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Dec 2018

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Accessing Olfactory Habituation in Drosophila melanogaster with a T-maze Paradigm
使用T迷宫范例模拟黑腹果蝇的嗅觉习性   

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Abstract

Habituation is the process whereby perceptual changes alter the value of environmental stimuli, enabling salience filtering. This behavioral response decrement is a form of non-associative learning, where the subject learns about the stimulus and does not involve sensory adaptation, sensory or motor fatigue. The range of behavioral responses in D. melanogaster led to the development of a number of habituation paradigms addressing various sensory modalities. Habituation of osmotactic responses has previously been measured with the Y-maze test and required 30 min of odor exposure. Here, we describe an olfactory habituation assay utilizing the widely used in associative learning paradigms T-maze. Continuous or repetitive odor exposure for 4 min is adequate to attenuate osmotactic responses both to attractive and aversive odors. Importantly, the decreased response conforms to habitation parameters, presenting dishabituation and spontaneous recovery. This assay allows the study of habituation after brief odor exposure, but also discriminates between the two distinct phases of the response, an initial habituation latency period followed by habituation. In addition, the characterization of the neuronal circuits implicated in each phase facilitates further study of the molecular components underlying this process.

Keywords: Olfactory habituation (嗅觉习惯), D. melanogaster (D. melanogaster), Drosophila (果蝇), Habituation (习惯), Olfaction (嗅觉)

Background

Habituation, the behavioral modification whereby responses to repeated inconsequential stimuli are attenuated, is highly conserved and has been studied in a wide range of species, from Aplysia to humans. In Drosophila melanogaster, various paradigms have been developed to study habituation to visual, mechanical, gustatory, and olfactory stimuli. The variety of fly responses to odor stimulation led to development of different habituation paradigms, including olfactory jump response habituation (Mihalek et al., 1997; Asztalos et al., 2007a and 2007b; Joiner et al., 2007; Sharma et al., 2009), olfactory startle response habituation (Cho et al., 2004; Wolf et al., 2007), unconditioned leg movement habituation (Chandra and Singh, 2005), and olfactory avoidance habituation (Das et al., 2011).

Previous studies on olfactory avoidance habituation demonstrated that exposure to a continuous odor for 30 min attenuates the response, evidenced by subsequent testing in a Y-maze. Independently and in parallel with these studies, we developed an olfactory habituation paradigm using the T-maze, which is widely used in olfactory associative learning experiments and had already been in use for such experiments in the lab. Short repetitive or continuous odor exposure for a total of 4 min results in decreased responses both to aversive and to attractive odors. Importantly, this attenuated response complies with the parameters of habituation, as it recovers spontaneously and can be dishabituated (Thompson and Spencer, 1966; Rankin et al., 2009). In addition, this paradigm facilitates the investigation of habituation latency, the initial process that precedes habituation, which may be linked to associative learning. In fact, because the equipment and mechanics of this olfactory habituation assay are similar to those used for classical odor discrimination-dependent associative learning, the paradigm is conducive to investigations of possible interdependence of these two processes. Lastly, this habituation paradigm has enabled identification of the neuronal subsets implicated in this process (Semelidou et al., 2018), allowing the further study of the molecular pathways underlying habituation, in a neuronal-specific manner.

Materials and Reagents

  1. 14 ml Falcon tubes
  2. Glass vials for odorants (diameter: 2.2 cm, height: 9.5 cm)
  3. Drosophila melanogaster
  4. 3-Octanol (CAS number: 589-98-0; ACROS Organics, catalog number: AC203770500), store at RT
  5. Benzaldehyde (CAS number: 100-52-7; Sigma, catalog number: 418099), store at 4 °C
  6. Ethyl acetate (CAS number: 141-78-6; Sigma, catalog number: 34858), store at RT
  7. Butanedione (CAS number: 431-03-8; Sigma, catalog number: B85307), store at RT
  8. Brewers Yeast (CAS number: 68876-77-7; ACROS Organics, catalog number: AC368080010), store at 4 °C
  9. Drosophila food (see Recipes)
    1. Semolina 
    2. Whole wheat flour
    3. Brown sugar
    4. Fructose
    5. Soy flour
    6. CaCl2
    7. Dry yeast
    8. Nipagen
    9. Propionic acid

Equipment

  1. T-maze (Figures 1-3, Video 1):
    1. One Plexiglass side panel with single and another of equal size but with two openings (height: 15 cm, width: 3.8 cm, thickness: 1.8 cm).
    2. One Plexiglass elevator panel with a single opening and a “training point” (height: 15 cm, width: 3.8 cm, thickness: 1.8 cm). All openings have 1.8 cm diameter and are fitted on the inner side of the side panels that come in contact with the elevator, with Teflon O-rings. The measurements between the openings on the sides and the elevator are depicted in Figure 1. The elevator “training point” (height: 1.3 cm) (Figures 1 and 3) consists of smaller openings in a configuration 3-7-9-9-10-10-10-9-9-7-3, both in width and height. Vacuum ports on the back of the elevator have 1 cm diameter. They are fitted with a plastic or teflon barbed vacuum port adaptor (length: 3.7 cm) and a piece of silicon rubber tubing (diameter 0.6 cm ID Small Parts inc # B-210015). If additional fine control of incoming vacuum is required, then the silicon tubing can be cut and the two parts connected together with polycarbonate/polyethylene one-way stopcocks (Small Parts Inc # B-LSCP-100C).
    3. One Plexiglass base (length: 10.5 cm, width: 5.2 cm, height: 1.8 cm) with four screw receptacles (upper diameter 1 cm, bottom diameter 0.6 cm, height: 1.6 cm) to receive aluminum screws as appropriate. The distance between two individual receptacles is 2.1 cm in length and 1.6 cm in width.
    4. Aluminum hex head cap screws (length with head: 2.4 cm), silicon tube (diameter: 1 cm), “male” part of the pair of Nalgene Quick Disconnect to connect to home vacuum, O-rings (2.2 cm diameter and 2.0 mm thick).


