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A Novel Task for Studying Memory of Occasional Events in Rats
大鼠偶尔事件记忆研究的新模型

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Abstract

Episodic memory has been defined in humans as the conscious recollection of unique personal past experiences often occurring singly during daily life, including remembrance of what happened, where and when it happened (Tulving, 1972). Here, we propose and describe in details a novel protocol we recently used to test the ability of rats to form and recollect episodic-like memory of previously encountered occasional episodes (Veyrac et al., 2015). During these episodes, the animals are briefly exposed to sets of specific odor–drink associations (what happened) encountered in specific locations (where it happened) within different multisensory enriched environments (in which context/occasion it happened). Memory of the episodes can be tested at relatively short (24 h) or much longer (24 d) delays in either a low or high interfering retrieval situation. This novel paradigm brought evidence for individual memory profiles of recall performance that might be correlated to different aspects of brain functional networks. More generally, it offers novel possibilities to explore cellular and network mechanisms that underlie memory of past events and memory dysfunction in brain pathologies.

Keywords: Episodic Memory(情景记忆), Animal Model(动物模型), Olfaction(嗅觉), Declarative Memory(陈述性记忆), Rodent(啮齿类动物)

Materials and Reagents

  1. Subjects
    All experiments were conducted in accordance with European guidelines for care of laboratory animals (2010/63/EU) and received approval from the Lyon 1 University Ethics Committee (permission DR2015-46). Adult male Long-Evans rats (Charles River Laboratories) aged 7-8 weeks (~300-350 g) at the start of the water deprivation protocol, are housed in groups of 2-4 per cage and kept in an environment with controlled temperature and humidity under a 12/12 h light/dark cycle with food ad libitum. Experiments are conducted during the light period.
    Note: So far these experiments have not been conducted with other strains of rats or mice, but we assume that usage of other strains shouldn’t introduce major difficulties to carry out the experiments. Nevertheless, according to the fact that contexts are characterized by visual stimulation, working with albinos rats might be more challenging. Regarding mice, some adjustments to the experimental device would be required.
  2. Odorants
    In order to be delivered in a fully controlled manner through a new generation of olfactometer (Sezille et al., 2013), each odorant is introduced in a U-shaped Pyrex® tube (volume: 10 ml; length: 50 mm; external diameter: 14 mm) (VS technologies) filled with microporous granules. All odors used in these experiments were obtained from Sigma-Aldrich, France (see a -f below).
    1. Geraniol (Sigma-Aldrich, catalog number: 163333 ) 20% of saturated vapor pressure
    2. Eugenol (Sigma-Aldrich, catalog number: E51791 ) 18% of saturated vapor pressure
    3. (S)-(+)-Carvon (Sigma-Aldrich, catalog number: 435759 ) 35% of saturated vapor pressure = Odor A
    4. Isoamylacetate (Sigma-Aldrich, catalog number: W205508 ) 15% of saturated vapor pressure = Odor B
    5. Trans-Anethole (Sigma-Aldrich, catalog number: 117870 ) 30% of saturated vapor pressure = Odor C
    6. Citral (cis+trans) (Sigma-Aldrich, catalog number: W230308 ) 20% of saturated vapor pressure = Odor D
      Note: Before the start of experiments, between 2 and 4 ml of pure odor solution are introduced gradually on several consecutive days into the U Shaped Glass Tubes until the microporous granules appear saturated (Figure 1) (for the experiments, the odors should be at saturated vapor pressure in the tube, at the same time in order to prevent olfactometer pollution it is important to avoid the accumulation of liquid at the bottom of the tube). The percentage of saturated vapor pressure introduced into the airflow is adjusted for each odor individually by the experimenter. The objective is to obtain an intensity that is perceived by the animal but moderate enough to prevent any avoidance behavior. For the different odors used in pairs we also try to equalize their perceived intensity. The choice of these particular odors and concentrations was mainly determined by previous experiments in the lab showing their easy discriminability and the absence of their natural attractiveness or repulsiveness to the rats at the concentrations used (Martin et al., 2004; Courtiol et al., 2014; Torquet et al., 2014).


      Figure 1. A photo of the U-shaped Pyrex tube filled with microporous granules showing different levels of saturation with Carvon odor solution. From left to right: pure granules without any added odor solution; partly saturated granules; fully saturated granules as used for the experiments.

  3. Drinking solutions
    1. 6% Sucrose in solution is used as positive reinforcement (Sigma-Aldrich, catalog number: number: 84097 )
    2. 0.06% Quinine hydrochloride dehydrate in solution is used as negative reinforcement (Sigma-Aldrich, catalog number: Q1125 )

Equipment

  1. Apparatus (EPISODICAGE)
    The experimental cage is a PVC rectangular box (60 x 35 x 40 cm) equipped with 4 devices for delivery of different odor and drinking solutions (Figure 2A) (Belkacem Messaoudi). On the two opposing walls, at 5 cm distance from each corner of the box, the cage contains an arrangement of odor and drinking ports. The odor port is a round indentation into the wall (5 x 5 cm in size), with a drinking port (small hole, 1 x 1 cm in size) placed 1cm below the odor port through which a drinking pipette can be inserted into the cage (Figure 1B).


    Figure 2. The EPISODICAGE. A. General view of the experimental cage showing the four cameras installed above each of the four odor ports. B. Details of an odor port (OP) with associated drinking pipette (P), view from the inside (left figure) or the outside (right figure) of the cage: odor injection (OI) and odor extraction (OE), motor (M), which allows introducing and withdrawing the pipette from the cage. C. Each nose poke first triggers an odor delivery for 13 sec. Three seconds after odor initiation, a pipette containing various drinking solutions (water, sugar or quinine) is introduced into the cage for 10 sec until the odor stimulation switches off. Each lick on the pipette made by the rat is detected and recorded by the computer devoted to the control of the experimental cage. Each trial starts with the nose poke and ends with withdrawal of the pipette and odor switch off. D. On the left, appearance of the cage in the version used in routine sessions. The two other pictures illustrate how the appearance of the cage can be modified by projecting visual patterns on the ground (center picture) or introducing objects (right picture). Figure adapted from Veyrac A. et al. (2015).

