Luis M. Zieher

DSP-4: a novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats.

The pharmacological actions of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) are compatible with a specific neurotoxic effect on both peripheral and central noradrenergic neurons. The systemic injection of DSP-4 to adult rats transiently alters sympathetic neurons in the periphery but in the central nervous system the compound determines a marked and prolonged reduction of noradrenaline (NA) levels in all brain regions studied. When DSP-4 was injected systemically to rats at birth in doses ranging from 6.25 to 100 micrograms/g, no changes were found in peripheral sympathetic neurons 40 days later. On the contrary, in the same conditions and in relation to the dose injected, there were marked and persistent changes in the levels of NA in different regions of the brain. In the cerebral cortex and the spinal cord, the neonatal injection of SDP-4 produced a marked and long-lasting depletion of NA levels, similar to that observed after injection of the compound to adult rats. These changes were accompanied by a moderate increase in brain stem NA and a marked elevation of the amine in the cerebellum. These changes, different from the depletion observed in both regions when the compound was given to adult rats, are however similar to those observed after the neonatal injection of the neurotoxic compounds 6-hydroxydopamine or its precursor amino acid, 6-hydroxydopa. This indicates that probably central noradrenergic neurons respond in the same manner after different chemical injuries. DSP-4 crosses the placental barrier because when it was given to pregnant rats at the end of gestation, long-term changes were found in brain NA levels in their offspring, similar to those produced by the neonatal administration of the compound. This new neurotoxic compound provides a very useful tool for the study of noradrenergic neurons both in adult animals and during ontogenesis.

NERVE ENDINGS IN METHIONINE SULPHOXIMINE CONVULSANT RATS, A NEUROCHEMICAL AND ULTRASTRUCTURAL STUDY*

THE FINDING that dogs underwent seizures after ingestion of flour from NC13-treated wheat (MELLANBY, 1946) led to the isolation of methionine sulphoximine (MSO) as the toxic convulsant factor (BEKTLEY, MCDEXMOTT, MORAN, PACE and WHITEHEAD, 1950; MISANI and REINER, 1950; REINER, MISANI and WEISS, 1950). Because of the similarity of the convulsions to those of human epilepsy this agent has been the object of numerous physiological and neurochemical studies (see WOLFE and ELLIOT, 1962). Several investigations pointed toward a possible causal relationship between MSO and a disequilibrium in glutamine-glutamic acid metabolism. PETERS and TOWER (1959) found a marked inhibition of glutamine synthesis in brain slices of MSO-treated cats, and a relationship between amidation of glutamic acid in brain and seizures was also postulated in rats after MSO or acoustic epileptogenic stimulation by KOLOUSEK, HORAK and JIRACEK (1959). SELLINGER and WEILER (1963) reported that in vitro MSO competitively inhibits glutamine synthetase (GS), and more recently LAMAR and SELLINGER (1965) found that in vivo the drug inhibits GS irreversibly. In this last paper some preliminary observations on the subcellular distribution of labelled MSO were made and evidence was reported that it is preferentially bound to the microsomal fraction isolated from an homogenate in water, which is particularly rich in GS (DE BALBIAN VERSTER, SELLINGER and HARKIN, 1965). Recently SALGANICOFF and DE ROBERTIS (1965) analysed the subcellular distribution of GS and other enzymes related to the metabolism of glutamate, glutamine and GABA. The fractionation procedure, carried out in sucrose solutions under standard conditions and with electron microscopic control, enabled them to follow the localization of certain enzymes in well defined subcellular fractions of the CNS such as mitochondria, myelin, and particularly nerve endings, synaptic vesicles and membranes. In agreement with the results of WAELSCH (1959) and SELLINGER and DE BALBIAN VERSTER (1962), GS was found to be concentrated in the microsomal fraction.

