Biochemical and behavioral effects of rosmarinic acid treatment in an animal model of Parkinson's disease induced by MPTP.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. The main therapeutic approach available nowadays relieves motor symptoms but does not prevent or stop neurodegeneration. Rosmarinic acid (RA), an ester of caffeic and 3,4-dihydroxyphenylacetic acids, is obtained from numerous plant species such as Salvia officinalis L. (sage) and Rosmarinus officinalis (rosemary). This compound has a wide spectrum of biological activities, such as antioxidant and anti-inflammatory, and could be an additional therapy for neurodegenerative disorders. Here we evaluated the potential neuroprotective effects of RA treatment in a murine model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice were separated into four groups: CN, Control/saline; RA, Rosmarinic acid/vehicle; MPTP, MPTP/saline; MPTP+RA, MPTP/RA. RA (20 mg/kg, or vehicle) was administered orally by intra-gastric gavage for 14 days, one hour before MPTP or saline injection. MPTP groups received the drug (30 mg/kg, intraperitoneally) once a day for five days (fourth to the eighth day of the experiment). MPTP-treated animals displayed hyperlocomotion behavior, which was significantly prevented by RA treatment. In addition, RA treatment increased dopaminergic signaling in the parkinsonian mice and improved the monoaminergic system in healthy animals. Analysis of alterations in the striatal mRNA expression of dopaminergic system components showed that MAO-A expression was increased in the MPTP+AR group. Overall, this study brings new evidence of the potential neuroprotective properties of RA not only in preventing behavioral features observed in PD, but also by improving neurotransmission in the healthy brain.

Molecular mechanisms underlying the neuroprotection of environmental enrichment in Parkinson's disease.

Parkinson's disease is the most common movement disorder, affecting about 1% of the population over the age of 60 years. Parkinson's disease is characterized clinically by resting tremor, bradykinesia, rigidity and postural instability, as a result of the progressive loss of nigrostriatal dopaminergic neurons. In addition to this neuronal cell loss, Parkinson's disease is characterized by the accumulation of intracellular protein aggregates, Lewy bodies and Lewy neurites, composed primarily of the protein α-synuclein. Although it was first described almost 200 years ago, there are no disease-modifying drugs to treat patients with Parkinson's disease. In addition to conventional therapies, non-pharmacological treatment strategies are under investigation in patients and animal models of neurodegenerative disorders. Among such strategies, environmental enrichment, comprising physical exercise, cognitive stimulus, and social interactions, has been assessed in preclinical models of Parkinson's disease. Environmental enrichment can cause structural and functional changes in the brain and promote neurogenesis and dendritic growth by modifying gene expression, enhancing the expression of neurotrophic factors and modulating neurotransmission. In this review article, we focus on the current knowledge about the molecular mechanisms underlying environmental enrichment neuroprotection in Parkinson's disease, highlighting its influence on the dopaminergic, cholinergic, glutamatergic and GABAergic systems, as well as the involvement of neurotrophic factors. We describe experimental pre-clinical data showing how environmental enrichment can act as a modulator in a neurochemical and behavioral context in different animal models of Parkinson's disease, highlighting the potential of environmental enrichment as an additional strategy in the management and prevention of this complex disease.

Neuronal scaffolding protein spinophilin is integral for cocaine-induced behavioral sensitization and ERK1/2 activation.

Spinophilin is a scaffolding protein enriched in dendritic spines with integral roles in the regulation of spine density and morphology, and the modulation of synaptic plasticity. The ability of spinophilin to alter synaptic strength appears to involve its scaffolding of key synaptic proteins, including the important structural element F-actin, AMPA/NMDA modulator protein phosphatase 1, and neuromodulatory G-protein coupled receptors, including dopamine receptor D2 and metabotropic glutamate receptor 5. Additionally, spinophilin is highly expressed in the striatum, a brain region that is fundamentally involved in reward-processing and locomotor activity which receives both glutamatergic and dopaminergic inputs. Therefore, we aimed to investigate the role of spinophilin in behavioral responses to cocaine, evaluating wild-type and spinophilin knockout mice followed by the examination of underlying molecular alterations. Although acute locomotor response was not affected, deletion of spinophilin blocked the development and expression of behavioral sensitization to cocaine while maintaining normal conditioned place preference. This behavioral alteration in spinophilin knockout mice was accompanied by attenuated c-Fos and ∆FosB expression following cocaine administration and blunted cocaine-induced phosphorylation of ERK1/2 in the striatum, with no change in other relevant signaling molecules. Therefore, we suggest spinophilin fulfills an essential role in cocaine-induced behavioral sensitization, likely via ERK1/2 phosphorylation and induction of c-Fos and ∆FosB in the striatum, a mechanism that may underlie specific processes in cocaine addiction.

Impairment of spatial working memory and oxidative stress induced by repeated crack cocaine inhalation in rats.

