Dissociable Roles of Prelimbic and Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression and Extinction of Conditioned Fear methods
Aim. Evidence-backed execution summary for Dissociable Roles of Prelimbic and Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression and Extinction of Conditioned Fear methods from Dissociable Roles of Prelimbic and Infralimbic Cortices, Ventral Hippocampus, and Basolateral Amygdala in the Expression and Extinction of Conditioned Fear.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Histology
reagent used in the protocol.
- Use
- Upon completion of experiments, rats were transcardially perfused with 0.9% saline followed by 10% buffered formalin. Brains were extracted and stored in a 30% sucrose/10% formalin solution. Coronal sections were cut 40 µm thick, mounted on slides, and stained for Nissl bodies.
Drug Infusions
reagent used in the protocol.
- Use
- Muscimol (MUS) was used to enhance GABA A receptor activity, thereby inactivating target structures, and was administered either 30 min prior to extinction training or immediately after extinction training (extinction consolidation). Obturators were removed and injectors were placed into the guide cannulas. In...
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 scra...
- Use
- 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 scra...
Behavior
Upon completion of behavioral experiments, a subset of rats were administered either MUS or SAL to assess the effects of localized inactivation on locomotor activity and anxiety levels in an open field apparatus (91.5 × 91.5 × 61 cm 3 ), which was divided into peripheral (within 15.25 cm of the...
- Use
- Upon completion of behavioral experiments, a subset of rats were administered either MUS or SAL to assess the effects of localized inactivation on locomotor activity and anxiety levels in an open field apparatus (91.5 × 91.5 × 61 cm 3 ), which was divided into peripheral (within 15.25 cm of the...
PL Inactivation Reduced Freezing, but Did Not Impair Extinction Memory
To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly reduced freezing from the start of the session ( ). Repeated-measures ANOVA of freezing during extinction training revealed a main effect of gro...
- Use
- To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly reduced freezing from the start of the session ( ). Repeated-measures ANOVA of freezing during extinction training revealed a main effect of gro...
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 va...
- Use
- 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 va...
vHPC Inactivation Reduced Freezing and Impaired Extinction Memory
Similar to PL and BLA, inactivation of vHPC ( ) prior to extinction training significantly reduced conditioned freezing ( ). Repeated-measures ANOVA revealed a main effect of group (F 1, 26 =10.53; p =0.003) and significant interaction of group with trial block (F 9, 234 =2.14; p =0.027). Post hoc compar...
- Use
- Similar to PL and BLA, inactivation of vHPC ( ) prior to extinction training significantly reduced conditioned freezing ( ). Repeated-measures ANOVA revealed a main effect of group (F 1, 26 =10.53; p =0.003) and significant interaction of group with trial block (F 9, 234 =2.14; p =0.027). Post hoc compar...
Data Collection and Analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- 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 va...
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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.
Measurement outputs
What raw and processed outputs should exist?
A total of 165 male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN; 270-320 g) were housed and handled as described previously ( ). Briefly, rats were re...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Auditory fear conditioning and extinction was performed in the same operant chambers (Coulbourn Instruments, Allentown, PA) located in sound-attenuating cubicles (Med Associates...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Upon completion of behavioral experiments, a subset of rats were administered either MUS or SAL to assess the effects of localized inactivation on locomotor activity and anxiety...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly redu...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2).
from paperScoring or quantification
Quantify the primary readouts for this experiment: A total of 165 male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN; 270-320 g) were housed and handled as described previously ( ). Briefly, rats were re...; Auditory fear conditioning and extinction was performed in the same operant chambers (Coulbourn Instruments, Allentown, PA) located in sound-attenuating cubicles (Med Associates...; Upon completion of behavioral experiments, a subset of rats were administered either MUS or SAL to assess the effects of localized inactivation on locomotor activity and anxiety...; To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly redu....
from paperStatistical comparison
To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly redu...; Behavior was recorded with digital video cameras (Micro Video Products, Bobcaygeon, Ontario, Canada) and freezing was quantified from digitized video images using commercially a...; Similar to PL, inactivation of BLA ( ) prior to extinction training caused a significant reduction in freezing ( ). Repeated-measures ANOVA of freezing during extinction trainin...; Similar to PL and BLA, inactivation of vHPC ( ) prior to extinction training significantly reduced conditioned freezing ( ). Repeated-measures ANOVA revealed a main effect of gr...
from paperReporting output
Report representative outputs alongside summary comparisons for A total of 165 male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN; 270-320 g) were housed and handled as described previously ( ). Briefly, rats were re..., Auditory fear conditioning and extinction was performed in the same operant chambers (Coulbourn Instruments, Allentown, PA) located in sound-attenuating cubicles (Med Associates..., Upon completion of behavioral experiments, a subset of rats were administered either MUS or SAL to assess the effects of localized inactivation on locomotor activity and anxiety..., To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly redu....
inferred from protocolStructured statistical methods
To evaluate the role of PL in fear expression and extinction memory, we inactivated PL ( ) prior to extinction training (Day 2). Unlike IL, inactivation of PL significantly redu...; Behavior was recorded with digital video cameras (Micro Video Products, Bobcaygeon, Ontario, Canada) and freezing was quantified from digitized video images using commercially a...; Similar to PL, inactivation of BLA ( ) prior to extinction training caused a significant reduction in freezing ( ). Repeated-measures ANOVA of freezing during extinction trainin...; Similar to PL and BLA, inactivation of vHPC ( ) prior to extinction training significantly reduced conditioned freezing ( ). Repeated-measures ANOVA revealed a main effect of gr...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
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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