Altered lymphocyte catecholamine reactivity in mice subjected to chronic mild stress

There is considerable evidence that the sympathetic nervous system influences the immune response via activation and modulation of beta(2)-adrenergic receptors (beta(2)R). Furthermore, it has been suggested that stress has effects on the sympathetic nervous system. In the present study, we analyzed the influence of catecholamines on the reactivity of lymphocytes from mice exposed to a chronic mild stress (CMS) model of depression (CMS-animals). The effects of the CMS treatment on catecholamine and corticosterone levels and on beta(2)R lymphoid expression were also assessed. For this purpose, animals were subjected to CMS for 8 weeks. Results showed that catecholamines (epinephrine and norepinephrine) exert an inhibitory effect on mitogen-induced normal T-cell proliferation and a stimulatory effect on normal B-cell proliferation in response to selective B lymphocyte mitogens. Specific beta- and beta(2)-antagonists abolished these effects. Lymphocytes from mice subjected to CMS had an increased response to catecholamine-mediated inhibition or enhancement of proliferation in T and B cells, respectively. Moreover, a significant increase in beta(2)R density was observed in animals under CMS compared to normal animals. This was accompanied by an increment in cyclic AMP production after beta-adrenergic stimulation. On the other hand, neither catecholamine levels, determined in both urine and spleen samples, nor serum corticosterone levels showed significant variation between normal and CMS-animals. These findings demonstrate that chronic stress is associated with an increased sympathetic influence on the immune response and may suggest a mechanism through which chronic stress alters immunity.

NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium‐3 (HC‐3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [32P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [l4C]HC‐3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC‐3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.

Cytochemistry of 5-hydroxytryptamine at the electron microscope level

SummaryThe nerves of rat pineal gland are known to contain norepinephrine and 5-hydroxytryptamine. With the glutaraldehyde-dichromate reaction for the cytochemical localization of unsubstituted catechol- and indoleamines, dense reactive granules could be demonstrated in such endings. A similar reaction was observed in the adrenergic nerves supplying the vas deferens and storing exclusively norepinephrine. Formaldehyde fixation, prior to the glutaraldehyde-dichromate treatment, interferes with the reaction given by catecholamines not affecting the indolic reactive sites. After this combined procedure pineal nerves still exhibited the dense reactive granules, while these were not found in the nerves of the vas deferens. Following bilateral cervical sympathectomy reactive granules disappeared from the perivascular processes of the pineal gland. No reaction could be observed in the cytoplasm of parenchymal cells neither in their perivascular processes.These cytochemical results suggest that both catecholamines and 5-hydroxytryptamine are contained within the granulated vesicles of pineal nerves.

Neurotoxicity of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP 4) on noradrenergic neurons is mimicked by its cyclic aziridinium derivative

Abstract The haloalkylamine N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP 4), impairs the ability of central and peripheral noradrenergic neurons to take up exogenous noradrenaline (NA) and produces a long-lasting reduction of endogenous NA levels. Previous work has shown that DSP 4 apparently binds to the NA carrier system where it is cyclized spontaneously to an aziridinium compound that seems to trigger the degenerative changes responsible for NA depletion as a result of the alkylation and irreversible inactivation of the carrier. To establish the importance of the binding of unchanged DSP 4 for the production of these changes, the compound was injected immediately after dissolving it or after its incubation under conditions known to favor its conversion into the aziridium derivative. Endogenous NA levels were studied in the brain and heart of adult mice. DSP 4, given immediately after being dissolved, depleted heart and brain NA. When injected after being incubated, DSP 4 reduced NA levels in the periphery but not in the central nervous system. This failure was due to the inability of the aziridinium ion to pass the blood-brain barrier because it could deplete NA when given directly into the brain. Pretreatment with the uptake blocker desmethylimipramine counteracted the effects of both DSP 4 and the aziridinium derivative. Thus, fixation of DSP 4 to the carrier is not a prerequisite for the activity of the aziridinium derivative as the derivative can interact directly with the membrane NA uptake system of central and peripheral noradrenergic neurons to produce the changes characteristic of DSP 4 administration.

