Prior Fear Learning Enables The Rapid Assimilation Of New Fear Memories Directly Into Cortical Networks Part 3

Experimental design

Data reproducibility was assessed with different replicates (S1 Table). The first CNQX inactivation experiments in Te2 (Fig 1A–1C) were performed in a higher number of replicates because they were the first experiments we conducted and served to test the major hypothesis of our study. Animals were a priori assigned to different behavioral groups in a weight-balanced manner. Male animals from the same brood were randomly assigned to each experimental group. We first addressed the main hypothesis of our study, i.e., the cortical inactivation may differently affect the consolidation of a new fear memory in experimentally naïve rats and in animals that had learned a prior fear event. 

Male animals have extremely strong memories because they have a stronger survival instinct and desire to reproduce. Animals surviving in the wild need to remember a lot of geographical information, food types, predator tracks, and the locations of group members to better adapt to the environment and protect life.

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In the case of statistical differences between these groups, we then eventually performed additional control groups through the injection of saline. A similar approach was applied to the optogenetic experiments, where the AAV-control group was performed only after statistical differences between naïve and previously trained rats were detected. These experimental schedules allowed us to avoid unnecessary control groups and the use of unnecessary animals, a key issue in the European and Italian legislation on animal experimentation (3 Rs principle).

Behavioral procedures

All the experiments were conducted during the light phase of the day (8 AM to 4 PM). Animals were transported singularly from the facility to the experimental rooms within different small transparent buckets depending on the experimental demands.

Auditory training: First behavioral session. Auditory fear-conditioned animals (CS1-CS2). In this group, rats were gently taken from their home cage and carried from the housing room to the soundproofed room. Once there, animals were placed inside the conditioning apparatus consisting of a rectangular black cage (35 × 40 cm) equipped with a stainless steel rods grid (1 cm in diameter, spaced 1.5 cm apart) connected to a shock delivery setup. 

Rats were left undisturbed for 1 minute. After this time, 7 conditioned stimuli (CSs) consisting of a pure tone of 15 kHz of frequency (15 s of duration each, 80 dB, 36 s trials interval) were administered. The last 1 s of each tone was paired with a painful US (0.5 mA, 1 s). At the end of the conditioning session, rats were brought back to their home cage.

Shock-only animals (shock-CS2). In this group, rats were similarly placed inside the same conditioning apparatus. Immediately afterward, each rat was subjected to 7-foot shocks (1 s, 0.5 mA) one immediately after the other. At the end of the stimulation, animals were brought back to their home cage. The time permanence in the conditioning cage was less than 9 seconds. Previous studies showed that this procedure allows for avoiding associative processes between painful stimuli and sensory stimuli [29,30].

Odor fear-conditioned animals (odor-CS2). In this group, rats were placed inside the same rectangular black cage employed in the above experimental groups and connected to a shock delivery setup. Rats were left undisturbed for 2 min. After this time, 7 CSs consisting of vanilla odors were administered (10 s of duration each, 24 s trial interval). The last 1 s of each odor was paired with a painful US (0.5 mA, 1 s). The conditioning module was placed near a ventilation source to avoid the persistence of the delivered stimuli after their offset. The cage was ensured with an upper grid. Odors were presented using a flow-dilution olfactometer. Clean air (1.5 L/min) was directed to a solenoid valve, which when operated, passed the air to a 15 ml bottle containing 10 ml of vanilla odor.

Tone-only animals (Tone-CS2). Rats in this group were placed inside the same black cage and were presented with the 15-kHz tone (7 stimuli, 15 s of duration, 36 s ITI) delivered in the absence of the US.

Pre-exposed animals (Tone-CS1-CS2). Rats were placed inside the conditioning cage and, 1 min later, they were presented 20 times with the 15-kHz tone alone, and 24 h later the same tone was paired with the US becoming the CS1.

White noise fear-conditioned animals (WN-CS2). In this group, rats were placed inside the rectangular black cage and left undisturbed for 1 minute. After this time, 7 CSs consisting of a WN (15 s of duration each, 75 dB, 36 s inter trials interval) were administered. The last 1 s of each tone was paired with a painful US (0.5 mA, 1 s). At the end of the conditioning session, rats were brought back to their home cage.

