This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.
Mange indblik i de molekylære mekanismer, der ligger til grund for læring og hukommelse, er blevet belyst ved brug af enkle adfærdsmæssige analyser i modelorganismer som frugtflyve , Drosophila melanogaster . Drosophila er nyttig til forståelse af de grundlæggende neurobiologiske underliggende kognitive underskud som følge af mutationer i gener forbundet med menneskelige kognitive lidelser, såsom intellektuel invaliditet (ID) og autisme. Dette værk beskriver en metode til afprøvning af læring og hukommelse ved hjælp af et klassisk paradigme i Drosophila kendt som kriminalitetskonditionering. Mand flyver domstol kvinder med et klart mønster af let genkendelige adfærd. Præsterede kvinder er ikke modtagelige for parring og vil afvise mannens copulationsforsøg. Som svar på denne afvisning reducerer mandlige fluer deres frieriadfærd. Denne lærte reduktion i retssagens adfærd måles over tid og tjener som indikator for læring og hukommelse. Den grundlæggende numéRical udgang af dette assay er domstolsindekset (CI), som defineres som den procentdel af tid, som en mand bruger hofning i løbet af et 10 min interval. Læringsindekset (LI) er den relative reduktion af CI i fluer, der har været udsat for en premieret kvinde sammenlignet med naive fluer uden tidligere sociale møder. Til statistisk sammenligning af LI'er mellem genotyper anvendes en randomiseringstest med bootstrapping. For at illustrere hvordan analysen kan anvendes til at adressere rollen som et gen relateret til læring og hukommelse, blev den pan- neuronale nedbrydning af dihydroxyacetonphosphatacyltransferase ( Dhap-at ) karakteriseret her. Den menneskelige ortholog af Dhap-at , glyceronphosphat O-acyltransferase ( GNPT ) er involveret i rhizomelic chondrodysplasia punctata type 2, et autosomalt recessivt syndrom kendetegnet ved svær ID. Ved anvendelse af kriminalitetskonditioneringsassayet blev det fastslået, at Dhap-at er påkrævet til langsigtet hukommelse, men ikke forKortvarig hukommelse. Dette resultat tjener som grundlag for yderligere undersøgelse af de underliggende molekylære mekanismer.
De molekylære mekanismer, der ligger til grund for læring og hukommelse, bevares gennem mange arter. Den banebrydende arbejde screening Drosophila melanogaster mutant linjer for defekter i olfaktorisk læring og hukommelse har givet nøgle molekylær indsigt i de processer, der ligger til grund for læring og hukommelse 1 . Disse undersøgelser identificerede nogle af de første gener involveret i læring og hukommelse , såsom rutabaga 2 , amnesiac 3 og dunce 4 , hvilket afslørede en kritisk rolle for signalering af cyclisk adenosinmonophosphat (cAMP) 5 .
Tidlige genetiske skærme til hukommelsesmutanter blev primært udført under anvendelse af olfaktorisk konditionering. Flere metoder til måling af andre former for læring og hukommelse er dog opstået over tid. Et af de mest anvendte lærings- og hukommelsesparadigmer, og analysen beskrevet her, er kendt som cOurtship conditioning, som først blev beskrevet af Siegel og Hall 6 og senere blev raffineret af flere andre forskningsgrupper 7 , 8 , 9 . Courtship-konditionering er afhængig af tilstedeværelsen af et specifikt feromon, cis- vaccenylacetat (cVA), på kvindens underliv, som aflejres af hanen under copulation. At mærke cVA på kvindens underliv reducerer naturligvis frieriadfærd, og når det kombineres med kvindens afvisning, er effekten af cVA på mandlig kropsreduktion dramatisk forbedret. Reaktionen hos hanflyvninger i denne analyse kan let kvantificeres ved at observere deres særskilte retfærdighedsadfærd, som er kendetegnet ved orientering mod og efter kvinden, tapping, udstrækning og vibrerende vingen, slikker og forsøger kopiering 10 ( figur 1A ). Mandlige fluer lærer at distinguis H mellem modtagelige, jomfruelige og ikke-modtagelige parede kvinder 11 og efter seksuel afvisning viser de nedsat frieri til ikke-modtagelige kvinder i op til 9 dage 8 . Denne naturlige opførsel kan bruges til at belyse de mekanismer, der ligger til grund for læring, kortvarig hukommelse (STM) og langvarig hukommelse (LTM) 8 , 9 , 12 . Læring er defineret som den øjeblikkelige reduktion i retssagens adfærd, der opstår i løbet af træningsperioden, og kaldes ofte som øjeblikkelig tilbagekaldshukommelse, målt 0 – 30 min efter udsættelse for en parret kvinde 6 , 8 . STM måles mellem 30 minutter og 1 time efter træning, mens LTM oftest måles 24 timer efter træning 8 ( figur 1B ). STM kan induceres ved hjælp af en 1-timers træningsperiode, men den varer kun i 2-3 timerS = "xref"> 6 , 8 . I de fleste læringsparadigmer kan LTM kun fremkaldes ved adskillige forsøg på gentagen træning. Mcbride et al . (1999) 8 viste at tre adskilte, 1-timers træningssessioner var tilstrækkelige til at fremkalde hukommelseshukommelse, der varede i op til 9 dage, i modsætning til de 2-3 timer, der blev fremkaldt af en enkelt 1-timers træningssession. McBride et al . 