Biology Reference
In-Depth Information
1.0-mA shock, can be purchased from some companies (e.g., Med
Associates, Columbus Instruments). Sound generator is used to
deliver broadband clicker sounds or low-frequency tones. Although
the fear conditioning chamber is commercially available, it can also
be made in your own laboratory.
2.2. Methods
Before we start to describe the detailed procedure, we introduce
four fear conditionings. Contextual fear conditioning refers that an
animal is placed in a novel chamber or cage and presented with a
US (electric foot shock). Cued fear conditioning, also called delayed
fear conditioning, refers that US occurs at the end of a cue (CS:
tone, light, odor). Trace fear conditioning is similar to cued fear
conditioning, except that US occurs at a period of time (up to 60 s)
after CS is terminated. Backward trace conditioning, as a control
group, is to ascertain that the freezing measured in normal fear
conditioning refl ects the learning of association.
The training procedure is carried out on day 1. The test com-
pounds are normally given before the training procedure. The
rodents are transferred to the test room in transfer cage 1-2 h prior
to training. The rodents are placed into the chamber for 2 min
and allowed for freely exploring the chamber. Then, a cued tone is
presented at a level of 5 KHz, 70-80 dB, for 15-30 s. The optimized
parameters of tone should be determined by pilot experiments
in each laboratory. An electric foot shock (0.17-2.0 mA, 1-2 s) is
delivered at the end of the tone and coterminated with the tone.
If multiple conditioning is required, repeat this procedure with a
60-210-s intertribal interval. Animals were removed from the
chambers 30 s after receiving the last shock. Place animals in the
transport cages and return to home cages. On day 2, animals are
subjected to test for contextual and cued fear conditioning. For
contextual fear conditioning, the animal is placed into the exactly
same chamber used in training session. The freezing behavior is
scored live or recorded for later analysis. Freezing is defi ned as the
total lack of movement, except for respiration. For cued fear con-
ditioning, the grid fl oor of the conditioning chamber is replaced by
a smooth plastic fl oor. The size or confi guration of the chamber
should also be changed. Olfactory cues are also changed by adding
food essence if preferred. This alteration is to minimize generaliza-
tion from the conditioning context. Place animal into the modifi ed
conditioning chamber to habituate for 3 min. Baseline freezing
time or freezing during a 20-s period prior to the fi rst tone presen-
tation was also measured to assay for generalization of fear. The
tone with same parameters used in training phase is activated after
3 min. The freezing behavior is captured live or recorded for analysis
later. For vehicle-treated animal, the percentage of freezing is
60-80% of total time observed. Another way to score the freezing
is to watch the animal at specifi c time interval to determine whether
there is freezing or not. Sample result showed that naive animal,
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