Auditory fear learning: Second behavioral training. Two weeks after the procedures described in the above paragraph, animals were trained in another different auditory fear conditioning task. Rats were put in a standard skinner box, as in our previous work [10], and left undisturbed for 2 min. After this time, 7 CSs consisting of pure tones of 3 kHz of frequency (8 s of duration each, 80 dB, 22 s inter-trial interval) were delivered. The last 1 s of each tone was paired with a painful US (0.5 mA, 1 s). At the end of the conditioning session, rats were brought back to their home cage.

Fear memory retention. The retention of auditory fear memory recently acquired to the CS2 (3 kHz) was tested 4 days later. For optogenetics experiments, the test of recent memory was performed with laser delivery 24 h after the CS2-US learning in analogy to the time interval at which we performed cortical inactivation through the administration of the CNQX (i.e., 24 h after training). Rats were habituated to an apparatus different from that used for conditioning and placed in a different room to avoid conditioned fear behavior to contextual cues [10,62]. 

The new apparatus consisted of a transparent plastic cage with a black painted side enclosed within a sound-attenuating box equipped with an exhaust fan, which eliminated odorized air from the enclosure and provided background noise of 60 dB. Animals were allowed to explore the cage for 5 min a day during the habituation session. On the day of the fear memory retention test, after 2 min of free exploration, we delivered 4 CS2 of 3 kHz (8 s—22 ITI) not followed by any US.

If required by the experimental demand, rats were then tested for the retention of the remote fear memory, acquired 2 weeks before the second auditory fear conditioning trial. To this aim, 7 days after the fear memory retention test to the 3-kHz tone, animals were put in a novel environment (a black and white striped cage) and then presented with the 15-kHz tone. Four tones were presented at 36s intervals.

Contextual training: First behavioral session. Contextual fear conditioning group (CtxA-CtxB). In this group, rats were gently taken from their home cage, placed in a bucket, and carried from the housing room to the soundproofed room. Once there, animals were placed inside the conditioning apparatus consisting of the aforementioned rectangular black cage, equipped with the stainless steel rods grid connected to a shock delivery setup. Rats were left undisturbed for 1 minute. After this time, 5 US (0.5 mA, 1 s) were administered with 51 s time intervals. At the end of the session, the animals were brought back to their home cage.

Shock-only group (shock-CtxB). Rats, once placed inside the conditioning apparatus, immediately received 5-foot shocks (1 s, 0.5 mA) one immediately after the other. The time permanence in the conditioning cage was less than 7 seconds. Previous studies showed that this procedure allows for avoiding associative processes between painful stimuli and sensory stimuli [29,30].

Shock-only group (shock-CtxB). Rats, once placed inside the conditioning apparatus, immediately received 5-foot shocks (1 s, 0.5 mA) one immediately after the other. The time permanence in the conditioning cage was less than 7 seconds. Previous studies showed that this procedure allows for avoiding associative processes between painful stimuli and sensory stimuli [29,30].

New contextual fear conditioning and recent fear memory retention. Two weeks after the procedures described in the above paragraph, all groups were trained to associate a new contextual environment (the skinner box module, placed in a different room) with a painful US (0.5 mA, 1 s). Each animal was placed inside the new chamber and left undisturbed for 2 min. Then, it was exposed to 5 US separated by intervals of 30 s.

The retention of contextual fear memory was tested 4 days after the fear conditioning procedure by putting rats again in the Skinner box chamber for 3 min. For optogenetics experiments, the test of recent memory was performed with laser delivery 24 h after the CtxB-US learning in analogy to the time interval at which we performed cortical inactivation through the administration of the CNQX (i.e., 24 h after training). If required by the experimental demand, rats were then tested for the retention of the remote fear memory by putting animals in the context paired to the US 2 weeks before the new association.

Animals were carried in 2 different buckets to the conditioning chambers according to the different contextual procedures.

Freezing measure. In all experimental procedures, the assessment of the fear memory retention was determined as a freezing response [10], analyzed as the complete absence of somatic mobility except for respiratory movements. For each animal, the amount of time (in seconds) spent in freezing was measured offline by 2 independent observers who were blinded to the animal groups.