8 viste også, at en enkelt 5-timers træningssession gav et lignende LTM respons i op til 9 dage. Fluer rømmer ikke konstant i løbet af denne 5-timers periode, og producerer faktisk deres egen indbyrdes træning for at fremkalde LTM i en enkelt træning. Dette er meget vigtigt fra et praktisk perspektiv, der øger den lethed, som denne analyse kan bruges til at undersøge LTM. Nuværende protokoller overvejende bruger en enkelt træningssession på 7-h for LTM 11 , 12 . Flere undersøgelser har undersøgt forskelligeMutante forhold, der har specifikke mangler i forskellige aspekter af courtship læring. For eksempel påvirker svampekroppens ablation STM og LTM, men ikke lærer 8 . Mutationer i amnesiacgenet , som først blev defineret som en specifik regulator af hukommelse ved hjælp af olfaktorisk konditionering 3 , påvirker STM, men ikke lærer 6 . Afbrydelse af oversættelsesregulatoren orb2 (oo18 RNA-bindende (orb) CPEB2-underfamilie) og ecdyson-signalering har udelukkende virkning på LTM 9 , 13 . Således er kriminalitetskonditionering et nyttigt paradigme til at opløse de mekanismer, der ligger til grund for de forskellige faser af læring og hukommelse.
Dette arbejde demonstrerer en optimeret eksperimentel opsætning, der muliggør den relativt høje gennemløbstest af kriminalitetskonditionering. Desuden beskriver den et statistisk analyse script og diskuterer kritiske faktorer i analysen. Det er shEjer her, at Drosophila – gendihydroxyacetonphosphatacyltransferasen ( Dhap-at ) Er påkrævet i neuroner for LTM, men ikke for STM. Den menneskelige ortholog af dette gen, glycerolphosphat-O-acyltransferase ( GNPAT ), muteres i rhizomelisk chondrodysplasia punctata type 2 14 , en autosomal-recessiv lidelse karakteriseret ved alvorlig intellektuel invaliditet, anfald og flere andre kliniske egenskaber 15 . I denne sammenhæng kan kriminalitetskonditionering anvendes til funktionelt validering af rollen som menneskelige sygdomsgener i læring og hukommelse, der danner grundlag for mekanistiske undersøgelser.
The courtship conditioning assay is a classic paradigm for the analysis of learning and memory in Drosophila. The protocol presented here follows the general methodology described previously6,7,8,9 but includes unique aspects such as practical guidelines, specialized equipment, and a data analysis script9,12 for randomization tests. Using this protocol, it is possible to analyze large numbers of flies in parallel using 96-well flat-bottom blocks (Figure 2A) to collect and train males. The blocks are sealed with PCR adhesive film, which makes the flies easily accessible when required. Additionally, the unique courtship chambers described here allow for the simultaneous pairing of 18 male-female pairs in a nearly two-dimensional space that is optimal for video analysis. The custom-designed courtship chambers are easy to use, and a building plan is provided (File S1, Figure 2D). This protocol, from the establishment of the cultures used to collect test subjects to the acquisition of video data, takes approximately 20 days (Table 1). Additional time is required for the analysis of video data. In our experience, the STM assay is extremely robust. The LTM assay is also quite robust, but it is more sensitive to confounding environmental variables and therefore can be more difficult to master.
Animal behavior can be quite variable. Therefore, critical steps in the protocol must be performed with care to reduce this variance. First, gentle use of the aspirator (Figure 2B) will reduce the stress that can be imposed by rough handling or by blowing out too strongly. A suggested method of transferring individual flies out of the aspirator is by using negative geotaxis. As flies tend to walk up, one can simply point the tip of the aspirator up; just before the fly reaches the tip, a gentle blow is sufficient to let the fly out. Additionally, to let the males into the courtship chambers before testing, a blow is often not necessary.
Another important step is the collection and generation of male test subjects. All males must be collected when they are very young and socially naïve. This can be achieved by frequent collection during the peak periods of eclosion (step 5.2). If males are not collected in this tight timeframe, they can have early social interactions, which may result in poor learning or high variability in CI. Another factor of male test subjects that should be assessed is the genetic background. Different genetic backgrounds will exhibit different levels of naïve courtship and may also differ in general activity or locomotor ability. When comparing multiple genotypes, care should be taken with regard to genetic background in order to avoid these confounding factors that may influence LI scores. Additionally, the distribution of CI data should be carefully assessed. CI data can be both parametric and non-parametric, depending on the genotype or other environmental factors. In some cases, if the distribution of CI is dramatically skewed away from a normal distribution, it may be better to use the median CI rather than the mean for the calculation of LIs. However, in our experience, the use of median or mean CI does not make a difference in the statistical interpretation of the data, and the use of the mean CI is the common practice in the literature.
