Histone Methyltransferase G9a Plays an Essential Role on Nicotine Preference in Zebrafish.

Psychostimulants regulate behavioral responses in zebrafish via epigenetic mechanisms. We have previously shown that DNA methylation and histone deacetylase (HDAC) inhibition abolish nicotine-induced conditioned place preference (CPP) but little is known about the role of histone methylation in addictive-like behaviors. To assess the influence of histone methylation on nicotine-CPP, zebrafish were treated with a histone (H3) lysine-9 (K9) dimethyltransferase G9a/GLP inhibitor, BIX-01294 (BIX), which was administered before conditioning sessions. We observed a dual effect of the inhibitor BIX: at high doses inhibited while at low doses potentiated nicotine reward. Transcriptional expression of α6 and α7 subunits of the nicotinic acetylcholine receptor and of G9a, DNA methyl transferase-3, and HDAC-1 was upregulated in zebrafish with positive scores for nicotine-CPP. Changes in relative levels of these mRNA molecules reflected the effects of BIX on nicotine reward. BIX treatment per sé did not affect transcriptional levels of epigenetic enzymes that regulate trimethylation or demethylation of H3. BIX reduced H3K9me2 protein levels in a dose-dependent manner in key structures of the reward pathway. Thus, our findings indicated that different doses of BIX differentially affect nicotine CPP via strong or weak inhibition of G9a/GLP activity. Additionally, we found that the lysine demethylase inhibitor daminozide abolished nicotine-CPP and drug seeking. Our data demonstrate that H3 methylation catalyzed by G9a/GLP is involved in nicotine-CPP induction. Dimethylation of K9 at H3 is an important epigenetic modification that should be considered as a potential therapeutic target to treat nicotine reward and perhaps other drug addictions.

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.

Purinergic signaling in the retina: From development to disease.

Retinal injuries and diseases are major causes of human disability involving vision impairment by the progressive and permanent loss of retinal neurons. During development, assembly of this tissue entails a successive and overlapping, signal-regulated engagement of complex events that include proliferation of progenitors, neurogenesis, cell death, neurochemical differentiation and synaptogenesis. During retinal damage, several of these events are re-activated with both protective and detrimental consequences. Purines and pyrimidines, along with their metabolites are emerging as important molecules regulating both retinal development and the tissue's responses to damage. The present review provides an overview of the purinergic signaling in the developing and injured retina. Recent findings on the presence of vesicular and channel-mediated ATP release by retinal and retinal pigment epithelial cells, adenosine synthesis and release, expression of receptors and intracellular signaling pathways activated by purinergic signaling in retinal cells are reported. The pathways by which purinergic receptors modulate retinal cell proliferation, migration and death of retinal cells during development and injury are summarized. The contribution of nucleotides to the self-repair of the injured zebrafish retina is also discussed.

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.

Injury-induced purinergic signalling molecules upregulate pluripotency gene expression and mitotic activity of progenitor cells in the zebrafish retina.

Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY1 messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY1 transcriptional and protein expression. The injury-induced upregulation of P2RY1 is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY1 endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPßS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY1 early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY1 antagonist. Ecto-ATPase activity inhibitors or P2RY1-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.

Behavioral and molecular analysis of nicotine-conditioned place preference in zebrafish.

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and ß2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine-associated reward in vertebrates.

Purinergic signals regulate daily S-phase cell activity in the ciliary marginal zone of the zebrafish retina.