Surgical procedures

To administer the selected substances in the target sites, rats were anesthetized with isoflurane: The induction was performed at 4% [vol/vol] in 2 L/min medical air and extended to a continuous exposure at 2% [vol/vol]) when rats were mounted in the stereotaxic apparatus.

An incision of the skull was made, and small burr holes were drilled to allow the penetration of a 28-gauge infusion needle. A 10-μl Hamilton syringe mounted on an infusion pump was used to deliver substances. After infusions, the needle was left in place for an additional 3 min. The incision was then closed with stainless steel wound clips, and the animal was given a subcutaneous injection of the analgesic/anti-inflammatory ketoprofen (2 mg/kg body weight), and the animal was kept warm and under observation until recovery from anesthesia.

As in previous studies [10,32–34], the cannulation of animals was unnecessary, with the active compounds being directly administered stereotaxically. This procedure is advantageous because the surgical trauma inherent to the permanent-cannulating procedure is avoided, thus restricting trauma to a single needle penetration [10,32–34]. Indeed, general anesthesia does not significantly affect memory consolidation [10,32–34]. To ensure that the timing of compounds administration related to the learning trial was accurate so that rats received the selected compounds around 1 h and around 1 day after training, isoflurane anesthesia was induced 5 min before the timing of the planned injection, e.g., at 55 min in rats treated 60 min after training and 23 h and 55 min in animals injected at 24 h after training. Each rat was conditioned and then operated separately. Animals belonging to the different behavioral groups were manipulated in an interleaved way.

The selective inhibitor of AMPA/kainate glutamate receptors CNQX (6-Cyano-7-nitroquinoxaline-2,3-dione) (Tocris, 10 ng/μl) [20,21] was dissolved in sterile saline solution (NaCl, 0.9%) and adjusted to pH 7.4 with HCl. The protein synthesis inhibitor anisomycin (Merck, 125 μg/μl) was dissolved in equimolar HCl, diluted with sterile saline, and adjusted to pH 7.4 with NaOH. Sterile saline (0.9% NaCl) was used as vehicle control. These substances were bilaterally injected at a rate of 0.1 μl/min and a volume of 0.5 μl per site at the following stereotaxic coordinates taken from Paxinos and Watson atlas [26], with Te2 cortical field refereed to the Zilles atlas [25]:

Secondary auditory cortex (Te2): 1) AP: −5,8 L: ±7,2 DV: −6. 2) AP: -6,8 L: ±7,2 DV: −6.

Anterior cingulate cortex (ACC): 1) AP: +2,5 L: ±0,6 DV: −2,2. 2) AP: +3,7 L: ±0,6 DV: −2,2.

The volume of injection (0.5 μl per site) was selected based on previous studies where active compounds were injected in Te2 [10,34] and ACC [55,63] in adult rats.

To inactivate the dorsal hippocampus, active compounds were injected at a volume of 0.6 μl per site at the following stereotaxic coordinates:

Dorsal Hippocampus: 1) AP: −2,8 L: ±1,6 DV: −3,3. 2) AP: −4,2 L: ±2,6 DV: −3.

The irreversible lesions of the dorsal hippocampal were induced by administering NMDA (Tocris, 18 μg/μl, dissolved in sterile saline solution, 0,4 μl per site) at a rate of 0.1 μl/min at points at the aforementioned coordinates. The same coordinates were used for saline-injected controls and sham-operated animals.

Red Retrobeads (Lumafluor, 1:2 dilution in saline, 0.6 μl) were injected in BLA according to the following coordinates: AP: −2.8; L: ±5.4; DV: −8.3.

At the end of the experiments, the needle tracks in the case of CNQX, anisomycin, or saline injections or the extension of the lesions in the case of NMDA injections were histologically verified. Rats were deeply anesthetized and intracardially perfused with 4% formaldehyde. Their brains were sectioned at 30 μm on a cryostat. Nissl-stained serial sections were prepared using the conventional procedure and the sections were histologically verified under a microscope magnified at 2.5×.

