Tomasz Lenda

The Antidepressant-Like Action of Metabotropic Glutamate 7 Receptor Agonist N,N′-Bis(Diphenylmethyl)-1,2-Ethanediamine (AMN082) Is Serotonin-Dependent

Behavioral studies show that modulation of the glutamatergic system might be an efficient way to achieve antidepressant activity. Among the group III metabotropic glutamate (mGlu) receptors, the mGlu7 receptor subtype seems to be the most promising target for potential antidepressants. It has been shown that a selective, allosteric mGlu7 receptor agonist, N,N′-bis (diphenylmethyl)-1,2-ethanediamine (AMN082), induced antidepressant-like action in behavioral tests in mice, although the mechanisms responsible for this action remained unknown. Here, we decided to investigate the possible role of the serotonergic system in the antidepressant-like activity of AMN082 in both the forced swim test (FST) in rats and the tail suspension test (TST) in mice. We found that AMN082 (1–10 mg/kg i.p.) induced a dose-dependent reduction in the immobility of rats and an increase in their swimming behavior, whereas there were not any changes in climbing behavior in the FST in rats. In the TST in mice we found that AMN082 (3 mg/kg i.p.) did not induce an antidepressant-like effect after depletion of serotonin (5-HT) with para-chlorophenylalanine. Moreover, we revealed that citalopram, but not reboxetine, when combined with AMN082 (all compounds used at low subeffective doses), induced a significant antidepressant-like effect in the TST. We also discovered that the 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridynyl) cyclohexane-carboxamide (WAY100635) (0.1 mg/kg s.c.), but not the 5-HT2A/2C receptor antagonist ritanserin (0.5 mg/kg i.p.), blocked the antidepressant-like action of AMN082. Altogether, the results of our studies show that the antidepressant-like action of the mGlu7 receptor-positive modulator AMN082 depends on the activation of the serotonergic system.

Antidepressant and antipsychotic activity of new quinoline- and isoquinoline- sulfonamide analogs of aripiprazole targeting serotonin 5-HT1A/5-HT2A/5-HT7 and dopamine D2/D3 receptors

A series of new quinoline- and isoquinoline-sulfonamide analogs of aripiprazole was synthesized to explore the influence of two structural features - replacement of ether/amide moiety with sulfonamide one, and localization of a sulfonamide group in the azine moiety. In contrast to aripiprazole, compound 33 (N-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)quinoline-7-sulfonamide) and 39 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-3-sulfonamide) displaying multireceptor 5-HT(1A)/5-HT(2A)/5-HT(7)/D(2)/D(3) profile, and behaving as 5-HT(1A) agonists, D(2) partial agonists, and 5-HT(2A)/5-HT(7) antagonists, produced significant antidepressant activity in FST in mice. On the other hand, their 4-isoquinolinyl analog 40 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoquinoline-4-sulfonamide), with similar receptor binding and functional profile, additionally displayed remarkable antipsychotic properties in the MK-801-induced hyperlocomotor activity in mice.

Different effects of intranigral and intrastriatal administration of the proteasome inhibitor lactacystin on typical neurochemical and histological markers of Parkinson's disease in rats

Impairment of the ubiquitin-proteasome system, responsible for clearing of misfolded and unwanted proteins, has been implicated in the loss of nigrostriatal dopaminergic neurons characteristic of Parkinsons disease (PD). Recently, proteasome inhibitors have been used to model parkinsonian-like changes in animals. In the present study, the effects of intrastriatal and intranigral injections of the selective proteasome inhibitor lactacystin on key markers of PD were examined in Wistar rats. Comparisons of these two different routes of lactacystin administration revealed that only a unilateral, intranigral injection of lactacystin at a dose of 0.5, 1, 2.5 and 5 μg/2 μl produced after 7 days distinct decreases in the concentrations of dopamine (DA) and its metabolites (DOPAC, 3-MT, HVA) in the ipsilateral striatum. The used doses of lactacystin (except for 0.5 μg/2 μl) significantly accelerated DA catabolism, i.e. the total, oxidative MAO-dependent and COMT-catalyzed pathways, as assessed by HVA/DA, DOPAC/DA and 3-MT/DA ratios, respectively, in the ipsilateral striatum. Such alterations were not observed in the striatal DA content and catabolism either 7, 14 or 21 days after a unilateral, intrastriatal high-dose lactacystin injection (5 and 10 μg/2 μl). Intranigrally administered lactacystin (1 μg/2 μl) caused a marked decline of tyrosine hydroxylase (TH) and α-synuclein protein levels in that structure. Neither TH nor α-synuclein protein levels in the substantia nigra (SN) were affected by high lactacystin doses injected intrastriatally. Moreover, stereological counting of TH-immunoreactive neurons and autoradiographic analysis of [(3)H]GBR 12,935 binding to dopamine transporter confirmed a loss of nigrostriatal dopaminergic neurons after an intranigral lactacystin (1 and 2.5 μg/2 μl) injection. An appearance of cardinal neurochemical and histological changes of parkinsonian type only after intranigral lactacystin injection indicates that DA cell bodies in the SN, but not DA terminals in the striatum are susceptible to proteasome inhibition.

