I have been working on fear conditioning memory for past 18 months on SD and wistar rats, the procedure goes like unconditioned shock (0.8 mA) stimulus preceeded by tone. On day 2 scored for duration of freezing. But could not find significant freezing with animals.
Did you actually confirm that the shock was being delivered, and that the magnitude delivered is right ?
Freezing is a species-specific response to fear, which has been defined as “absence of movement except for respiration.” This may last for seconds to minutes depending on the strength of the aversive stimulus, the number of presentations, and the degree of learning achieved by the subject.
So if you do not see freezing and you are sure that conditioning protool was followed this could be due to 1) The strenght of the aversive stimulus, your animals could be conditioned already to the shock stimulus,or the aversive stimuli is insufficient to induce fear. 2).the number of presentations to this dose of footshock is insufficient to condition fear. 3) your animals are unable the condition to this stimulus.
The number of shocks are 6 and the shock being delivered is confirmed by animal behaviour like jumping and magnitude is measured by milliammeter.
A few things to consider: Are these wild type animals? If they are some kind of mutants / disease model you have to verify that 1) they feel the pain 2) they hear the tone. Do they show any behavioural reaction to the tone (jumping, digging, etc.)? Is the tone loud enough? Do you use exactly the same tone for training and recall? Is it possible that your animal facility is a very stressful environment so that a fear conditioning is not able to frighten you animals more?
Thanks for the response Krisztina, rats are not wild type and there is jumping once after animals receive 3 out of total 6 shocks on the day of training. on the day of recall where same tone is being used animal does not show any jumping or digging but we could sense calmness in the animals for just afew seconds (
I have several questions:
1. After conditioning, do you return the animals to the home cage or conduct additional tests/manipulations?
2. Do you house them alone or in pairs/groups?
3. Do you do anything else before the test on day 2?
4. Is the test box cleaned before use?
5. Do you conduct the conditioning and test in the same box?
Thanks for the response Shira,
1. Yes, we return the animals to the home cage and no addtional experiments are conducted.
2. Animals are housed 4-5 animals/cage
3. Nothing will be done before the test on day 2
4. yes, the box will be cleaned.
5. The conditioning and test are conducted in the same box.
Hi Sumanth,
Thanks for the answers. I was asking them because I thought chronic stress (e.g. due to isolation) or contextual changes (between conditioning and test box) might play a role, but according to your answers it seems to be fine.
Are those males (I guess) or females? How old? Are they completely naive before conditioning?
@shira meir Drexler. These are male rats and 8-10 weeks old. Animals are naive before conditioning.
@ Imam. Thanks for the response. The animals are subjected to conditioned sound stimulus paired with a shock stimulus on the day on conditioning. On the day of test animals are introduced in to same chamber and are not subjected to shock or sound stimulus. we expect animal to remember the shock it has received the before day and be aware of that shock and show fear for the shock in the form of freezing behaviour.
I asked about the memory aspect of your study because your responses to other questions suggested to me that perhaps something else was going on and wanted to check that some other issue may not be at play. It is not uncommon for people to mistake behavioral processes for cognitive ones, partly because of pervasive mentalism in the field (a whole other topic, but don't fall victim to it).
Fear conditioning (using classical conditioning procedures) does not require an appeal to memory to account for the acquisition, or lack thereof, of fear responses that may include jumping, freezing, etc. Best to think of what you are observing in terms of the strength of fear conditioning, rather in terms of the rat "forgetting and/ or remembering" the CS (tone). Perhaps the number of pairings (of CS-US or tone-shock) you have implemented have not been sufficient enough to maintain the fear response you are looking for. This possibility is more likely than EXT (which could be another reason the rat would exhibit little or no fear responses) because your test occurs only the day after your training session without extended expose to the tone without the shock (which would be required for EXT to occur). In this regard, I would recommend that you explore (empirically) various number of pairings to see how many would maintain the level of fear response you desire.
If your interest is solely memory, however, there are other paradigms/preparations for studying memory and memory processes in the lab with rats or pigeons. I would most certainly not use responses to the CS in your current preparation as a measure of "memory."
Hope this helps...
@A.Imam. Thanks for the response. Really informative. what you said could well be factual. I will try different combinations and would also try to see the freezing behaviour with tone and without shock on day2.
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