Anna Czarnecka

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.

Role of nitric oxide in the regulation of motor function. An overview of behavioral, biochemical and histological studies in animal models.

A compelling body of evidence suggests that nitric oxide (NO), a unique gaseous neurotransmitter and neuromodulator plays a key role in the regulation of motor function. Recently, the interest of researchers concentrates on the NO - soluble guanylyl cyclase (sGC) - cyclic GMP (cGMP) signaling pathway in the striatum as a new target for the treatment of Parkinsons disease (PD). The aim of the study is to review the available literature referring to the role of NO in the integration of basal ganglia functions. First, attention has been focused on behavioral effects of NO donors and neuronal nitric oxide synthase (nNOS) inhibitors in the modulation of motor behavior. Then, disturbances in the nitrergic neurotransmission in PD and its 6-OHDA animal model have been presented. Moreover, the most current data demonstrating the contribution of both dopamine and glutamate to the regulation of NO biosynthesis in the striatum have been analyzed. Finally, the role of NO in the tonic and phasic dopamine release as well as in the regulation of striatal output pathways also has been discussed.

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.

Asymmetric Dimethylarginine and Hepatic Encephalopathy: Cause, Effect or Association?

The methylated derivative of l-arginine, asymmetric dimethylarginine (ADMA) is synthesized in different mammalian tissues including the brain. ADMA acts as an endogenous, nonselective, competitive inhibitor of all three isoforms of nitric oxide synthase (NOS) and may limit l-arginine supply from the plasma to the enzyme via reducing its transport by cationic amino acid transporters. Hepatic encephalopathy (HE) is a relatively frequently diagnosed complex neuropsychiatric syndrome associated with acute or chronic liver failure, characterized by symptoms linked with impaired brain function leading to neurological disabilities. The l-arginine—nitric oxide (NO) pathway is crucially involved in the pathomechanism of HE via modulating important cerebral processes that are thought to contribute to the major HE symptoms. Specifically, activation of this pathway in acute HE leads to an increase in NO production and free radical formation, thus, contributing to astrocytic swelling and cerebral edema. Moreover, the NO-cGMP pathway seems to be involved in cerebral blood flow (CBF) regulation, altered in HE. For this reason, depressed NO-cGMP signaling accompanying chronic HE and ensuing cGMP deficit contributes to the cognitive and motor failure. However, it should be remembered that ADMA, a relatively little known element limiting NO synthesis in HE, may also influence the NO-cGMP pathway regulation. In this review, we will discuss the contribution of ADMA to the regulation of the NO-cGMP pathway in the brain, correlation of ADMA level with CBF and cognitive alterations observed during HE progression in patients and/or animal models of HE.

Brain extracellular levels of dimethylarginines (ADMA, SDMA) and cGMP and their modulation by exogenous S-adenosylmethionine in rats with acute liver failure

Acute liver failure (ALF) instantly evokes symptoms of hepatic encephalopathy (HE), mainly attributed to hyperammonemia. Ammonia neurotoxicity is related to disturbances in the nitric oxide (NO)/cGMP pathway. The methylated derivative of L-arginine (MDALs) - asymmetric dimethylarginine (ADMA) but not symmetrically methylated arginine (SDMA) is an endogenous inhibitor of nitric oxide synthase. Elevated blood and brain ADMA was reported in HE patients and experimental animals. The question arose whether ALF evokes changes in the incorporation of the methyl donor S-adenosylmethionine (SAM) to MDALs, and how they affect cGMP accumulation. To this end, we investigated the effect of intracortical perfusion of SAM on the brain extracellular levels of ADMA, SDMA and cGMP in rats with ALF in the thioacetamide (TAA) model. Sprague Dawley rats (250–270 g) received three i.p. injections of TAA (300 mg/kg) at 24 h intervals. Bilateral microdialysis of the prefrontal cortex was carried out 24 h after the last TAA administration. SAM at 2 mM concentration in artificial cerebrospinal fluid was infused for 40 min and then the medium was changed back. The extracellular levels of ADMA and SDMA were analyzed using positive mode electrospray LC–DMS–MS/MS and cGMP was determined with cGMP enzyme immunoassay. ALF resulted in the increased extracellular levels of ADMA (by ~800%) and SDMA (by ~250%). Moreover, in ALF rats infusion of SAM decreased the ADMA and SDMA (~30%) as compared to the basal value. It seems reasonable that an excessive amount of substrate inhibited the enzymes synthesizing MDALs. On the other hand, the cGMP level did not differ between control and TAA rats. SAM increased by ~90% cGMP only in the control group, what indicates affected NO/cGMP pathway in TAA model. The study demonstrates, that ALF modulates methylation of arginine to MDALs in a manner affecting cGMP accumulation.