Alterations in the expression of nNOS in the substantia nigra and subthalamic nucleus of 6-OHDA-lesioned rats: the effects of chronic treatment with l-DOPA and the nitric oxide donor, molsidomine.

Recently, it has been strongly suggested that reciprocal interactions between nitrergic and dopaminergic systems play a crucial role in the control of the nigrostriatal pathway. Degeneration of dopaminergic neurons in the substantia nigra (SN) in Parkinsons disease leads to disturbances in the nitrergic transmission in the basal ganglia. In the present study, we aimed to compare regional distribution of nNOS immunoreactivity and NADPH-diaphorase activity in the SN and subthalamic nucleus (STN) of unilaterally 6-OHDA-lesioned rats treated chronically with l-DOPA (25mg/kg) and the nitric oxide donor, molsidomine (2 or 4mg/kg). Our results showed that degeneration of dopaminergic neurons in the ipsilateral SN resulted in a 25% decrease in the number of nNOS-immunoreactive neurons in that structure and in nNOS protein level determined by Western blot. We also found that nNOS was present in about 70% of all SN neurons. NADPH-d histochemistry did not reveal nNOS activity in the SN of any studied groups. Furthermore, the stereological analysis of the SN volume showed that chronic administration of l-DOPA evoked a hypertrophy of the ipsilateral SN when compared to the contralateral side. Such difference between sides was abolished in the group receiving l-DOPA in combination with molsidomine. Degeneration of the nigrostriatal pathway had no influence on the number of nNOS-ir neurons in the STN. NADPH-histochemistry revealed nNOS activity only in a part of neurons of that structure. Our results make an essential contribution to the research on the role of nitric oxide in the regulation of basal ganglia function.

Chronic l-DOPA treatment attenuates behavioral and biochemical deficits induced by unilateral lactacystin administration into the rat substantia nigra

The aim of the study was to determine whether the dopamine (DA) precursor l-DOPA attenuates parkinsonian-like symptoms produced by the ubiquitin-proteasome system inhibitor lactacystin. Wistar rats were injected unilaterally with lactacystin (2.5 μg/2 μl) or 6-OHDA (8 μg/2 μl) into the substantia nigra (SN) pars compacta. Four weeks after the lesion, the animals were treated chronically with l-DOPA (25 or 50 mg/kg) for two weeks. During l-DOPA treatment, the lactacystin-treated rats were tested for catalepsy and forelimb asymmetry. Rotational behavior was evaluated after apomorphine (0.25 mg/kg) and l-DOPA in both PD models. After completion of experiments, the animals were killed and the levels of DA and its metabolites in the striatum and SN were assayed. We found that acute l-DOPA administration effectively decreased catalepsy and increased the use of the compromised forelimb in the cylinder test. However, the lactacystin group did not respond to apomorphine or acute l-DOPA administration in the rotational test. Repeated l-DOPA treatment produced contralateral rotations in both PD models, but the number of rotations was much greater in the 6-OHDA-lesioned rats. Both toxins markedly (>90%) reduced the levels of DA and its metabolites in the striatum and SN, while l-DOPA diminished these decreases, especially in the SN. By demonstrating the efficacy of l-DOPA in several behavioral tests, our study confirms the usefulness of the lactacystin lesion as a model of PD. However, marked differences in the rotational response to apomorphine and l-DOPA suggest different mechanisms of neurodegeneration evoked by lactacystin and 6-OHDA.

