SarcTrack.

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

Estudio farmacognóstico preliminar de tallo y raíz de la especie Moringa Oleífera lam cosechada en Machala / Preliminary pharmacognostic study of the stem and root of the species Moringa Oleifera lam grown in Machala

Introducción: la composición química de las especies vegetales está sujeta a cambios, dependiendo, entre otros factores, de la localización geográfica. Moringa oleífera Lam., que crece en Machala, Ecuador, puede diferir de especies de otras regiones geográficas. Objetivo: realizar un estudio farmacognóstico preliminar del tallo y raíz (corteza y pulpa) de la planta M. oleífera cultivada en las áreas de la Unidad Académica de Ciencias Agropecuarias, de la Universidad Técnica de Machala. Métodos: se desarrolla el control de la calidad de la droga cruda según la metodología establecida por la Organización Mundial de la Salud, mediante determinación de la humedad residual, el porciento de cenizas y el porciento de sustancias solubles en el tallo y la raíz. Se cuantificaron algunos metales mediante espectrometría de emisión óptica con plasma acoplado inductivamente. El estudio químico preliminar se efectuó a través de ensayos de tamizaje fitoquímico y mediante cromatografía en capa delgada. Resultados: la humedad residual para ambos órganos y los valores de cenizas obtenidos para la raíz se encuentran dentro de los límites establecidos. Las cenizas totales para el tallo resultaron elevadas. La determinación de metales descartó la presencia de metales tóxicos en los órganos estudiados. Los valores de sustancias solubles indicaron mayor poder extractivo para el agua. La evaluación mediante tamizaje fitoquímico sugirió triterpenos y esteroides, azúcares reductores, alcaloides, flavonoides, aminoácidos y saponinas, en los extractos de la raíz. En el tallo se detectaron, además, catequinas, mucílagos y compuestos fenólicos, no así flavonoides. La cromatografía en capa delgada sugirió la existencia de alcaloides derivados de la fenilmetilamina. Conclusiones: el estudio permitió establecer parámetros de calidad de la droga cruda para la especie estudiada; sugerir, en principio, semejanzas en composición química de la planta analizada con otras de orígenes geográficos diferentes, y comprobar la ausencia de metales tóxicos en los órganos estudiados(AU)

Introduction: The chemical composition of plant species is subject to changes which depend, among other factors, on their geographic location. The Moringa oleifera Lam. growing in Machala, Ecuador, may differ from species from other geographic regions. Objective: Conduct a preliminary pharmacognostic study of the stem and root (bark and pulp) of the plant M. oleifera grown in areas from the Agricultural Sciences Academic Unit of the Technical University of Machala. Methods: Quality control was performed of the crude drug following the methodology set up by the World Health Organization to determine residual humidity, percentage of ashes and percentage of soluble substances in the stem and the root. Several metals were quantified by inductively coupled plasma atomic emission spectroscopy. The preliminary chemical study was conducted by phytochemical screening testing and thin layer chromatography. Results: Both the residual humidity for both organs and the ash values obtained for the root are within the limits established. Total ashes for the stem were high. Metal determination discarded the presence of toxic metals in the organs studied. Values for soluble substances awarded a greater extraction capacity to water. Phytochemical screening pointed to the presence of triterpenes and steroids, reducing sugars, alkaloids, flavonoids, amino acids and saponins in root extracts. The stem was found to also contain catechins, mucilages and phenolic compounds, but not flavonoids. Thin layer chromatography pointed to the presence of alkaloids derived from phenyl methylamine. Conclusions: The study made it possible to set up crude drug quality parameters for the study species, make preliminary suggestions about similarities between the chemical composition of the plant analyzed and other plants of different geographic origin, and verify the absence of toxic metals in the organs studied(AU)

Estudio farmacognóstico preliminar de tallo y raíz de la especie Moringa Oleífera lam cosechada en Machala / Preliminary pharmacognostic study of the stem and root of the species Moringa Oleifera lam grown in Machala