    1. Odor port
      When a rat makes a nose poke into the port, a capacitance change is detected by the system that immediately triggers for 13 sec the introduction of odor-saturated vapor into a constant airflow (air flow overall: 1 liter/min). The proportion of odorized air and the quality of the odorant are controlled through a custom made olfactometer connected to the odor port (OI) (Figure 1B). In a given experimental session, five different odors can be delivered at each of the four odor ports. A venturi-based system of aspiration allows extraction of the odorized air through a separate channel, so that the odor remains limited to the port (OE) (Figure 1B). A permanent compensation of OI and OE prevents any change in overall air pressure due to odor switch.
      Note: Rats are highly sensitive to changes in air pressure. To prevent any behavioral response to mechanical stimulation, air pressure must be kept at the same level during the presence and absence of odors.
    2. Drinking port
      Each movable drinking pipette is connected to a pump. The pipette is introduced into the cage 3 sec after the first nose poke of the animal and is withdrawn from the cage 10 sec later simultaneously with termination of odor release (Figure 1C). This ensures that the odor stimulation is delivered during the entire liquid consumption. A second capacitance change sensor integrated into the drinking pipette detects every single lick of the animal and triggers the pump which in turn delivers a calibrated amount of 8-9 µl of drinking solution. The licks made by the rats are recorded as text files for later analysis of the results.
      Note: The amount of liquid delivered upon each lick from the drinking ports must be equal between the ports (the variation should be within 8±1 µl), in order to avoid any preference of the animals for a particular port.
      Different drinking solutions can be delivered by the system, water, sucrose or quinine solutions according to the different phases of the protocol and the odor-port configurations. After each trial and retraction of the pipette, the drinking solution device is purged with the solution of the following trial.
      Note: Purge duration is set in a way that allows the complete wash out of the drinking solution of the last trial. This was verified through addition of ink into the water before the start of experiments.
  2. Items for contextual enrichment during episodes
    Acoustic context
    Different types of sounds can be played through two loudspeakers symmetrically placed above the experimental arena to enhance the discriminability of the episodes: nature sounds, bird songs, piano music …etc. The general guideline for choosing these sounds was their discriminability as validated in spectrograms and the animals/experimenter’s well-being.
    Tactile and visual context
    Different types of floors can be used with distinct tactile and visual characteristics (Figure 1D). As color vision in rats is poor, combinations of high-contrast black and white coloring of materials are preferred. Additionally, visual patterns can also be displayed on the floor by a video projector and various objects can be placed into the box.
  3. Video recording
    Five cameras are used to monitor precisely the behavior of the rats, one placed centrally above the experimental box and four others each placed above the four odor ports. The position of the rat is detected online via a video tracking software from the signal recorded from the central camera. When the animal approaches a port, the corresponding camera is selected for signal acquisition. This allows us to save space occupied by the video files.

Software

  1. VOLCAN (Marc Thevenet)

Procedure

All the essential steps of the protocol are summarized in Figure 3. Prior to the start of the shaping procedures, rats are habituated to the experimenter and water deprivation is established.

  1. Water deprivation protocol
    During the experiments, and most critically during the shaping period, the animals have to be motivated enough to activate the drinking device. This requires a controlled reduction of their access to water in the animal housing facility. It must not be too severe to preserve the animal in a good physiological state, but sufficient enough to keep them motivated in a controlled and stable way. One week after their arrival in the animal facility, when the animals are habituated to their new housing environment, we quantify daily ad lib water consumption for each individual cage by measuring the bottle weight before and after drinking. Water restriction is introduced progressively one week later: during the first 2 d of deprivation, a drinking pipette is accessible in their home cage for 2 h in the morning (between 9 and 11 am) and in the evening (4 to 6 pm) respectively, then the access is reduced to 1h during the next 2 d and to 40 min during the following 7 d. All along this period, we control that the total amount of water consumed is close to the previous consumption during the ad lib period and consequently adjust the duration of the evening access. Additionally, the weight of the animals is periodically checked to ensure that it remains steady (±5%) across the entire protocol. During behavioral experiments, if rats are tested in the morning, they receive water for 20-40 min only in the evening, but if they are tested in the afternoon, they receive water during ~5 min in the morning and for 20-40 min in the evening. Moreover, evening access to water may be adjusted all along the experiments depending on the amount of water consumed by the animals in the experimental arena.
    Note: Within a 20 min period, rats are expected to perform at least 10 trials. For animals that do not perform the expected number of trials, and do not exhibit other signs than a lack of motivation, the access time to drinking water is reduced (e.g. from 40 to 20 min). Additionally, it is helpful to decrease the amount of drinking time on the day which precedes the first episode to increase the motivation of the rats and overcome stress which arises through exposure to a novel situation.
    Water consumption of the animals when water restriction has been established is estimated to around 20 ml/d. Upon water or sugar delivery, the rats make approximately 60 licks per trial, drinking 8 µl solution per lick, thus consuming around 10 ml in 20 trials. The time of free water access in the evening during the experimental period can therefore be adjusted in such a way that rats can consume at least 10-15 ml when the bottle is accessible for drinking.
    For the long-term test performed 24 days after the last episode presentation, water deprivation is interrupted at the end of the episode presentations and reinstalled one week prior to the test. It should also be slightly stricter than during exposure to the episodes, because by that time the rats spontaneously decrease their water consumption.

  2. Shaping
    Note: Because the time during which rats are exposed to the episodes is relatively limited, it is essential to minimize stress as much as possible in order to maximize behavioral exploration during episodes. Additionally, during shaping the protocol is adapted to each individual rat in order to start the episodic phase with a relatively homogenous group of rats in terms of their behavioral exploration.
    Habituation to the experimenter
    One week after arrival of the rats the experimenter starts handling and playing with the rats for 5 min twice a day (once in the morning and once in the afternoon), first in the animal facility and later in the experimental room, which continues for 2-3 weeks untill the start of the experiment. Handling should also be done for one week before the long-term test to ensure that the rats get used again to the experimenter (this time only in the animal facility).
    Note: Preferably the same person should carry out the experiments, since it is an episodic memory task and the experimenters might be associated to the context of the episode by the animals. If several experimenters are doing the experiments, one should then pay attention that the same experimenter is performing the episode session and corresponding test (e.g. Episode E1 and Test in the context E1).


    Figure 3. The different phases of the protocol. A. Shaping: Rats are submitted to 11 sessions (1 per day) to habituate them to the cage and the pipette operating system (a nose poke into any of the four odor ports triggers, after a delay, a pipette delivering water (Apparatus shaping, 7 d). During a second phase of shaping, rats are also habituated to receive odor stimulations associated with sugar or quinine from the pipettes (Odors shaping, 4 d). Once shaping is completed, rats undergo 3 routine sessions (R, 1 session a day) with no odor, no enriched context, and only water available from the 4 ports. B. Episodes exposure: During each of these sessions (1 per day) the animals are exposed to 2 distinct episodes [left, Episode 1 (E1) configuration; right, Episode 2 (E2) configuration] with a 1 d routine (R) session in between, or one day rest in home cages with maintained water deprivation. Episodes are presented once or twice in independent groups of rats. They were each characterized by a unique combination of odor-place-context associations rewarded with sugar solution (S): odor A at port 2 for E1; odor C at port 4 for E2, while the 3 other incorrect odor-place associations were associated with quinine solution (Q): odor B at port 2 and odors A and B at port 3 for E1; Odor D at port 4 and odors C and D at port 1 for E2. During episode exposures, in a given context (“in which context”), rats encoded at which port location (“where”), one of the odors (“what”) was associated with sugar solution. This configuration is referred to as P+O+. C and D. Episodic memory tests. During the retrieval test 24 h or 24 days after the last episode session, rats are placed again in one of the context (E2, in this case) to evaluate what type of information they are able to recollect. The 2-port test (C) completely matches the episode in terms of context, odors and locations, except that only water (W) is delivered whatever place-odor configuration is experienced. The challenging 4-port test (D) is a more complex situation since it takes place in the context of E2, but with 4 accessible ports [2 previously associated with context E2 (“In context”, IC, in pink) and 2 associated with context E1 (“out of context”, OC, in blue)]. Each port is associated with a pair of odors that correspond to their respective episode.