Crack cocaine is a highly toxic drug with great potential to induce addiction. It produces more intense effects than cocaine powder, with its use having grown worldwide. However, few studies have focused on the cognitive and biochemical consequences that result from crack cocaine inhalation. This study examined the effects of direct crack cocaine inhalation on spatial working memory and brain oxidative stress parameters in rats. Male adult Wistar rats, well-trained in an eight-arm radial maze (8-RM), underwent five sessions of crack cocaine inhalation (crack cocaine group) once a day or inhalation simulation (sham group), being tested in 1-h delayed tasks 24 h after the last inhalation. An additional inhalation session was carried out the following day, with the prefrontal cortex, hippocampus and striatum being removed five minutes afterwards in order to assess oxidative damage such as lipid peroxidation, thiobarbituric acid-reactive species (TBARS) levels, and advanced oxidation protein products (AOPP), as well as the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Animals from the crack cocaine group showed more errors (p <  0.01) in the 1-h post-delay performance in the 8-RM when compared to the sham group. The crack cocaine group showed decreased (p <  0.05) lipid peroxidation in the hippocampus and increased (p <  0.001) levels of AOPP and SOD activity (p < 0.05) in the striatum when compared to the sham group. Therefore, the repeated inhalation of crack cocaine impaired long-term spatial working memory and elicited oxidative stress in the hippocampus and striatum of rats.

Atropine counteracts the depressive-like behaviour elicited by acute exposure to commercial chlorpyrifos in rats.

Acute organophosphate (OP) poisoning induces well-known signs of toxicosis related to acetylcholinesterase (AChE) inhibition. However, the relationship between acute OP poisoning and the onset of psychiatric disorders remains unclear. Thus, we investigated behavioural and biochemical consequences of acute exposure to the OP chlorpyrifos in male rats and also the effectiveness of the antidotes atropine and pralidoxime on reversing these changes. A sub-lethal dose of commercial chlorpyrifos (20 mg/kg, i.p.) elicited signs of acute toxicosis during the first hours after its injection in rats. Twenty-four hours after treatment, this single dose of chlorpyrifos induced a depressive-like behaviour in the rat forced swimming test without impairing locomotor activity. At this time (24 h), chlorpyrifos decreased plasma butyrylcholinesterase (BChE) activity and hippocampal, striatal and prefrontal cortical AChE activity in rats. The behavioural and biochemical consequences of acute chlorpyrifos poisoning do not seem to be long lasting, since 30 days later they were absent. We evaluated whether these behavioural and biochemical consequences of acute chlorpyrifos treatment would be reversed by the antidotes atropine (10 mg/kg i.p.) and/or pralidoxime (40 mg/kg; i.p.) given 1 h after poisoning. Pralidoxime partially reactivated the AChE activity in the prefrontal cortex, but not in the hippocampus and striatum. Atropine attenuated the depressive-like behaviour induced by chlorpyrifos in rats. Our results suggest that acute chlorpyrifos poisoning induces a transient depressive-like behaviour possible related to hippocampal AChE inhibition. They suggest that treatment with atropine and pralidoxime seems to be insufficient to counteract all the effects of OP acute poisoning, at least in rats.

Environmental enrichment cognitive neuroprotection in an experimental model of cerebral ischemia: biochemical and molecular aspects.

Stroke is considered a major cause of global morbidity. Currently, there are no effective treatments for post-stroke cognitive impairment. Enriched environment (EE) has been brought forward as a preconditioning method to induce cerebral tolerance in an ischemic event. However, the subjacent mechanisms involved in this tolerance are not yet clear. Herein we aimed to identify the mechanisms of neuroprotection triggered by EE preconditioning in a murine model of ischemic stroke. In order to do so, C57Bl/6 mice were kept for five weeks either in EE or in standard environment (SC) prior to ischemic injury through bilateral carotid occlusion (BCCAo) or sham surgery. To evaluate cognitive deficits resulting from ischemia, animals were subjected to a set of behavioral test to assess short-term (STM), long-term (LTM) and working memory (WM) performance. Despite no effect of EE having been observed in LTM and WM, EE preconditioning was able to prevent short-term deficits in response to ischemia. This improvement was accompanied by a reduction in the infarct volume in animals following EE pre-exposure. Next, we aimed to analyze the expression of genes involved in cholinergic (M1 and alpha 7 receptors) and glutamatergic (NMDA subunits GluN1, GluN2A, GluN2B and GluN2C) neurotransmission, inflammatory mediators (GFAP and IL-1ß) and of the neurotrophin BDNF. Animals tested for STM did not present alterations in the expression of glutamatergic or cholinergic receptors; however, EE was shown to prevent increased expression of IL1-ß. On the other hand, in animals submitted to LTM task, EE exposure lead to increased GFAP expression in EE animals that underwent ischemic injury, affecting also the expression of NMDA subunits. In spite of that, no alterations in glutamate content were observed in either group. Altogether, this study suggests that the changes observed in the expression of glutamatergic receptors, the reduction of the inflammatory cytokine IL1-ß expression and the increased expression of GFAP in ischemic animals might contribute to the cognitive improvement induced by the EE paradigm.