Regeneration and growth that occur in the adult teleost retina have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. Here, it is reported that S-phase cell number, in the ciliary marginal zone (CMZ) of the adult zebrafish retina, exhibits day-night variations with a mid-light phase peak. Oscillations persist for 24 h in constant darkness (DD), suggesting control by a circadian component. However, variations in the S-phase nuclei number were rapidly dampened and not present during and after a second day in DD. An ADPßS treatment significantly enhanced S-phase activity at night to mid-light levels, as assessed by in vivo BrdU incorporation in a 2-h interval. Moreover, daylight increase in S-phase cell number was completely abolished when extracellular nucleotide levels or their extracellular hydrolysis by ectonucleoside triphosphate diphosphohydrolases (NTPDases) were significantly disrupted or when a selective antagonist of purinergic P2Y1 receptors was intraocularly injected before BrdU exposure. Extracellular nucleotides and NTPDase action were also important for maintaining nocturnal low levels of S-phase activity in the CMZ. Finally, we showed that mRNAs of NTPDases 1, 2 (3 isoforms), and 3 as well as of P2Y1 receptor are present in the neural retina of zebrafish. NTPDase mRNA expression exhibited a 2-fold increment in light versus dark conditions as assessed by quantitative RT-PCR, whereas P2Y1 receptor mRNA levels did not show significant day-night variations. This study demonstrates a key role for nucleotides, principally ADP as a paracrine signal, as well as for NTPDases, the plasma membrane-bound enzymes that control extracellular nucleotide concentration, for inducing S-phase cell entry in the CMZ-normally associated with retinal growth-throughout the light-dark cycle.

Extracellular ADP regulates lesion-induced in vivo cell proliferation and death in the zebrafish retina.

Regeneration and growth that occur in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. In this report, we demonstrate that endogenous purinergic signals regulate cell proliferation induced by a cytotoxic injury of the adult zebrafish retina which mainly damages inner retinal layers. Particularly, we found that ADP but not ATP or adenosine significantly enhanced cell division as assessed by 5-bromo-2'-deoxyuridine incorporation following injury, during the degenerative and proliferative phase of the regeneration process. This effect of ADP occurs via P2Y1 metabotropic receptors as shown by intra-ocular injection of selective antagonists. Additionally, we describe a role for purinergic signals in regulating cell death induced by injury. Scavenging of extracellular nucleotides significantly increased cell death principally seen in the inner retinal layers. This effect is partially reproduced by blocking P2Y1 receptors suggesting a neuroprotective function for ADP, which is derived from extracellular ATP probably released by dying cells as a consequence of the ouabain treatment. This study demonstrates a crucial role for ADP as a paracrine signal in the repair of retinal tissue following injury.

Immunocytochemical localization of NTPDases1 and 2 in the neural retina of mouse and zebrafish.

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of membrane-bound enzymes that hydrolyze extracellular di- and triphosphate nucleosides. E-NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E-NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Müller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Müller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto-ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated-signaling in vertebrate retinas.

Odor increases [3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion.

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.

Cellular processing of cone photoreceptor cyclic GMP-gated ion channels: a role for the S4 structural motif.

We examined cellular protein processing and functional expression of photoreceptor cyclic nucleotide-gated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel alpha subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG beta subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane.

Mitotic activation of proliferative cells in the inner nuclear layer of the mature fish retina: regulatory signals and molecular markers.

New neurons continuously differentiate within the otherwise mature retina of teleost fish, both under normal conditions and in response to injury. We investigated the effects of surgical injury and intraocular injection of neurotrophic factors on the mitotic rate of proliferative inner nuclear layer cells (PINC). PINC are continually born in the inner nuclear layer and then migrate to the outer nuclear layer (ONL). Surgical excision of a part of a retina activates PINC mitotic activity near and far from the lesion. In the injured eye, up-regulation of PINC cells is largest in the dorsonasal sector of the retina, regardless of the site of lesion. Up-regulation extends even to the unlesioned contralateral eye, where it occurs in the same dorsonasal sector. Intraocular injection of ciliary neurotrophic factor mimics the effect of injury on PINC in the treated eye but not on the untreated contralateral retina. We searched for the expression in PINC of Pax6, a transcription factor linked to retinal progenitor cells and found that less than 0.5% of all PINC cells express it. Importantly, the number of Pax6-expressing PINC does not change significantly in the retinas subjected to any of the experimental manipulations tested. Under normal conditions, the default fate of PINC cells is to migrate to the ONL and, likely, replenish the rod progenitor pool. PINC respond to injury (both surgical and light-dependent) by increasing their mitotic rate; this increase is long lived, but there are no changes in the expression level of Pax6. PINC probably are a heterogenous cell population that can be specified for ultimate, different purposes: creating rod precursors, creating founder cells, creating cone precursors. Several fates are recognized now, but others may also be possible.