Monica Armida

Adenosine A2A receptors are required for normal BDNF levels and BDNF-induced potentiation of synaptic transmission in the mouse hippocampus

Brain‐derived neurotrophic factor (BDNF), a member of neurotrophin family, enhances synaptic transmission and regulates neuronal proliferation and survival. Both BDNF and its tyrosine kinase receptors (TrkB) are highly expressed in the hippocampus, where an interaction with adenosine A2A receptors (A2ARs) has been recently reported. In the present paper, we evaluated the role of A2ARs in mediating functional effects of BDNF in hippocampus using A2AR knock‐out (KO) mice. In hippocampal slices from WT mice, application of BDNF (10 ng/mL) increased the slope of excitatory post‐synaptic field potentials (fEPSPs), an index of synaptic facilitation. This increase of fEPSP slope was abolished by the selective A2A antagonist ZM 241385. Similarly, genetic deletion of the A2ARs abolished BDNF‐induced increase of the fEPSP slope in slices from A2AR KO mice The reduced functional ability of BDNF in A2AR KO mice was correlated with the reduction in hippocampal BDNF levels. In agreement, the pharmacological blockade of A2Rs by systemic ZM 241385 significantly reduced BDNF levels in the hippocampus of normal mice. These results indicate that the tonic activation of A2ARs is required for BDNF‐induced potentiation of synaptic transmission and for sustaining a normal BDNF tone in the hippocampus.

Nonmotor symptoms in Parkinson's disease: Investigating early-phase onset of behavioral dysfunction in the 6-hydroxydopamine-lesioned rat model

To investigate the psychiatric symptoms accompanying the early phases of Parkinsons disease (PD), we injected adult rats with 10.5 μg 6‐hydroxydopamine (6‐OHDA) bilaterally into the dorsal striatum. The resulting neurodegeneration led, 12 weeks after injection, to a mild (36%) reduction of striatal dopamine. We tested the behavioral response of sham and 6‐OHDA‐lesioned animals at different time points after injection to evaluate the onset and progression of behavioral abnormalities. The results showed that such a mild reduction of dopamine levels was associated with a decrease in anxiety‐like behavior, an increase in “depression”‐like behavior, and a marked change in social behavior. Learning and memory abilities were not affected. Overall, the PD rat model used here displays behavioral alterations having face validity with psychiatric symptoms of the pathology and thus appears to be a valuable tool for investigating the neural bases of the early phases of PD.

Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS): the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG), a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising therapeutic strategy by interfering with the neuroinflammatory component of the disease.

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease.

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntingtons disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.

Neuroprotective Effects of Thymosin β4 in Experimental Models of Excitotoxicity

Abstract:  The aim of this study was to evaluate the possible neuroprotective effects of thymosin β4 in different models of excitotoxicity. The application of thymosin β4 significantly attenuated glutamate‐induced toxicity both in primary cultures of cortical neurons and in rat hippocampal slices. In in vivo experiments, the intracerebroventricular administration of thymosin β4 significantly reduced hippocampal neuronal loss induced by kainic acid. These results show that thymosin β4 induced a protective effect in models of excitotoxicity. The mechanisms underlying such an effect, as well as the real neuroprotective potential of thymosin β4, are worthy of further investigations.

Striatal 6-OHDA lesion in mice: Investigating early neurochemical changes underlying Parkinson's disease

Early phases of Parkinsons disease (PD) are characterized by a mild reduction of dopamine (DA) in striatum and by emergence of psychiatric disturbances that precede overt motor symptoms. In order to characterize the neurochemical re-arrangements induced by such striatal impairment, we used a mouse model in which a low dose of 6-hydroxydopamine (6-OHDA) was bilaterally injected into the dorsal striatum. These mice showed a DA reduction of about 40% that remained stable up to 12 weeks after injection. This reduction was accompanied by changes in DA metabolite levels, such as HVA, transiently reduced at 4 weeks, and DOPAC, decreased at 12 weeks. No change in the 5-hydroxytryptamine (5-HT) levels was found but the 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was increased at 4 weeks. In addition, at the same time-point, the levels of 15-F(2t)-IsoP, an index of oxidative stress, and of PGE(2), a major product of cyclooxygenase-2, were decreased in different brain areas while BDNF levels were increased. These neurochemical changes were accompanied by altered behavioral responses concerning the emotional reactivity. Overall, the present findings suggest that a change of 5-HT metabolism and a modification of oxidative stress levels may play a role in the early PD degeneration phases.

