However, depletion of 5-HT by combined treatment with p-chlorophenylalanine (PCPA) and reserpine provides substantial protection against the neurotoxic effects of PCA. These results indicate that release of 5-HT is a necessary step in the neurotoxicity of PCA and that a peripheral source of 5-HT is involved. We suggest that 5-HT release from platelets into the peripheral circulation may result in the formation of a neurotoxic 5-HT metabolite.

Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a potent and highly selective neurotoxin which induces degeneration of noradrenergic axons. The effects of DSP-4 vary considerably in different brain regions: the drug produces nearly complete depletion of noradrenaline in neocortex, hippocampus, cerebellum and spinal cord, but only partial depletion in hypothalamus and brainstem. In this study we have employed an immunohistochemical method to assess the neurotoxic effects of DSP-4 on the structural integrity of central noradrenergic neurons in the rat, and to identify those noradrenergic axons that remain in the central nervous system 2-4 weeks after DSP-4 treatment. The staining results identified noradrenergic axon terminals as the principal site of action of DSP-4; noradrenergic cell bodies and preterminal axons were not noticeably affected. DSP-4 produced an almost all or none neurotoxic effect on noradrenergic axon terminals in different brain regions. Nearly all noradrenergic axon terminals were destroyed in the neocortex, hippocampus, olfactory bulb, thalamus, tectum, cerebellum and spinal cord dorsal horn. In contrast, most noradrenergic axons were unaffected in the basal forebrain, hypothalamus, reticular formation, brainstem motor nuclei and spinal cord ventral horn. These remaining noradrenergic axon terminals differed morphologically from sensitive axons by their thickness, size and spacing of their varicosities and their dense arborizations within terminal fields. The distribution of noradrenergic axons susceptible to DSP-4 correlates very closely with the distribution of locus coeruleus axons and possibly all regions in which noradrenergic terminals are unaffected by DSP-4 receive their major noradrenergic input from non-locus coeruleus neurons. This study provides the first direct evidence that DSP-4 destroys noradrenergic axon terminals from the locus coeruleus, but not those from non-locus coeruleus neurons. This profound differential sensitivity of noradrenergic axons to DSP-4 is matched by distinct differences in their morphology and their topographic projections. The results support the view that locus coeruleus and non-locus coeruleus noradrenergic neurons constitute two separate subsystems, which differ not only in their projections but also with respect to the pharmacological properties of their axon terminals.