Abstract
This century-old observation is still valid today, despite everything we have learned about the mammal nervous system, especially in the area of neurobiology of learning and memory. After “training” an experimental animal, such as a rat or a mouse, the only way to be sure that a “memory” was formed is by evoking it back, i.e., by recalling it in a “test” session: this “memory” is expressed by a behavior that differs from that one emitted in the training session. Until proof to the contrary, the best explanation for this new response to the same context is that some kind of internal modification—a “record”—mediates it inside the animal: this is what we call “memory”. Everything else is consequence: if recalling depends upon the established memory trace intensity, it will be a function of the experience intensity during the acquisition, or “training”, session, and so on.
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Notes
- 1.
By the other hand, when interpreting behavioral results we must avoid to anthropomorphize them; remember that human memory has at least two very different, “nontranslatable” aspects (to animal models), symbolic language and conscience.
- 2.
Some authors mention another type of long-lasting memory, sometimes dubbed Remote M. or even Persistent LTM (see, e.g., Bekinschtein et al. 2008).
- 3.
Hence, the term “consolidation”, created by Miller and Pilzecker in 1900 (McGaugh, 2000).
- 4.
This treatment must be applied in the first few minutes, preferably in less than 1 min (usually termed a “0 min” treatment), in order to still act upon its targets while possible.
- 5.
In intraparenchymal (intracerebral injections) and intracerebroventricular administrations, special attention must be paid to the vehicle’s chemical characteristics in order to assure it is as functionally “neutral” as possible: phosphate buffered saline solution (PBS—a buffered isosmotic 0.9 g % NaCl solution) of pH 7.4 are strongly recommended. If the drug is somewhat lipophylic, trouble may be avoided if the substance is first dissolved in a hydrophobic medium such as ethanol or DMSO, and then suspended in PBS to a reasonable percent: control groups in this case must be exactly like the drug’s solvent, just without the drug. Systemic administration (endovenous, intraperitoneal, intramuscular or intradermic) must at least avoid osmotic effects, i.e., plain, distilled water is never an acceptable vehicle!
- 6.
On this subject, we recommend two introductory books, Norman and Streiner (1994) and Callegari-Jacques (2003)—this last, in Portuguese; for advanced information, one of the best manuals in biostatistical analysis is Zar (1999). Finally, since nonparametric statistics is frequently necessary to analyze behavioral data, one excellent reference is Siegel and Castellan (1988).
- 7.
This must be done both for systemic and/or intraparenchymal injections, and every targeted structure into the brain may display its own dose-response curve due to histological particularities.
- 8.
Even when this (chosen) dose is known, it is recommended to produce a dose-response curve centered in this value.
- 9.
Intracerebral doses may sometimes be defined taking concentrations effective in in vitro experiments and administering a volume containing the substance in a 10–20 times larger concentration: this supposes a reasonable diffusion volume in the quite compact brain parenchyma (but this may vary in different regions, in the presence of nerve fibers, etc.).
- 10.
The expression immediately posttraining is usually employed for a treatment delivered (such as drug infused) in less than 1 min; despite the resistance of some authors, since this procedure starts to take place well between 0 and 59 s (and frequently ends in less than 2–3 min), we may term it as a “0 min” treatment.
- 11.
Supposing we are sure they are specifically mnemonic effects, and not motor, sensory, attentional or emotional memory-masking effects (see item 3.3). In the case of pre/postreactivation treatments, we may speak in the blocking or facilitation of extinction or of reconsolidation (it is also accepted to talk about blocking or facilitation of the consolidation of extinction).
- 12.
The following classification lacks generality due the specific needs of this chapter: a more encompassing classification would employ Skinner’s terminology and mention negative reinforces, positive reinforces and punishments (Skinner 1953).
- 13.
Rewarding stimuli comprise a broader category, involving not only appetitive, but other types of pleasant stimuli, such as sexual stimuli.
- 14.
This may be considered a “neutral” environment, but it is well-known that even the novelty of a new environmental may be stressful for the animal, with intensities that vary according to its intrinsic sensibility.
- 15.
Linoleum is recommended, because it is easy to clean up: a 70 % alcohol solution is ideal, since it is still somewhat volatile, and yet it does not smells too intensely. Some authors employ different floor textures in order to create subtle context modification.
- 16.
By partial amnesia we understand a situation in which memory was formed, but its trace is less prominent (so, measured memory indexes display lower values).
- 17.
An even stronger amnesia is the one observed when these two variables does not change their values and are taken together.
- 18.
The operant factors are described as follows: “In one-trial inhibitory avoidance (IA), a fear-motivated learning task (Gold 1986), rats associate a conditioned stimulus (CS; an elevated platform present in a given context) with an unconditioned stimulus (US; a shock given to the foot when they step down from that platform)” (Cammarota et al. 2003).
- 19.
Small variations around these values are possible.
- 20.
The variation may be larger than this: usually the platform should cover ¼ (or 1/3) of the grid-floor, but depending on the experimental design, it can be larger (e.g., to measure animal activity, Netto and Izquierdo 1985, have used a platform that covered ½ of the floor).
