Can rats sense your fear?

These experiments establish that rats can communicate fear and induce specific odor fear learning via pheromone information.

nature.com - Pheromone-Induced Odor Associative Fear Learning in Rats - Nature

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Subjects

Sprague Dawley rats (8–10 weeks old, weight 200–300 g, in good health) of both sexes (n = 110 total) were assigned randomly to groups. Rats were housed in polycarbonate cages on a 12 h light/dark cycle, given ad libitum access to food and water, with all behavioural manipulation completed during the light phase of the light cycle. Odor + shock conditioned rats (O+/S+) were housed with same sex companion rats one week before the experiments and for the duration of the experiments, while other groups were housed alone. All procedures were approved by the Memorial University Institutional Animal Care Committee and carried out in compliance with the guidelines of the Canadian Council on Animal Care.

Experimental designs and statistical analysis

Experiment 1: Stressed cage mate-induced odor associative learning

Behavioral study: Odorants: Odorants were diluted with mineral oil to specific concentrations. Odorants used were terpinene (6.63%) and octanol (2.67%). These odorants were chosen as they are neither innately appetitive nor aversive to adult rats, and the concentrations were chosen so that the odors would emit a vapor-phase partial pressure of 1 Pascal17. Apparatus: All behavioural training and testing was completed with a custom-made olfactometer for air and odorant delivery attached to the shock chamber: a plexiglass chamber that sits on top of an electrified grid, connected to a shock generator/scrambler (Muromachi Kikai Model SGS-003DX). Polyvinyl carbonate bottles were used for each odor and connected to the olfactometer by C-flex tubing pinched shut when not in use. Evacuation tubing with a fan was attached to the top lid of the shock chamber to promote odor removal. Odor conditioning and testing: All rats were habituated to the shock chamber for one 30 min session each on two consecutive days with clean air pumped through the shock chamber. On the third day, rats were trained individually with four separate exposures to either odor, shock, or odor and shock, depending on their respective groups at 5, 15, 20, and 30 min during a 30 min training session. Odorant (terpinene) was delivered for 1 min at each time point. Shock was delivered at the last sec of the odor delivery (0.5 mA for 1 sec). Between each experiment, the shock chamber and grids were thoroughly cleaned with 70% ethanol and clean paper towels. There was a 15 min interval between the chamber cleaning and next experiment, where residual smell and ethanol were removed via the evacuation tubing. The fourth day consisted of a 30 min behavioural testing session in the same conditioning chamber. Medical air was delivered in the first half of the session and an odorant was delivered in the second half. Rats were tested with terpinene and octanol (a control odor) on the same day, and the order of the odorant testing was randomized and counter-balanced. The percentage of freezing time in response to the terpinene and octanol exposure was measured. Six groups were examined: (1) O−/S+, rats were housed alone and received shock only, no odor during the experiment; (2) O+/S−, rats were housed alone and received terpinene odor but no shock during the training; (3) O+/S+, rats received both terpinene odor and shock; and (4) O+/Comp (companion), rats were housed with O+/S+ rats and exposed to terpinene only during the training. O+/S+ rats were returned to the cages with O+/Comp rats immediately following the odor/shock conditioning. Ten minutes later, O+/Comp rats were subjected to the odor only conditioning. A subset of the O+/Comp rats in this group received saline (50 µl, i.p.) during the habituation and 40 min before the training; (5) O+/Comp + Prop, O+/Comp rats received saline during the habituation and propranolol (20 mg/kg, i.p.) 40 min before the training. (6) O−/Comp, companion rats were housed with the O+/S+ rats but not exposed to the conditioned odor. Neural circuit mapping: A separate cohort trained identically to groups 1–4 as described above underwent tissue collection for Arc and H1a mRNA visualization on the 4th day. Animals were given a final odor exposure in lieu of behavioural testing. Rats were placed in a sealed plexiglass container ventilated with a continuous flow of charcoal-filtered air for 1.5 hrs. Rats were then given a 5 min exposure to octanol, another 20 min of charcoal-filtered air, then a 5 min exposure to the conditioned odor, terpinene, followed by immediate isoflurane anaesthesia and decapitation. Brains were collected and flash frozen in 2-methylbutane immersed in an ethanol/dry ice slurry and kept at −80 °C. Fluorescence in situ hybridization (FISH): Brains were trimmed so that the cerebellum was discarded, and only the right hemisphere was analyzed. The right hemispheres of rats from each behavioural group were arranged so that the rostral end of their olfactory bulbs touched a razor blade to align them at the same rostral-caudal level. Brains were then arranged in a custom-made plastic box filled with OCT medium at −20 °C in a cryostat and frozen in a block. Coronal sections of 20 μm were collected on 2% 3-aminopropyltriethoxysilane treated slides. Five representative slides over the rostral-caudal range of each of the MOB, AOB, aPC/tubercle, and pPC/amygdala were chosen for FISH and stored at −20 °C.