For successful courtship conditioning, the active rejection of male courtship attempts by premated females is crucial during the training period. It is important to ensure that the premated females used in this assay have been efficiently mated and are thus not allowing copulation. This premating is established in the mating vials prepared in step 4, where male and females flies are housed together for 4 days (Table 1). Subsequently, mating can be monitored by regular examination of testing videos and by observing male-female pairs during training. If mating does occur, there are several measures that can be taken during the preparation of premated females. First, premated females should be reared under optimal breeding conditions. Vials can be supplemented with yeast paste and a folded filter paper to increase potential mating surfaces. The incubation of flies under the conditions described here has produced robust premated females in the past, but this may vary in different labs and with the use of different genetic strains. Therefore, it may be necessary to optimize the generation of premated females by varying the incubation time and conditions.
Quantification of courtship behavior is another critical step in this protocol. This can be done manually or automatically using specialized software programs9. Automated quantification is fast and, in principle, unbiased. Several programs have been published21,22,23; however, they are not straightforward to use, often requiring specialized video formats and advanced computational skills. Manual quantification is easy and accurate, but it is highly labor intensive and subject to individual variability and bias. It is important to emphasize that this protocol does not address the requirements for video formatting that are potentially required for the automated quantification of CIs. For manual quantification, use any simple video recording device that has the potential to produce a video of sufficient quality to accurately observe courtship behavior. For automated quantification, there will likely be different requirements depending on the software used, and users should investigate this thoroughly if automated quantification is desired.
In combination with the extensive tools that are available for the genetic manipulation of flies, the courtship conditioning assay provides a robust readout that can be used to dissect molecular mechanisms and neuronal networks involved in learning and memory.
The authors have nothing to disclose.
We acknowledge the Vienna Drosophila Resource Center for providing the Drosophila strains. Additionally, stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This research was supported in part by the European Union’s FP7 large-scale integrated network Gencodys to K.K., H.v.B., and A.S. and by NSERC Discovery and CIHR Project Grants to J.M.K.
P{KK101437}VIE-260B | VDRC | 101437 | Dhat-at-RNAi in 60100 background |
P{KK108109}VIE-260B | – | – | Control-RNAi in 60100 background (gift from K. Keleman) |
w+, UAS-dcr2/yhh;;elav-Gal4 (III) | – | – | panneuronal driver line |
Containers for plant tissue culture | VWR | 960177 | 175 mL plastic vials |
Folded filters | Whatman | 10311643 | Filter paper to enlarge area flies can pupate on |
Flat-bottom blocks (96-wells) | Qiagen | 19579 | Used for housing blocks |
MicroAmp Clear Adhesive Film | Applied Biosystems | 4306311 | PCR adhesive film as lid on flat-bottom blocks |
Razor blade | – | – | Any sharp will do |
Needle | – | – | 0.8 mm diameter |
Aspirator | – | – | Cut a 1mL pipet tip with scissors in order to have two pieces. The narrow tip of the pipettip is placed as fly entrance in a ~80 cm flexible hose. To prevent a fly from getting in the hose, a normal piece of cotton or small mesh gaze is placed in between the tip and the hose. The other half of the pipettip can be used as mouth piece at the end of the hose. |
Courtship chambers | – | – | file S1 can be opened with indicated CAD software |
Camcorder | Sony | – | camera specification: >4M pixels, full HD. For manual quantification, any simple video recording device has the potential to produce a video of sufficient quality to observe courtship behavior accurately. For automated quantification, there will likely be different requirements depending on the software to be used, and users should investigate this thoroughly if automated quantification is desired. |
Name | Company | Catalog Number | Comments |
power food | |||
Agar | Sigma | A7002 | |
Yeast | Bruggeman | – | |
Yeast extract | MP biomedicals | 0210330391 | |
Peptone | Sigma | P6838 | |
Sucrose | Sigma | S9378 | |
Glucose | Sigma | G7021 | |
MgSO4 | Sigma | M2643 | |
CaCl2 | Merck | 1023780500 | |
Methylparabene (CAUTION) | Sigma | H5501 | |
Propionic acid (CAUTION) | Sigma | P1386 | |
Demineralized water | – | ||
Yeast paste | – | yeast grains and water mixture in a 1:1 ratio | |
Name | Company | Catalog Number | Comments |
normal food | |||
Agar | MP biomedicals | 215017890 | |
Yeast | bruggeman | – | |
Corn flour | de Molen | – | |
Sugar | de Molen | – | |
Methylparabene (CAUTION) | Sigma | H5501 | |
Propionic acid (CAUTION) | Sigma | P1386 | |
Demineralized water | – |