Animals

Male C57BL/6 mice (Charles River Laboratories) were used in all behavioural tests. All mice were 8–10 weeks old at the time of surgery and 12–14 weeks old at the time behavioural testing began. Before surgery, mice were group-housed in a temperature-controlled room on a 12 h light/dark cycle with ad libitum access to food and water. Following surgery, mice were individually housed. A total of 60 mice were distributed into two groups for hM4D experiments (hM4D-mCherry: n = 10, mCherry control: n = 8) and three groups for ArchT experiments (GFP-control: n = 14, iHPC-lAON: n = 12, vHPC-mAON: n = 16). An additional 29 animals were used for c-Fos immunoreactivity experiments. All procedures were performed in accordance with the guidelines of the Canadian Council on Animal Care and the University of Toronto Animal Care Committee.

Surgical procedures

CAV2-Cre viral vector was purchased from the Plateforme de Vectorologie de Montpellier, AAV2/8-hSyn-FLEX-hM4D-mCherry (hM4D) and AAV2/8-hSyn-DIO-mCherry (mCherry control) from the vector core at University of North Carolina at Chapel Hill, and AAV2/5-hSyn-hChR2-mCherry (ChR2-mCherry), AAV2/5-hSyn-hChR2-eYFP (ChR2-YFP), AAV2/5-CaMKIIa-ArchT-eYFP (ArchT), and AAV2/8-CB7-CI-EGFP-RBG (GFP-control) from the University of Pennsylvania Vector Core. Stereotaxic surgery was conducted on mice anaesthetized with isoflurane and administered ketoprofen (5 mg/kg) for pain management. For chemogenetic experiments, CAV2-Cre viral vector was bilaterally infused into the mAON (10° angle toward midline; anterior/posterior (AP): + 2.90, medial/lateral (ML): ± 1.10, dorsal/ventral (DV): − 3.42) and lAON (no angle; AP: + 3.20, ML: ± 1.10, DV: − 3.90) at a volume of 0.1–0.2 μL and hM4D into the iHPC (no angle; AP: − 2.70, ML: ± 2.20, DV: − 2.00) and vHPC (10° angle away from midline; AP: − 2.92, ML: ± 2.15, DV: − 4.90) at a volume of 0.3–0.4 μL. The position immediate to the rhinal fissure was used as a landmark to delineate intermediate and ventral parts. For optogenetic experiments, ArchT or GFP-control was bilaterally infused into the iHPC or vHPC in a volume of 0.3–0.4 μL, and optical fibres (200 μm core diameter, 0.39 NA; Thorlabs, Newton, NJ, USA) threaded through 1.25 mm-wide zirconia ferrules (Thorlabs) were bilaterally implanted into the lAON or mAON, respectively. For anterograde tracing experiments, ChR2-mCherry was infused into the vHPC and ChR2-YFP was infused into the contralateral iHPC. All infusions were made by means of pressure ejection at a rate of 0.1 μL/min through a cannula connected by Tygon tubing to a 10 μL Hamilton syringe (Hamilton, Reno, NV). A 15 min interval was allotted after each infusion to limit the viral spread.

Drugs

CNO obtained from the NIH was dissolved in a solution of 10% dimethyl sulphoxide and 0.9% saline. A dose of 5 mg/kg of CNO was used in all behavioural experiments using hM4D-mCherry- and mCherry-only-expressing animals. All CNO treatments were separated by a minimum of 72 h.

Apparatus for optogenetic experiments

Inhibition of hippocampal terminals at the AON was conducted by illumination with green light (532 nm, 12 mW) generated by a diode-pumped solid state laser (Laserglow, Toronto, ON, Canada). The laser was connected to a 1 × 2 optical commutator (Doric Lenses, Quebec, QC, Canada), which divided the light path into two arena patch cables attached to the implanted optical fibres.

Experimental design

Unless otherwise noted, all tests took place in a 50 cm × 25 cm × 20 cm plexiglass open-topped cage. Odours were presented mixed with woodchip bedding in 3 cm wide, 1 cm high aluminium cups. Multiple identical odour cups were used such that an animal never investigated the same cup twice. The odours used included nutmeg, vanillin, coriander, banana, garlic, cinnamon, thyme, almond, onion, curry, ginger, savoury, cumin, dill, jasmine, coffee, oregano, sage, and rosemary. The odours presented and the order of their presentation between animals was pseudorandomized. For habituation, mice were given 15 min of exploration time for each unique context prior to initial exposure. Each exposure was 5 min in length and inter-trial intervals were 15 min. All tests were video-recorded at 60 fps using a NIKON D5200 equipped with a 30 mm lens. An additional overhead video was recorded using a Logitech webcam. All videos were subsequently scored blind to the treatment groups. Exploration was strictly defined as head up sniffing, directed towards and within 1 cm of the odour source. This definition excludes the use of the odour cup for sitting or as support during rearing.