  2. Figure 1. Depiction of the T-maze parts


    Figure 2. Olfactory habituation T-maze with fitted training and testing arms


    Figure 3. Side view of the T-maze. The training point and receptacle of the elevator are indicated.

  3. 14 ml polypropylene Round bottom tubes (Falcon, catalog number: 352006)
  4. Custom-made T-maze arms using Falcon 352006 tubes cut at the 1 ml mark (Figures 2 and 6)
    These are fit at the now open “bottom” end with the “female” part of the pair of Nalgene Quick Disconnect, HDPE size 3/8 to 7/16 inch, which has been fitted on its wide end with monofilament cloth Nylon Mesh (1 mm opening, Small Parts Inc. #B-CMN-1000 or -500) (Figures 4 and 6) and attached to the Falcon tube with a piece 3-3.5 cm in length silicon rubber tubing (diameter: 1.4 cm ID, Small Parts Inc .# B-210025) (Figure 4) 
  5. Glass vials (diameter: 2.2 cm, height: 9.5 cm) (Figure 5)
  6. Odor vial caps: Two-hole rubber stoppers #2 for the 14 ml tubes (Top: 20 mm, Bottom: 16 mm, Length: 25 mm) and #4 for the glass vials (Top: 25 mm, Bottom: 20 mm, Length: 25 mm), penetrated with two 0.3 cm glass tubes (diameter: 3 mm, length 5.5 cm for the long and 2 cm for the short). Silicon tubing (diameter: 4 mm Small Parts Inc # B-210010) to cover the upper part of the glass tubes and a “male” and “female” part of the pair of Nalgene Quick Disconnect, HDPE size 3/8 to 7/16 inch (Figure 5)
  7. Custom made Copper grids (width: 5.9 cm, length: 8.5 cm) (Figure 6)
  8. Adjustable house vacuum
  9. Gilmont flowmeter (Thermo Scientific, catalog number: GF-2200)
  10. Astro-Med/Grass technologies S48 Square Purse Stimulator
    Note: Different versions of T-mazes can be found and purchased from http://www.celexplorer.com/.


    Figure 4. The “female” part of the pair of Nalgene Quick Disconnect, fitted on its wide end with monofilament cloth Nylon Mesh (1 mm opening). The silicon tubing is used to attach it to the Falcon tubes used as arms.


    Figure 5. Odor vial cap made with rubber stopper #4 fitted into the odor glass vial, and the two parts demonstrated separately


    Figure 6. Shock dishabituation arm. The shock dishabituation arm consists of a copper grid, a cut Falcon tube and the “female” part of the pair of Nalgene Quick Disconnect with the silicon tubing attached. A new copper grid is demonstrated on the right.

Software

  1. JMP (by SAS, https://www.jmp.com/en_us/home.html), or any other statistics software

Procedure

  1. T-maze assembly (Video 1)

    Video 1. Demonstration of the T-maze construction

    The plexiglass components of the maze are assembled by first attaching the side panels and the elevator to the base with the aluminum screws. When all four screws are in place, tighten them crosswise to the point that they are tight without forcing them further. If forced too tight it may damage the plexiglass screw threads. Once the sides are fastened on the base, attach the vacuum adaptors onto the vacuum ports. Make sure that the elevator is snug, but it can move up and down without difficulty.

  2. Fly preparation
    1. Backcross control flies carrying the w1118 mutation to the Canton-S for at least 10 generations to obtain the Cantonised-w1118. Outcross all the Drosophila lines with Cantonised-w1118 for six generations to obtain the same genetic background for all animals used in the behavioral experiments.
    2. Raise the flies in standard wheat-flour-sugar food supplemented with soy flour and CaCl2 under a 14:10 h light-dark cycle, 60% relative humidity, at 25 °C, unless you use the TARGET system. In that case, raise the flies at 18 °C until hatching.
    3. To collect flies for the experiment, anesthetize them under CO2 at least one day before the experiment and separate them in groups of 50-60 flies. Place each group of flies in food vials at 25 °C, 14:10 h light-dark conditions and 60% humidity, if no transgene will be expressed during the experiment (for control experiments or mutants). For experiments with Gal4 lines, place the flies at 30 °C overnight to enhance transgene expression. For experiments using the TARGET system, place the flies at 30 °C for 2 or 3 days prior to testing. The days of transgene induction depend on the transgene used in each experiment. For neuronal silencing experiments with Shibirets, place the flies at 32-33 °C for 30 min before the experiment, while for experiments with TRPA1 for neuronal activation, transfer the flies at 30-31 °C for the time period you want to keep the neurons activated.