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The double FISH protocol was established previously18. Briefly, full length Arc riboprobes conjugated to digoxigenin and H1a riboprobes conjugated to fluorescein were obtained using commercial transcription kits (Maxiscript) and RNA labeling mixes (Roche). Riboprobes were purified using RNA mini quickspin columns (Roche) and verified via agarose gel. Slides were thawed for 30 minutes at room temperature, fixed in 4% paraformaldehyde at 4 °C, bathed in acetic anhydride and acetone/methanol (Fisher Scientific), and treated with pre-hybridization buffer and hybridization buffer (Sigma-Aldrich) containing Arc and H1a probes. Slides were hybridized overnight in a 56 °C oven. All steps until this point were performed in the absence of RNAse. Slides were washed in a series of sodium citrate solutions followed by cleavage of any remaining single-stranded RNA using RNAse A. Endogenous peroxidases were quenched with H 2 O 2 and slides blocked with 5% sheep serum (Sigma-Aldrich). Arc riboprobe was detected with anti-digoxigenin-POD (Roche) and a TSA cyanine-3 substrate kit (Perkin Elmer). Following Arc detection slides were dipped in 2% H 2 O 2 solution to quench any residual HRP activity. H1a riboprobe was detected with anti-fluorescein-POD (Roche) and a TSA Fluorescein Tyramide substrate kit (Perkin Elmer). Nuclei were counterstained with DAPI (Sigma-Aldrich). Slides were coverslipped with Vectashield antifade medium (Vector Laboratories), sealed with clear nail polish, and kept at 4 °C before confocal microscopy scanning. Image Acquisition and Analysis: All slides were scanned in a Fluoview FV1000 confocal microscope (Olympus). All images were taken at 20X magnification. The photomultiplier tube assignments, confocal aperture size, and contrast remained constant for each slide. The z-stacks (optical thickness: 1.0 μm) were taken throughout the thickness of the section and were acquired from 3–4 slides for each animal. The mitral cell layer was analyzed in the olfactory bulbs, including the dorsolateral and ventral medial regions in the MOB. Layer II was analyzed in the PC, and the dense cell layer was analyzed in the OT. Images were analyzed from the center of each of the amygdala subdivisions. ImageJ software was used for counting cells in the scanned images. In all areas except the OBs total cell counting was done automatically for the DAPI stained nuclei; images were cropped to include only the area of analysis, transformed to binary images (black and white), and cells were counted using the “Analyze Particles” function in ImageJ. For the H1a+ and Arc+ cells, counting was done manually by checking 20% of the mid-range of the stack that comprised each cell. Average cell counts of Arc+ cells were divided by the average cell counts of H1a+ cells to compute a ratio of cells active to the conditioned odor versus cells active to the control odor for each animal.

