Dissociable Roles of Prelimbic and Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression and Extinction of Conditioned Fear methods
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Surgery
After bar-press training, rats were anesthetized with intraperitoneal injections of a mixture of ketamine (80 mg/kg)-xylazine (10 mg/kg) and were chronically implanted with a 26-gauge bilateral guide cannula (Plastics One, Roanoke, VA) in one of the four sites: IL (+2.8 mm AP; ±3.1 mm ML; -3.8 mm DV; angled at 30°); PL (+2.9 mm AP; ±0.60 mm ML; -2.6 mm DV); BLA (-2.8 mm AP; ±5.0 mm ML; -7.6 mm DV); or ventral HPC (vHPC; -6.30 mm AP; ±5.00 mm ML; -5.5 mm DV) ( ). For preextinction infusions into IL, an angled placement was used to avoid the overlying PL cortex. For post-extinction infusions into IL, bilateral cannula targeted IL vertically, with no angle (+2.8 mm AP; ±0.60 mm ML; -4.2 mm DV). Acrylic cement w...
Behavior
Auditory fear conditioning and extinction was performed in the same operant chambers (Coulbourn Instruments, Allentown, PA) located in sound-attenuating cubicles (Med Associates, Burlington, VT) throughout all phases of the experiment. The floor of the chambers consisted of stainless steel bars that delivered a scrambled electric footshock. Between experiments, shock grids and floor trays were cleaned with soap and water, and chamber walls were cleaned with wet paper towels. On Day 1, rats received five habituation tones (30 s, 4 kHz, 77 dB; 3 min intertrial interval (ITI)), immediately followed by seven conditioning tones that co-terminated with footshocks (0.5 s, 0.54 mA). On Day 2, rats were returned to the chambers for extinction training, which consisted of 20 tones in the absence of footshock. On Day 3, rats were returned to the chambers and p...
Data Collection and Analysis
Behavior was recorded with digital video cameras (Micro Video Products, Bobcaygeon, Ontario, Canada) and freezing was quantified from digitized video images using commercially available software (Freezescan, Clever Systems, Reston, VA). Trials were averaged in blocks of two, on which repeated-measures analysis of variance (ANOVA) or Student's two-tailed t -tests were made, followed by Tukey's post hoc comparisons (STATISTICA; Statsoft, Tulsa, OK). The amount of time freezing to the tone was expressed as a percentage of the tone presentation. In addition to freezing, suppression of bar pressing was also used as a measure of conditioned fear ( ) and analyzed using Student's t -test. A suppression ratio comparing pretone press rates with tone press rates was calculated as follows: (pretone-tone)/(pretone+tone). A value of 0 represents no suppression (low fear), whereas a value of +...
Post-Training Inactivation had no Effect in any Structure
Impaired retrieval of extinction in the IL, BLA, and vHPC experiments suggests that processing within these structures during, and/or after, extinction training is necessary for long-term extinction memory. To dissociate between acquisition vs consolidation processes, we ran a separate set of experiments in which the same doses of muscimol infused prior to extinction were infused immediately after extinction training in either IL, BLA, or vHPC. Also, the duration of the extinction training session was shortened (from 60 to 30 min) in an attempt to interrupt the earliest stages of extinction consolidation. In no structure, however, did post-training inactivation impair extinction memory (Supplementary Figure 2). Repeated-measures ANOVA of freezing during extinction retrieval showed no effect of group for any structure (all p values >0.4), nor a significant interaction between gro...
vHPC and BLA Are Necessary for Both Fear Expression and Extinction Memory
Inactivation of vHPC reduced fear expression, similar to what has been observed with lesions of vHPC ( ). It has been previously shown that vHPC lesions reduce anxiety ( ), but this is unlikely to explain reduced freezing given that we observed no effect of vHPC inactivation in our measure of anxiety. Thus, unlike dHPC ( ), the vHPC has a key role in expression of auditory conditioned fear.
