NMDA Receptor Activation and Ca2+/PKC Signaling in Nicotine-Induced GABA Transport Shift in Embryonic Chick Retina.

Nicotinic receptors are present in the retina of different vertebrates, and in the chick retina, it is present during early development throughout to post-hatching. These receptors are activated by nicotine, an alkaloid with addictive and neurotransmitter release modulation properties, such as GABA signaling. Here we evaluated the mechanisms of nicotine signaling in the avian retina during the development of neuron-glia cells at a stage where synapses are peaking. Nicotine almost halved [3H]-GABA uptake, reducing it by 45% whilst increasing more than two-fold [3H]-GABA release in E12 embryonic chick retinas. Additionally, nicotine mediated a 33% increase in [3H]-D-aspartate release. MK-801 50 µM blocked 66% of nicotine-induced [3H]-GABA release and Gö 6983 100 nM prevented the nicotine-induced reduction in [3H]-GABA uptake by rescuing 40% of this neurotransmitter uptake, implicating NMDAR and PKC (respectively) in the nicotinic responses. In addition, NO-711 prevented [3H]-GABA uptake and release induced by nicotine. Furthermore, the relevance of calcium influx for PKC activation was evidenced through fura-2 imaging. We conclude that the shift of GABA transport mediated by nicotine promotes GABA release by inducing transporter reversal via nicotine-induced EAA release through EAATs, or by a direct effect of nicotine in activating nicotinic receptors permeable to calcium and promoting PKC pathway activation and shifting GAT-1 activity, both prompting calcium influx, and activation of the PKC pathway and shifting GAT-1 activity.

Tabagismo na pandemia de Covid-19: aspectos neurobiológicos do abuso de drogas em condições de estresse prolongado / Smoking on Covid-19 pandemic: neurobiological aspects of drug abuse in lasting stress conditions / Fumar en la pandemia de Covid-19: aspectos neurobiológicos del abuso de drogas en condiciones de estrés duradero

No fim de 2019 iniciou-se uma das maiores crises da saúde pública global em Wuhan, China. Essa emergência foi o aparecimento do SARS-CoV-2 e da doença COVID-19, uma síndrome respiratória aguda de alta transmissibilidade. A declaração da pandemia pela OMS em março de 2020 fez com que o mundo tomasse diversas medidas para o combate e contenção da doença. Inicialmente o isolamento social e lockdown foram as principais iniciativas, já que não havia formas de tratamento ou prevenção da doença. Essas medidas restritivas geraram uma mudança de hábito da população que deflagrou sérios comprometimentos físicos e psicológicos. Uma das consequências foi o aumento do uso de substâncias de abuso e, consequentemente, do transtornos por uso de substâncias, dentre elas o tabaco. Durante a pandemia o consumo de cigarro aumentou de 10 a 30% no mundo, o tabagismo é a principal causa de morte evitável e fator de risco para diversas doenças. Conjuntamente ao álcool, a nicotina têm um poder aditivo superior a muitas drogas ilícitas. A combinação dos transtornos por uso de substâncias e a COVID-19 acabam por ter um efeito sinérgico, dessa forma, buscamos integrar aspectos neuroquímicos, cognitivos e comportamentais que levaram ao aumento do consumo e/ou recaída nicotina e a terapêutica utilizada.

One of the biggest global public health crisis began in Wuhan, China at the end of 2019. That emergency was the emergence of SARS-CoV-2 and the disease COVID-19, a highly transmissible acute respiratory syndrome. The pandemic declaration by the WHO in March 2020 caused the world to take on several measures to combat and contain the virus. Initially, social isolation and lockdown were the main initiatives, as there were no forms of treatment or prevention of the disease. These restrictive measures generate a change in the habit of the population that triggered serious physical and psychological impairments. One of the consequences was the increase in the use of substances of abuse and, consequently, substance use disorder, including tobacco. During the pandemic, cigarette consumption increased from 10 to 30% worldwide, whereas smoking is the main cause of preventable death and a risk factor for several diseases. Along with alcohol, nicotine has a greater addictive power than illicit drugs. Substance use disorders and COVID-19 have a synergistic effect, in this way, we seek to integrate neurochemical, cognitive and behavioral aspects that led to increased consumption and/or relapse in nicotine consumption and the used therapy.