CYTOCHEMISTRY OF 5-HYDROXYTRYPTAMINE AT THE ELECTRON MICROSCOPE LEVEL I. STUDY OF THE SPECIFICITY OF THE REACTION IN ISOLATED BLOOD PLATELETS

Blood platelets obtained from normal rabbits and those isolated from reserpine-treated animals and subsequently incubated in vitro with 5-hydroxytryptamine, norepinephrine and histamine were assayed for amine content or processed for examination under the electron microscope. With the glutaraldehyde-dichromate reaction for unsubstituted catechol- and indoleamines, reactive granules were observed in normal platelets. Formaldehyde fixation prior to the glutaraldehyde-dichromate reaction resulted in a similar image under the electron microscope. In platelets obtained from animals treated with reserpine a decrease of the amine content with a corresponding reduction in the number of dense granules was observed. Following incubation with 5-hydroxytryptamine the concentration of the amine increased markedly and the number of dense granules that reacted with both techniques became practically normal. In norepinephrine-incubated platelets dense granules were demonstrated with the glutaraldehyde-dichromate reaction, but no reactive products were observed using prefixation with formaldehyde. Histamine was also incorporated into depleted platelets but gave no reaction. It is concluded that prefixation with formaldehyde renders negative the reaction with catecholamines, leaving unaffected indoleamine-reactive sites. The previous assumption that the dense granules contain 5-hydroxytryptamine has been confirmed by such a cytochemical approach. The possibility that these organelles constitute a common storage site for different amines is discussed.

Chronic Corticosterone Impairs Inhibitory Avoidance in Rats: Possible Link With Atrophy of Hippocampal CA3 Neurons

The aim of our work was to evaluate the effect of a chronic (22 days) administration of corticosterone, which induces supraphysiological serum levels of the hormone, on an inhibitory avoidance learning in rats (one-trial step-through learning task, footshock: 0.5 mA, 2 s). We also studied hippocampal markers of neuroanatomical CA3 pyramidal neuron atrophy by using the Golgi staining method. Chronic exposure to high CORT serum levels induced a significant impairment of inhibitory avoidance learning. The CORT group also showed hippocampal glucocorticoid receptor (GR) downregulation and the decrease of hippocampal CA3 branch points and total dendritic length in the apical tree that would be causally related with the learning impairment.

Regional differences in the long-term effect of neonatal 6-hydroxydopa treatment on rat brain noradrenaline

Abstract 6-Hydroxydopa (6-OH-DOPA), the precursor amino acid in the biosynthesis of 6-hydroxydopamine, traverses the blood-brain barrier and determines a permanent alteration of central adrenergic neurons after its systemic administration. When the drug is injected into newborn rats, it produces a long-lasting depletion of whole brain noradrenaline (NA). However, if different brain regions are separated for study, a marked and persistent increase in brain stem NA is observed, while in the tele-diencephalon the content of the amine is strongly reduced. A similar effect is observed in the offspring of rats injected with 6-OH-DOPA during gestation. These results most probably reflect either the impaired growth or the destruction of adrenergic nerve terminals in the forebrain as well as the accumulation of NA in preterminal axons, anomalous sprouts or cell bodies in the brain stem region.

Noradrenaline and dopamine content of normal, decentralized and denervated pineal gland of the rat☆

Abstract Noradrenaline and dopamine were determined with flourimetric methods in normal, decentralized and denervated pineal glands of the rat. A high content in both amines was found in the normal gland. While noradrenaline decreases 44.5 per cent with decentralization and almost disappears with bilateral superior cervical gangliectomy, the dopamine content is not significantly affected with either procedure. These results give support to the concept that the granulated vesicles of adrenergic nerves contain noradrenaline 3 . They suggest the existence of a central regulation for the content of noradrenaline in the peripheral sympathetic neurons. They also prove that there is a rich extraneuronal pool of dopamine in the pineal gland of the rat.