Behavioral, Biochemical and Molecular Characterization of a Parkinson's Disease Mouse Model Using the Neurotoxin 2'-CH3-MPTP: A Novel Approach.

The neurotoxin MPTP has long been used to create a mouse model of Parkinson's disease (PD). Indeed, several MPTP analogues have been developed, including 2'-CH3-MPTP, which was shown to induce nigrostriatal DA neuronal depletion more potently than MPTP. However, no study on behavioral and molecular alterations in response to 2'-CH3-MPTP has been carried out so far. In the present work, 2'-CH3-MPTP was administered to mice (2.5, 5.0 and 10 mg/kg per injection, once a day, 5 days) and histological, biochemical, molecular and behavioral alterations were evaluated. We show that, despite a dose-dependent-like pattern observed for nigrostriatal dopaminergic neuronal death and dopamine depletion, dose-specific alterations in dopamine metabolism and in the expression of dopaminergic neurotransmission-associated genes could be related to specific motor deficits elicited by the different doses tested. Interestingly, 2'-CH3-MPTP leads to increased DAT and MAO-B transcription, which could explain, respectively, its higher potency and the requirement of higher doses of MAO inhibitors to prevent nigrostriatal neuronal death when compared to MPTP. Also, perturbations in dopamine metabolism as well as possible alterations in dopamine bioavailability in the synaptic cleft were also identified and correlated with strength and ambulation deficits in response to specific doses. Overall, the present work brings new evidence supporting the distinct effects of 2'-CH3-MPTP when compared to its analogue MPTP. Moreover, our data highlight the utmost importance of a precise experimental design, as different administration regimens and doses yield different biochemical, molecular and behavioral alterations, which can be explored to study specific aspects of PD.

Crack cocaine inhalation induces schizophrenia-like symptoms and molecular alterations in mice prefrontal cortex.

Crack cocaine (crack) addiction represents a major social and health burden, especially seeing as users are more prone to engage in criminal and violent acts. Crack users show a higher prevalence of psychiatric comorbidities - particularly antisocial personality disorders - when compared to powder cocaine users. They also develop cognitive deficits related mainly to executive functions, including working memory. It is noteworthy that stimulant drugs can induce psychotic states, which appear to mimic some symptoms of schizophrenia among users. Social withdraw and executive function deficits are, respectively, negative and cognitive symptoms of schizophrenia mediated by reduced dopamine (DA) tone in the prefrontal cortex (PFC) of patients. That could be explained by an increased expression of D2R short isoform (D2S) in the PFC of such patients and/or by hypofunctioning NMDA receptors in this region. Reduced DA tone has already been described in the PFC of mice exposed to crack smoke. Therefore, it is possible that behavioral alterations presented by crack users result from molecular and biochemical neuronal alterations akin to schizophrenia. Accordingly, we found that upon crack inhalation mice have shown decreased social interaction and working memory deficits analogous to schizophrenia's symptoms, along with increased D2S/D2L expression ratio and decreased expression of NR1, NR2A and NR2B NMDA receptor subunits in the PFC. Herein we propose two possible mechanisms to explain the reduced DA tone in the PFC elicited by crack consumption in mice, bringing also the first direct evidence that crack use may result in schizophrenia-like neurochemical, molecular and behavioral alterations.

Cholinergic and Dopaminergic Alterations in Nigrostriatal Neurons Are Involved in Environmental Enrichment Motor Protection in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, being characterized by dopaminergic neurodegeneration of substantia nigra pars compacta. PD pharmacotherapy has been based on dopamine replacement in the striatum with the dopaminergic precursor 3,4-dihydroxyphenylalanine (L-DOPA) and/or with dopaminergic agonists, alongside anticholinergic drugs in order to mitigate the motor abnormalities. However, these practices neither prevent nor stop the progression of the disease. Environmental enrichment (EE) has effectively prevented several neurodegenerative processes, mainly in preclinical trials. Several studies have demonstrated that EE induces biological changes, bearing on cognitive enhancement, neuroprotection, and on the attenuation of the effects of stress, anxiety, and depression. Herein, we investigated whether EE could prevent the motor, biochemical, and molecular abnormalities in a murine model of PD induced by 1-methyl-4-phenyl-2,3-dihydropyridine (MPTP). Our results show that EE does not prevent the dopaminergic striatal depletion induced by MPTP, despite having averted the MPTP-induced hyperlocomotion. However, it was able to slow down and avoid, respectively, the 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) depletion. Analysis of dopaminergic mRNA alterations in the midbrain showed that D1R expression was increased by MPTP, while the normal expression level of this receptor was restored by EE. As for the cholinergic system, MPTP led to a decrease in the ChAT gene expression while increasing the expression of both AChE and M1R. EE attenuated and prevented-respectively-ChAT and M1R gene expression alterations triggered by MPTP in the midbrain. Overall, our data brings new evidence supporting the neuroprotective potential of EE in PD, focusing on the interaction between dopaminergic and cholinergic systems.