Una de las mayores crisis mundiales de salud pública comenzó en Wuhan (China) a finales de 2019. Esa emergencia fue la aparición del SARS-CoV-2 y la enfermedad COVID-19, un síndrome respiratorio agudo altamente transmisible. La declaración de pandemia por parte de la OMS en marzo de 2020 hizo que el mundo adoptara varias medidas para combatir y contener el virus. Inicialmente, el aislamiento social y el encierro fueron las principales iniciativas, ya que no existían formas de tratamiento o prevención de la enfermedad. Estas medidas restrictivas generaron un cambio en los hábitos de la población que desencadenó graves alteraciones físicas y psicológicas. Una de las consecuencias fue el aumento del consumo de sustancias de abuso y, en consecuencia, el trastorno por consumo de sustancias, incluido el tabaco. Durante la pandemia, el consumo de cigarrillos aumentó del 10 al 30% en todo el mundo, mientras que el tabaquismo es la principal causa de muerte evitable y un factor de riesgo de varias enfermedades. Junto con el alcohol, la nicotina tiene un mayor poder adictivo que las drogas ilícitas. Los trastornos por uso de sustancias y la COVID-19 tienen un efecto sinérgico, de esta manera, buscamos integrar los aspectos neuroquímicos, cognitivos y conductuales que llevaron al aumento del consumo y/o recaída en el consumo de nicotina y la terapia utilizada.

Caffeine Improves GABA Transport in the Striatum of Spontaneously Hypertensive Rats (SHR).

The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.

Ensino emergencial remoto: uma perspectiva da neurofisiologia / Remote emergency teaching: a neurophysiology perspective

No ano de 2020, iniciou-se uma emergência de saúde pública mundial. A fim de frear a transmissão do SARSCoV-2, foram aplicadas medidas de distanciamento social, que reduziram o contato interpessoal e deflagraram mudanças profundas em comportamentos relacionados à interação social. Os sistemas educacionais ao redor do mundo têm se adaptado a esta nova realidade, adotando protocolos emergenciais caracterizados, em grande parte, pela substituição das atividades presenciais pelas virtuais. O objetivo deste artigo é dialogar sobre os impactos do distanciamento social no ensino superior através de uma abordagem neurofisiológica, conectando estresse, empatia e o contexto histórico do ensino emergencial quando comparado ao Ensino à Distância, além das ferramentas à nossa disposição e as complexidades do ambiente virtual. Os professores devem estar conscientes do impacto do estresse e do uso de ferramentas digitais e de ensino à distância na formação dos estudantes, buscando formas responsáveis de passar por este momento histórico e estratégias para lidar eficientemente com os desafios no ensino evocados pela pandemia.

In 2020, a worldwide public health emergency began. In order to curb the transmission of the SARS CoV-2, measures of social distancing were applied, which reduced interpersonal contact and triggered profound changes in behaviors related to social interaction. Educational systems around the world have adapted to this new reality, adopting emergency protocols characterized, in large part, by replacing classroom activities with virtual ones. The objective of this article is to discuss the impacts of social distancing in higher education through a neurophysiological approach, connecting stress, empathy and the historical context of emergency education when compared to Distance Learning, in addition to the tools at our disposal and the complexities of the virtual environment. Teachers must be aware of the impact of stress and the use of digital and distance learning tools on student training, looking for responsible ways to go through this historical moment and strategies to deal effectively with the teaching challenges evoked by the pandemic.

Single Cocaine Exposure Inhibits GABA Uptake via Dopamine D1-Like Receptors in Adolescent Mice Frontal Cortex.

Cocaine (COC) is a psychostimulant that acts by increasing catecholaminergic neurotransmission mainly due to its effects on the dopamine transporter (DAT). However, other neurotransmitter systems may also be regulated by COC, including the GABAergic system. Since the effect of COC in modulating gamma-aminobutyric acid (GABA) reuptake is not defined, we investigated the molecular mechanisms related to the increase in GABA uptake induced by acute COC exposure and its effects on locomotor activity in adolescent mice. Behavioral experiments showed that COC increased locomotor activity and decreased immobilization time in mice. A single COC exposure reduced both GABA uptake and GAT-1 protein levels. On the other hand, cyclic adenosine monophosphate (cAMP) levels increased after a COC challenge. The major changes induced by acute COC on behavioral and neurochemical assays were avoided by previous treatment with the selective D1 receptor antagonist SCH-23390 (0.5 mg/kg). Our findings suggest that GABA uptake naturally decreases during mice development from preadolescence until adulthood and that dopamine (DA) D1-like receptors are key players in the regulation of GABA uptake levels following a single COC exposure in adolescent mice.

