Methionine Supplementation Abolishes Nicotine-Induced Place Preference in Zebrafish: a Behavioral and Molecular Analysis.

In zebrafish, nicotine is known to regulate sensitivity to psychostimulants via epigenetic mechanisms. Little however is known about the regulation of addictive-like behavior by DNA methylation processes. To evaluate the influence of DNA methylation on nicotine-induced conditioned place preference (CPP), zebrafish were exposed to methyl supplementation through oral L-methionine (Met) administration. Met was found to reduce dramatically nicotine-induced CPP as well as behaviors associated with drug reward. The reduction was associated with the upregulation of DNA methyltransferases (DNMT1 and 3) as well as with the downregulation of methyl-cytosine dioxygenase-1 (TET1) and of nicotinic receptor subunits. Met also increased the expression of histone methyltransferases in nicotine-induced CPP groups. It reversed the nicotine-induced reduction in the methylation at α7 and NMDAR1 gene promoters. Treatment with the DNMT inhibitor 5-aza-2'-deoxycytidine (AZA) was found to reverse the effects of Met in structures of the reward pathway. Interestingly, Met did not modify the amount of the phospho-form of CREB (pCREB), a key factor establishing nicotine conditioning, whereas AZA increased pCREB levels. Our data suggest that nicotine-seeking behavior is partially dependent on DNA methylation occurring probably at specific gene loci, such as α7 and NMDAR1 receptor gene promoters. Overall, they suggest that Met should be considered as a potential therapeutic drug to treat nicotine addiction.

P2X7 and A2A receptor endogenous activation protects against neuronal death caused by CoCl2 -induced photoreceptor toxicity in the zebrafish retina.

Injured retinas in mammals do not regenerate and heal with loss of function. The adult retina of zebrafish self-repairs after damage by activating cell-intrinsic mechanisms, which are regulated by extrinsic signal interactions. Among relevant regulatory extrinsic systems, purinergic signaling regulates progenitor proliferation during retinogenesis and regeneration and glia proliferation in proliferative retinopathies. ATP-activated P2X7 (P2RX7) and adenosine (P1R) receptors are involved in the progression of almost all retinopathies leading to blindness. Here, we examined P2RX7 and P1R participation in the retina regenerative response induced by photoreceptor damage caused by a specific dose of CoCl2 . First, we found that treatment of uninjured retinas with a potent agonist of P2RX7 (BzATP) provoked photoreceptor damage and mitotic activation of multipotent progenitors. In CoCl2 -injured retinas, blockade of endogenous extracellular ATP activity on P2RX7 caused further neurodegeneration, Müller cell gliosis, progenitor proliferation, and microglia reactivity. P2RX7 inhibition in injured retinas also increased the expression of lin28a and tnfα genes, which are related to multipotent progenitor proliferation. Levels of hif1α, vegf3r, and vegfaa mRNA were enhanced by blockade of P2RX7 immediately after injury, indicating hypoxic like damage and endothelial cell growth and proliferation. Complete depletion of extracellular nucleotides with an apyrase treatment strongly potentiated cell death and progenitor proliferation induced with CoCl2 . Blockade of adenosine P1 and A2A receptors (A2A R) had deleterious effects and deregulated normal timing for progenitor and precursor cell proliferation following photoreceptor damage. ATP via P2RX7 and adenosine via A2A R are survival extracellular signals key for retina regeneration in zebrafish.

Pre-Exposure to Nicotine with Nocturnal Abstinence Induces Epigenetic Changes that Potentiate Nicotine Preference.