Olfactory spatial memory test: In this paradigm adapted from Eacott and Norman53, mice were tested for memory of odour location in context. The test chamber was altered to produce two distinct contexts. Zebra-patterned paper was used to line the walls of context A, whereas context B had transparent walls surrounded by red plastic cups and bedding on the floor. Each animal underwent two encoding phases and one retrieval phase. During the first encoding phase, mice explored context A where two highly distinct odours were placed at opposite ends of the chamber (odour 1 on the left and odour 2 on the right). Next, mice were removed from the chamber and placed in a holding cage. The mice were then returned to the chamber, except it was now configured as context B and contained both odours in opposite positions (odour 2 on the left and odour 1 on the right). The animals were allowed to explore both odours in their new positions before being placed back into the holding cage. For the retrieval phase, the chamber was reconfigured as context A but now two copies of one odour were presented on both sides of the chamber. Time spent investigating the odour cups was measured. The novel configuration consists of the familiar odour in a novel position within the original context. The initial context and left/right position of the odour cups were pseudorandomized.

Olfactory temporal order memory test: This paradigm is based on similar tests used previously to measure memory for the temporal order of objects54,55. Mice were tested in a transparent chamber with spatial cues kept constant throughout the session. Each animal underwent three encoding phases and one retrieval phase. In the first exposure, mice were placed in the chamber with two copies of one odour presented on opposite sides of the arena. After exploring both copies, the animal was removed from the chamber and placed in a holding cage. This process was repeated two more times using different odours each time. During the retrieval phase the animal was returned to the chamber, but this time earlier and recently explored odours were presented on opposite sides. In this case, the odour explored earlier is more novel given its TD compared with the recently explored odour. The left–right positions of the first and last odours during the test were pseudorandomized. Time spent investigating both odours was measured.

Novel odour recognition test: This test was given 72 h after examining performance on temporal order memory and followed a similar paradigm. The animals underwent three encoding phases where two copies of a unique odour were presented in each. On the retrieval phase, mice were presented with the initially encountered odour and a previously unexplored odour on opposite sides of the chamber. Time spent investigating each odour was measured.

Olfactory episodic memory test: Animals were first habituated to the apparatus, which consisted of a 50 cm × 50 cm × 20 cm transparent plexiglass open field for a 30 min period. The animals were then exposed to two encoding phases and one retrieval phase each separated by a 1 h delay. In the first encoding phase, animals were given 10 min to explore two different odours located at two adjacent corners of the arena. In the second encoding phase, the animals were given an additional 10 min to explore another set of unique odours presented on the opposite adjacent corners. During the retrieval phase, all four odours were presented with the spatial position of one odour from each set exchanged. This presentation results in each odour possessing a unique spatiotemporal configuration—NL/TD, FL/TD, NL/TR, and FL/TR. Successful memory for an integrated (what, when, and where) memory results in a pattern of exploration such that the odour with the NL/TD configuration is preferentially investigated the most while the FL/TR configuration is investigated the least. Time spent investigating all four odours was measured for 5 min in the retrieval phase. Overhead videos were analysed using the ANY-maze software to produce average heat maps of each treatment group’s position within the arena.

Context-driven odour recall test: In this paradigm adapted from Mandairon et al.10, mice were trained to associate a visually distinct context with an odour and subsequently tested for recollection of the odour when exposed to the context alone. The testing apparatus consists of a 50 cm × 30 cm × 20 cm plexiglas cage with colourful visual patterns pasted on the outside of the walls. A wooden applicator with a cotton swab tip was positioned 3 cm from the floor and 5 cm from one end of the chamber. Before introducing mice into the chamber, 100 μL of a pure odourant was applied to the cotton tip. Each mouse was randomly assigned a monomolecular odourant to be trained with among limonene, isoamyl acetate, nonane, and 1-pentanol. Mice were allowed to explore the context and the odorized cotton swab for 30 min per day for 9 consecutive days. On Day 10, mice were once again placed into the cage, however no odour was added to the cotton swab. Investigation time of the cotton swab was measured on day 9 and 10 for the first 5 min of their exposure to the context. Upon failure to detect the expected odour, mice behaviourally expressed memory by spending a greater amount of time investigating the cotton swab compared to their investigation time when the odour was present on Day 9.

General histology

After behavioural testing, mice were transcardially perfused with phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in phosphate buffer. Brain tissue was extracted and postfixed overnight at 4 °C. The brains were then cryoprotected using a 30% sucrose in PBS solution. Coronal 40 µm-thick sections were collected using a cryostat (Leica, Germany). The sections were slide-mounted, counterstained with 4′,6-diamidino-2-phenylindole 135 for 5 min and subsequently coverslipped with Aquamount (Polysciences, Inc., Warrington, PA). Wide-field fluorescent images were captured using a 4 × objective lens on a fluorescent microscope (Olympus, Japan). Confocal images were captured using a × 20 and × 60 objective through a Quorum spinning disk confocal microscope (Zeiss, Germany). Adobe Photoshop CS6 (Adobe Systems, Incorporated, San Jose, CA) was used to adjust the brightness and contrast of representative sections.