Caffeine regulates GABA transport via A1R blockade and cAMP signaling.

Caffeine is the most consumed psychostimulant drug in the world, acting as a non-selective antagonist of adenosine receptors A1R and A2AR, which are widely expressed in retinal layers. We have previously shown that caffeine, when administered acutely, acts on A1R to potentiate the NMDA receptor-induced GABA release. Now we asked if long-term caffeine exposure also modifies GABA uptake in the avian retina and which mechanisms are involved in this process. Chicken embryos aged E11 were injected with a single dose of caffeine (30 mg/kg) in the air chamber. Retinas were dissected on E15 for ex vivo neurochemical assays. Our results showed that [3H]-GABA uptake was dependent on Na+ and blocked at 4 °C or by NO-711 and caffeine. This decrease was observed after 60 min of [3H]-GABA uptake assay at E15, which is accompanied by an increase in [3H]-GABA release. Caffeine increased the protein levels of A1R without altering ADORA1 mRNA and was devoid of effects on A2AR density or ADORA2A mRNA levels. The decrease of GABA uptake promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA) but not by A2AR activation with CGS 21680. Caffeine exposure increased cAMP levels and GAT-1 protein levels, which was evenly expressed between E11-E15. As expected, we observed an increase of GABA containing amacrine cells and processes in the IPL, also, cAMP pathway blockage by H-89 decreased caffeine mediated [3H]-GABA uptake. Our data support the idea that chronic injection of caffeine alters GABA transport via A1R during retinal development and that the cAMP/PKA pathway plays an important role in the regulation of GAT-1 function.

The role of striatum and prefrontal cortex in the prevention of amphetamine-induced schizophrenia-like effects mediated by nitric oxide compounds.

Pharmacological manipulation of nitric oxide (NO) has been suggested as a promising treatment for schizophrenia symptoms. A single infusion of sodium nitroprusside, a NO donor with short half-life, was found to improve schizophrenia symptoms. However, an increasing number of preclinical studies have demonstrated the potential beneficial effects of both NO donors and inhibitors. We investigated the potential synergistic effect of sub-effective doses of the NO donor sodium nitroprusside or the NO inhibitor 7-Nitroindazole (7NI) combined with clozapine, a standard atypical antipsychotic, on counteracting amphetamine or MK-801-induced psychosis-like behaviors. The impact of sodium nitroprusside and 7NI on cAMP regulation in the prefrontal cortex and striatum was also evaluated. Confirming previous results, we found that both NO donors and inhibitors prevented amphetamine-induced effects (prepulse inhibition [PPI] disruption and hyperlocomotion). In addition, we observed a synergistic effect of sodium nitroprusside and clozapine on antagonizing the disruptive effects of amphetamine, but not MK-801, in the PPI test. The sub-effective dose of 7NI tested did not prevent amphetamine or MK-induced PPI effects when combined with clozapine. Interestingly, cAMP levels were significantly decreased in the prefrontal cortex after treatment with sodium nitroprusside. In the striatum, both sodium nitroprusside and 7NI blocked the amphetamine-induced increase of cAMP. Our data corroborate previous findings on the dopaminergic mechanisms involved in the action of sodium nitroprusside. It is likely that the differential effects of sodium nitroprusside are related to its ability to modify cAMP levels in the prefrontal cortex.

Caffeine alters glutamate-aspartate transporter function and expression in rat retina.