Prior exposure to drugs of abuse may facilitate addiction. It has been described that pre-exposure to nicotine can increase or, contrarily, prevent conditioned place preference (CPP). Here, we evaluated the effect of nicotine pre-exposure on CPP performance using an original protocol mimicking smokers' behaviour in zebrafish. We simulated nicotine withdrawal at sleep time by exposing zebrafish to nicotine during daylight but not at night (D/N) for 14 days and then performed nicotine-CPP in zebrafish. D/N-nicotine-treated zebrafish obtained the highest CPP score, whereas zebrafish pre-exposed continuously to nicotine did not show nicotine-CPP. Evaluation of locomotor activity, seeking and anxiety-like behaviours supported the CPP findings. Nicotinic receptor subunit gene expression showed significant increases in the brain of zebrafish exposed to nicotine. Zebrafish exposed to D/N-nicotine showed further increases of α6- and α7-subunit expression after CPP establishment. Inhibition of histone acetylation by phenylbutyrate prevented nicotine-CPP. Transcriptional expression of epigenetic enzymes controlling histone acetylation/deacetylation and DNA methylation/demethylation was widely modified in brain portions containing reward areas of zebrafish exposed to D/N-nicotine after CPP. Zebrafish exposed to D/N-nicotine showed high levels of acetylated histone 3 and pCREB immunoreactivity differentially found in nuclei of the dopaminergic reward circuit in zebrafish homologous to the ventral tegmental area, nucleus accumbens and dorsal habenula. Our findings demonstrated that repetitive abstinent periods are risky factors for drug abuse that potentiate nicotine-environment associations and seeking. Brain modifications can persist long after nicotine use and are likely due to changes in the transcriptional expression of enzymes regulating drug reward-related gene expression via epigenetic modifications.

Characterization of proliferative, glial and angiogenic responses after a CoCl2 -induced injury of photoreceptor cells in the adult zebrafish retina.

The adult zebrafish is considered a useful model for studying mechanisms involved in tissue growth and regeneration. We have characterized cytotoxic damage to the retina of adult zebrafish caused by the injection of cobalt chloride (CoCl2 ) into the vitreous cavity. The CoCl2 concentration we used primarily caused injury to photoreceptors. We observed the complete disappearance of cones, followed by rods, across the retina surface from 28 to 96 hr after CoCl2 injury. The loss of 30% of bipolar cells was also observed by 50 hr after lesion (hpl). CoCl2 injury provoked a strong induction of the proliferative activity of multipotent Müller glia and derived progenitors. The effect of CoCl2 on retina cells was significantly reduced by treatment with glutamate ionotropic receptor antagonists. Cone photoreceptor regeneration occurred 25 days after injury. Moreover, a single dose of CoCl2 induced vascular damage and regeneration, whereas three injections of CoCl2 administered weekly provoked neovascular-like changes 20 days after injury. CoCl2 injury also caused microglial reactivity in the optic disc, retina periphery and fibre layer. CoCl2 -induced damage enhanced pluripotency and proneural transcription factor gene expression in the mature retina 72 hpl. Tumour necrosis factor alpha, vascular endothelial growth factor (VEGF) and VEGF receptor mRNA levels were also significantly enhanced by 72 hpl. The injury paradigm we have described in this work may be useful for the discovery of signalling molecules and pathways that participate in the regenerative response and it may serve as a model to screen for compounds that could potentially treat aberrant angiogenesis.

Anticonvulsant effect of sodium cyclamate and propylparaben on pentylenetetrazol-induced seizures in zebrafish.

Screening for novel anticonvulsant drugs requires appropriate animal seizure models. Zebrafish provide small, accessible, and cost-efficient preclinical models applicable to high-throughput small molecule screening. Based on previous results in rodents, we have here examined the effects of artificial sweetener sodium cyclamate and antimicrobial agent sodium propylparaben on a model of pentylenetetrazole (PTZ)-induced seizures in zebrafish. Sodium cyclamate reduced the bursts of hyperactivity, the spasms, increased the latency to spasms, and the latency to seizure, while propylparaben increased the latency to spasms. The results show the potential of zebrafish to detect novel anticonvulsant compounds while they also demonstrate the ability of two commonly ingested chemical compounds to modify the seizure threshold when were administrated at low concentration.