Optogenetic experiments

Virus injection. The adeno-associated virus AAV5:CaMKIIα::eNpHR3.0-cherry and the control vector AAV5:CaMKIIα-cherry were obtained from the University of North Carolina Vector Core (Chapel Hill, North Carolina, United States of America). The thesentenceViraltiterwas5:8 Viral titer was 5.8 × 10^12 vg/ml for both viruses. The use of CaMKII promoter enables transgene expression favoring pyramidal neurons. Viruses were housed in an −80˚C freezer. Viral infusions targeting the Te2 or the ACC were performed at the above stereotaxic coordinates at the volumes of 0.5 μl for each hole. The virus was injected at a rate of 0.1 μl/min, and the needle was left in place for an additional 5 min. Viral injections were performed 4 weeks before optogenetics experiments.

Illumination. 

The optic fibers (Plexon, 200/230 μm core; 10 mm length) were implanted bilaterally in the BLA (AP = −2.8, L = ± 5.4, V = −8.2 mm from bregma). Optogenetic inhibition of Te2 projections to BLA or of ACC projections to BLA was obtained by using the PlexBright Optogenetic Stimulation System coupled to a laser diode (Laserglow Technologies). Yellow light (589 nm) is generated and passed through an optical fiber. The power density estimated at the tip of the optic fiber was 10 to 15 mW for the illumination of projection sites. During fear memory retention, light emission was initiated 4 s before tone onset, persisted throughout the tone, and was stopped 4 s after the tone offset. Animals belonging to contextual fear conditioning groups received light stimulation during the entire context exploration (3 min). Rats were familiarized with the patch cord for 2 days before the memory retention trial.

Immunohistochemistry. Upon completion of optogenetics experiments, rats were deeply anesthetized and perfused intracardially with 4% PAF to examine the diffusion of the virus. The brains were dissected, stored overnight at 4˚C, and finally transferred to 30% sucrose. Coronal sections (30 μm) were cut on a cryostat and collected in PBS. Free-floating sections were incubated in a blocking solution for 1 h at RT. Then, they were incubated in primary monoclonal mouse antibody anti mCherry (1:500 dilution, Abcam) in the blocking solution overnight at RT. Subsequently, sections were washed with PBS and incubated for 1 h at RT with a secondary fluorescent AlexaFluor-568 anti-mouse antibody (1:600, Invitrogen) diluted in PBS. Sections were washed in PBS, mounted with mounting media containing DAPI (Vector), and the coverslipped.

The brains of rats that underwent NMDA injections were similarly collected. Free-floating sections, after several rinses, were incubated with primary monoclonal mouse anti-Neun (1:1,000 dilution, Merck) antibody in the blocking solution overnight at room temperature. Subsequently, sections were washed with PBS and incubated for 1 h at room temperature with biotinylated horse anti-mouse antibody (1:200 dilution, Vector). The avidin-biotin complex (ABC complex 1:100, Vector, 2 h and half of incubation) was coupled to diaminobenzidine (0.03%, Merck) to stain Neun. Sections were then rinsed in PBS and transferred to gelatin-coated slides, dehydrated, and covered with a coverslip.

Te2 sections of retro beads-injected rats underwent incubation with primary polyclonal rabbit anti-Fos antibody (1:2,000, Cell Signaling) in the blocking solution overnight at RT. Subsequently, slices were washed with PBS and incubated for 1 h at RT with anti-rabbit Alexa 488 (1:1,000, Invitrogen, in PBS). Finally, immunolabeled sections were washed in PBS, mounted on gelatin-coated slides, and covered with a DAPI-supplemented mounting medium.

Microscopy

mCherry labeling was examined by using a Zeiss Airyscan confocal microscope: Two lasers were used (405 and 561 nm), each corresponding to the peak emission spectrum for DAPI (Nissl stain for cell nuclei) and Alexa 568 (mCherry), respectively. To analyze the diffusion of the virus at the injection sites, micrographs of Te2 and ACC were acquired as mosaic images (each single was acquired by using a 10× objective). Axon terminals into BLA were analyzed by using a 40× objective as a z-stack of 10 sections, spaced 1 μm apart (159 μm square; zoom fraction, 1.0).