l-Glutamate and l-aspartate are the main excitatory amino acids (EAAs) in the Central Nervous System (CNS) and their uptake regulation is critical for the maintenance of the excitatory balance. Excitatory amino acid transporters (EAATs) are widely distributed among central neurons and glial cells. GLAST and GLT1 are expressed in glial cells, whereas excitatory amino acid transporter 3/excitatory amino acid carrier 1 (EAAT3/EAAC1) is neuronal. Different signaling pathways regulate glutamate uptake by modifying the activity and expression of EAATs. In the present work we show that immature postnatal day 3 (PN3) rat retinas challenged by l-glutamate release [3H]-d-Aspartate linked to the reverse transport, with participation of NMDA, but not of non-NMDA receptors. The amount of [3H]-d-Aspartate released by l-glutamate is reduced during retinal development. Moreover, immature retinae at PN3 and PN7, but not PN14, exposed to a single dose of 200 or 500µM caffeine or the selective A2A receptor (A2AR) antagonist 100nM ZM241385 decreased [3H]-d-Aspartate uptake. Caffeine also selectively increased total expression of EAAT3 at PN7 and its expression in membrane fractions. However, both EAAT1 and EAAT2 were reduced after caffeine treatment in P2 fraction. Addition of 100nM DPCPX, an A1 receptor (A1R) antagonist, had no effect on the [3H]-d-Aspartate uptake. [3H]-d-Aspartate release was dependent on both extracellular sodium and Dl-TBOA, but not calcium, implying a transporter-mediated mechanism. Our results suggest that in the developing rat retina caffeine modulates [3H]-d-Aspartate uptake by blocking adenosine A2AR.

Exposure to tobacco smoke containing either high or low levels of nicotine during adolescence: differential effects on choline uptake in the cerebral cortex and hippocampus.

INTRODUCTION: There is a lack of experimental studies that investigate the effects of tobacco smoke exposure during adolescence. Here, we investigated the effects of tobacco smoke generated from cigarettes containing either high or low levels of nicotine on the cholinergic system. METHODS: From postnatal day (PN) 30 to 45, 18 C57BL/6 (inbred) and 16 Swiss (outbred) mice of both sexes were exposed to tobacco smoke (whole body exposure for 8 hr/day and 7 days/week) generated from one of two reference research cigarettes: type 3R4F (HighNIC group-nicotine = 0.73 mg/cigarette) or type 4A1 (LowNIC group-nicotine = 0.14 mg/cigarette). Control mice (CT) were exposed to air. On PN 45, cotinine (nicotine metabolite) serum levels and [(3)H]choline uptake in the cerebral cortex and hippocampus were assessed. RESULTS: Cotinine serum levels were eight times higher in HighNIC mice (C57BL/6:142.0 +/- 16.7 ng/ml and Swiss: 197.6 +/- 11.1 ng/ml) when compared with LowNIC ones (C57BL/6:17.4 +/- 7.4 ng/ml and Swiss: 24.6 +/- 2.2 ng/ml). Only HighNIC mice presented a significant increase in [(3)H]choline uptake in the hippocampus (C57BL/6: HighNIC > CT and HighNIC > LowNIC, p < .001 and Swiss: HighNIC > CT and HighNIC > LowNIC, p < .001), whereas in the cerebral cortex, both HighNIC and LowNIC mice presented increased [(3)H]choline uptake (C57BL/6: HighNIC > CT and LowNIC > CT, p < .05 and Swiss: HighNIC > CT and LowNIC > CT, p < .001). DISCUSSION: Our results indicate that tobacco smoke exposure during adolescence increases [(3)H]choline uptake. However, the effects are dependent on the type of cigarette and on the brain region.

Pituitary adenylate cyclase-activating polypeptide (PACAP) can act as determinant of the tyrosine hydroxylase phenotype of dopaminergic cells during retina development.

In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.

Expression of functional receptors and transmitter enzymes in cultured Muller cells.

Glia represents the most numerous group of nervous system cells and CNS development and function depend on glial cells. We developed a purified Muller glia culture to investigate the expression of several neurotransmitter markers on these cells, such as dopaminergic, cholinergic, GABAergic and peptidergic receptors or enzymes, based on functional assays measuring second messenger levels or Western blot for specific proteins. Purified Muller cell culture was obtained from 8-day-old (E8) embryonic chick. Glial cells cultured for 15 days (E8C15) expressed D1A and D1B receptors mRNAs, but not D1D, as detected by RT-PCR. The binding of [3H]-SCH 23390 revealed an amount of expressed receptors around 40 fmol/mg protein. Dopamine (100 microM), PACAP (50 nM) and forskolin (10 microM) induced a 50-, 30- and 40-fold cAMP accumulation on glial cells, respectively, but not ip3 production. The dopamine-promoted cAMP accumulation was blocked by 2 microM SCH 23390. Carbachol stimulated a 3-fold ip3 accumulation. Western blot analysis also revealed the expression of tyrosine hydroxylase, L-dopa decarboxylase, PAC1 receptor, GAD67 and beta2-nicotinic receptor subunit by these cells. These results indicate that several components of neurotransmitter signaling and metabolism are found in cultured Muller cells.