Male AS and wild-type mice were subjected to fear conditioning testing as described in Supplemental Experimental Procedures . On training day, mice were injected intraperitoneally (i.p.) with apamin (0.4 mg/kg) 30 min before training. Training was conducted in a fear-conditioning chamber (H10-11M-TC, Coulbourn Instruments) and behavior was recorded with the Freezeframe software and analyzed with Freezeview software (Coulbourn Instruments).
H10 11m tc
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The H10-11M-TC is a specialized laboratory equipment designed for precise temperature control. It features a compact and durable construction, making it suitable for various applications in research and scientific settings. The core function of this product is to provide accurate and stable temperature regulation for experimental samples or processes.
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15 protocols using h10 11m tc
1
Fear Conditioning in Male AS and Wild-Type Mice
2
Fear Conditioning Paradigm for Mice
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Fear conditioning was conducted by the Emory University Rodent Behavioral Core by trained personnel who were blinded to the mouse condition. Fear conditioning occurred over 3 consecutive days in a chamber (H10-11M-TC, Coulbourn Instruments) equipped with a house light, a speaker, a ceiling-mounted camera, and an electric grid shock floor that could be replaced with a non-shock wire mesh floor. Fear conditioning training on day 1 began with a 3-min acclimatization period followed by 3 tone-shock pairings during which the tone lasted 20 s and was co-terminated with a 3-s, 0.5-mA foot shock. Mouse behavior was recorded for 60 s after a tone-shock pairing before the next round. Contextual fear testing on day 2 was conducted in the same chamber as day 1 without any tone or shock. Cued fear testing on day 3 was conducted in a different chamber with a non-shock wire mesh floor and began with a 3-min acclimatization period followed by a 5-min tone without any shock. FreezeFrame software (Coulbourn Instruments) was used to record freezing behavior and the percentage of freezing time was determined.
3
Auditory Fear Conditioning and Extinction
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For auditory fear conditioning, after 5-min of habituation, mice received three pairings of a tone (conditioned stimulus, CS, 75 dB, 10,000 Hz, 30 s) and a foot shock (US, 0.6 mA, 2 s) that was finished with the tone with 20–40 s of inter-stimulus intervals (ISIs) in the conditioning context (H10-11M-TC, Coulbourn Instruments, Whitehall, PA, USA). After the last shock, mice stayed in the context for another 30 s and were then moved back to their home cages (HCs). Fear extinction was performed in the extinction context (acrylic hexagonal prism with an apothem of 11 cm and height of 29 cm) 24 h later. Mice were acclimated for 2 min to the context and were then exposed to a shock-free tone (CS, 75 dB, 10,000 Hz, 30 s) 30 times with 30-s ISIs. After the last tone presentation, mice stayed in the extinction context for 30 s and were then returned to their HCs. The same extinction protocol was repeated 24 h later to complete a 2-day extinction protocol. Freezing behavior was analyzed every 2 s manually by assessing movements excluding respiration (N = 4 for each strain).
4
Contextual Fear Conditioning and Extinction
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For contextual fear conditioning, after 5-min of habituation, mice were exposed to a foot shock (US, 0.6 mA, 2 s) three times with 30-s ISIs in the conditioning context (H10-11M-TC, Coulbourn Instruments). After the last shock, mice stayed in the context for an additional 30 s and were then returned to their HCs. Fear extinction was conducted 24 h later in the same conditioning context for two consecutive days. Mice were placed in the context without shocks for 30 min. The protocol was repeated the next day. Freezing behavior was assessed every 2 s manually (N = 6 for each strain).
5
Spatial Memory and Fear Conditioning Assays
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Behavioral tests were conducted 2 months after injection. Spontaneous alteration performance in the Y‐maze was used to test the spatial working memory of the mice. The test was performed as described previously 25 . Contextual fear‐conditioning testing was carried out in a fear‐conditioning chamber (H10‐11M‐TC, Coulbourn Instruments, Whitehall, PA,
http://www.coulbourn.com/ ) fitted with metal shock grid. The test was conducted over 3 days. During the habituation period, cages containing the mice were kept in the testing room for 3 hours. During the training session, each mouse was allowed to explore the conditioning chamber for 4 minutes 30 seconds and received two electric foot shocks (0.65 mA, 2 seconds). For the context session on day 3, mice were placed in the chamber for 5 minutes, during which time no shock was applied. Time spent freezing was recorded with a chamber‐mounted camera. Freezing time was analyzed by FreezeFrame software (Coulbourn Instruments) connecting the conditioning chamber.
