Normal Ethanol Sensitivity and Rapid Tolerance Require the G Protein Receptor Kinase 2 in Ellipsoid Body Neurons in Drosophila.

BACKGROUND: G protein signaling pathways are key neuromodulatory mechanisms for behaviors and neurological functions that affect the impact of ethanol (EtOH) on locomotion, arousal, and synaptic plasticity. Here, we report a novel role for the Drosophila G protein-coupled receptor kinase 2 (GPRK2) as a member of the GRK4/5/6 subfamily in modulating EtOH-induced behaviors. METHODS: We studied the requirement of Drosophila Gprk2 for naïve sensitivity to EtOH sedation and ability of the fly to develop rapid tolerance after a single exposure to EtOH, using the loss of righting reflex (LORR) and fly group activity monitor (FlyGrAM) assays. RESULTS: Loss-of-function Gprk2 mutants demonstrate an increase in alcohol-induced hyperactivity, reduced sensitivity to the sedative effects of EtOH, and diminished rapid tolerance after a single intoxicating exposure. The requirement for Gprk2 in EtOH sedation and rapid tolerance maps to ellipsoid body neurons within the Drosophila brain, suggesting that wild-type Gprk2 is required for modulation of locomotion and alertness. However, even though Gprk2 loss of function leads to decreased and fragmented sleep, this change in the sleep state does not depend on Gprk2 expression in the ellipsoid body. CONCLUSION: Our work on GPRK2 has established a role for this GRK4/5/6 subfamily member in EtOH sensitivity and rapid tolerance.

The FlyBar: administering alcohol to flies.

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Neurophysiological studies of Heteropteris glabra Hok. & Arn. (Malpighiaceae) in DBA/2J mice.

Fruits of Heteropterys glabra (Malpighiaceae) are used in South-American folk medicine as a sedative and anxiolytic agent. In present research, we studied neurophysiological effects of the plant. Ethanolic extract was assayed in DBA/2J mice in order to evaluate the sleep wakefulness cycle, electroencephalogram (EEG) and visual evoked potentials (VEP). The results of our experiments indicated that the EtOH extract of the plant induced a reduction of motor activity and alterations of EEG and VEP parameters, supporting the possibility that the plant acts as an anxiolytic/sedative agent, thus, confirming its usual assumption and the traditional use.

Cytogenetic and nucleolar meiotic cycle analyses in Dysdercus imitator Blöte, 1931 (Pyrrhocoridae, Heteroptera) from Argentina.

So far, only seven and five species of Dysdercus from the Old and New Worlds, respectively, have been cytogenetically analysed. They all have holokinetic chromosomes and a pre-reductional type of meiosis. In the present study the chromosome complement, male meiosis and nucleolar meiotic cycle of Dysdercus imitator were analyzed. During male meiosis several cytogenetic features are remarkable, namely the presence of a long diffuse stage after pachytene, the finding of one or two ring bivalents per cell in almost all specimens, and the presence of several prenucleolar bodies lasting up to telophase II. The origin and function of these prenucleolar bodies could be related to a particular physiological cycle of the meiocytes.

Meiotic studies in Dysdercus Guérin Meneville, 1831 (Heteroptera: Pyrrhocoridae). II. Evidence on variations of the diffuse stage between wild and laboratory-inbred populations of Dysdercus chaquensis Freiberg, 1948.

Dysdercus Guerin Méneville, 1831 comprises insect species that are often serious pests of cotton both in the Old and New World, representing the only taxon from Pyrrhocoridae in the Neotropical Region. The genus is cytologically characterized by possession of holokinetic chromosomes and a prereductional type of meiosis. So far, only seven species from the Old World and five species from the Neotropical Region have been cytogenetically described. In the present report we compare the male meiosis from both natural and inbred populations of Dysdercus chaquensis Freiberg, 1948. Our results demonstrated that even though both populations share the same diploid chromosome number, the presence of a diffuse stage was found to be committed to the wild population of the species. Furthermore, the diffuse stage was found in a high frequency in all analysed wild specimens. indicating the long duration of this period among the regular meiosis of D. chaquensis. Taking into account that the diffuse stage is connected with an intense and long period of cellular growth, and with an important transcriptional activity, the absence of this stage in all the inbred specimens of D. chaquensis could be related with the lack of unfavourable physiological conditions due to the environmental uniformity along seven years of inbreeding.