  3. Preparation before the experiment
    1. Transfer the flies in new food vials approximately 1 h before the experiment. Place the vials in a dark box and keep it at the temperature flies were kept before. For experiments with Shibirets, transfer the flies in new pre-warmed vials, kept at 32-33 °C, 30 min before the experiment. To ensure that neuronal transmission is blocked for the same time interval for all groups of flies, transfer the flies at 32-33 °C sequentially during the experiment.
    2. Prepare the behavior room. Clean the T-mazes and arms with soft cloth. Ensure that the arms fit tightly on the maze and the air flow is stable at 500 ppm (0.5 ml/min). If you use the T-maze for the first time, run a control experiment without odors to verify that there is no bias towards one arm of the maze. Check that humidity in the room ranges from 60% to 70% and the temperature from 23 to 24 °C. Experiments are performed under dim red light (photography dark room grade, Figure 7).
    3. Prepare the odor and connect it to the T-maze set up. Let the odor flow for 30 min, to prime the system for the experiment. For Octanol add 1 ml 3-Octanol in a glass vial. For experiments conducted with Benzaldehyde, add 100 μl of Benzaldehyde in a 14 ml Falcon. Similarly, for experiments with ethyl acetate or 2,3-butanedione prepare priming in a 14 ml Falcon with 10 μl of a 0.1% dilution of ethyl acetate in water and for 2,3-butanedione with 10 μl of a 0.5% dilution. The concentration of each compound and the surface area of the container (odorant meniscus) were determined empirically to produce the optimal response. Previous studies have demonstrated that the same odor compound can be either aversive or attractive, depending on the concentration employed (Wang et al., 2003). For odorants different than the ones described herein, avoidance experiments are required prior to habituation to standardize the odor concentrations. These odorants can be diluted either in water (ethyl acetate and 2,3-butanedione) or isopropyl myristate (Octanol, Benzaldehyde [Gouzi et al., 2018])


      Figure 7. The equipment set under the conditions of the experiment. All experiments must be conducted under dim red light.

  4. Odor Avoidance and Attraction Testing
    1. Use a clamp to hold the parts of the T-maze together tightly (Figure 2). Load the flies in a clean arm and connect it on the upper part of the maze. Move the middle part of the maze (the elevator) so that the elevator receptacle (Figure 3) will be aligned with the arm. Tap the maze gently to transfer the flies into the receptacle and slide the elevator down quickly to trap the flies inside the maze.
    2. Connect the air and odor tubing, so that air will flow on one side of the maze and odor on the other.
    3. Connect the vacuum.
    4. Slide the elevator down and let the flies choose between the two arms for 90 s for aversive odors and 180 s for attractive odors. Make sure that during testing the flow remains at 500 ppm.
    5. Move the elevator up to trap the flies inside the two arms.
    6. Transfer the content of each arm to separate (numbered) tubes.
    7. Clean the elevator receptacle to remove any remaining flies, attach the arms back on the maze and proceed with the next n.
    8. Repeat the procedure until you finish with all the repetitions of the experiment. 
    9. Transfer the rack where you have collected the flies to -80 °C. Wait for approximately 15 min and then count the flies. Note the number of flies trapped in the air-arm and the odor-arm, as well as their genotype.

  5. Habituation Training and Testing (Video 2)

    Video 2. Olfactory Habituation Training and Testing

    1. Load the flies in an arm used specifically for training exposure to a particular odor and connect the arm on the upper part of the maze. Make sure you use different arms for each odor used in the assay if more than one odor is to be used.
    2. Slide the elevator so that the flies will stay trapped in the arm (elevator training point, Figure 3), but the odor can flow through it.
    3. Connect the vacuum tubing and then the odor tubing. Make sure that the odor flow is at 500 ppm.
    4. Leave the flies in the arm with the odor flow for the designated amount of time–1 min for habituation latency experiments, and 4 min or 30 min for habituation experiments. For odor pulse experiments substitute 1 min odor exposure with 2 x 30 s (with 8 s interstimulus interval), 4 min with 4 x 1 min (with 15 s interstimulus interval), and 30 min with 3 x 10 min (with 2.5 min interstimulus interval) odor pulses.
    5. Disconnect the odor tubing but leave the vacuum tubing connected such that air will flow through the arm. Wait for 30 s.
    6. Slide the elevator so that the receptacle will be aligned with the training arm. Tap the maze gently to transfer the flies in the receptacle and slide the elevator down quickly to trap the flies inside the maze (Video 2).
    7. Continue the procedure from “Step C2”.

  6. Dishabituation with electric shock
    1. Set the Grass Stimulator to 1.2 s stimulus duration at 45 V.
    2. Load the flies in an arm with a custom-made copper grid (Figure 6) and connect the arm on the upper part of the maze. 
    3. Train the flies with 1, 4, or 30-min odor exposure as above. During this interval, connect the crocodile clips holding the electric shock wires with the extending parts of the copper grid.
    4. Disconnect the odor tubing and immediately apply one electric shock.
    5. Disconnect the crocodile clips and wait for 30 s with the vacuum on, so that air will flow through the arm.
    6. Slide the elevator so that the receptacle will be aligned with the copper-grid arm. Tap the maze gently to transfer the flies in the hollow of the elevator. Slide the elevator down quickly to trap the flies inside the maze.
    7. Continue the procedure from “Step C2”.
    Previous studies have shown that concurrent exposure to an odor and twelve 45 V electric shocks results in associative learning formation (Berry et al., 2018). Dishabituation, however, requires stimulation with a single electric shock following the odor exposure and therefore no associative learning formation is anticipated from the application of the protocol.

  7. Dishabituation with vortex
    1. Train the flies with 1, 4, or 30-min odor exposure as above.
    2. Disconnect the odor tubing and remove the arm from the maze, sealing it with your hand. Apply vortex for 3 s at maximum speed.
    3. Connect the arm on the maze again and connect the vacuum tubing so that air will flow through the arm. Wait for 30 s.
    4. Slide the elevator so that the receptacle will be aligned with the training arm. Tap the maze gently to transfer the flies in the elevator receptacle and slide the elevator down quickly to trap the flies inside the maze.
    5. Continue the procedure from “Step C2”.

