Measuring functional brain recovery in regenerating planarians by assessing the behavioral response to the cholinergic compound cytisine.

Planarians are traditional model invertebrates in regeneration and developmental biology research that also display a variety of quantifiable behaviors useful to screen for pharmacologically active compounds. One such behavior is the expression of seizure-like movements (pSLMs) induced by a variety of substances. Previous work from our laboratory showed that cocaine, but not nicotine, induced pSLMs in intact but not decapitated planarians. Interestingly, as decapitated planarians regenerated their heads, they gradually recovered their sensitivity to cocaine. These results suggested a method to assess planarian brain regeneration and a possible way of identifying compounds that could enhance or hold back brain regeneration. In the present work, we demonstrate that the cholinergic agent cytisine is a suitable reference compound to apply our method. Cytisine induces pSLMs in a concentration-dependent manner in intact (but not decapitated) planarians of the species Girardia tigrina. Based on our data, we developed a behavioral protocol to assess planarian brain regeneration over time. We tested this method to measure the effect of ethanol on G. tigrina's brain regeneration. We found that ethanol slows down the rate of planarian brain regeneration in a concentration-dependent manner, consistently with data from other research groups that tested ethanol effects on planarian brain regeneration using different behavioral protocols. Thus, here we establish a general method using cytisine-induced pSLMs as an indicator of brain regeneration in planarians, a method that shows potential for assessing the effect of pharmacologically active compounds in this process.

Cotinine antagonizes the behavioral effects of nicotine exposure in the planarian Girardia tigrina.

Nicotine is one of the most addictive drugs abused by humans. Our laboratory and others have demonstrated that nicotine decreases motility and induces seizure-like behavior in planarians (pSLM, which are vigorous writhing and bending of the body) in a concentration-dependent manner. Nicotine also induces withdrawal-like behaviors in these worms. Cotinine is the major nicotine metabolite in humans, although it is not the final product of nicotine metabolism. Cotinine is mostly inactive in vertebrate nervous systems and is currently being explored as a molecule which possess most of nicotine's beneficial effects and few of its undesirable ones. It is not known whether cotinine is a product of nicotine metabolism in planarians. We found that cotinine by itself does not seem to elicit any behavioral effects in planarians up to a concentration of 1mM. We also show that cotinine antagonizes the aforementioned nicotine-induced motility decrease and also decreases the expression of nicotine-induced pSLMs in a concentration-dependent manner. Also cotinine prevents the manifestation of some of the withdrawal-like behaviors induced by nicotine in our experimental organism. Thus, we obtained evidence supporting that cotinine antagonizes nicotine in this planarian species. Possible explanations include competitive binding of both compounds at overlapping binding sites, at different nicotinic receptor subtypes, or maybe allosteric interactions.

Transcription factor Sp1 regulates SERCA2 gene expression in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium.

Pressure-overload hypertrophy results in downregulation of the sarcoplasmic reticulum Ca(2+)-ATPase pump encoding SERCA2 gene that regulates Ca(2+) uptake and myocardial relaxation. We previously characterized a proximal promoter region containing four Sp1 element consensus sequences (-284 to -72 base pairs (bp)) that was responsible for pressure-overload-induced transcriptional regulation. The purpose of the present study was to determine which of the Sp1 sites was responsible for the downregulation of SERCA2 gene transcription under pressure overload. Using an in vivo direct gene transfer assay, SERCA2 gene transcriptional activity was measured under pressure overload. Site-directed mutagenesis of the four Sp1 sites (I-IV) in the SERCA2 gene promoter (-284 to -72 bp) was performed. Wild-type and Sp1 mutant-luciferase reporter constructs were injected into the left-ventricular apices of pressure overload or sham-operated rats, and Sp1 mRNA and SERCA2 gene-luciferase activity was measured sequentially from 3 to 14 d after surgery. At 5 d, Sp1 mRNA in the pressure-overload rats increased to 124 +/- 7% of sham group levels, and pressure-overload-induced SERCA2 transcriptional activity was 15 +/- 4% of sham group when all four Sp1 sites remained intact. Mutation of the Sp1 mutant sites I (-196 to -191 bp) and III (-118 to -113 bp) blocked the inhibitory effect of pressure overload and resulted in SERCA2 gene transcriptional activity of 54 +/- 15% and 56 +/- 7% of sham group, respectively. We conclude that the pressure-overload-induced decrease in SERCA2 mRNA is mediated by Sp1 sites I and III.