The preferential nNOS inhibitor 7-nitroindazole and the non-selective one NG-nitro-l-arginine methyl ester administered alone or jointly with l-DOPA differentially affect motor behavior and monoamine metabolism in sham-operated and 6-OHDA-lesioned rats

Reciprocal interactions between nitrergic and dopaminergic systems play a key role in the control of motor behavior. In the present study, we performed a comparative analysis of motor behavior (locomotor activity, catalepsy, rotational behavior) and monoamine metabolism in the striatum and substantia nigra of unilaterally sham-operated and 6-OHDA-lesioned rats treated with the preferential neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) or the non-selective one N(G)-nitro-L-arginine methyl ester (L-NAME), alone or in combination with L-DOPA. Each NOS inhibitor given alone (50mg/kg) induced a distinct catalepsy 30 min after injection but only 7-NI impaired spontaneous locomotion after 10 min. In 6-OHDA-lesioned rats, chronic L-DOPA (25mg/kg) induced 2.5-h long contralateral rotations. 7-NI (30 and 50mg/kg) markedly reduced the intensity of L-DOPA-induced contralateral rotations while extending their duration until 4.5h whereas L-NAME (50 and 100mg/kg) only tended to attenuate their intensity without affecting the duration. 7-NI but not L-NAME significantly increased endogenous tissue DA levels in the nigrostriatal system of both sham-operated and 6-OHDA-lesioned rats. In L-DOPA-treated group, 7-NI significantly enhanced the L-DOPA-derived tissue DA content in this system and decreased the level of the intracellular DA metabolite DOPAC produced by monoamine oxidase (MAO). In contrast to 7-NI, L-NAME decreased markedly DA content and did not affect DOPAC level in the ipsilateral striatum. It means that the differences in 7-NI and L-NAME-mediated modulation of L-DOPA-induced behavioral and biochemical effects resulted not only from the inhibition of NOS activity but also from differences in their ability to inhibit MAO.

Cerebrovascular reactivity and cerebral perfusion of rats with acute liver failure: role of L-glutamine and asymmetric dimethylarginine in L-arginine-induced response

Cerebral blood flow (CBF) is impaired in acute liver failure (ALF), however, the complexity of the underlying mechanisms has often led to inconclusive interpretations. Regulation of CBF depends at least partially on variations in the local brain L‐arginine concentration and/or its metabolic rate. In ALF, other factors, like an increased concentration of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor and elevated level of L‐glutamine, may contribute to CBF alteration. This study demonstrated strong differences in the reactivity of the middle cerebral arteries and their response to extravascular L‐arginine application between vessels isolated from rats with thioacetamide (TAA)‐induced ALF and control animals. Our results also showed the decrease in the cerebral perfusion in TAA rats measured by arterial spin labeling perfusion magnetic resonance. Subsequently, we aimed to investigate the importance of balance between the concentration of ADMA and L‐arginine in the CBF regulation. In vivo, intraperitoneal L‐arginine administration in TAA rats corrected: (i) decrease in cerebral perfusion, (ii) decrease in brain extracellular L‐arginine/ADMA ratio and (iii) increase in brain L‐glutamine concentration. Our study implicates that impaired vascular tone of cerebral arteries is most likely associated with exposure to high ADMA and L‐glutamine levels resulting in limited availability of L‐arginine and might be responsible for reduced cerebral perfusion observed in ALF.