  8. Dishabituation with yeast puff
    1. Prepare a 30% solution of yeast in water, an arm and tubing that will be used specifically for this odor. Make sure that the odor flows with 500 ppm.
    2. Train the flies with 1, 4, or 30-min odor exposure as above. 
    3. Disconnect the odor tubing and remove the arm from the maze, sealing it with your hand. Transfer the flies to the new arm, specifically used for yeast puff dishabituation. Connect the tubing for yeast puff for 3 s.
    4. Remove the yeast puff tubing and leave the vacuum tubing on so that air will flow through the arm. Wait for 30 s.
    5. Slide the elevator so that the receptacle will be aligned with the training arm. Tap the maze gently to transfer the flies in the receptacle and slide the elevator down quickly to trap the flies inside the maze.
    6. Continue the procedure from “Step C2”.

  9. Spontaneous recovery
    Proceed as described in ‘Procedure D: Habituation training and testing’. After Step D5, transfer the
    flies to food vials for 6 min. Continue with Step D6.

Data analysis

  1. Open JMP and create a new data table.
  2. Name the columns as Genotype-Air-[Odor name]–PI [Odor] *100 (Figure 8).


    Figure 8. JMP data table for Habituation to OCT and Dishabituation with shock

  3. Add formulas for ‘PI [Odor] *100’. To add a formula on a column, right click on the name of the column and choose ‘Formula’. The formula for ‘PI [Odor] *100’ is: [(AIR - OCT)/(AIR + OCT)] * 100.
  4. Add the genotypes and the type of training (Avoidance, [minutes of odor] for habituation training, [minutes of odor + shock] for dishabituation training with electric shock, etc.).
  5. Transfer your results in the data table. ‘Air’ indicates the number of flies that chose the arm with air, while [Odor] indicates the number of flies that chose the arm with the odor.
  6. When you have completed enough experiments and have approximately 10 ns/genotype for each treatment you can proceed with the statistical analysis. 
  7. For the statistical analysis concatenate the JMP files from all experiments and create a ‘Fit X by Y’ distribution by the ‘Analyze’ tool. Add ‘Genotype’ as the X, Factor and PI [Odor] *100 as the Y, Response (Figure 9). In the graphic representation choose ‘Means/Anova’ from the top left red arrow. In case ANOVA shows P < 0.01, proceed with further analysis. For Dunnett’s test, choose ‘Compare means’ from the top left red arrow and then ‘With Control, Dunnett’s’. Dunnett’s must be applied for each genotype individually.


    Figure 9. Example of Fit Y by X analysis of a 4 m Habituation experiment and Dishabituation with electric shock

Notes

Perform experiments by random, pseudorandom or blind but balanced design of ns for Avoidance, Habituation and Dishabituation testing.

Recipes

  1. Drosophila food
    140 g semolina
    180 g whole wheat flour
    180 g brown sugar
    72 g fructose
    15 g soy flour
    6 g CaCl2
    210 g dry yeast
    150 ml Nipagen (10% in EtOH)
    25 ml Propionic acid
    Bring up to 8.5 L final volume with house distilled water

Acknowledgments

This work has been co-financed by the European Union (European Social Fund–ESF) and Greek National funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework 2007-2013, Research Funding Program: THALES- Investing in knowledge society through the European Social Fund, MIS: 376898. We also acknowledge support by the project ‘Strategic Development of the Biomedical Research Institute ‘Alexander Fleming’’ (MIS 5002562) which is implemented under the ‘Action for the Strategic Development on the Research and Technological Sector’, funded by the Operational Programme ‘Competitiveness, Entrepreneurship and Innovation’ (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund). Parts of this work were also supported by Fondation Sante.

Competing interests

The authors have no conflict of interest to declare.

References

  1. Asztalos, Z., Arora, N. and Tully, T. (2007a). Olfactory jump reflex habituation in Drosophila and effects of classical conditioning mutations. J Neurogenet 21(1-2): 1-18.
  2. Asztalos, Z., Baba, K., Yamamoto, D. and Tully, T. (2007b). The fickle mutation of a cytoplasmic tyrosine kinase effects sensitization but not dishabituation in Drosophila melanogaster. J Neurogenet 21(1-2): 59-71.
  3. Berry, J. A., Phan, A. and Davis, R. L. (2018). Dopamine neurons mediate learning and forgetting through bidirectional modulation of a memory trace. Cell Rep 25: 651-662 e5.
  4. Chandra, S. B. and Singh, S. (2005). Chemosensory processing in the fruit fly, Drosophila melanogaster: generalization of a feeding response reveals overlapping odour representations. J Biosci 30(5): 679-688.
  5. Cho, W., Heberlein, U. and Wolf, F. W. (2004). Habituation of an odorant-induced startle response in Drosophila. Genes Brain Behav 3(3): 127-137.
  6. Das, S., Sadanandappa, M. K., Dervan, A., Larkin, A., Lee, J. A., Sudhakaran, I. P., Priya, R., Heidari, R., Holohan, E. E., Pimentel, A., Gandhi, A., Ito, K., Sanyal, S., Wang, J. W., Rodrigues, V. and Ramaswami, M. (2011). Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci U S A 108(36): E646-654.
  7. Gouzi J. Y., Bouraimi, M., Roussou, I. G., Moressis, A. and Skoulakis, E. M. C. (2018). The Drosophila receptor tyrosine kinase alk constrains long-term memory formation. J Neurosci, 38, 7701-7712.
  8. Joiner, M. A., Asztalos, Z., Jones, C. J., Tully, T. and Wu, C. F. (2007). Effects of mutant Drosophila K+ channel subunits on habituation of the olfactory jump response. J Neurogenet 21(1-2): 45-58.
  9. Mihalek, R. M., Jones, C. J. and Tully, T. (1997). The Drosophila mutation turnip has pleiotropic behavioral effects and does not specifically affect learning. Learn Mem 3(5): 425-444.
  10. Rankin, C. H., Abrams, T., Barry, R. J., Bhatnagar, S., Clayton, D. F., Colombo, J., Coppola, G., Geyer, M. A., Glanzman, D. L., Marsland, S., McSweeney, F. K., Wilson, D. A., Wu, C. F. and Thompson, R. F. (2009). Habituation revisited: an updated and revised description of the behavioral characteristics of habituation. Neurobiol Learn Mem 92(2): 135-138.
  11. Semelidou, O., Acevedo, S. F. and Skoulakis, E. M. (2018). Temporally specific engagement of distinct neuronal circuits regulating olfactory habituation in Drosophila. Elife 7: e39569.
  12. Sharma, P., Keane, J., O'Kane, C. J. and Asztalos, Z. (2009). Automated measurement of Drosophila jump reflex habituation and its use for mutant screening. J Neurosci Methods 182(1): 43-48.
  13. Thompson, R. F. and Spencer, W. A. (1966). Habituation: a model phenomenon for the study of neuronal substrates of behavior. Psychol Rev 73(1): 16-43.
  14. Wang, Y., Chiang, A. S., Xia, S., Kitamoto, T., Tully, T. & Zhong, Y. (2003). Blockade of neurotransmission in Drosophila mushroom bodies impairs odor attraction, but not repulsion. Curr Biol 13(21): 1900-4.
  15. Wolf, F. W., Eddison, M., Lee, S., Cho, W. and Heberlein, U. (2007). GSK-3/Shaggy regulates olfactory habituation in Drosophila. Proc Natl Acad Sci U S A 104(11): 4653-4657.