For retrobeads-injected animals that underwent cFos immunolabeling, images were acquired at a 40× magnification (159 μm square; zoom fraction, 1.0) with 3 different lasers, corresponding to the peak emission spectrum for DAPI (Nissl stain for cell nuclei), AlexaFluor 488 (cFos), and Texas Red (Retrobeads), respectively. Sections of 0.7 μm were acquired along a 10-μm z-stack. The number of nuclei expressing cFos, beads-labeled, and double-positive (cFos+ beads labeled) was quantified for each animal in the Te2 region at the anteroposterior coordinates from 6.7 to 7.3 mm from the bregma [10]. Data were then averaged to produce the mean of each animal and the results were statistically compared. Images of sections with DAB staining of Neun were analyzed using Neurolucida software connected to a microscope via a color CCD camera [10].

Data analysis and exclusion criteria

The n for each group was established a priori according to our previous studies [10,16,17], previously published works in the field (see as references for ACC [6,22,55] and Te2 [10,59]), and through G-power estimations, according to the following table (Table 1):

For each analysis, we estimated a final mean number of 8 to 10 animals for each group. Groups were run with internal controls in the same experimental session (S1 Table). Given the variability of surgical and behavioral procedures, we included a priori a greater number of experimental subjects (up to 15% more) that in some cases met the experimental criteria and were included, thus avoiding an arbitrary exclusion. In the case of hippocampal studies, to assess the effects of NMDA and CNQX injections at distinct time intervals, we balanced the total number of control animals between vehicle and sham-operated rats among the different groups.

Behavioral analysis, histological inspections, and cell counts were performed blindly. Animals with inadequate localization of the needle track or the lesion in the case of NMDA-irreversible lesions were excluded from data analysis (S1 Table). In pharmacological studies, we excluded 57 animals over a total of 465 animals because of an incorrect needle placement (22/255 for Te2, 31/179 for ACC, and 4/31 for hippocampus). In tracing studies, on 21 animals, we eliminated 3 subjects in which the injection of retrobeads missed the BLA. In Te2 optogenetics studies, over a total of 30 animals, we excluded 1 rat because the placement of the optic fibers was not above the target region, 1 rat. After all, the virus was absent in Te2, and 2 rats because the virus diffusion was respectively less than 4.7% and 5.3% of the Te2 area at the injection sites. In rats included in the statistical analysis, the minimal virus diffusion was 39.6% at the more anterior stereotaxic coordinate and 50.2% at the more posterior stereotaxic coordinate.

Similarly, in the ACC optogenetics experiments, over a total of 32 animals, 2 rats were eliminated because the placement of optic fibers was not above the target region. Two rats were excluded because the virus in ACC was absent, 1 animal because the virus was into the adjacent secondary motor cortex, and 1 rat because the virus diffusion was less than 2.3% of the ACC area at the injection sites. In the remaining rats, the minimal virus diffusion was 33.3% at the more anterior stereotaxic coordinate and 42.2% at the more posterior coordinate.

In the NMDA lesions studies, lesions mostly targeted the CA1 region of the dorsal hippocampus. The minimal (red) and maximal extension (pink) of hippocampal lesions over the entire area of the dorsal hippocampus were respectively 23.2% and 53,5% at the more anterior stereotaxic coordinate and 28.3% and 62.3% at the more posterior coordinate. Over a total of 73 animals, 4 rats were eliminated because the lesion was absent, while an additional 3 animals were discarded because the extension of the lesions was less than 5.0% (namely 4.8%, 4.3%, 3.1%) concerning the hippocampal area at the 2 coordinates.

Area contour analysis was performed through visual inspection and manual quantification using the region-contour tool of ZEN 3.0 software. Area values were then normalized on the total area of the region. Stereotaxic coordinates of Te2, ACC, and hippocampus were based on the Paxinos atlas [26] and, in the case of Te2, with cortical field refereed to Zilles atlas [25].

Statistical analysis

All data are presented as mean ± SEM. All data passed Levene’s test for equality of variances. Thus, parametric statistics were employed throughout all the experiments. Data from 2 groups were compared using 2-tailed unpaired Student t-tests. Multiple-group comparisons were assessed using a 1-way ANOVA test with Tukey’s post hoc test. To address the between and within groups differences, we computed a 3 × 2 mixed-design ANOVA model with a group (CS1-CS2, Tone-CS1-CS2, WN-CS2) as the between-subjects variable and condition (before and after recent conditioning +injection) as the within-subjects variable. Where the group × condition interaction was significant, we performed a simple main effects analysis and we adjusted each p-value with the Bonferroni correction. For each mixed ANOVA model, we assessed the Sphericity assumption through Mauchly’s Test of Sphericity. 