    Shaping consists of two phases completed in an average of 11 daily sessions lasting 20 min at maximum. During this period, the appearance of the cage is relatively neutral (see Figure 3A): The floor is black and smooth and no odors, sounds or visual stimulation are used.
    1. Phase1 (apparatus shaping)
      The objective of this phase (5-7 d) is to teach the animal how to access water in the experimental environment. It includes several steps described in details below:
      Step 1: Rats housed in the same home cage are placed together into the experimental arena for 15 min with pipettes from all ports already introduced into the arena and ready to deliver water upon licking. This collective exploration period considerably reduces stress and facilitates the discovery of how to operate the drinking pipettes. This session is repeated the next day with the difference that rats are now placed into the experimental arena individually for 15 min. Once a rat licks from the pipette, we proceed to step B2, otherwise we repeat step B1.
      Step 2: At this stage, rats need to learn to trigger the introduction of the pipette into the cage by making a nose poke to the odor port. Odor ports are made as indentations into the walls of the cage, and the rats naturally come to explore them. At this stage the each pipette becomes accessible for drinking immediately after a nose-poke to the odor port and remains available for drinking for 20 sec. After this the pipette is automatically withdrawn from the cage and has to be triggered by the rat again after a refractory period (purge, duration: a few sec) which is set by the experimenter.
      Note: It is better to choose a short refractory period at the beginning of shaping procedures (e.g. 5 sec) in order to accelerate learning of the link between the nose-poke and the pipette accessibility. For the same reason, it is important for the pipettes to be accessible for a sufficient amount of time (20 sec) to be discovered by the rats. Indeed during this period, rats usually do not wait at a particular port, but are moving around from port to port. Once they have understood how to trigger the pipettes, the amount of time during which the pipettes are accessible is reduced to 10 sec for the following session. If at this stage a rat still does not know how to trigger the pipette, this step can be repeated. This learning unit is acquired once rats perform 10-20 trials (pipette triggering) in 20 min.
      Step 3: A 3 sec offset between nose-poke and pipette entry into the cage is introduced at this stage to habituate the rats to wait for the pipette in front of the port. 10-20 trials should be initiated by the rats in a 20 min session.
      Any of the 3 steps can be repeated for individual rats depending on their performance.
    2. Phase 2 (odor shaping)
      The objective of this phase is to habituate the animals to receive odor stimulations after the nose poke and use it as an indication for the nature of the drinking solution that will be delivered a few seconds later. During this phase, two different odors are used: Eugenol and Geraniol. Hereby, geraniol is always associated with sugar solution, while eugenol is always announcing the distribution of a bitter quinine solution. For a given trial, the same odor-solution combination is delivered in the four ports present in the experimental arena. Each daily session consisted of 15 to a maximum of 24 trials initiated by the rats in 20 min. The session consists of a sequence of pseudo-randomly distributed geraniol and eugenol trials and is repeated on four consecutive days. The 3 sec delay between a nose-poke coupled with odor release and delivery of the pipette gives the animal some time to process the odor without being distracted by the pipette moving into the cage. Geraniol and eugenol are used uniquely in this shaping procedure.
      During this phase, the rats progressively learn that the odor coming from the odor port can be predictive of a positive or negative reward. As a consequence, their behavioral response is modified and they begin to avoid licking the quinine solution on the basis of eugenol cue. Depending on the rat, the odor shaping phase will take from 3 to a maximum of 4 d before such behavioral change is observed.
      Note: The first three trials of every shaping session are always positively reinforced in order to ensure that the animal receives sugar before quinine drinking solution and remains motivated throughout the session.

  3. Episodic task
    1. Routine (Figure 3A)
      Prior to exposure to the episodes, rats undergo routine sessions for 3 consecutive days, with one session per day (Figure 3A). Each session consists of 12-24 trials performed in the same condition as step 3 of phase 1 of shaping. Routine sessions are thus characterized by a contextual environment with which rats are already familiar from shaping procedures (smooth black floor, no specific acoustic and visual ambiance), without any odors being released from the odor ports and with water as the only drinking solution. The objectives of the routine sessions are: First, to verify that there is an even licking distribution among the ports without any preference for a particular place; second, to further monitor and adjust water deprivation (especially when there is a delay between the shaping and the experimental phase due to e.g. some surgical procedures); and finally, to increase the memorability of the following episodes, by contrasting them to accustomed and highly familiar routine sessions.
    2. Episodes (Figure 3B)
      Three essential characteristics distinguish episodes from routine sessions:
      1. The context: The simple black and smooth floor of the cage used during shaping and routine sessions is replaced by various materials specific to each episode. Distinct visual patterns are displayed on the floor of the arena by a projector to increase discrepancy between different episodes. Acoustic ambiance is enriched through the playing of natural sounds or music through the loudspeakers. For example, Episode 1 is characterized by a smooth white floor, projection of white triangles on a dark background and sounds of crickets. Episode 2 is characterized by a granular white floor, projection of white background with black circles and sounds of birds singing in the rain.
      2. Only two of the four ports of the cage can be activated by the rat in each episode, ports 2 and 3 for E1, and ports 4 and 1 for E2 (Figure 3). When a rat makes a nose poke to any of the two remaining non-active ports, nothing happens.
      3. On the active ports of a given episode two novel odors are delivered. For simplicity, odor A and B in Episode 1 and odors C and D in Episode 2. As a consequence, in each episode, four different combinations of odor-place can be encountered. Among these possibilities only one odor-place combination is rewarded with sugar solution whereas the three others are associated with quinine. For example, in E1, sugar will be delivered when odor A is presented in port 2 (referred to as P+O+, positively reinforced place and odor). When odor B is presented on the same port, the animal will receive quinine (combination referred to as P+O-). The two other combinations involving port 3 will also lead to quinine: Odor A on port 3 is referred as P-O+ and odor B on the same port as P-O-.
        Each episode session consists of 12-24 trials and lasts 20-40 min. As during the routine session, the animal is free to explore the environment. Consequently, the number of trials can vary depending on the exploratory motivation of the rat. The odors are presented at the assigned ports in a pseudorandom sequence with three repetitions of the same odor-port configurations every 12 trials. Episode sessions are separated by either one routine session (R) or one day rest in home cages with maintained water deprivation. Depending on the experiments, each episode can be repeated once before the retention test, when more homogenous performance of the group is needed (e.g. when the effect of a drug is tested on episodic memory performance).
        To summarize:


        Episode 1. Odor A (O+) is associated with a sugar drinking solution at port 2 (P+O+) and with quinine solution at port 3 (P-O+). Odor B (O-) is associated with quinine solution at port 2 (P+O-) and port 3 (P-O-).
        Episode 2. Odor C (O+) is associated with sugar drinking solution at port 4 (P+O+) and with quinine solution at port 1 (P-O+). Odor D (O-) is associated with quinine solution at port 4 (P+O-) and port 1 (P-O-).