Introducción: la composición química de las especies vegetales está sujeta a cambios, dependiendo, entre otros factores, de la localización geográfica. Moringa oleífera Lam., que crece en Machala, Ecuador, puede diferir de especies de otras regiones geográficas. Objetivo: realizar un estudio farmacognóstico preliminar del tallo y raíz (corteza y pulpa) de la planta M. oleífera cultivada en las áreas de la Unidad Académica de Ciencias Agropecuarias, de la Universidad Técnica de Machala. Métodos: se desarrolla el control de la calidad de la droga cruda según la metodología establecida por la Organización Mundial de la Salud, mediante determinación de la humedad residual, el porciento de cenizas y el porciento de sustancias solubles en el tallo y la raíz. Se cuantificaron algunos metales mediante espectrometría de emisión óptica con plasma acoplado inductivamente. El estudio químico preliminar se efectuó a través de ensayos de tamizaje fitoquímico y mediante cromatografía en capa delgada. Resultados: la humedad residual para ambos órganos y los valores de cenizas obtenidos para la raíz se encuentran dentro de los límites establecidos. Las cenizas totales para el tallo resultaron elevadas. La determinación de metales descartó la presencia de metales tóxicos en los órganos...(AU)

Introduction: The chemical composition of plant species is subject to changes which depend, among other factors, on their geographic location. The Moringa oleifera Lam. growing in Machala, Ecuador, may differ from species from other geographic regions. Objective: Conduct a preliminary pharmacognostic study of the stem and root (bark and pulp) of the plant M. oleifera grown in areas from the Agricultural Sciences Academic Unit of the Technical University of Machala. Methods: Quality control was performed of the crude drug following the methodology set up by the World Health Organization to determine residual humidity, percentage of ashes and percentage of soluble substances in the stem and the root. Several metals were quantified by inductively coupled plasma atomic emission spectroscopy. The preliminary chemical study was conducted by phytochemical screening testing and thin layer chromatography. Results: Both the residual humidity for both organs and the ash values obtained for the root are within the limits established. Total ashes for the stem were high. Metal determination discarded the presence of toxic metals in the organs studied. Values for soluble substances awarded a greater extraction capacity to water. Phytochemical screening pointed to the presence of triterpenes and steroids, reducing sugars, alkaloids, flavonoids, amino acids and saponins in root extracts. The stem was found to also contain catechins, mucilages and phenolic compounds, but not flavonoids. Thin layer chromatography pointed to the presence of alkaloids derived from phenyl methylamine. Conclusions: The study made it possible to set up crude drug quality parameters for the study species, make preliminary suggestions about similarities between the chemical composition of the plant analyzed and other plants of different geographic origin, and verify the absence of toxic metals in the organs studied(AU)

Tyramine and benzylamine partially but selectively mimic insulin action on adipose differentiation in 3T3-L1 cells.

Biogenic amines like tyramine, methylamine and the non-naturally occuring amine, benzylamine, have been described to promote adipose conversion of murine 3T3 preadipocytes. To further investigate these novel effects of amines, we studied whether they selectively mimic the long-term adipogenic action of insulin. To this aim, we decided to use the 3T3-L1 cell line since this model needs a complex combination of inducers to trigger the differentiation programme: insulin, isobutylmethylxanthine (IBMX, an activator of cAMP-signal transduction pathway) and the synthetic glucocorticoid, dexamethasone. A cell culture protocol was designed, by which each component of the differentiation cocktail was replaced with either benzylamine or tyramine, in order to determine whether these amine oxidase substrates could substitute any of the differentiation inducers in 3T3-L1 cells. The incomplete lipid accumulation found in cells grown under IBMX- or dexamethasone-free conditions was not improved by the daily addition of amines to the culture medium. Insulin was the only component of adipose differentiation cocktail of 3T3-L1 that could be replaced, although partially, by tyramine or benzylamine. When used at 0.5 mM, these amines resulted in a significant increase of triacylglycerol accumulated eight days after confluence, when compared to cells kept without insulin. This partial insulin replacement was totally abolished by SSAO-inhibitors, while MAO-blockade did not reduce lipid accumulation. As previously reported for other insulin-sensitive processes, such as stimulation of glucose transport or lipolysis inhibition in mature adipocytes, the stimulation of adipogenesis by tyramine and benzylamine was an SSAO-dependent mechanism that apparently shared common signaling pathways with insulin.

Combined treatment with benzylamine and low dosages of vanadate enhances glucose tolerance and reduces hyperglycemia in streptozotocin-induced diabetic rats.

Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAO-inhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 micromol x kg(-1) x day(-1) i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.

Neuropeptide Y attenuates the effect of locus coeruleus denervation by DSP-4 treatment on social behaviour in the rat.