- 21.
Visible signs of reaction to the shock may include piloerection, back-arching, eyeball-protrusion and even jumping and squeaking, according to the intensity of the shock and/or the sensibility of the animal; extreme reactions such as freezing should be avoided.
- 22.
As described for the OF habituation, test session may be done after an interval of 24 h (for long-term memory), less than 6 h (for short-term memory), or up to 3 min (for working memory).
- 23.
Actually, the same IA apparatus may be used for this task, provided only that a dividing wall is positioned in order use only one side of the box (without a platform).
- 24.
It is highly recommended to measure freezing behavior at the end of the experiment, preferably by a person who was blind in relation to the treatment applied to each animal (and videotaping is important).
- 25.
In some cases, the delimitation is made by a wall with an opening (say, 7 × 10 cm) situated on the grid-floor level, and each side is independently illuminated by a 5 W lamp inside the compartment.
- 26.
Some variation may be introduced in these values without problems, according to the experimental design.
- 27.
Higher footshock values may be used, up to 1.0 mA.
- 28.
Delimited by the hurdle. In the automated shuttle-box IR photocells constantly monitor the side the animal is.
- 29.
The alternative would be the (less employed) One-Way Active Avoidance in which the tone-shock pairing is done in just one and the same side (and the task would be much easier to learn).
- 30.
Some authors use an elaborate protocol in order to habituate the animals to the situational cues of the apparatus: the pre-habituation may last up to 10 min and be repeated for two consecutive days (Savonenko et al.2003); in this case, the training session takes place in the following day and the tone-footshock pairings start sooner, after 20 s. Another modification consists of limiting the shock duration up to 30 s.
- 31.
Escape latency in the shuttle box may be affected by the modality of the CS, be it a tone or the illumination level.
- 32.
A lot of practical information about water maze techniques can be find at http://www.hvsimage.com/documents/watermaze_tips.pdf
- 33.
If you use albino or white strains, a black pool maximizes visual contrast for video recording; for dark-haired animals, use white-painted pool. In this last case, some authors, including Morris himself whitens the water with skim powder milk or titanium dioxide in order to increase animal-background contrast and prevent animal from seeing thru the water. However, this may be an overcare, since shuttling the animal from home cage to pool goes by swiftly, and, when on water, the animal cannot easily see anything under waterline; in our experience, it suffices to have a transparent platform.
- 34.
Although colder water would encourage activity, it may induce hypothermia, known to impair learning; warmer water would favor animal relaxation and decrease exploration.
- 35.
Swimming for more than 12–15 min without finding any escape is, otherwise, stressful; actually, this is a classic stress model called “forced swimming”.
- 36.
To avoid recue expectation from the animal.
- 37.
Dr. Morris’ tips (note 23 above) suggest that it is much better to put the animal in a litter of tissues, so it can dry itself.
- 38.
For instance, the HVS tracking system for water maze, from Dr. Morris’ lab (www.hvsimage.com).
- 39.
Since rodents are good at spatial learning, do this only on trained animals, and not too often: when done at the start, it tests for spatial bias; when done after trainings, it tests for spatial learning.
- 40.
These two variants of the WM task have demonstrated a double dissociation of the mnemonic functions of the hippocampus (with the Spatial WM task) and the dorsal striatum (with the Cued WM task), a phenomenon also observed with the win-shift and win-stay radial-maze tasks (Packard et al. 1989), and, to some extent, with the allocentric vs. egocentric maze tasks (Kesner et al. 1993).
- 41.
Tracking programs may assist in measuring animal’s running speed, useful to evaluate nonspecific effects upon motor performance.
- 42.
These may consist of palatable pellets such as peanuts, Froot loops (Kellogg’s sweet pellets of wheat and corn starch and sucrose) or even special brands, such as Noyes Formula A pellets.
- 43.
Regular spacing favours search strategies that do not involve long-lasting forms of memory (only, of course, working memory), such as, “entering every other arm to the left”.
- 44.
As far as it could be ascertained, the objects should have no natural significance for the rats, to avoid being associated with a reinforcer (or an aversive stimulus).
- 45.
By the other hand, place recognition tasks are more versatile and may allow the investigation of long-term memory processes (with “delays” of more than 6 h).
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Acknowledgements
Science involves a long apprenticeship and, at last, always remains a collective endeavor. Anyway, some people deserve to be mentioned with special care: I would like to thank my former tutor, Dr. Ivan Izquierdo (PUCRS, Brazil), for the privilege of his friendship. I would also like to acknowledge Dr. James McGaugh (UCI, USA) for all the precious lessons over the years. Finally, I would like to thank my good friends Dr. Diana Jerusalinsky (UBA, Argentina) and Victor Molina (UNC, Argentina). I also thank each one of them for kindly reading this manuscript, but I, alone, am responsible for any errors found here.
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Quillfeldt, J.A. (2016). Behavioral Methods to Study Learning and Memory in Rats. In: Andersen, M., Tufik, S. (eds) Rodent Model as Tools in Ethical Biomedical Research. Springer, Cham. https://doi.org/10.1007/978-3-319-11578-8_17
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