The statistical parameters (i.e., the exact value of n for each group, SEM, the statistical test, the effect size, and the exact p-value) are reported in the legends. For equal sample sizes, effect sizes for unpaired t-tests were determined by calculating Cohen’s d or Glass’s d according to the similarity of SDs while, for different sample sizes, Hedges’g. Correlations between cell counts and freezing were calculated using Pearson’s coefficient. To determine whether the data met the assumptions of the statistical approach, we rejected the null hypothesis at the P < 0.05 level. All statistical analyses were performed using SPSS Statistics 22 (IBM).

Supporting information

S1 Fig. Magnification of the needle tracks of Te2 (A) and ACC cortex (B) injected with CNQX, selected as examples. (C) When represented with the same context 2 weeks after the procedure, shock-only animals showed a low fear response, demonstrating that the procedure did not elicit conditioned freezing in the context where the shock was delivered. (D) Similar results as in Fig 1 were obtained by counterbalancing the 2 tones employed as CSs (CS1, 3 kHz and CS2, 15 kHz) (Student t test, t(14) = 3.44, p = 0.0040, Glass’s d = 4.14). (E) In the odor-CS conditioned rats freezing to the odor, 2 weeks after conditioning, was high even if tested in a different environment concerning the conditioning context, thereby showing that fear was specifically associated with this cue delivery. Scale bars, 300 μm. ��P < 0.01. All data are mean and SEM. The summary data for S1 Fig can be found in supporting information in the file named S1 Supporting Figure Data. (TIF)

S2 Fig. CNQX injection impaired the retention of recent auditory fear memories in rats where fear generalization was lowered by a tone alone pre-exposure or by employing a white noise tone as CS1. A 3 × 2 mixed-design ANOVA (main effect of group: F(2,42) = 6.478, p = 0.004, η2 = 0. 236, main effect of condition: F(1,42) = 0.161, p = 0.691, η2 = 0.004, group × condition interaction F(2,42) = 3.942, p = 0.027, η2 = 0.158) showed that freezing to the 3 kHz tone before its association to the US was lower in animals that received the 15 kHz tone pre-exposure before the 15 kHz-US pairing (n = 10, p = 0.001) or in rats conditioned to a white noise (n = 13, p = 0.008) as compared to CS1-CS2 animals (n = 22) that showed a variable fear generalization response. However, after CS-US learning and cnqx injections in the Te2 cortex, recent fear memory was impaired in all groups (p > 0.05). Simple main effect within groups (before and after CS-US learning followed by cnqx injection): CS1-CS2, p = 0.025; Tone-CS1-CS2, p = 0.189; WN-CS2, p = 0.347) �P < 0.05, ��P < 0.01. All data are mean and SEM. The summary data for S2 Fig can be found in Supporting information in the file named S1 Supporting Figure Data. (TIF)

S3 Fig. (A) Random example of retrobeads injection targeting BLA. (B and C) Examples of optical fiber placements above the BLA of animals injected with AAV vectors in Te2 (B) and ACC cortex (C). Scale bars, 500 μm.

Acknowledgments

We thank Dr. E. Manassero for their continuous support and advice.

Author Contributions

Conceptualization: Giulia Concina, Annamaria Renna, Benedetto Sacchetti.

Data curation: Giulia Concina, Luisella Milano, Benedetto Sacchetti.

Formal analysis: Giulia Concina, Annamaria Renna, Luisella Milano.

Funding acquisition: Giulia Concina, Benedetto Sacchetti.

Investigation: Giulia Concina, Annamaria Renna, Luisella Milano.

Methodology: Giulia Concina, Annamaria Renna, Luisella Milano.

Supervision: Benedetto Sacchetti.

Validation: Benedetto Sacchetti.

Writing – original draft: Giulia Concina, Benedetto Sacchetti.

Writing – review & editing: Annamaria Renna, Luisella Milano.

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