简介

习惯化是感知变化改变环境刺激的价值,从而实现显著性过滤的过程。这种行为反应的减少是一种非联想学习的形式,在这种学习中,受试者学习刺激,不涉及感觉适应、感觉或运动疲劳。行为反应范围D。melanogaster导致了针对各种感官模式的许多习惯化范式的发展。渗透反应的习惯化以前已经用y迷宫实验测量过,需要30分钟的气味暴露。在这里,我们描述了一个嗅觉习惯测试,利用广泛使用的联想学习范式t迷宫。连续或重复的气味暴露4分钟足以减弱对诱人和令人厌恶的气味的渗透反应。重要的是,降低的响应符合居住参数,表现出去适应和自发恢复。本实验允许研究短暂接触气味后的习惯化,但也区分了反应的两个不同阶段,最初的习惯化潜伏期之后是习惯化。此外,每个阶段所涉及的神经元回路的特性有助于进一步研究这一过程背后的分子成分。
【背景】习惯化是一种行为改变,对反复出现的无关紧要的刺激的反应会减弱,它是高度保守的,已经在从海兔到人类的各种物种中得到了广泛的研究。在黑腹果蝇中,已经发展了各种范式来研究视觉、机械、味觉和嗅觉刺激的习惯化。果蝇对气味刺激反应的多样性导致了不同的习惯化范式的发展,包括嗅觉跳跃反应习惯化(Mihalek et al., 1997;Asztalos 等, 2007a, 2007b;Joiner et al., 2007;Sharma et al., 2009),嗅觉惊吓反应习惯化(Cho et al., 2004;Wolf et al., 2007), unleg movement habituation (Chandra and Singh, 2005), and olfactory avoidance habituation (Das et al., 2011)。



之前关于嗅觉回避习惯化的研究表明,持续暴露在气味中30分钟会减弱反应,随后在y形迷宫中进行的测试证明了这一点。独立和与这些研究,我们开发了一个使用性嗅觉习惯化范式,这是广泛应用于嗅觉联想学习实验,已经使用了这些实验在实验室里。短的重复或连续气味暴露共有4分钟导致减少反应厌恶和迷人的气味。重要的是,这种减弱的反应符合习惯化的参数,因为它会自动恢复,可以被消除习惯(Thompson and Spencer, 1966;Rankin 等。, 2009)。此外,这种范式有助于研究习惯形成的潜伏期,即习惯形成之前的初始过程,这可能与联想学习有关。事实上,由于这个嗅觉习惯测试的设备和机制与经典气味识别依赖联想学习的方法相似,该范式有助于研究这两个过程之间可能的相互依赖关系。最后,这种习惯化范式已经能够识别这一过程中涉及的神经元亚群(Semelidou et al., 2018),允许以神经元特异性的方式进一步研究潜在习惯化的分子通路。

关键字:嗅觉习惯, D. melanogaster, 果蝇, 习惯, 嗅觉

材料和反应

  1. 14毫升猎鹰管
  2. 香水玻璃通道(直径2.2厘米,体重9.5厘米)
  3. 果蝇电位
  4. 3-Octanol (CAS编号589-98-0;ACROS有机,目录编号:AC203770500), store at RT
  5. 苯甲醛(CAS编号:100-52-7;西格玛,目录编号418099),商店4c
  6. 乙酸乙酯(CAS编号:141-78-6;目录编号:3458),商店at RT
  7. 案件编号431-03-8;目录编号:B85307), store at RT
  8. (CAS编号:68876-77-7;ACROS有机,目录编号:AC368080010),第四C商店
  9. 果蝇食物(见模式)
    1. Semolina
    2. 整个小麦流
    3. 红糖
    4. Fructose
    5. 大豆flour
    6. CaCl <潜水>潜水2 < / >
    7. 干yeast
    8. Nipagen
    9. Propionic酸