    3. Memory recall test (Figure 3C or 2D)
      The test can be performed 24 h or 24 days after the last exposure to Episode 2 to probe recent or remote long-term memory, respectively. In both tests, quinine and sugar are replaced by water and performance of the rats is estimated by taking into account only the 12 first trials of the test. Two versions of the test that differ in their levels of difficulty can be carried out.
      2-Port Test: The test situation matches completely Episode 2 (Figure 3C). The same contextual environment as during the episode exposure is present. The same two ports are accessible and deliver the same two odors as during the corresponding episode (odors C and D, at ports 1 and 4).
      4-Port Test: The test takes place in the context corresponding to Episode 2 (same visual, auditory and tactile information), but this time all 4 ports are accessible for drinking, each delivering the same odors as during the corresponding episodes (Figure 3D). As during exposure to episode 2, ports 1 and 4 are releasing odors C and D and are therefore referred to as “In context” (IC) combinations, additionally ports 2 and 3 are releasing odors A and B, as during exposure to episode 1 and are referred to as “out of context” (OC) combinations.
      Note: One modification introduced to the original protocol is the removal of the routine sessions between the episodes. Instead of routine sessions the rats are kept in their home cages on the same water deprivation schedule as used before the shaping procedures (~30 min access to water in the morning and evening). We didn’t notice a difference in performance between the two protocols (with either a routine session between the episodes, or one day rest in the home cage with maintained water deprivation) and recommend therefore the simpler version of the protocol which doesn’t include routine sessions between the episodes.
      Note: The level of difficulty of both tests can be additionally adjusted by using more overlapping or distinct contextual features, such as e.g. the flooring material of the experimental cage, or the chemical proximity of the odors.

Analysis

The output files of the experiments contain the data about the total amount of licks done for each odor-port configuration on every single trial.

  1. To normalize the measures of licking behavior across rats, the licks of all the trials that correspond to a specific odor-port configuration (e.g. P+O+) are summed up for a given rat and divided by the total amount of licks made by this rat in the respective session (lick ratio).
  2. The group performance is determined by calculating the group mean of the ratios (calculated for each individual rat in step B1) by odor-port configuration, which is referred to as the licks index.
  3. The same calculations can be performed by analyzing the number of times rats encountered each of the different odor-port configurations (referred to as visits). For this, trials performed on a given odor-port configuration are summed up and divided by all the trials made in a session for each individual rat, from which the group mean performance is calculated.

Representative data

Figure 4 depicts a group performance of 6 rats that experienced two exposures to episodes E1 and E2 according to the experimental protocol described above. Recollection memory of Episode 2 was tested after a retention interval of 24 h in a 2-port test. As a group, rats remembered the correct, combined odor-place information linked to the context of the previously experienced E2 episode, since they were making significantly more licks at P+O+ configuration of the E2 context compared to all other odor-port configurations. Nonparametric Friedman test, followed by Wilcoxon test were used for statistical analysis (* p < 0.05).


Figure 4. Group memory performance in a 2-port test. 24 h after two exposures to the episodes. In the group as a whole there are more visits to positively reinforced port P+, which delivered sugar during the initial exposure to the episode, compared to P- where no sugar is delivered irrespective of the odor (visits ratio: blue bars). Despite the fact that during the test session water is delivered in every encountered configuration, the group shows a significantly higher lick ratio for the previously positively reinforced odor-port configuration (P+O+) compared to the three other odor-port configurations in the given context (lick ratio: black bars) (*p<0.05). Figure adapted from Veyrac et al. (2015). As an illustration, Videos 1 and 2 are provided below.

Video 1. Example of a rat exhibiting a drinking behavior. The animal puts his nose into the odor port, waiting the release of the pipette and drinks until the end of the trial.

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Video 2. Example of a rat with an avoidance behavior. The animal puts his nose into the odor port, samples the odor associated with quinine and avoids drinking.

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Beside the group analysis, individual results are also considered to determine the memory recall profile of each animal according to respective test situation. Two examples are presented in Figure 5. These individual profiles allow classification of each animal according to the type of information it is able to retrieve in the different test situations.


Figure 5. Examples of licking profiles for 4 different rats during the recall test of episode 2, in a 2-port (A and B) and a 4-port test condition (C). A. Performance of a rat presenting a typical “What-Where” profile, This rat drinks more on the good port associated with the tested context and with the correct odor (P+O+), thus showing a good episodic recollection. B. Performance of a rat presenting a typical “Where” profile. This rat drinks on the good port associated with the tested context (P+) but does not exhibit any discrimination between the two odors, thus showing a good memory only for the place information associated with this episode. C. The 4-port test situation allows it to test explicitly if the animal is able to retrieve the whole episode. Examples of two different memory profiles observed in the 4-port test performed in the context of episode 2. WW-IC: this rat is categorized as “What-Where-in context”, the rat is licking mainly at the P+O+ configuration of the port which delivered sugar in the tested context of episode 2. WW-OC: This rat is categorized as “What-Where-out of context”, the rat is licking at the P+O+ configuration corresponding to episode 1 that was not currently tested.

Acknowledgments

The protocol presented here is adapted from Veyrac et al. (2015). This work was supported by CNRS, University Lyon 1, University Paris-Sud 11, and by grants from the “Agence Nationale de la Recherche” (ANR-2010-BLAN-1413-01) to SL and NR, from the “Fondation pour la recherche médicale” (FDT20140930863) to AG and from LABEX CORTEX (ANR-11-LABX-0042) to NR.

References

  1. Courtiol, E., Lefevre, L., Garcia, S., Thevenet, M., Messaoudi, B. and Buonviso, N. (2014). Sniff adjustment in an odor discrimination task in the rat: analytical or synthetic strategy? Front Behav Neurosci 8: 145.
  2. Herlitz, A. and Viitanen, M. (1991). Semantic organization and verbal episodic memory in patients with mild and moderate Alzheimer's disease. J Clin Exp Neuropsychol 13(4): 559-574.
  3. Martin, C., Gervais, R., Hugues, E., Messaoudi, B. and Ravel, N. (2004). Learning modulation of odor-induced oscillatory responses in the rat olfactory bulb: a correlate of odor recognition? J Neurosci 24(2): 389-397.
  4. Sezille, C., Messaoudi, B., Bertrand, A., Joussain, P., Thevenet, M. and Bensafi, M. (2013). A portable experimental apparatus for human olfactory fMRI experiments. J Neurosci Methods 218(1): 29-38.
  5. Torquet, N., Aime, P., Messaoudi, B., Garcia, S., Ey, E., Gervais, R., Julliard, A. K. and Ravel, N. (2014). Olfactory preference conditioning changes the reward value of reinforced and non-reinforced odors. Front Behav Neurosci 8: 229.
  6. Veyrac, A., Allerborn, M., Gros, A., Michon, F., Raguet, L., Kenney, J., Godinot, F., Thevenet, M., Garcia, S., Messaoudi, B., Laroche, S. and Ravel, N. (2015). Memory of occasional events in rats: individual episodic memory profiles, flexibility, and neural substrate. J Neurosci 35(19): 7575-7586.