The influence of acute and chronic administration of 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline on the function of the nigrostriatal dopaminergic system in rats.

The contribution of (R)-enantiomer of N-methyl-salsolinol (1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; NMSal) to the degeneration of dopaminergic neurons in the course of Parkinsons disease (PD) has been predominantly suggested by in vitro experiments in cell culture and by an in vivo study in which this compound has been directly injected into the rat striatum. The aim of the present study was to examine the effects of racemic NMSal (50 and 100 mg/kg) administered systemically, acutely and chronically for 21 days to rats, on the neurochemical and behavioral markers of PD. Our results showed that racemic NMSal easily penetrated the blood-brain barrier. Its brain level was relatively high 2-6 h after a single injection than gradually decreased. NMSal was quickly eliminated from the rat brain, its concentration 24 h after withdrawal from chronic treatment was very low. NMSal at both examined doses did not affect striatal and nigral levels of dopamine (DA) 2 h after the first and last chronic injections, however, it markedly changed DA catabolism. In the striatum both its doses evoked a significant acceleration of the total and oxidative, monoamine oxidase (MAO)-dependent DA catabolism without affecting the catechol-O-methyltransferase (COMT)-dependent O-methylation. In the substantia nigra (SN), only the higher dose of NMSal produced such effect. DA catabolism in either structure was the same as in control 24 h after cessation of chronic treatment. The second characteristic marker of PD, the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SN, was not affected by chronic NMSal treatment as revealed by the stereological counting. In the behavioral study, it was found that racemic NMSal significantly suppressed spontaneous locomotor activity and effectively prevented that stimulated by apomorphine. Our results suggest that NMSal may play an important role in the regulation of dopaminergic activity rather than in inducing changes of parkinsonian type.

Concentration-Dependent Dual Mode of Zn Action at Serotonin 5-HT1A Receptors: In Vitro and In Vivo Studies.

Recent data has indicated that Zn can modulate serotonergic function through the 5-HT1A receptor (5-HT1AR); however, the exact mechanisms are unknown. In the present studies, radioligand binding assays and behavioural approaches were used to characterize the pharmacological profile of Zn at 5-HT1ARs in more detail. The influence of Zn on agonist binding to 5-HT1ARs stably expressed in HEK293 cells was investigated by in vitro radioligand binding methods using the agonist [3H]-8-OH-DPAT. The in vivo effects of Zn were compared with those of 8-OH-DPAT in hypothermia, lower lip retraction (LLR), 5-HT behavioural syndrome and the forced swim (FST) tests. In the in vitro studies, biphasic effects, which involved allosteric potentiation of agonist binding at sub-micromolar Zn concentrations and inhibition at sub-millimolar Zn concentrations, were found. The in vivo studies showed that Zn did not induce LLR or elements of 5-HT behavioural syndrome but blocked such effects induced by 8-OH-DPAT. Zn decreased body temperature in rats and mice; however, Zn failed to induce hypothermia in the 5-HT1A autoreceptor knockout mice. In the FST, Zn potentiated the effect of 8-OH-DPAT. However, in the FST performed with the 5-HT1A autoreceptor knockout mice, the anti-immobility effect of Zn was partially blocked. Both the binding and behavioural studies suggest a concentration-dependent dual mechanism of Zn action at 5-HT1ARs, with potentiation at low dose and inhibition at high dose. Moreover, the in vivo studies indicate that Zn can modulate both presynaptic and postsynaptic 5-HT1ARs; however, Zn’s effects at presynaptic receptors seem to be more potent.

