Cannabinoid Receptor Type 1 Expression in the Developing Avian Retina: Morphological and Functional Correlation With the Dopaminergic System

The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212–2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation

Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats

<div><p>Methylphenidate (MPD) is one of the most prescribed drugs for alleviating the symptoms of Attention Deficit/Hyperactivity Disorder (ADHD). However, changes in the molecular mechanisms related to MPD withdrawal and susceptibility to consumption of other psychostimulants in normal individuals or individuals with ADHD phenotype are not completely understood. The aims of the present study were: (i) to characterize the molecular differences in the prefrontal dopaminergic system of SHR and Wistar strains, (ii) to establish the neurochemical consequences of short- (24 hours) and long-term (10 days) MPD withdrawal after a subchronic treatment (30 days) with Ritalin® (Methylphenidate Hydrochloride; 2.5 mg/kg orally), (iii) to investigate the dopaminergic synaptic functionality after a cocaine challenge in adult MPD-withdrawn SHR and Wistar rats. Our results indicate that SHR rats present reduced [³H]-Dopamine uptake and cAMP accumulation in the prefrontal cortex (PFC) and are not responsive to dopaminergic stimuli in when compared to Wistar rats. After a 24-hour withdrawal of MPD, SHR did not present any alterations in [³H]-Dopamine Uptake, [³H]-SCH 23390 binding and cAMP production; nonetheless, after a 10-day MPD withdrawal, the results showed a significant increase of [³H]-Dopamine uptake, of the quantity of [³H]-SCH 23390 binding sites and of cAMP levels in these animals. Finally, SHR that underwent a 10-day MPD withdrawal and were challenged with cocaine (10 mg/kg i.p.) presented reduced [³H]-Dopamine uptake and increased cAMP production. Wistar rats were affected by the 10-day withdrawal of MPD in [³H]-dopamine uptake but not in cAMP accumulation; in addition, cocaine was unable to induce significant modifications in [³H]-dopamine uptake and in cAMP levels after the 10-day withdrawal of MPD. These results indicate a mechanism that could explain the high comorbidity between ADHD adolescent patients under methylphenidate treatment and substance abuse in adult life.</p></div

Glutathione-Induced Calcium Shifts in Chick Retinal Glial Cells

Submitted by Sandra Infurna ([email protected]) on 2016-12-13T15:06:03Z No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-13T15:23:31Z (GMT) No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5)Made available in DSpace on 2016-12-13T15:23:31Z (GMT). No. of bitstreams: 1 leonardo_ferreira_etal_IOC_2016.pdf: 3073404 bytes, checksum: bcdeb147a5807f576266e60f4c1d2e0e (MD5) Previous issue date: 2016Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Ciências Biológicas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neurogeneses. Rio de Janeiro, RJ, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inflamação. Rio de Janeiro, RJ. Brasil.Universidade Federal Fluminense. Departamento de Fisiologia e Farmacologia. Laboratório de Neurofarmacologia. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxoplasmose. Rio de Janeiro, RJ, Brasil.Universidade Federal do Pará. Instituto de Biologia. Belém, PA, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Neuroquímica. Rio de Janeiro, RJ, Brasil.Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10 mM) showed that 5-10 mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50 mM KCl (labeled as βIII tubulin positive cells). BBG 100 nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70 μM and MK-801 20 μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5 mM failed to induce calcium mobilization in glia cells, indicating that the antioxidant and/or structural features of GSH are essential to promote elevations in cytoplasmic calcium levels. Indeed, a short GSH pulse (60s) protects Müller glia from oxidative damage after 30 min of incubation with 0.1% H2O2. Finally, GSH induced GABA release from chick embryonic retina, mixed neuron-glia or from Müller cell cultures, which were inhibited by BBG or in the absence of sodium. GSH also induced propidium iodide uptake in Müller cells in culture in a P2X7 receptor dependent manner. Our data suggest that GSH, in addition to antioxidant effects, could act signaling calcium shifts at the millimolar range particularly in Müller glia, and could regulate the release of GABA, with additional protective effects on retinal neuron-glial circuit

[³H]-Dopamine uptake and cAMP accumulation in MPD-withdrawn SHR and Wistar rats after a challenge with cocaine.

<p>Wistar (A) and SHR (B) [³H]-Dopamine uptake after a challenge with cocaine (10 mg/kg i.p.) in vehicle-treated and MPD-withdrawn rats (n = 5–8). Wistar (C) and SHR (D) cAMP levels in vehicle-treated and MPD-withdrawn rats after a single administration of cocaine (n = 3–6). Results are expressed as means ± S.E.M. * = p < 0.05; ** = p < 0.01; *** = p < 0.001, vs. Control. a = p < 0.01, b = p < 0.001, vs. COC. c = p < 0.01, d = p < 0.001, vs. MPD.</p

Characterization of [³H]-Dopamine uptake and cAMP accumulation in naïve Wistar and SHR rats.

<p>(A) [³H]-Dopamine uptake after 60 min in the PFC (n = 6–8). (B) Time curve of [³H]-Dopamine uptake (n = 3–6). (C) cAMP levels in PFC slices in basal conditions and after exogenous dopamine stimulation (200 μM) (n = 4–8). Results are expressed as means ± S.E.M. A and B: * = p < 0.05; ** = p < 0.01; *** = p < 0.001, SHR vs. Wistar. C: ** = p < 0.01, Control vs. DA.</p

[³H]-SCH 23390 binding and cAMP accumulation after MPD withdrawal in SHR PFC.

<p>(A) [³H]-SCH 23390 binding in the 24-h (PN56) and 10-day (PN65) MPD withdrawal and control groups (n = 4). (B) Basal cAMP accumulation in the 24-h (PN56) and 10-day (PN65) withdrawal group and after SCH 23390 exposure (50 μM; n = 4–7). Results are expressed as means ± S.E.M. ** = p < 0.01; *** = p < 0.001, vs. Control. a = p < 0.001, vs. SCH. b = p < 0.001, vs. MPD.</p

Analysis of SHR [³H]-Dopamine uptake.

<p>[³H]-Dopamine uptake was analyzed 24 h (PN56) and 10 days (PN65) after chronic treatment with MPD, as well as 30 min after a single treatment with MPD in vehicle-treated animals (n = 5–6). Results are expressed as means ± S.E.M. * = p < 0.05; *** = p < 0.001, vs. Control.</p

Western Immunoblotting for DAT and D1R.

<p>PFC samples of SHR rats were incubated with anti-DAT (1:500) or D1R (1:500) and anti-Tubulin (1:25000) at PN56 (A and C) or PN65 (B and D) after chronic MPD treatment. Results of densitometry indicated in arbitrary units (n = 3). Results are expressed as means ± S.E.M.</p