简介

在人类中已经定义了特殊记忆,因为对日常生活中经常发生的独特个人过去经历的自觉记忆,包括记忆发生的事情,发生的时间和地点(Tulving,1972)。在这里,我们提出并详细描述了一种我们最近用来测试大鼠形成和记忆以前遇到的偶发事件的情景样记忆的能力的新方案(Veyrac等,2015)。在这些情节中,动物短暂地暴露于不同的多感官富集环境(在其中发生的情况/场合)中遇到的特定气味饮料组合(发生了什么事)。可以在低或高干扰检索情况下相对较短(24小时)或更长时间(24天)延迟测试剧集的记忆。这种新颖的范例为召回性能的个人记忆特征提供了可能与脑功能网络的不同方面相关的证据。更一般来说,它提供了新的可能性来探索细胞和网络机制,这些机制是脑病变过去事件和记忆功能障碍的记忆。

关键字:情景记忆, 动物模型, 嗅觉, 陈述性记忆, 啮齿类动物

材料和试剂

  1. 主题
    所有实验根据欧洲实验动物护理指南(2010/63/EU)进行,并且获得了里昂1大学伦理委员会(允许DR2015-46)的批准。在脱水方案开始时,7-8周龄(?300-350g)的成年雄性Long-Evans大鼠(Charles River Laboratories)以2-4只/笼的形式圈养,并保持在具有控制温度的环境和湿度在12/12小时光照/黑暗循环下,随意食用。实验在光照期间进行。
    注意:到目前为止,这些实验还没有对其他品系的大鼠或小鼠进行,但我们假设其他品系的使用不应该带来进行实验的主要困难。然而,根据上下文的特点是视觉刺激的事实,使用白化病大鼠可能更具挑战性。对于鼠标,需要对实验设备进行一些调整。
  2. 气味
    为了以完全受控的方式通过新一代的嗅觉仪递送(Sezille等人,2013),将每种气味剂引入U形Pyrex (体积:10ml;长度:50mm;外径:14mm)(VS技术)填充微孔颗粒。在这些实验中使用的所有气味获自Sigma-Aldrich,France(参见下文的a-f)。
    1. 香叶醇(Sigma-Aldrich,目录号:163333)20%饱和蒸气压
    2. 丁香酚(Sigma-Aldrich,目录号:E51791)18%饱和蒸气压
    3. (S) - (+) - Carvon(Sigma-Aldrich,目录号:435759)35%饱和蒸汽压=气味A
    4. 异戊基乙酸酯(Sigma-Aldrich,目录号:W205508)15%饱和蒸汽压=气味B
    5. Trans-Anethole(Sigma-Aldrich,目录号:117870)30%饱和蒸汽压=气味C /
    6. 柠檬醛(顺式+反式)(Sigma-Aldrich,目录号:W230308)20%饱和蒸汽压=气味D
      注意:在开始实验之前,在2和4ml之间的纯气味 溶液在连续几天逐渐引入U ?成形玻璃管直到微孔颗粒出现饱和 (图1)(对于实验,气味应当处于饱和蒸汽 压力在管中,同时为了防止嗅觉仪 污染重要的是避免液体在液体中的积聚 底部)。饱和蒸汽压的百分比 引入气流中的每种气味单独调节 实验者。目的是获得一个强度 感觉到动物,但足够温和,以防止任何回避 行为。对于成对使用的不同气味,我们也尝试均衡 他们的感知强度。选择这些特殊的气味和 浓度主要通过实验室中的先前实验确定 显示他们容易辨别和缺乏他们的自然 在所使用的浓度下对大鼠的吸引力或排斥力 (Martin et al。,2004; Courtiol et al。,2014; Torquet et al。,2014)。


      图1.填充有微孔的U形Pyrex管的照片 颗粒显示不同水平的饱和度与Carvon气味 解。 从左到右:纯颗粒,没有任何额外的气味 解;部分饱和颗粒;完全饱和的颗粒 用于实验
  3. 饮用溶液
    1. 6%蔗糖溶液用作阳性增强剂(Sigma-Aldrich,目录号:数量:84097)
    2. 0.06%盐酸奎尼汀脱水物溶液用作负增强物(Sigma-Aldrich,目录号:Q1125)

设备

  1. 装置(EPISODICAGE)
    实验笼是配备有用于递送不同气味和饮用溶液(图2A)(Belkacem Messaoudi)的4个装置的PVC矩形盒(60×35×40cm)。在两个相对的壁上,距离盒的每个角5cm的距离,笼子包含气味和饮用端口的安排。气味口是进入壁(5×5cm大小)的圆形缺口,具有饮用口(小孔,尺寸为1×1cm),位于气味口下方1cm处,饮用吸管可通过该口插入笼(图1B)。


    图2.EPISODICAGE。 A.实验笼子的一般视图,显示在四个气味端口上方安装的四个摄像机。 B.具有相关联的饮用移液管(P)的气味端口(OP)的细节,从内部(左图)或外部(右图)看到的气味注入(OI)和气味提取(M),其允许将吸液管从保持架中引入和抽出。 C.每个鼻子戳首先触发气味传递13秒。在气味发生后三秒钟,将含有各种饮用溶液(水,糖或奎宁)的移液管引入笼中10秒钟,直到气味刺激关闭。由大鼠制成的移液管上的每个舔子由专用于控制实验笼的计算机检测和记录。每次试验从鼻子开始,结束吸管和气味关闭的撤除。 D.在左边,在常规会话中使用的版本中的笼子的外观。另外两张图片说明了如何通过在地面上投影视觉图案(中心图片)或引入对象(右图片)来修改笼子的外观。图改编自Veyrac A. 等人(2015)。