Noradrenaline (NA) has been implicated in both increase and reduction of anxiety. Selective destruction of nerve endings of the locus coeruleus projections by DSP-4 has been shown to reduce active behaviour in novel situations by enhancing anxiety. In the present study, DSP-4 (50 mg/kg) treatment reduced locomotor activity and time spent in social interaction in rats placed into a novel environment together with an unfamiliar rat, indicating an anxiogenic-like effect. The effect of DSP-4 on time spent in social interaction was completely antagonized by intracerebroventricular administration of neuropeptide Y (NPY) (1 microg) which had no effect of its own on this measure. The present study thus supports the idea that DSP-4 pretreatment is anxiogenic in novel situations and suggests a functional relationship of NA- and NPY-using neural mechanisms in the regulation of social behaviour.

Salbutamol overcomes the effect of the noradrenergic neurotoxin DSP-4 on memory function in the day-old chick.

These experiments investigated the effect of the relatively selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on memory formation in day-old chicks trained on a discriminated passive avoidance task. A time course study showed that DSP-4 treatment resulted in amnesia as early as 20 min post-learning. In a second study, a series of alpha- and beta-adrenergic agonists (noradrenaline; the alpha 1 agonist phenylephrine; the beta 1 agonist dobutamine; and the beta 2 agonist salbutamol) were applied immediately after the training trial. Both noradrenaline and salbutamol were effective in ameliorating the memory deficits caused by DSP-4 treatment, and in consolidating weakly reinforced training. These studies support the notion that noradrenaline subserves a vital role in the consolidation of memory in the chick, and that the beta 2 receptor subtypes are principally involved in the intermediate phase of memory formation.

[Effect of selegiline against selective neurotoxins]. / Effekt selegilina protiv selektivnykh neirotoksinov.

The complexity of the pharmacological activity of selegiline cannot be considered only as a result of a simple MAO-B inhibition. The mechanism of its neuroprotective action against the noradrenergic neurotoxin DSP-4 was widely studied (-)-p-fluoro-deprenyl (PFD), the chemical derivative of selegiline, with its possible metabolites were also involved into these studies. The results suggested that the uptake inhibitory effect of selegiline, and mainly that of its metabolite (-)-methylamphetamine (MA), played an essential role in the protection. MA was more potent to inhibit the uptake of noradrenaline and dopamine, than the parent compound. Neither selegiline nor its metabolite inhibited the reuptake of serotonin. In respect of the protection against DSP-4 induced toxicity PFD and its metabolites behaved similarly to selegiline, but their effects were more lasting than that of selegiline. After oral treatment selegiline undergoes an intensive "first pass" metabolism, which leads to an enhanced formation of MA. The better understanding of the fate of selegiline in the body, including its pharmacokinetic behaviour and metabolism, may contribute to a better knowledge of the complex pharmacological activity of the drug. The results could be summarised as follows. a) MAO-B inhibition-which is due to the parent compound-is an irreversible "hit and run" effect, the level of which after an initial phase is independent of the presence of the substance which caused it. b) The uptake inhibition is a reversible process and strictly proportional to the concentration of the substance responsible for the effect. In this respect the uptake inhibitory action of the metabolites exceeds that of the parent compounds. The role of the reversible uptake inhibition in neuroprotection may partly explain the need of the daily administration of selegiline to parkinsonian patients in spite of the irreversible MAO-B inhibitory action of the drug.

Immunohistochemical evidence of neuroprotection by R(-)-deprenyl and N-(2-hexyl)-N-methylpropargylamine on DSP-4-induced degeneration of rat brain noradrenergic axons and terminals.

DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] is a potent neurotoxin highly selective to the locus coeruleus noradrenaline (NA) system. Previous biochemical studies have shown that the monoamine oxidase-B (MAO-B) inhibitors, R(-)-deprenyl and (+/-)2-HxMP [N-(2-hexyl)-N-methylpropargylamine], are able to prevent DSP-4 induced NA depletion in the mouse hippocampus. It is not quite certain, however, whether this actually represents neuroprotection of NA axons or a metabolic effect due to inhibition of MAO activity. Employing dopamine-beta-hydroxylase immunohistochemical and image analysis methods, we have shown that 92% and 84% of NA nerve fibers in the rat hippocampus are spared from DSP-4 neurotoxicity by a single pretreatment dose of either R(-)-deprenyl or (+/-)2-HxMP respectively. Similar neuroprotective effects of R(-)-deprenyl and (+/-)2-HxMP were also observed in the cerebral cortex, thalamus, amygdaloid complex and cerebellum. This is the first morphological evidence demonstrating that R(-)-deprenyl and (+/-)2-HxMP can indeed protect noradrenergic axons of locus coeruleus origin against DSP-4 neurotoxicity.

