Bainton and colleagues made a study about the behavioral response of mutant Drosophila melanogaster with altered acute responses to cocaine.
Cocaine is known to be one of the best examples of a psychostimulant which result to behavioral activation, act as discriminative stimulus and support cocaine self-administration(Lau et al.) Functionally, it works as an indirect dopamine antagonist by blocking dopamine transporter sites in the terminal buttons of neurons in the brain hence hinders the reuptake of the dopamine. This in turn results into concentration build up of dopamine in the synapse for a longer period of time. The blocking of the re-use of the dopamine can affect movement, attention, learning and the reinforcing effects of the drug use as it affects the particular the part of the brain that specifically control the aforementioned functions.
Through the years here have been lots of studies elucidating the mechanism of action of psychostimulants such as cocaine. A study conducted by (Sora et al.) on mice with absent dopamine transporter and (DAT) and serotonin transporter (SERT) resulted into the mice not stimulated by the cocaine administration. Thus conditioned preference for cocaine was not also developer. On the contrary, enhanced cocaine preference was the observed result of (Hiroi et al.) when mice lacking FosB or ever express fosB, which are both highly sensitive with the psychostimulatory effect of cocaine.
Drosophila melanogaster has been used as model to elucidate the genes that are responsible for the control of behavioral responses to addictive drugs such as cocaine. Furthermore, it is also a good specimen for it also reacts the same way as mammals specially the motor behavior induced by cocaine. Also it is able to demonstrate behavioral sensitization as in induced by repeated cocaine administration and this is a behavioral plasticity that models some kind of addiction. But what takes a greater importance is the discovery of the genes and pathways that manifested unexpected roles in cocaine-related behaviors such that of (Abarca, UAlbrecht and Spanagel) findings. It was found out in their study that mice carrying mutations in the homologous per genes showed cocaine sensitization and conditioned place preference.
In the study of Bainton and his colleagues, flies were maintained on standard cornmeal molasses agar at 25°C and 70% humidity. The control group (w1118 strain) includes lines that showed phenotypes a 1.5 standard deviation from the mean and underwent a retesting for cocaine sensitivity. It also includes the group of lines that fall near the mean of the distribution of phenotype. Line EP369 on the other is used as the wild-type control and this group exhibited similar cocaine sensitivity with that of the control group with w1118 strain. All these groups were crossed to w118 for five generations prior to behavioral testing. Excisions of EP1529 were generated by using standard crosses and with the use of PCR analysis and DNA sequencing this stain demonstrated normal cocaine sensitivity. In the process, two lethal excision lines, D17 and D18, were shown to be allelic and to carry imprecise excisions of the EP1529 element and adjacent genomic DNA.
For the behavioral assays conducted, groups of 15 male flies were collected under CO2 anesthesia 0–2 days posteclosion and tested 2–3 days later. Prior to behavioral testing, the flies were equilibrated at 20°C for an hour. The flies were then to volatilize cocaine and nicotine and after which were then transferred to a glass cylinder to quantify startle-induced negative geotaxis. For five minutes with a minute interval the flies that are on the bottom of the cylinder were counted and the drug effect score was obtained by averaging the number of flies. Inebriometer was used to measure alcohol sensitivity. Student’s paired t tests one-way ANOVAs with Newman-Keuls post hoc tests were used to establish significance of the results obtained. All genotypes were tested on several different days. Baseline locomotion and startle-induced climbing were all tested in genotypes and were found to be normal and found to be normal. Testing of all genotypes was conducted on several separate days.
For the isolation of moody cDNAs and generation of UAS constructs, the head of the Drosophila was obtained and was identifies a unique probe to the fourth exon of moody. Clones that exhibited a positive in the probe test were isolated and sequenced and from here moody-? and moody-? transcripts were identified. The moody transcripts were cloned using a pUAST vector and this generated UAS-a or UAS-b and germline transformants were generated through the standard protocol use
In the construction of genomic rescue and RNAi constructs, a 9.4 kb genomic EcoRV(partial)-StuI (partial) fragment which contains moody gene and intragenic sequences was cooled to obtain gen-ab construct. The constructs with gen-a and gen-b were obtained by replacing a 3 kb SacII-XbaI fragment of the genomic clone with an equivalent fragment from either the moody-a or -b cDNAs. UAS-RNAi construct was obtained though PCR of the moody fragments and one of the fragments is 1.3 kb of genomic DNA with exon 3, intron 3, exon 4, and intron 4 while the other garment contained 0.6 kb of exon 4.
Northern blots was carried out as cited by (Sullivan, Ashburner and Hawley) in which the heads or bodies of flies of various genotypes was used as the genome source and this was done to verify the presence of moody-a and -b transcripts in the heads of adult flies.
For the mapping of the moody deletions a PCR product that spanned the deletion was obtained and was cloned into the TOPO TA vector. After which, it was sequenced and the exact extent of each deletion was obtained.
Though PCR, the predicted C-terminal regions encoding Moody-? and Moody-? were amplified and cloned into the pGEX4T-1 vector to produce in-frame fusions with GST. For Western blots, fly heads, or bodies were homogenized in 5 _l of loading buffer (0.125 M Tris base, 2% SDS) per fly and samples run on 8% polyacrylamide gels. Electroblotting was used to transfer proteins to PVDF membranes and the membranes were incubated with affinity-purified primary antibody (1:100) overnight at 4°C and with secondary antibody (1:1000) for 1 hr at room temperature. LumigenPS chemiluminescence was used to visualize the moody protein.