    1. 气味端口
      当大鼠使鼻子捅进端口,电容 改变被系统检测到立即触发13秒 将气味饱和蒸汽引入恒定气流(空气 总流速:1升/min)。加臭空气的比例 通过定制的嗅觉计控制气味剂的质量 ?连接到气味端口(OI)(图1B)。在给定的实验中 会话,五种不同的气味可以在每种四种气味下交付 端口。基于文丘里管的抽吸系统允许抽取 加味的空气通过单独的通道,使气味残留 限于端口(OE)(图1B)。 OI和的永久补偿 ?OE防止由于气味开关导致的总气压的任何变化。
      注意:大鼠对气压的变化非常敏感。防止 任何对机械刺激的行为反应,空气压力必须 在存在和不存在气味的情况下保持在相同的水平。
    2. 饮水港
      每个可移动饮用移液管连接到泵。移液器是 在动物的第一鼻子戳后3秒引入笼中 并在10秒后同时从笼中取出 终止气味释放(图1C)。这保证了气味 刺激在整个液体消耗期间递送。一秒 集成在饮用吸管中的电容变化传感器检测 动物的每一个舔和触发泵,反过来 提供校准量的8-9μl的饮用溶液。舔 由大鼠做出的记录作为文本文件用于以后的分析 结果。
      注意:每个舔的液体量 饮用端口必须在端口之间相等(变化应该 ?在8±1μl内),以避免动物对a的任何偏爱 ?特定端口。
      不同的饮用溶液可以通过 系统,水,蔗糖或奎宁溶液 不同阶段的协议和气味端口配置。后 ?每次试验和收缩移液管,饮用溶液装置 用以下试验的溶液吹扫 注意:清除 持续时间以允许完全洗出的方式设置 饮用溶液的最后一次试验。这通过加入来验证 的墨水进入实验开始之前的水。
  2. 在剧集中上下文丰富的项目
    语境
    不同类型的声音可以通过对称地放置在实验舞台上方的两个扬声器播放,以增强剧集的可辨别性:自然声音,鸟歌曲,钢琴音乐...等。选择这些声音的一般准则是它们在频谱图和动物/实验者的健康方面的可辨别性 触觉和视觉语境
    可以使用具有不同触觉和视觉特性的不同类型的地板(图1D)。由于大鼠的颜色视觉差,优选材料的高对比度黑白着色的组合。另外,视觉图案也可以通过视频投影仪显示在地板上,并且各种物体可以放置在箱体中。
  3. 视频录制
    五个照相机用于精确地监测大鼠的行为,一个放置在实验箱的中央上方,另外四个放置在四个气味端口上方。通过视频跟踪软件从中央摄像机记录的信号在线检测大鼠的位置。当动物接近端口时,选择相应的摄像机用于信号采集。这使我们可以节省视频文件占用的空间

软件

  1. VOLCAN(Marc Thevenet)

程序

该方案的所有必要步骤总结在图3中。在成形程序开始之前,将大鼠习惯于实验者,并建立脱水。

  1. 水剥夺程序
    在实验期间,并且最关键的是在成形期间,动物必须被激励到足以激活饮用装置。这需要控制地减少他们在动物住房设施中的水的获得。它不能太严重以保持动物在良好的生理状态,但足够保持他们以受控和稳定的方式动机。在它们到达动物设施之后一周,当动物习惯于他们的新的住房环境时,通过测量饮用前后的瓶重量,对每个笼子的每日自来水消耗进行量化。一个星期后逐步引入水限制:在第一个2d的剥夺期间,在早晨(上午9点至上午11点)和晚上(下午4点至下午6点),在他们的家笼中可以使用饮用移液器2小时,则在接下来的2d期间访问被减少到1h,在随后的7d期间被减少到40分钟。在此期间,我们控制在自由使用期间消耗的水的总量接近先前的消耗,并且因此调整晚上访问的持续时间。此外,定期检查动物的重量,以确保其在整个方案中保持稳定(±5%)。在行为实验期间,如果大鼠在早上测试,他们只在晚上接受水20-40分钟,但如果他们在下午测试,他们在早上约5分钟和20-40分钟接受水晚上。此外,根据实验场地中的动物消耗的水量,可以在实验中一直调整晚上取水。
    注意:在20分钟内,大鼠预期进行至少10次试验。对于不进行预期数量的试验并且不表现出缺乏动机的其他迹象的动物,减少了对饮用水的存取时间(例如从40至20分钟)。另外,减少在第一次发作之前的那天的饮酒时间量有助于增加大鼠的动机,并克服由于暴露于新的情况而产生的压力。
    当水限制已经建立时,动物的水消耗估计为约20ml/d。在水或糖递送时,大鼠每次试验大约60次舔,每次舔饮用8μl溶液,因此在20次试验中消耗大约10ml。因此,在实验期间晚上自由饮水的时间可以以这样的方式调节,即当瓶子可接近饮用时,大鼠可以消耗至少10-15ml。
    对于在最后一次发作后24天进行的长期测试,在发作结束时中断水分剥夺,并在测试前一周重新安装。它也应该比暴露于情节稍微更严格,因为那时大鼠自发地减少他们的水消耗。

  2. 整形
    注意:因为大鼠暴露于发作的时间相对有限,所以尽可能最小化压力以便在发作期间最大限度地进行行为探索是必要的。另外,在成形期间,将方案适应于每只大鼠,以便在其行为探索方面用相对同质的大鼠组开始发作阶段。
    习惯给实验者
    在大鼠到达后一周,实验者开始处理和与大鼠一起播放5分钟,每天两次(一次在早晨和下午一次),首先在动物设施中,然后在实验室中,继续2 -3周直到实验开始。处理也应在长期测试前一周进行,以确保大鼠再次用于实验者(此次仅在动物设施中)。
    注意:优选地,同一个人应该进行实验,因为它是一种情景记忆任务,并且实验者可以与动物的情节的上下文相关联。如果几个实验者正在进行实验,那么应该注意同一个实验者正在进行情节会话和相应的测试(例如,在上下文E1中的Episode E1和Test)。


    图3.协议的不同阶段。A.整形:大鼠被提交到11次会议(每天1次),以使他们习惯于笼子和移液器操作系统(鼻子戳入任何在成型的第二阶段期间,大鼠还习惯于从移液管接收与糖或奎宁相关的气味刺激(气味成型,4 d。一旦成型完成,大鼠进行3次常规会话(R,每天1次),没有气味,没有富集的背景,并且只有来自4个端口的水B.暴露在这些期间每天),将动物暴露于2个不同发作[左,第1组(E1)构型;右,第2组(E2)构型],其间有1天的常规(R)持续的水剥夺在独立的大鼠组中呈现一次或两次,每个特征在于得到糖溶液(S)的气味 - 地方 - 环境关联的独特组合:E1口2处的气味A;气味C在端口4为E2,而其他3个不正确的气味 - 位置关联与奎宁溶液(Q):端口2的气味B和端口3的气味A和B为E1; E2端口4的气味D和端口1的气味C和D。在发作期间,在给定的上下文("在哪个上下文中"),大鼠在哪个端口位置("where")编码,气味("what")之一与糖溶液相关联。该配置称为P + O +。 C和D.情景记忆测试。在最后一次发作后24小时或24天的恢复测试期间,将大鼠再次放置在上下文(在这种情况下为E2)中的一个中,以评价它们能够记住什么类型的信息。 2端口测试(C)在上下文,气味和位置方面完全匹配该情节,除了只有水(W)被递送任何地方 - 气味配置经历。具有挑战性的4端口测试(D)是更复杂的情况,因为它发生在E2的上下文中,但是具有4个可访问端口[2]先前与上下文E2("在上下文中",IC,粉色)与上下文E1("不在上下文",OC,在蓝色)]。每个端口与一对对应于它们各自的情节的气味相关联。