Experiment 2: Pheromone-induced odor associative learning

Behavioral study: Four groups were included: (1) O+/S− (terpinene odor only); (2) Ph-T (pheromone paired with terpinene). Rats were housed alone and exposed to the clean bedding with a piece of filter paper soaked with 0.75 mL 4-methylpentanal (1.3 × 10−6 M) and hexanal (8.7 × 10−6 M) binary mixture (dissolved in purified water)2 on top of the bedding and received terpinene as the conditioned odor. (3) SB-T (soiled-bedding conditioned with terpinene); (4) SB-Oc (soiled-bedding conditioned with octanol). Rats were housed alone and exposed to the soiled bedding. A donor rat was shocked to release pheromone in the shock chamber (4 shocks during 30 min). The soiled bedding was woodchip bedding placed underneath the shock chamber during the donor rat shock and was subsequently left untouched for the conditioning of the SB rat. Habituation, odor delivery during the training, and testing were carried out in the same manner as in Experiment 1, except testing lasted 10 min (5 min in clean air, 5 min in an odorant), instead of 30 min. Additionally, Experiment 1 and 2 were carried out in two different rooms with different experimenters.

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To study the role of NMDA receptors in the basolateral amygdala, a separate cohort underwent cannular implantations. Cannular surgeries were performed 1 week before the behavioral experiments. During surgeries, rats were anesthetized with isoflurane gas and secured in a stereotaxic apparatus. Two holes were drilled 2.5 mm posterior, and 4.9 mm bilateral relative to bregma for the BLA. Guide cannulae were inserted 7.8 mm ventral to the skull surface. Guide cannulae were secured by dental cement to two skull screws. The skin was sutured and the rats were returned to their cages for recovery. O+/S+ and pheromone molecule conditioned (O+/Ph) rats were infused with either saline or D-APV (5 mM; 1 µl) bilaterally into the BLAs 30 min before the conditioning experiments. Infusion tubing and cannular attachment were performed during habituation for animals to become acclimated to the attachment of the infusion tubing.

Statistics

OriginPro 9.0 was used to analyze the datasets. One-way ANOVAs plus post-hoc Bonferroni tests were used to compare different groups in Figs 1–3. A two sample t-test (2-tail) was used in Fig. 4. Data are presented as mean ± SEM in Results and Figures. Figure 1 Conditioned fear can be transmitted to conspecifics in the absence of an external aversive stimulus. (A) Schematics of the odor conditioning and testing paradigm. (B) Percentage freezing time during the testing to the conditioned odor terpinene. (C) Percentage freezing time during the testing to the novel control odor octanol. O−/S+, shock only rats; O+/S−, odor only rats that were caged alone; O+/S+, odor/shock conditioned rats; O+/Comp, odor only rats that were caged with odor/shock conditioned rats; O+/Comp + Prop: O+/S− comp rats that were injected propranolol before training; O−/Comp: companion rats without subsequent odor exposure. *p < 0.05, **p < 0.01. Error bars, mean ± SEM. Full size image Figure 2 Pheromone odor conditioning and classical odor conditioning activate distinct but converging circuitries in the brain. (A) An example of Arc and H1a mRNA staining. “1” indicates a double labeled cell (green and red in the nucleus) that was activated by both terpinene and octanol; “2” indicates a cell expressing H1A (green; activated by octanol); “3” indicates a cell expressing Arc (red; activated by terpinene). Scale bars, 100 and 20 µm. (B–K) Ratios of Arc+/H1A+ cells in various olfactory and limbic structures. O−/S+, shock only rats; O+/S−, odor only rats that were caged alone; O+/S+, odor/shock conditioned rats; O+/Comp, odor only rats that were caged with odor/shock conditioned rats. *p < 0.05, **p < 0.01. Error bars, mean ± SEM. Full size image Figure 3 Alarm pheromone mediates the fear learning in companion rats. (A) Schematics of the odor conditioning and testing paradigm. (B) Percentage freezing time during the testing to the odor terpinene. (C) Percentage freezing time during the testing to the odor octanol. O+/S−, odor only rats that were caged alone; Ph-T, terpinene odor exposed rats that were conditioned with previously identified alarm pheromone molecules; SB-T, terpinene exposed rats that were conditioned with soiled bedding; SB-O, octanol exposed rats that were conditioned with soiled bedding. *p < 0.05, **p < 0.01. Error bars, mean ± SEM. Full size image

nature.com - Pheromone-Induced Odor Associative Fear Learning in Rats - Nature