6
Fear Conditioning Apparatus for Mice
7
Auditory Fear Conditioning and Extinction
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After 5 days of handling (3 min/day), auditory fear conditioning and extinction procedures were conducted. In the conditioning protocol, after 5 min of acclimation, mice were presented with three pairings of a neutral tone (conditioned stimulus [CS], 75 dB, 10,000 Hz, 30 s) with a foot shock (unconditioned stimulus [US], 0.6 mA, 2 s, overlapped and terminated with the CS), along with 20–40 s of random inter-stimulus intervals (ISIs). During this protocol, the mice were placed in a hexahedral conditioning chamber having a width of 18 cm, depth of 18 cm, and height of 30 cm (H10-11M-TC, Coulbourn Instruments, PA, USA). After the last foot shock, the mice were kept in the conditioning chamber for another 30 s and then moved back to their home cages. After 24 h, fear extinction was performed. The mice were subjected to the novel extinction context (acrylic hexagonal prism with an apothem of 11 cm and height of 29 cm). After 2 min of acclimation, a US-free CS (a neutral tone, 75 dB, 10,000 Hz, 30 s) was delivered 30 times, along with 30 s of ISIs. After the last tone, the mice were retained in the extinction context for another 30 s and then returned to their home cages. The same extinction protocol was conducted for two consecutive days. Freezing behaviors were manually analyzed by observing whether mice moved or not (except for respiration) every 2 s.
8
Fear Conditioning in Transgenic Mice
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AS mice and their WT littermates were randomly assigned to either control or p18 siRNA groups and blinded to the examiner. Four weeks after AAV injection, mice were placed in the fear-conditioning chamber (H10-11M-TC, Coulbourn Instruments). The conditioning chamber was cleaned with 10% ethanol to provide a background odor. A ventilation fan provided a background noise at ∼55 dB. After a 2 min exploration period, three tone-footshock pairings separated by 1 min intervals were delivered. The 85 dB 2 kHz tone lasted 30 s and co-terminated with a footshock of 0.75 mA and 2 s. Mice remained in the training chamber for another 30 s before being returned to home cages. Context test was performed 1 day after training in the original conditioning chamber with 5 min recording. On day 3, animals were subjected to cue/tone test in a modified chamber with different texture and color, odor, background noise, and lighting. After 5 min recording, mice were exposed to a tone (85 dB, 2 kHz) for 1 min. Mouse behavior was recorded with the Freezeframe software and data were analyzed using the Freezeview software (Coulbourn Instruments). Motionless bouts lasting more than 1 s were considered as freezing. The percent of time animal froze was calculated and group means with SEM were analyzed.
9
Automated Fear Conditioning in Mice
10
Contextual and Cued Fear Memory Assessment
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The shock‐associated training context A and the similar context B without shock shared many features, including an exposed stainless‐steel grid floor and roof. The similar context B differed from training context A in that four plastic inserts were used to cover the walls. A nonalcoholic antiseptic solution was used to clean the grids between trials. On the first day, mice were allowed to explore the conditioning chamber (context A) (29 × 29 × 24 cm; H10‐11M‐TC, Coulbourn Instruments) for 180 s before the onset of a tone that lasted 30 s at 80 dB and 5 kHz. A foot shock (0.75 mA, 2 s) was administered during the last 2 s of the tone presentation and co‐terminated with the tone. The tone‐shock pairing was delivered three times, with an intertrial interval of 30 s. Mice were returned to the home cage after the conditioning session was completed. The freezing level of the last tone–shock pairing was measured to evaluate their learning. One day later, mice were placed into context A without shock and tone for 5 min to evaluate the contextual fear memory. On the third day, the tone‐cued fear memory was assessed by placing them into context B with a tone that lasted 30 s at 80 dB and 5 kHz but without shock. The freezing levels were measured by a video camera (Freezeframe, Coulbourn Instruments).
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