    整形由两个阶段完成,平均每天11次,最多持续20分钟。在此期间,笼子的外观相对中性(见图3A):地板是黑色和光滑的,没有使用气味,声音或视觉刺激。
    1. 阶段1(设备整形)
      这一阶段(5-7 d)的目标是 ?教导动物如何在实验环境中获得水。 它包括以下详细描述的几个步骤:
      步骤1:大鼠 安置在同一个家笼里一起放入实验 竞技场15分钟用移液器从所有港口已经引入 竞技场和准备在舔时交付水。这个集体 探索期大大减少压力和便利 发现如何操作饮用吸液管。这个会话是 重复第二天与大鼠现在放入的区别 实验竞技场单独15分钟。一旦大鼠舔 移液器,我们进行步骤B2,否则我们重复步骤B1 步骤2:在这个阶段,大鼠需要学会触发引入 ?通过使鼻子戳到气味端口而将移液管插入笼中。气味 端口做成凹陷进入笼子的壁,和老鼠 自然来探索他们。在这个阶段,每个移液器变成 在鼻子戳到气味端口后立即饮用 并保持可饮用20秒。之后,移液器 ?自动从笼中抽出并且必须由 大鼠在不应期后(清除,持续时间:几秒)再次 由实验者设置 注意:最好选择一个短 在成形过程开始时(例如5秒)的不应期 ?为了加速学习鼻嗅之间的联系 移液器无障碍。出于同样的原因,重要的是 移液器可以接近足够的时间(20秒) ?发现由老鼠。事实上在这期间,老鼠通常不会 在特定端口等待,但正在从端口移动到端口。一旦 ?他们已经了解如何触发移液器,时间量 在此期间移液管可接近的时间减少到10秒 下次会议。如果在这个阶段一个老鼠仍然不知道如何 触发移液器,此步骤可重复。这个学习单位是 获得一旦大鼠在20分钟内进行10-20次试验(移液管触发)。
      步骤3:鼻尖和移液管之间偏移3秒 在该阶段引入笼子以习惯大鼠等待 移液管在口岸前面。 10-20次试验应由 大鼠在20分钟的会话 对于单个大鼠,可以根据其性能重复3个步骤中的任何一个
    2. 阶段2(气味整形)
      这个阶段的目的是习惯动物接受气味 ?鼻子刺激后使用它作为指示 饮用溶液的性质将被交付几秒钟 后来。在这个阶段,使用两种不同的气味:丁香酚和 Geraniol。因此,香叶醇总是与糖溶液相关, 而丁香酚总是宣布苦瓜的分布 解。对于给定的试验,相同的气味 - 溶液组合是 交付在实验舞台上的四个港口。每 每天的会话由15个到最多24个试验发起 ?大鼠在20分钟。会话由伪随机序列组成 分布的香叶醇和丁香酚试验,并重复四次 连续多日。鼻涕与气味之间的3秒延迟 释放和递送移液器给动物一些时间 处理气味,而不会被移液管移动而分心 ?笼。香叶醇和丁子香酚在该成型过程中独特地使用 在这个阶段,大鼠逐渐学到气味来了 从气味端口可以预测正或负的奖励。 因此,他们的行为反应被修改,他们开始 以避免基于丁香酚提示舔奎宁溶液。 根据大鼠,气味形成阶段将从3至a 在观察到这种行为变化之前最多4天。
      注意: 每个成型阶段的前三个试验总是积极的 加强,以确保动物之前收到糖 奎宁饮用溶液,并在整个会话期间保持动力。

  3. 情节任务
    1. 例程(图3A)
      在暴露于发作之前,大鼠经历 常规会话连续3天,每天一次 (图3A)。每个会议包括在同一个12-24个试验 ?条件作为成形阶段1的步骤3。因此,例行会议 其特征在于与大鼠已经在其中的语境环境 熟悉成形程序(光滑的黑色地板,无具体 声学和视觉氛围),没有任何气味释放 ?气味口和用水作为唯一的饮用溶液。目标 ?的例行会话是:第一,验证是否有均匀 舔分配在港口之间没有任何偏爱 特殊地方第二,进一步监测和调节水 剥夺(特别是当成形和之间存在延迟时) 由于例如一些外科手术的实验阶段);和 最后,提高下面剧集的记忆力 将它们与习惯和高度熟悉的日常会话进行对比
    2. 剧集(图3B)
      三个基本特征将剧集与日常会话区分开来:
      1. 上下文:使用笼子的简单黑色和光滑的地板 在成型和常规会话期间被各种材料替代 特定于每一集。不同的视觉模式显示在 地板的竞技场由投影仪增加之间的差异 不同的情节。声音环境通过演奏丰富 ?自然声音或音乐通过扬声器。例如,第1集 ?其特点是平滑的白色地板,投影的白色三角形 ?在蟋蟀的黑暗的背景和声音。第2集的特点 ?由一个粒状白色地板,白色背景的投射与黑色的 圈子和在雨中唱歌的鸟的声音
      2. 只有两个 笼子的四个端口可以由大鼠在每一集中激活, 端口2和3用于E1,端口4和1用于E2(图3)。当一只老鼠 使鼻子捅到两个剩余的非活动端口中的任一个,什么也没有 发生。
      3. 在一个给定的剧集的活跃港口两个新颖的气味 交付。为简单起见,第1集中的气味A和B和气味C和 ?D在第2集。因此,在每一集,四个不同 可能遇到气味地点的组合。在这些可能性中 ?只有一个气味 - 地方组合奖励糖溶液,而 ?其他三个与奎宁相关。例如,在E1中,糖 ?将在气味A出现在端口2(称为 P + O +,积极增强的地方和气味)。当气味B出现时 ?同样的港口,动物会收到奎宁(组合指 ?作为P + O-)。涉及端口3的两个其他组合也将导致 ?奎宁:在端口3上的气味A在相同上被称为P-O +和气味B. 端口为P-O-。
        每集包含12-24个试验 持续20-40分钟。在例行会话期间,动物是自由的 探索环境。因此,试验的数量可以变化 取决于大鼠的探索动机。气味是 在具有三个的伪随机序列中在所分配的端口处呈现 每12次试验重复相同的气味端口配置。 剧集会话由一个例程会话(R)或一个例程会话分隔 日休息在家庭笼子与维护的水剥夺。取决于 实验,每次发作可以重复一次保留 当需要更均匀的群体性能时(例如) ?测试药物对情景记忆性能的影响)。
        总结:


        第1集。气味A(O +)与糖饮用溶液有关 端口2(P + O +)和在端口3的奎宁溶液(P-O +)。 气味B(O-)是 ?与端口2(P + O-)和端口3(P-O-)的奎宁溶液相关 第2集。气味C(O +)与糖饮用溶液有关 端口4(P + O +),并在端口1(P-O +)使用奎宁溶液。气味D ?与端口4(P + O-)和端口1(P-O-)的奎宁溶液相关。