Effects of chronic caffeine intake in a mouse model of amyotrophic lateral sclerosis

Caffeine is a nonselective adenosine receptor antagonist; chronic consumption has proved protective toward neurodegenerative diseases such as Parkinsons and Alzheimers diseases. The present study was designed to determine whether caffeine intake affected survival and/or motor performance in a transgenic model of amyotrophic lateral sclerosis (ALS). SOD1G93A mice received caffeine through drinking water from 70 days of age until death. Body weight, motor performance and survival were evaluated. Furthermore, the expression of adenosine A2A receptors (A2ARs), glial glutamate transporter (GLT1), and glial fibrillar acidic protein (GFAP) were evaluated by Western blotting. The results showed that caffeine intake significantly shortened the survival of SOD1G93A mice (log rank test, P = 0.01) and induced a nonsignificant advancing of disease onset. The expression of A2AR, GLT1, and GFAP was altered in the spinal cords of ALS mice, but caffeine did not influence their expression in either wild‐type or SOD1G93 mice. These data indicate that adenosine receptors may play an important role in ALS.

Influence of CGS 21680, a selective adenosine A2A receptor agonist, on NMDA receptor function and expression in the brain of Huntington's disease mice

The effect of chronic treatment with the selective adenosine A(2A) receptor agonist CGS 21680 on N-Methyl-d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntingtons disease (HD). Starting from 8weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5mg/kg i.p.) for 3weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A(2A) receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.

Striatal adenosine–cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors

Adenosine A2A receptors (A2ARs) and cannabinoid CB1 receptors (CB1Rs) are highly expressed in the striatum, where they functionally interact and form A2A/CB1 heteroreceptor complexes. We investigated the effects of CB1R stimulation in a transgenic rat strain over‐expressing A2ARs under the control of the neural‐specific enolase promoter (NSEA2A rats) and in age‐matched wild‐type (WT) animals. The effects of the CB1R agonist WIN 55,212‐2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2AR agonist (CGS 21680) and antagonists (ZM 241385, KW‐6002 and SCH‐442416) in WT animals, the A2AR antagonists failed to influence WIN‐mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over‐expression of A2ARs, the effects mediated by CB1R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2AR forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2ARs and CB1Rs, playing a fundamental role in the regulation of striatal functions.

Aberrant self-grooming as early marker of motor dysfunction in a rat model of Huntington's disease

In the study of neurodegenerative diseases, rodent models provide experimentally accessible systems to study multiple pathogenetic aspects. The identification of early and robust behavioural changes is crucial to monitoring disease progression and testing potential therapeutic strategies in animals. Consistent experimental data support the translational value of rodent self-grooming as index of disturbed motor functions and perseverative behaviour patterns in different rodent models of brain disorders. Huntingtons disease (HD) is a progressive neurodegenerative disorder, characterized by severe degeneration of basal ganglia, cognitive and psychiatric impairments and motor abnormalities. In the rat species, intrastriatal injection of the excitotoxin quinolinic acid (QA) mimics some of the neuroanatomical and behavioural changes found in HD, including the loss of GABAergic neurons and the appearance of motor and cognitive deficits. We show here that striatal damage induced by unilateral QA injection in dorsal striatum of rats triggers aberrant grooming behaviour as early as three weeks post-lesion in absence of other motor impairments: specifically, both quantitative (frequency and duration) and qualitative (the sequential pattern of movements) features of self-grooming behaviour were significantly altered in QA-lesioned rats placed in either the elevated plus-maze and the open-field. The consistent abnormalities in self-grooming recorded in two different experimental contexts support the use of this behavioural marker in rodent models of striatal damage such as HD, to assess the potential effects of drug and cell replacement therapy in the early stage of disease.