设备

  1. t -迷宫(数字1-3,视频1):
    1. 一组有机玻璃,只有一组,另一组大小相等,但有两个开口(height: 15厘米,width: 3.8厘米,thickness: 1.8厘米)。
    2. 一个有机玻璃升降机面板,只有一个开口和一个训练点(height: 15厘米,width: 3.8厘米,thickness: 1.8厘米)。所有的openings都有1.8厘米的直径,并且适合于与电梯接触的一侧panels,与Teflon O-rings。边开口和电梯之间的测量如图1所示。训练点升降机(高:1.3厘米)(图1和图3)由3-7-9-9-10-10-10-9- 7-3型的小型开口组成,两者都有。电梯后面的空隙门有1厘米直径。它们是用塑料或半空端口自适应器(length: 3.7 cm)和一块硅橡胶软管(直径0.6 cm ID Small Parts inc . B-210015)填充的。如果对进入真空的额外精细控制是必需的,那么硅管可以被切断,并与聚碳酸/单路聚乙二醇连接(B-LSCP-100C)。
    3. 一个有机玻璃基底(长:10.5厘米,宽:5.2厘米,高:1.8厘米),有四个螺丝接受(上直径1厘米,底部直径0.6厘米,高:1.6厘米)。两个个体受体之间的距离是2.1厘米长,1.6厘米宽。
    4. 铝头裂(长2.4厘米),硅管(直径1厘米),纳尔根快速断开连接到家庭真空,O-rings(2.2厘米直径和2.0毫米厚)。
  2. 14ml环底管(猎鹰,目录编号352006)
  3. 海关制造t -迷宫武器使用猎鹰352006 这是fit at the now open“底部”终点with the兼职“女”of the pair of Nalgene奎克Disconnect HDPE码的3 to 7/16 8寸,往fitted on其终点with 1mm布尼龙网状(1毫米后援,小部件公司B-CMN-1000前500)(主要4和6)and attached到猎鹰输卵管with 3 - 3一部分。5厘米length硅橡皮油管(直径波动:1。4厘米ID,小部件公司B-210025)(图4)
  4. 玻璃樽(直径:2.2厘米,高度:9.5厘米)(图5)
  5. 气味瓶帽:两眼橡胶瓶塞的14 # 2毫升管(上图:20毫米,底部:16毫米,长度:25毫米)和# 4的玻璃小瓶(上图:25毫米,底部:20毫米,长度:25毫米),渗透和两个0.3厘米玻璃管(直径:3毫米,长度5.5厘米长和短2厘米)。硅管(直径:4mm Small Parts Inc # B-210010)用于覆盖玻璃管上部和一对Nalgene快速断开的“公”和“母”部分,HDPE尺寸为3/8到7/16英寸(图5)
  6. 定制铜制网格(宽度5.9 cm,长度8.5 cm)(图6)
  7. 可调房子真空
  8. 吉尔蒙特流量计(Thermo Scientific,目录号:GF-2200)
  9. Astro-Med/Grass technologies S48方形钱包刺激器 注:不同版本的T-mazes可以在http://www.celexplorer.com/. < br / > 图1 。T-maze部分的描述 < br / > 图2 。嗅觉习惯t型迷宫配备训练和测试臂 < br / > 图3 。t形迷宫的侧面图。指出电梯的训练点和插座。< br / > < br / > 图4 。对“女性”部分的Nalgene快速断开,安装在其宽端与单丝布尼龙网(1毫米开口)。硅管用于将其连接到用作武器的猎鹰管上。< br / > < br / > 图5 。气味瓶盖由橡胶塞4号制成,装入气味玻璃瓶中,两部分分别显示 < br / > 图6。冲击dishabituation手臂。减震除习惯臂由铜栅极、切割的猎鹰管和一对Nalgene快速断开连接的硅管的“女性”部分组成。右边是一个新的铜栅极。