Neuroprotection by R(-)-deprenyl and N-2-hexyl-N-methylpropargylamine on DSP-4, a neurotoxin, induced degeneration of noradrenergic neurons in the rat locus coeruleus.

N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a neurotoxin and capable of selectively depleting noradrenergic axons and subsequently causing lesions of locus coeruleus (LC) noradrenergic neurons in the rat. R(-)-deprenyl and N-(2-hexyl)-N-methylpropargylamine (2-HxMP) have been previously shown to be quite effective in protecting NA nerve fibers in different brain tissues against DSP-4. The present report reveals the neuroprotective effect of these drugs on the LC noradrenergic cell bodies using a histochemical method. Neurons were quantitatively assessed using Nissl-stained sections. DSP-4 induced a 34% loss of LC perikarya neurons 3 months after a single systemic administration in comparison to control animals. Approximately 90% and 88% of neurons in the same regions survived against DSP-4 induced insult following multiple injections of R(-)-deprenyl and 2-HxMP, respectively. The neuroprotective effect towards the LC neurons against DSP-4 is probably due to prevention of retrograde degeneration of NA axons.

Depletion of NOS activity in the rat dentate gyrus neurons by DSP-4 and protection by deprenyl.

DSP-4 is a potent and highly selective neurotoxin of noradrenergic axons of locus coeruleus origin. The authors found that in addition to depletion of the hippocampal noradrenergic terminals the histochemical reactivity of nitric oxide synthase (NOS, NADPH-diaphorase) was lost from neurons in the subgranule zone and hilar region of the dentate gyrus 2 weeks after a systemic administration of this toxin. Pretreatment with R(-)-deprenyl and 2-HxMP (2-hexyl-N-methylpropargylamine, which protects hippocampal noradrenergic axons against DSP-4 neurotoxicity, led to a complete prevention of the loss of NADPH-diaphorase activity.

Differential effects of angiotensin II and eledoisin on monoamine oxidase A and B activities in rat brain.

Intracerebroventricular injections of angiotensin II caused 108, 62, and 54% increases in monoamine oxidase A activities in rat hippocampus, hypothalamus, and striatum, respectively. These activatory effects were abolished by simultaneous injections of eledoisin. No significant changes of monoamine oxidase B activities were found under the same experimental conditions. Neither angiotensin II nor elodoisin changed substrate/inhibitor affinities of both isoenzymes. These data indicate that angiotensin II and tachykinin transmitter systems may exert opposite, long-term regulatory effects on monoaminergic neurons in rat brain.

Protection against DSP-4-induced neurotoxicity by deprenyl is not related to its inhibition of MAO B.

Clinical studies suggest that deprenyl may retard the progression of Parkinson's disease, an effect that may be related to its monoamine oxidase (MAO) inhibiting properties. Deprenyl also protects against the neurodegenerative effects of the noradrenergic toxin DSP-4. In this study we investigated the role of MAO B inhibition in this protection. C57BL/6 mice were given DSP-4 (50 mg/kg i.p.) 1 h. 24 h or 4 days after the administration of deprenyl (10 mg/kg i.p.) or the selective MAO B inhibitor MDL 72974 (1.25 mg/kg), and then killed 1 week later for assay of hippocampal norepinephrine. The MAO B inhibiting effects of deprenyl or MDL 72974 were also determined after these same intervals of time. Deprenyl and MDL 72974 produced comparable degrees of enzyme inhibition 1 h (greater than 95%), 24 h (greater than 90%) or 4 days (greater than 70%) after their administration. Given 1 h before, deprenyl totally blocked the norepinephrine-depleting effects of DSP-4, but this protection declined sharply when 24 h or 4 days was allowed to elapse between deprenyl and DSP-4 administration. MDL 72974 failed to protect at any time point. In vitro, we detected no activity using DSP-4 as a substrate for MAO. These findings suggest that the ability of deprenyl to protect against DSP-4-induced neuronal degeneration may not depend on its MAO B inhibiting properties.

The inhibitory effect of thioproline on carcinogenesis induced by N-benzylmethylamine and nitrite.