Immunohistochemistry

For c-Fos immunostaining, free-floating tissue sections were obtained and washed with PBS with Triton X-100 (PBS-T) and then blocked with normal donkey serum (5 in 0.1% PBS-T) for 1 h. The sections were subsequently incubated with rabbit polyclonal anti-c-Fos antibody (1:1000 in PBS-T; Santa Cruz Biotechnology, California) for 72 h on a nutating mixer at 4 °C. After incubation with the primary antibody the sections were submerged in Alexa Flour 594-conjugated donkey anti-rabbit secondary antibody (1:500 in PBS-T; Invitrogen) for 90 min at room temperature.

Validation of chemo- and optogenetic inhibition

To characterize the AON’s response patterns we exposed groups of three animals to one of four conditions: homecage only, homecage with an applied odour (100 µL of limonene on a cotton ball), novel context only, or novel context with an applied odour. With the exception of the homecage control group, all animals were put into each condition for a total duration of 30 min.

To validate our chemogenetic and optogenetic manipulations, we prepared two groups of four animals—one group received unilateral CAV2-Cre infusions into the mAON and Cre-responsive hM4D in the vHPC; another received unilateral ArchT infusions into the vHPC and optic fibre implantations in the mAON. Each group was exposed to a novel odour-context pairing for 10 min (reduced from 30 min to prevent prolonged illumination in the optogenetic experiment). hM4D-expressing mice were injected with CNO 15 min prior to exposure whereas ArchT-expressing mice were illuminated with green light throughout the exposure. In the subsequent tissue analysis, the mean density of c-Fos-labelled neurons were compared between each hemisphere.

To examine context-driven AON activity, two groups of three mice were trained on a context-odour association by placing the animals in a context (50 × 30 × 20 cm plexiglas closed-lid cage with colourful visual patterns pasted on the outer walls) with an applied odour for 1 h over three consecutive days. The mice were killed the following day after exposure to the context-odour pair or the context alone for 30 min. c-Fos density was measured and compared to a third group trained and exposed to a context in the absence of an applied odour.

All animals were transcardially perfused 75 min after initial exposure to their testing condition.

Cell counting

Every other section from bregma + 3.20 to + 2.34 was collected and immunostained for c-Fos. Sections were mounted and scanned using a 4 × objective lens on a fluorescent microscope. c-Fos-positive cell counting was performed using the cellSens software (Olympus). The area of each AON section was outlined to form a region of interest (ROI) and the number of mCherry-expressing cells were counted. Calibration parameters were established using randomly chosen prominent neurons and adjusted by raising the threshold of detection to a conservative level, excluding the majority of false positives. Once set, the calibration was kept constant. The density of neurons was calculated by dividing the number of labelled cells in the ROI by the area for each AON section. The mean density was determined for each animal and for all animals in each experimental group.

Calculations and statistical analysis

The discrimination ratios were derived from the exploration time of odour-context pairings during the retrieval phase of each test. For all tests based on the spontaneous novelty preference paradigm, the discrimination ratio was calculated as the difference between the times spent exploring the novel and familiar odour-context configurations divided by the total amount of time investigating both odours. Here, a value of zero indicates that the animal investigated both odours to an equal extent. Positive values up to one indicate preference for the novel combination, whereas a negative value indicates greater investigation of the familiar odour-context pair. The discrimination ratios of each group obtained using the behavioural paradigms detailed in Fig. 1 and Fig. 2 were compared to zero (chance performance) using a (two-tailed) one-sample t-test.

For the episodic memory test, percent investigation time was calculated by dividing the amount of time spent investigating an individual odour-spatiotemporal configuration by the time investigating all odours and multiplied by 100%:

$$\frac{{{\mathrm{NL/TD}}}}{{\left( {{\mathrm{NL/TD + NL/TR}}} \right) + \left( {{\mathrm{FL/TD + FL/TR}}} \right)}} \times 100{\mathrm{\% }}$$

For analysing spatial memory, the difference in the amount of time spent investigating odours with novel and familiar positions was divided by the total investigation time:

$$\frac{{\left( {{\mathrm{NL/TD + NL/TR}}} \right) - \left( {{\mathrm{FL/TD + FL/TR}}} \right)}}{{\left( {{\mathrm{NL/TD + NL/TR}}} \right) + \left( {{\mathrm{FL/TD + FL/TR}}} \right)}}$$

For temporal memory, the difference in the time spent investigating odours experienced earlier and later was divided by the total investigation time:

$$\frac{{\left( {{\mathrm{NL/TD + FL/TD}}} \right) - \left( {{\mathrm{NL/TR + FL/TR}}} \right)}}{{\left( {{\mathrm{NL/TD + NL/TR}}} \right) + \left( {{\mathrm{FL/TD + FL/TR}}} \right)}}$$

Performance in novelty preference-based paradigms was compared using t-test in hM4D experiments and a one-way analysis of variance (ANOVA) with testing group as the factor in optogenetic experiments. Percent investigation time data collected in the episodic memory test was analysed using a two-way ANOVA with testing group and odour-spatiotemporal configuration as factors. A two-way ANOVA was used to analyse data in the contextually cued odour recall test for hM4D and optogenetic experiments, respectively, using experimental day and treatment group as factors. Where appropriate, Tukey’s multiple comparisons test was used for post hoc comparisons. Significance was defined as *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Data availability

All relevant data are available from the corresponding authors upon reasonable request.