RESUMEN
Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.
Asunto(s)
Astrocitos , Enfermedades Neurodegenerativas , Animales , Humanos , Ratones , Anciano , Astrocitos/metabolismo , Glutamina/genética , Glutamina/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Regulación hacia Arriba , Sistema de Transporte de Aminoácidos X-AG/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácido D-Aspártico/genética , Ácido Glutámico/metabolismo , Hipocampo/metabolismoRESUMEN
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.
Asunto(s)
Nicotina , Receptores Nicotínicos , Animales , Nicotina/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Calcio/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Receptores Nicotínicos/metabolismo , RetinaRESUMEN
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.
Asunto(s)
Humanos , Tabaquismo/epidemiología , Pandemias/historia , COVID-19/epidemiología , Ansiedad , Recurrencia , Epidemiología , Trastornos Relacionados con Sustancias/epidemiología , Vareniclina/uso terapéutico , Distrés PsicológicoRESUMEN
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.
Asunto(s)
Cafeína/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Cuerpo Estriado/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Western Blotting , Cuerpo Estriado/metabolismo , Femenino , Masculino , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
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.
Asunto(s)
Humanos , Educación a Distancia , COVID-19 , Aprendizaje , Estrés Psicológico , EmpatíaRESUMEN
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.
Asunto(s)
Cocaína/farmacología , Dopamina/metabolismo , Lóbulo Frontal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/efectos de los fármacos , Animales , Estimulantes del Sistema Nervioso Central/farmacología , Cocaína/administración & dosificación , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/farmacología , Lóbulo Frontal/metabolismo , Ratones , Actividad Motora/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80â¯mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.
Asunto(s)
Benzoxazinas/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Lóbulo Frontal/efectos de los fármacos , Proteínas Transportadoras de GABA en la Membrana Plasmática/efectos de los fármacos , Morfolinas/farmacología , Naftalenos/farmacología , Animales , Endocannabinoides/metabolismo , Lóbulo Frontal/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ratones , Receptor Cannabinoide CB1/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/efectos de los fármacosRESUMEN
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.
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Antipsicóticos/farmacología , Cuerpo Estriado/efectos de los fármacos , Óxido Nítrico/metabolismo , Corteza Prefrontal/efectos de los fármacos , Esquizofrenia/tratamiento farmacológico , Anfetamina , Animales , Cuerpo Estriado/metabolismo , AMP Cíclico/metabolismo , Maleato de Dizocilpina , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Corteza Prefrontal/metabolismo , Inhibición Prepulso/efectos de los fármacos , Esquizofrenia/metabolismoRESUMEN
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.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Corteza Cerebral/metabolismo , Colina O-Acetiltransferasa/metabolismo , Hipocampo/metabolismo , Nicotina/farmacología , Sistema Nervioso Parasimpático/efectos de los fármacos , Adolescente , Animales , Conducta Animal/efectos de los fármacos , Depresores del Sistema Nervioso Central/administración & dosificación , Corteza Cerebral/efectos de los fármacos , Colinérgicos/farmacología , Femenino , Hipocampo/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Maduración Sexual/efectos de los fármacos , HumoRESUMEN
Several mechanisms underlying ethanol action in GABAergic synapses have been proposed, one of these mechanisms is on GABA release. Here, we report that in ovo exposure to ethanol induces an increase on GABA release in the embryonic chick retina. Eleven-day-old chick embryos (E11) received an injection of either phosphate buffer saline (PBS) or ethanol (10%, v/v, diluted in PBS), and were allowed to develop until E16. A single glutamate stimulus (2 mM) showed approximately a 40% increase on GABA release in E16 retinas when compared to controls. The effect was dependent on NMDA receptors and GAD65 mRNA levels, which were increased following the ethanol treatment. However, the numbers of GABA-, GAD-, and NR1-immunoreactive cells, and the expression levels of these proteins, were not affected. We conclude that ethanol treatment at a time point when synapses are being formed during development selectively increases GABA release in the retina via a NMDA receptor-dependent process.
Asunto(s)
Etanol/administración & dosificación , Retina/embriología , Retina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Embrión de Pollo , Pollos , Relación Dosis-Respuesta a DrogaRESUMEN
GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.
Asunto(s)
Neuroglía/fisiología , Retina/citología , Ácido gamma-Aminobutírico/metabolismo , Análisis de Varianza , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Cloruros/metabolismo , Técnicas de Cocultivo/métodos , Relación Dosis-Respuesta a Droga , Antagonistas del GABA/farmacología , Neuroglía/efectos de los fármacos , Neuronas/química , Ácidos Nipecóticos/farmacología , Oximas/farmacología , Sodio/metabolismo , Temperatura , Tritio/metabolismoRESUMEN
Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.
Asunto(s)
Agonistas de Dopamina/farmacología , Norepinefrina/farmacología , Receptores Adrenérgicos beta 1/metabolismo , Receptores de Dopamina D1/agonistas , Retina/efectos de los fármacos , Animales , Benzazepinas/metabolismo , Células Cultivadas , Embrión de Pollo , Ensayo de Unión Radioligante , Retina/citología , Retina/embriologíaRESUMEN
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.
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Dopamina/metabolismo , Factores de Crecimiento Nervioso/fisiología , Neuronas/metabolismo , Neuropéptidos/fisiología , Neurotransmisores/fisiología , Retina , Tirosina 3-Monooxigenasa/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Factores de Edad , Animales , Animales Recién Nacidos , Western Blotting/métodos , Recuento de Células/métodos , Técnicas de Cultivo de Célula , Embrión de Pollo , Colforsina/farmacología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Factores de Crecimiento Nervioso/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuropéptidos/antagonistas & inhibidores , Neurotransmisores/antagonistas & inhibidores , Fenotipo , Inhibidores de Fosfodiesterasa/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Retina/citología , Retina/embriología , Retina/enzimología , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Tirosina 3-Monooxigenasa/genéticaRESUMEN
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.