Thioproline, which is readily nitrosated to form nitrosothioproline, is expected to act as a nitrite scavenger. The effect of thioproline as an inhibitor of the carcinogenesis induced by N-nitroso-N-benzylmethylamine precursors was examined. Two groups of male F-344 rats were given diet containing 0.25% N-benzylmethylamine (group I) or 0.25% N-benzylmethylamine plus thioproline (0.25% until wk 17 and then 0.5%; group II). Both groups were given drinking-water containing sodium nitrite (0.1% until wk 17 and then 0.2%). The experiment was continued for 717 days. Squamous cell carcinoma of the forestomach developed in six out of seven rats in group I and in significantly fewer, two out of nine rats, in group II. The degree of invasion by the tumours was also less in group II rats, given thioproline, than in group I. Thus thioproline suppressed carcinogenesis induced by N-benzylmethylamine and nitrite, possibly by inhibiting the in vivo nitrosation of N-benzylmethylamine.

Inhibition of MAO B, but not MAO A, blocks DSP-4 toxicity on central NE neurons.

The neurotoxin, DSP-4, (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) specifically and significantly reduces norepinephrine (NE) and its metabolite, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), in cortical, hippocampal and cerebellar brain regions. Pretreatment with the selective monoamine oxidase (MAO) B inhibitor, deprenyl, and the non-specific MAO inhibitor, pargyline, effectively blocked NE depletion. Pretreatment with the selective MAO A inhibitor, clorgyline, had no effect on central NE DSP-4 toxicity. Thus formation of a toxic metabolite by the action of MAO B may be involved in DSP-4 induced neural damage.

Pharmacological modifications of the neurotoxic action of the noradrenaline neurotoxin DSP4 on central noradrenaline neurons.

Systemic treatment with the noradrenaline neurotoxin DSP4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine; 7 days) led to a marked and quantitatively similar reduction (-80%) of endogenous noradrenaline, [3H]noradrenaline uptake in vitro and [3H]desipramine binding in the frontal cortex of adult rats. Inhibition of monoamine oxidase, and/or 1-dopa administration 1 week after DSP4 produced very small changes in brain noradrenaline and dopamine levels. These results are all consistent with the view that DSP4 produces an acute and selective degeneration of central noradrenaline nerve terminals. Pretreatment with the noradrenaline uptake blocker desipramine prevented the action of DSP4 almost completely, while treatment after DSP4 had minute effects on DSP4-induced reduction of endogenous noradrenaline and [3H]noradrenaline uptake. The data suggest that the irreversible neurotoxic actions of DSP4 are very rapid and largely complete within 0.5 h after DSP4 administration. Measurement of catecholamine turnover using monoamine oxidase inhibition by pargyline indicated an increased noradrenaline turnover in the remaining nerve terminals innervating cerebral cortex and hippocampus after DSP4, while dopamine turnover appeared to be decreased. Pretreatment with d-amphetamine and clonidine or subsequent treatment with oxotremorine were without effect on the DSP4-induced reductions of the regional brain noradrenaline levels. Morphine pretreatment was also ineffective, while repeated morphine administration after DSP4 produced a significant potentiation of the DSP4-induced noradrenaline depletion in the frontal cortex, cerebellum and the spinal cord. Pretreatment with the monoamine oxidase inhibitor pargyline led to a very pronounced counteraction of the DSP4-induced noradrenaline depletion in all brain regions analysed, in particular in the occipital cortex. The data suggest that morphine can potentiate the neurotoxic action of DSP4 while pargyline can counteract it.

A parametric study of the effects of the noradrenaline neurotoxin DSP4 on avoidance acquisition and noradrenaline neurones in the CNS of the rat.

The effects of various doses of DSP4 on two-way active avoidance acquisition in rats and on central noradrenaline neurones were compared. Doses of DSP4 from 3 mg kg-1 i.p. and upwards injected one week before the onset of the avoidance trials significantly impaired two-way avoidance learning. The learning impairment caused by DSP4 (50 mg kg-1 i.p.) lasted for at least 10 weeks. Desipramine (20 mg kg-1) injected either 30 or 60 min before DSP4 (50 mg kg-1) antagonized the active avoidance impairment. A high dose of DSP4 (50 mg kg-1 i.p.) produced profound decreases in dopamine-beta-hydroxylase activity in the frontal cortex and in the concentrations of noradrenaline in various brain regions indicating degeneration of the locus coeruleus noradrenaline system. Low doses of DSP4 (3 and 6 mg kg-1 i.p.) produced small but significant decrease in the concentrations of noradrenaline (NA) in some regions, e.g. cerebral cortex, hippocampus, olfactory bulb and spinal cord. The avoidance impairment caused by the low dose of DSP4 (3 mg kg-1) was absent when rats were tested 10 weeks after treatment nor was NA depletion present when NA was analysed 3 months after treatment.