软件

  1. JMP(通过SAS, https://www.jmp.com/en_us/home.html),或任何其他统计软件

过程

  1. T-maze assembly(视频1) < br / > < br / >视频1。T-maze构建演示 < br / > 迷宫的有机玻璃组件是通过先用铝螺丝将侧板和电梯连接到基座上组装而成的。当所有四个螺丝都到位后,将它们横向拧紧,使之紧到一定程度,而不要强迫它们进一步拧紧。如果用力太紧,可能会损坏有机玻璃螺纹。一旦两侧固定在底座上,将真空适配器连接到真空端口上。确保电梯是舒适的,但它可以毫无困难地上下移动。< br / > < br / >
  2. 飞行准备
    1. 回交对照果蝇携带w1118突变体到canicon - s至少10代,获得广州化-w1118。将所有果蝇系与广州化-w1118杂交6代,获得所有行为实验动物相同的遗传背景。
    2. 除非使用目标系统,否则在14:10 h的明暗循环,60%相对湿度,25°C下,在添加大豆粉和CaCl2的标准小麦面粉-糖食品中饲养苍蝇。在这种情况下,把苍蝇放在18°C的温度下直到孵化。
    3. 为了收集实验用的果蝇,至少在实验前一天将其麻醉在CO2下,并将其分成50-60只果蝇的组。将每组果蝇置于25°C, 14:10 h明暗条件下,60%湿度的食物瓶中,如果实验期间没有转基因表达(用于对照实验或突变体)。对于Gal4系的实验,将果蝇放置在30℃过夜,以增强转基因表达。对于使用目标系统的实验,在测试前将果蝇放置在30°C的温度下2 - 3天。转基因诱导的天数取决于每个实验中使用的转基因。对于Shibirets的神经元沉默实验,在实验前将果蝇置于32-33℃静置30 min,而对于TRPA1的神经元激活实验,在30-31℃静置30-31℃静置,静置时间为神经元激活时间。< br / > < div风格= "空白:nowrap;} " > < br / > < / div >
  3. 实验前准备
    1. 在实验前约1小时将果蝇转移到新的食物瓶中。把瓶子放在一个黑暗的盒子里,并保持它的温度苍蝇之前被保存。对于Shibirets的实验,将果蝇移入新的预热瓶中,置于32-33℃,实验前30min。为保证所有果蝇组神经元传递被阻断的时间间隔相同,在实验过程中,将果蝇按32-33℃顺序转移。
    2. 准备好行为室。用软布清洁t形迷宫和扶手。确保手臂与迷宫紧密贴合,空气流量稳定在500ppm (0.5 ml/min)。如果你第一次使用t型迷宫,进行一个没有气味的控制实验,以验证迷宫的一只手臂没有偏向。检查房间湿度在60% - 70%之间,温度在23 - 24℃之间。实验在暗红色灯光下进行(摄影暗室等级,图7)。
    3. 准备好气味并将其与设置好的T-maze连接起来。让气味流动30分钟,以启动实验系统。对于辛醇,在玻璃瓶中加入1毫升3-辛醇。对于苯甲醛的实验,增加100μl苯甲醛的14毫升猎鹰。同样,对于实验与乙酸乙酯或2,3-butanedione准备启动与10μl 14毫升猎鹰稀释0.1%乙酸乙酯在水和2,3-butanedione 10μl 0.5%的稀释。实验确定了各化合物的浓度和容器的表面积(气味半月板),以获得最佳反应。以前的研究表明,相同的气味化合物可以是令人厌恶的,也可以是吸引人的,这取决于使用的浓度(Wang et al., 2003)。对于不同于本文描述的气味,在习惯化之前需要避免实验来标准化气味浓度。这些气味剂可以在水中(乙酸乙酯和2,3-丁二酮)或肉豆蔻酸异丙酯(辛醇、苯甲醛[Gouzi et al., 2018]) 中稀释 < br / > 图7。该设备是在实验条件下设置的。所有实验必须在暗红色灯光下进行。< br / >< div风格= "空白:nowrap;} " > < br / > < / div >
  4. 气味避免和吸引测试
    1. 用夹子把t型迷宫的各个部分紧紧地夹在一起(图2)。用一只干净的手臂把苍蝇装进去,连接到迷宫的上部。移动迷宫的中间部分(电梯),使电梯插座(图3)与手臂对齐。轻轻地敲击迷宫,将苍蝇转移到容器中,然后快速滑下电梯,将苍蝇困在迷宫中。
    2. 连接空气和气味管道,让空气在迷宫的一边流动,气味在另一边。
    3. 连接真空。
    4. 把电梯滑下来,让苍蝇在两只手臂之间做出选择,90秒时发出令人讨厌的气味,180秒时发出诱人的气味。确保在测试期间流量保持在500ppm。
    5. 把电梯往上移动,把苍蝇困在两只手臂里。
    6. 将每只手臂的内容转移到单独的(编号的)管子上。
    7. 清洁电梯插座,清除所有剩余的苍蝇,把手臂放回迷宫,继续下一个n。
    8. 重复这个过程,直到你完成所有重复的实验为止。
    9. 将收集苍蝇的架子移至-80°C。等待大约15分钟,然后数苍蝇。注意被困在空气臂和气味臂中的苍蝇的数量,以及它们的基因型。< br / > < div风格= "空白:nowrap;} " > < br / > < / div >
  5. 习惯训练和测试(视频2) < br / > < br / > 视频2。嗅觉习惯训练与测试 < br / >
    1. 将果蝇装进一只专门用来训练它们接触特定气味的手臂中,然后将这只手臂连接到迷宫的上半部分。如果要使用一种以上的气味,请确保对试验中使用的每种气味使用不同的手臂。
    2. 滑动电梯使苍蝇停留在手臂上(电梯训练点,图3),但是气味可以通过它。
    3. 连接真空管和气味管。确保气味的浓度为500ppm。
    4. 让果蝇在手臂上随气味流动指定时间- 1分钟用于适应潜伏期实验,4分钟或30分钟用于适应实验。气味脉冲实验用2×30 s (8 s间期)、4 min(4×1 min (15 s间期)、30 min(3×10 min (2.5 min间期)气味脉冲代替1 min。
    5. 断开气味管,但让真空管连接,这样空气就会流经手臂。等30秒。
    6. 滑动电梯,使插座与训练臂对齐。轻轻地点击迷宫,将苍蝇转移到容器中,然后快速滑下电梯,将苍蝇困在迷宫中(视频2)。
    7. 继续执行“步骤C2”中的步骤。< br / > < div风格= "空白:nowrap;} " > < br / > < / div >
  6. 电击使人不适应
    1. 将草地刺激器设置为1.2 s,刺激持续时间为45 V。
    2. 用定制的铜网格(图6)将苍蝇装入一个手臂中,并连接迷宫上部的手臂。
    3. 训练果蝇1、4或30分钟以上的气味暴露。在此期间,将装有电击线的鳄鱼夹与铜网的延伸部分连接。
    4. 断开气味管,立即施加一次电击。
    5. 断开鳄鱼夹,在真空状态下等待30秒,让空气流过手臂。
    6. 滑动电梯,使插座与铜栅臂对齐。轻轻敲击迷宫,将苍蝇转移到电梯的中空处。迅速滑下电梯,把苍蝇困在迷宫里。
    7. 继续执行“步骤C2”中的步骤。
    之前的研究表明,同时暴露于一种气味和12次45 V的电击会导致联想学习形成(Berry et al., 2018)。然而,去习惯化需要在气味暴露后用单一的电击进行刺激,因此该方案的应用没有预期会形成联想学习。< br / > < br / >
  7. Dishabituation与涡
    1. 训练果蝇1、4或30分钟以上的气味暴露。
    2. 断开气味管,将手臂从迷宫中取出,用手密封。以最大速度涡旋3秒。
    3. 再次连接迷宫上的机械臂,并连接真空管,使空气流经机械臂。等30秒。
    4. 滑动电梯,使插座与训练臂对齐。轻轻地敲击迷宫,将苍蝇转移到电梯的插座上,然后迅速将电梯滑下,将苍蝇困在迷宫中。
    5. 继续执行“步骤C2”中的步骤。< br / > < div风格= "空白:nowrap;} " > < br / > < / div >
  8. 用酵母泡芙使人不适应
    1. 准备30%的酵母溶液在水中,一个手臂和管道将专门用于这种气味。确保气味以500ppm的浓度流动。
    2. 训练果蝇1、4或30分钟以上的气味暴露。
    3. 断开气味管,将手臂从迷宫中取出,用手密封。将苍蝇转移到新手臂,特别用于酵母泡芙的去适应。连接酵母泡芙管3秒。
    4. 取下酵母吹气管,让真空管开着,这样空气就会通过手臂。等30秒。
    5. 滑动电梯,使插座与训练臂对齐。轻轻地敲击迷宫,将苍蝇转移到容器中,然后快速滑下电梯,将苍蝇困在迷宫中。
    6. 继续执行“步骤C2”中的步骤。< br / > < div风格= "空白:nowrap;} " > < br / > < / div >
  9. 自发恢复< br / > 按照“程序D:习惯训练和测试”中的描述进行。步骤D5之后,传输 飞向食物瓶6分钟。继续步骤D6。< br / >