Third-instar CNSs or cryosections of adult fly heads were fixed in 3.7% paraformaldehyde with sodium phosphate buffer (pH 7.2). The samples were transferred into primary antibody and incubated overnight at 4°C after washed with PBS + 0.3% Triton X-100. Incubation of samples with secondary antibodies for 4 hr at room temperature follwed after additional washes. Samples were stored and mounted in 80% glycerol. The primary antibodies used were as follows: affinity-purified rabbit anti-Moody, affinity-purified rat anti-Moody, mouseanti-GFP, mouse anti-Repo (Xiong et al., 1994). FITCgoat anti-rabbit, Rhodamine goat anti-rat, and Texas Red goat antimouse alexa-Fluor 488 goat were used as Secondary antibodies. Confocal images were acquired and an image analysis was performed.
CO2-anesthetized adult flies were injected with fluorescent dyes and immobilized after. Approximately 0.3 µl of dye was injected into the soft tissue between two abdominal segments of the exoskeleton. The flies were injected with 50 mg/ml tetramethylrhodamine dextran and photographed to visualize the intactness of the eyes of the animals. For quantification of dye absorption into the brain, flies were injected with 50 mg/ml eosin dextran. Brains were dissected in PBS 18 hr after injection and placed in Corning Costar Special and fluorescence was measured using after. For the group of other group of flies heat shock was done five times within 2 days. Twenty-four after the last heat shock, the dye was injected and after 18 hr dye penetration into the retina
Through the screening of 400 fly mutants, EP1529 was characterized among mutant flies with EP elements in their x chromosome. EP1529 is characterized to have an increased sensitivity to cocaine as well as to volatile nicotine exposure however it exhibited a resistance with the acute intoxicating effects of alcohol. It was found out in the study that the gene disrupted by EP1529 is CG4322 which is named as moody.
Through the sequence analysis of the cDNA library obtained from the heads of the flies it was found out that alternative splicing of the fourth intron generates an mRNA which differs by two nucleotide base. From this it was found out that moody even exist in two forms: the one with the longer mRNA transcript is termed as moody- ? while the shorter one is termed as moody-?.
An evaluation of the complete loss of moody function is made by imprecise excision of EP1529 element and recovery of the two deletions that remove entire moody gene. Mutations of this kind are lethal as supported by the survival of only 1% homozygous females and hemizygous males while flies that survived proved to be sickly with severe motor abnormalities.
It was also shown in the results that moody encodes two stable proteins that are expressed in glial cells surrounding the embryonic and adult nervous system. Furthermore, moody proteins which are localized to the plasma membrane and are highly enriched with areas of cell-cell contact among surface glia. As for the elucidation of role of moody – ? and moody-? for the normal sensitivity to cocaine, both play a distinct role. It was determined that both proteins are expressed in their normal regulatory sequences; hence both are required for the normal cocaine sensitivity.
In terms of the exhibition of normal blood-brain-barrier (BBB) in conjunction with the role of moody in regulating cocaine sensitivity, it was postulated that there is no direct link between the two. This is strengthened by the result that EP1529 with abnormal cocaine sensitivity still manifested normal BBB formation. Also, this would mean that either protein would be sufficient to confer a normal BBB. Moreover, the expression of moody in surface glia is necessary for normal development and to maintain the BBB.
From the study it was known that moody locus encodes two proteins that exhibit difference in the C-terminal and that at this region moody- ? is 271 amino acids long while moody-? is 230 amino acids long. These proteins are expressed during embryonic development and adulthood in glia at an approximately equal amount. One of the questions from the result obtained is the reason behind the need for the two proteins in cocaine sensitivity while either of the two is enough already for proper insulation of the nervous system. The authors postulated the possibility that the two proteins must interact with distinct downstream signaling pathways. Also this may be due to need for the formation of heterodimers that renders maximized function, maturation and stability.
In Drosophila, their nervous system is protected by a glial-dependent BBB from the humoral environment. This protective insulation structure is believed to be owed to septate junctions located in between glial cells. (Bellen et al.) mentioned that this structure contain cell adhesion molecules gliotactin, Neurexin IV, and Contactin. The septate junctions can be signaled by the moody protein resulting into the regulation of cortical actin cytoskeleton (Schwabe et al.). moody also take a function in strengthening the BBB that is why it is continuously expressed in the surface glial proteins. The authors came up also with the proposal of a moody-mediated signaling pathway functions in glia which serve as a regulatory process in the insulation of the nervous system and drug sensitivity.
Another resolution made in the study, is the discounting of the association that the abnormal behavior observed among flies with defective moody protein consequently altering the accessibility of t he drug t o the central nervous system. Instead alterations in observed behavior were due to the impaired function of BBB which in turns alters the normal functioning of the nervous system. Cocaine enters the body via the respiratory system specifically it takes a path in the tracheal system which allows entry through the spiracles. The tracheal network successively divides into tubes smaller tubes eventually leading to tracheoles. Tracheoles terminate to the hem lymph and to end organs such as brain (Manning and Krasnow). It is further asserted that moody does not affect the delivery of the drugs to the tracheal system for it is not expressed in these cells. Instead moody takes effect in the development of surface glia which insulates the nervous system from the blood lymph. Moreover, cocaine and nicotine have relatively low molecular weight hence can readily cross the BBB.
It is recommended for further studies that a clearer mechanism of moody signaling as well as its downstream effects in glia should be elucidated at the molecular level. In this way BBB along with the molecules that regulate it its permeability would associate with the nervous system to regulate behavior.
The conclusions made by the author are acceptable for these were all supported by the evidences obtained in the experimental part of the study. Also sufficient literatures were utilized to support all the arguments.
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