    3. 记忆回忆测试(图3C或2D)
      该测试可以在最后一次暴露后24小时或24天进行 第2集分别探测最近或远程长期记忆。在 两项试验中,奎宁和糖都被水和性能所取代 大鼠估计只考虑12次第一次试验 ?考试。两个版本的测试,其水平不同 难度可以进行。
      2端口测试:测试情况 完全匹配第2集(图3C)。相同的上下文 环境,如在发作暴露期间存在。同样的两个 端口是可访问的,并提供相同的两种气味 相应的剧集(气味C和D,在端口1和4) 4端口 测试:测试发生在对应于剧集2的上下文中 (相同的视觉,听觉和触觉信息),但这一次全部4 端口可以??饮用,每个提供相同的气味 (图3D)。在曝光期间 第2集,端口1和4释放气味C和D,因此 称为"在上下文"(IC)组合,另外端口2和3 ?释放气味A和B,如在暴露于第1集和暴露期间 称为"不在上下文中"(OC)组合 注意:一 修改引入原来的协议是删除 在剧集之间的例行会话。而不是日常会话 大鼠在相同的剥水时间表中保持在它们的家笼中 ?如在成型程序之前使用(?30分钟获取水中的水 早晚)。我们没有注意到性能的差异 之间的两个协议(与之间的例行会话 发作,或一天休息在家庭笼子里与维持水 剥夺),因此建议更简单的协议版本 ?其中不包括 情节之间的例行会话。
      注意: 两个测试的难度水平可以通过另外调整 使用更多重叠或不同的上下文特征,例如的 ?实验笼的地板材料或化学接近度 的气味。

分析

实验的输出文件包含关于每次试验中每个气味端口配置所进行的舔的总量的数据。

  1. 为了使大鼠的舔行为的测量标准化,对于给定的大鼠,将对应于特定气味端口配置(例如


    P + O +)的所有试验的舔总数除以总数该鼠在各个阶段中舔的虱数(舔比率)。
  2. 通过计算气味口配置(称为舔指数)计算比值的组平均值(步骤B1中每只大鼠的计算值)来确定组的性能。
  3. 通过分析大鼠遇到的每种不同的气味口配置(称为访问)的次数,可以进行相同的计算。为此,对给定的气味口配置进行的试验被总结并除以对于每个大鼠在会话中进行的所有试验,从中计算组平均表现。

代表数据

图4描述了根据上述实验方案经历两次暴露于发作E1和E2的6只大鼠的组表现。在2端口测试中在24小时的保留间隔之后测试了第2集的回忆记忆。作为一组,大鼠记住与先前经历的E2发作的背景相关联的正确的组合气味 - 位置信息,因为与所有其他气味端口配置相比,它们在E2上下文的P + O +配置下显着更多舔。使用非参数Friedman检验,接着进行Wilcoxon检验用于统计分析(* p <0.05)。


图4. 2端口测试中的组内存性能。两次曝光后的24小时。在整个组中,更多的访问积极加强端口P +,在最初的暴露于发作期间传送糖相比,其中没有糖没有传递,无论气味(访问率:蓝色酒吧)。尽管事实上在测试期间在每个遇到的配置中输送水,与给定的三种其它气味端口配置相比,该组显示了先前积极增强的气味端口配置(P + O +)的显着更高的舔气比率上下文(舔比:黑条)(* p <0.05)。图改编自Veyrac等人(2015)。例如,下面提供了视频1和2
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视频1.表现出饮酒行为的大鼠的实例。 动物将他的鼻子放入气味口,等待释放移液器和饮料,直到试验结束。
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视频2.具有回避行为的老鼠的示例。 动物将他的鼻子放入气味口,取样与奎宁相关的气味,避免饮酒。
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除了组分析之外,还考虑个体结果以根据相应的测试情况确定每只动物的记忆回忆曲线。在图5中示出了两个示例。这些单独的简档允许根据能够在不同的测试情况中检索的信息的类型来对每只动物进行分类。


图5.在2端口(A和B)和4端口测试条件(C)下在第2集的召回试验期间4只不同大鼠的舔曲曲线的实例。 的大鼠呈现典型的"什么地方"的轮廓,该大鼠喝更多的与测试上下文和正确的气味(P + O +)相关联的良好端口,从而显示良好的情景记忆。 B.呈现典型"地点"概况的大鼠的性能。该鼠在与测试的上下文(P +)相关联的良好端口上饮用,但是不表现出两种气味之间的任何区别,因此仅对与该剧集相关联的地点信息显示良好的记忆。 C. 4端口测试情况允许它明确测试动物是否能够检索整个剧集。在第2集的上下文中进行的4端口测试中观察到的两种不同的记忆概况的实例.WW-IC:该大鼠被分类为"在什么情况下",大鼠主要在P + O +构型在第2集的测试环境中递送糖的WW口。WW-OC:该大鼠被分类为"在什么情况下上下文",大鼠在对应于第1集的P + O +构型舔that目前测试。

致谢

这里提出的方案改编自Veyrac等人(2015)。这项工作得到了国家科学研究中心(CNRS),里昂大学1,巴黎大学11大学以及来自"Agence Nationale de la Recherche"(ANR-2010-BLAN-1413-01)至SL和NR,"Fondation pour (FDT20140930863)到AG,从LABEX CORTEX(ANR-11-LABX-0042)到NR。

参考文献

  1. Courtiol,E.,Lefevre,L.,Garcia,S.,Thevenet,M.,Messaoudi,B.and Buonviso,N。 大鼠气味辨别任务中的嗅觉调整:分析或综合策略? < em> Front Behav Neurosci 8:145.
  2. Herlitz,A。和Viitanen,M。(1991)。 轻度和中度阿尔茨海默病患者的语义组织和言语情景记忆。 J Clin Exp Neuropsychol 13(4):559-574
  3. Martin,C.,Gervais,R.,Hugues,E.,Messaoudi,B.and Ravel,N。(2004)。 学习调节大鼠嗅球中气味诱发的振荡反应:气味识别的相关性?/a> J Neurosci 24(2):389-397
  4. Sezille,C.,Messaoudi,B.,Bertrand,A.,Joussain,P.,Thevenet,M.and Bensafi,M.(2013)。 用于人嗅觉fMRI实验的便携式实验装置 J Neurosci Methods 218(1):29-38。
  5. Torquet,N.,Aime,P.,Messaoudi,B.,Garcia,S.,Ey,E.,Gervais,R.,Julliard,A.K.and Ravel,N。(2014)。 嗅觉偏好调节改变了强化和非强化气味的奖励值。 Front Behav Neurosci 8:229.
  6. Veyrac,A.,Allerborn,M.,Gros,A.,Michon,F.,Raguet,L.,Kenney,J.,Godinot,F.,Thevenet,M.,Garcia,S.,Messaoudi, Laroche,S。和Ravel,N。(2015)。 记忆大鼠偶发性事件:个别情景记忆情况,灵活性和神经基质。 J Neurosci 35(19):7575-7586。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Allerborn, M., Gros, A., Messaoudi, B., Gervasoni, D., Garcia, S., Thevenet, M., Laroche, S., Veyrac, A. and Ravel, N. (2016). A Novel Task for Studying Memory of Occasional Events in Rats. Bio-protocol 6(5): e1740. DOI: 10.21769/BioProtoc.1740.
  2. Veyrac, A., Allerborn, M., Gros, A., Michon, F., Raguet, L., Kenney, J., Godinot, F., Thevenet, M., Garcia, S., Messaoudi, B., Laroche, S. and Ravel, N. (2015). Memory of occasional events in rats: individual episodic memory profiles, flexibility, and neural substrate. J Neurosci 35(19): 7575-7586.
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