数据分析

  1. 打开JMP并创建一个新的数据表。
  2. 将列命名为gene - type- air -[Odor Name] - pi [Odor] *100(图8)。< br / > < br / > 图8。JMP数据表,用于OCT的适应和休克 的适应 < br / >
  3. 添加“PI [Odor] *100”的公式。若要在列上添加公式,请右键单击列的名称并选择“公式”。PI [Odor] *100的公式是:[(AIR - OCT)/(AIR + OCT)] *100。
  4. 添加基因型和训练类型(回避,[气味分钟]适应训练,[气味分钟+电击]适应训练,等.)。
  5. 在数据表中传输结果。“Air”表示选择有空气的手臂的苍蝇数量,而“Odor”表示选择有气味的手臂的苍蝇数量。
  6. 当你完成了足够的实验,并且每种治疗都有大约10个ns/基因型时,你就可以进行统计分析了。
  7. 在统计分析中,将所有实验的JMP文件连接起来,使用“Analyze”工具创建一个“Fit X by Y”分布。将“gene type”作为X, Factor和PI [Odor] *100作为Y, Response(图9)。在图形表示中,从左上角的红色箭头中选择“Means/Anova”。如果方差分析显示P <0.01,进行进一步分析。对于Dunnett的测试,从左上角的红色箭头中选择“Compare means”,然后选择“With Control, Dunnett’s”。Dunnett’s必须单独应用于每个基因型。< br / > < br / > 图9 。用X线拟合Y的例子对一个4 m的习惯化实验和电击 的反习惯化进行了分析

笔记

采用随机、伪随机或盲均衡设计神经网络进行实验,进行回避、适应和反适应测试。

食谱

  1. 果蝇食物< br / > 粗粒小麦粉 180克全麦面粉 180克红糖 果糖 15克大豆粉 6g CaCl2 210 g干酵母 150ml尼泊根(10% EtOH) 25 ml丙酸 将室内蒸馏水 调至8.5 L

致谢

这项工作由欧盟(欧洲社会基金esf)和希腊国家基金共同资助,通过2007-2013年国家战略参考框架“教育和终身学习”运营项目,研究资助项目:泰勒斯——通过欧洲社会基金投资于知识社会,MIS: 376898。我们也承认支持项目的战略发展生物医学研究所的亚历山大·弗莱明”(MIS 5002562)实施下的行动战略发展研究和技术部门的,由运营计划“竞争力、创业和创新”(NSRF 2014 - 2020)和希腊和欧盟联合(欧洲区域发展基金)。这项工作的一部分也得到了Sante基金会的支持。

相互竞争的利益

作者没有任何利益冲突需要声明。

参考文献

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  2. Asztalos, Z。巴巴,K。,山本,D.和塔利,T. (2007b)。细胞质酪氨酸激酶的易变突变对果蝇 >有致敏作用,但不影响其去适应作用。。jneurogenet 21(1-2): 59-71。
  3. 浆果,j . A。潘,A.和戴维斯,R. L.(2018)。多巴胺神经元通过双向调节记忆线索介导学习和遗忘。 Cell Rep 25: 651-662 e5。
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Copyright Semelidou et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Semelidou, O., Acevedo, S. F. and Skoulakis, E. M. (2019). Accessing Olfactory Habituation in Drosophila melanogaster with a T-maze Paradigm. Bio-protocol 9(11): e3259. DOI: 10.21769/BioProtoc.3259.
  2. Semelidou, O., Acevedo, S. F. and Skoulakis, E. M. (2018). Temporally specific engagement of distinct neuronal circuits regulating olfactory habituation in Drosophila. Elife 7: e39569.
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