Decreased behavioral response to intranigrally administered GABAA agonist muscimol in the lactacystin model of Parkinson's disease may result from partial lesion of nigral non-dopamine neurons: Comparison to the classical neurotoxin 6-OHDA

Lactacystin is a selective UPS inhibitor recently used to destroy dopamine (DA) neurons in animal models of Parkinsons disease (PD). However, both in vitro and in vivo studies show discrepancies in terms of the sensitivity of non-DA neurons to its toxicity. Therefore, our study was aimed to examine the toxic effect of intranigral administration of lactacystin on DA and non-DA neurons in the rat substantia nigra (SN), compared to the classic neurotoxin 6-OHDA. Tissue DA levels in the striatum and SN and GABA levels in the SN were also examined. Moreover, behavioral response of nigral GABAA receptors to locally administered muscimol was evaluated in these two PD models. We found that both lactacystin and 6-OHDA induced a strong decrease in DA level in the lesioned striatum and SN but only lactacystin slightly reduced GABA levels in the SN. A stereological analysis showed that both neurotoxins highly decreased the number of DA neurons in the SN, while only lactacystin moderately reduced the number of non-DA ones. Finally, in the lactacystin group, the number of contralateral rotations after intranigrally administrated muscimol was decreased in contrast to the increased response in the 6-OHDA model. Our study proves that, although lactacystin is not a fully selective to DA neurons, these neurons are much more vulnerable to its toxicity. Partial lesion of nigral non-DA neurons in this model may explain the decreased behavioral response to the GABAA agonist muscimol.

Early increase in dopamine release in the ipsilateral striatum after unilateral intranigral administration of lactacystin produces spontaneous contralateral rotations in rats

Since the discovery of the role of the ubiquitin-proteasome system (UPS) in the pathogenesis of Parkinsons disease, UPS inhibitors, such as lactacystin have been used to investigate the relationship between UPS impairment and degeneration of dopamine (DA) neurons. However, mostly long-term neurotoxic effects of lactacystin have been studied in animal models. Therefore, the aim of our study was to investigate behavioral and biochemical changes related to the DA system during the first week following unilateral intranigral injection of lactacystin to rats. We found that lactacystin produced early spontaneous contralateral rotations which were inhibited by combined administration of DA D1 and D2 receptor antagonists. Simultaneously, an increase in the extracellular level of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was found in the ipsilateral striatum. In contrast, one week after lesion, when turning behavior was no longer visible, a decrease in the extracellular level of DA, DOPAC and HVA was demonstrated. It was accompanied by a substantial reduction in the tissue levels of DA and its metabolites in the lesioned substantia nigra and striatum. We concluded that unilateral intranigral administration of lactacystin produces an early increase in DA neurotransmission which precedes a decrease in the striatal and nigral tissue DA content. It is manifested by the appearance of spontaneous contralateral rotations and an elevation of the extracellular DA level in the ipsilateral striatum. Since similar behavior was previously observed after intranigral administration of rotenone and MPP(+) but not 6-hydroxydopamine (6-OHDA), it may indicate a common mechanism of action shared by these neurotoxins.

The significance of rotational behavior and sensitivity of striatal dopamine receptors in hemiparkinsonian rats: A comparative study of lactacystin and 6-OHDA

A growing body of evidence indicates that impairment of the ubiquitin-proteasome (UPS) system in the substantia nigra (SN) plays an important role in the pathogenesis of Parkinsons disease (PD). The aim of our study was to compare two unilateral rat models, one produced by intranigral administration of the UPS inhibitor lactacystin or the other induced by 6-OHDA, in terms of their effect on the amphetamine- and apomorphine-induced rotational behavior, striatal dopamine (DA) D1 and D2 receptor sensitivity and tissue levels of DA and its metabolites. We found that these models did not differ in the intensity of ipsilateral rotations induced by amphetamine. In contrast, apomorphine produced contralateral rotations only in 6-OHDA-lesioned rats, and, depending on the dose, it induced either no or moderate ipsilateral rotations in the lactacystin-lesioned group. In addition, lactacystin induced a strong reduction in the tissue DA level and its metabolites in the lesioned striatum and SN when measured three weeks after the administration which was aggravated six weeks post-lesion, reaching the level comparable to the 6-OHDA group. Binding of [3H]raclopride to D2 receptors was increased in the lesioned striatum in both investigated (PD) models six weeks after lesion. In turn, binding of [3H]SCH23390 to the striatal D1 receptors was not changed in the lactacystin group but was increased bilaterally in the 6-OHDA group. The present results add a new value to the study of DA receptor sensitivity and are discussed in the context of the validity of the lactacystin model as a suitable model of Parkinsons disease.