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After bar-press training, rats were anesthetized with intraperitoneal injections of a mixture of ketamine (80 mg/kg)-xylazine (10 mg/kg) and were chronically implanted with a 26-gauge bilateral guide cannula (Plastics One, Roanoke, VA) in one of the four sites: IL (+2.8 mm AP; ±3.1 mm ML; -3.8 mm DV; angled at 30°); PL (+2.9 mm AP; ±0.60 mm ML; -2.6 mm DV); BLA (-2.8 mm AP; ±5.0 mm ML; -7.6 mm DV); or ventral HPC (vHPC; -6.30 mm AP; ±5.00 mm ML; -5.5 mm DV) ( ). For preextinction infusions into IL, an angled placement was used to avoid the overlying PL cortex. For post-extinction infusions into IL, bilateral cannula targeted IL vertically, with no angle (+2.8 mm AP; ±0.60 mm ML; -4.2 mm DV). Acrylic cement was used to affix cannulas to the skull. After surgery, a triple antibiotic was applied, and an analgesic (Buprenorphine; 0.05 mg/kg) was injected intramuscularly. Rats were allowed 7 days to recover from surgery prior to behavioral testing. Stainless steel obturators (33 gauge) were inserted i...
Auditory fear conditioning and extinction was performed in the same operant chambers (Coulbourn Instruments, Allentown, PA) located in sound-attenuating cubicles (Med Associates, Burlington, VT) throughout all phases of the experiment. The floor of the chambers consisted of stainless steel bars that delivered a scrambled electric footshock. Between experiments, shock grids and floor trays were cleaned with soap and water, and chamber walls were cleaned with wet paper towels. On Day 1, rats received five habituation tones (30 s, 4 kHz, 77 dB; 3 min intertrial interval (ITI)), immediately followed by seven conditioning tones that co-terminated with footshocks (0.5 s, 0.54 mA). On Day 2, rats were returned to the chambers for extinction training, which consisted of 20 tones in the absence of footshock. On Day 3, rats were returned to the chambers and presented with 15 tones to test for extinction retrieval (the last retrieval trial was omitted from the analyses and figures, which show blocks of two trials). Food was available on the trained VI-60 schedule throughout all phases of the experiment. For post-extinction infusion experiments, the same...
Behavior was recorded with digital video cameras (Micro Video Products, Bobcaygeon, Ontario, Canada) and freezing was quantified from digitized video images using commercially available software (Freezescan, Clever Systems, Reston, VA). Trials were averaged in blocks of two, on which repeated-measures analysis of variance (ANOVA) or Student's two-tailed t -tests were made, followed by Tukey's post hoc comparisons (STATISTICA; Statsoft, Tulsa, OK). The amount of time freezing to the tone was expressed as a percentage of the tone presentation. In addition to freezing, suppression of bar pressing was also used as a measure of conditioned fear ( ) and analyzed using Student's t -test. A suppression ratio comparing pretone press rates with tone press rates was calculated as follows: (pretone-tone)/(pretone+tone). A value of 0 represents no suppression (low fear), whereas a value of +1 represents complete suppression of bar pressing (high fear). Total line crosses in the open field were counted to determine locomotor activity, and the percent of time spent in the center was used to determine anxiety, both of which were analyzed using Student's t -test.
Impaired retrieval of extinction in the IL, BLA, and vHPC experiments suggests that processing within these structures during, and/or after, extinction training is necessary for long-term extinction memory. To dissociate between acquisition vs consolidation processes, we ran a separate set of experiments in which the same doses of muscimol infused prior to extinction were infused immediately after extinction training in either IL, BLA, or vHPC. Also, the duration of the extinction training session was shortened (from 60 to 30 min) in an attempt to interrupt the earliest stages of extinction consolidation. In no structure, however, did post-training inactivation impair extinction memory (Supplementary Figure 2). Repeated-measures ANOVA of freezing during extinction retrieval showed no effect of group for any structure (all p values >0.4), nor a significant interaction between group with trial block (all p values >0.3). Bar-press suppression data largely mirrored freezing results, except for the fact that post-training inactivation of BLA caused reduced fear expression the following day (see Supplementary Figure 2). Together, these data suggest that activity necessary for ex...
Inactivation of vHPC reduced fear expression, similar to what has been observed with lesions of vHPC ( ). It has been previously shown that vHPC lesions reduce anxiety ( ), but this is unlikely to explain reduced freezing given that we observed no effect of vHPC inactivation in our measure of anxiety. Thus, unlike dHPC ( ), the vHPC has a key role in expression of auditory conditioned fear.
Machine-readable layer
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