A novel role for the lateral habenula in fear learning

Abstract

Fear is an extreme form of aversion that underlies pathological conditions such as panic or phobias. Fear conditioning (FC) is the best-understood model of fear learning. In FC the context and a cue are independently associated with a threatening unconditioned stimulus (US). The lateral habenula (LHb) is a general encoder of aversion. However, its role in fear learning remains poorly understood. Here we studied in rats the role of the LHb in FC using optogenetics and pharmacological tools. We found that inhibition or activation of the LHb during entire FC training impaired both cued and contextual FC. In contrast, optogenetic inhibition of the LHb restricted to cue and US presentation impaired cued but not contextual FC. In either case, simultaneous activation of contextual and cued components of FC, by the presentation of the cue in the training context, recovered the conditioned fear response. Our results support the notion that the LHb is required for the formation of independent contextual and cued fear memories, a previously uncharacterized function for this structure, that could be critical in fear generalization.

Fig. 1. Inactivation of the LHb during FC training blocks contextual FC.

A Diagram of the experimental setup: bilateral vehicle/muscimol intra LHb infusions were performed 30 min before FC training. Contextual FC was tested 7 days later. During training, subjects freely explored the cage for a baseline period of 197 s. After that they were exposed to 4 foot-shocks (0.6 mA, 3 s) interspaced by 87 s. In the recall session animals were re-exposed to the training context for 180 s and freezing was quantified. B Test of contextual FC memory. Left panel: freezing over time. Right panel: average baseline freezing during FC training and memory recall session 7 days later. During recall session freezing in the muscimol group was lower than in the control group (t₍₂₀₎ = 4.294, p = 0.0004, n_vehicle = 11, n_muscimol = 12). Additional statistics information can be found in the Statistics details section of Supplementary Material.

Fig. 2. Inactivation of the LHb during training blocks cued FC.

A Diagram of the experimental setup: bilateral vehicle/muscimol intra LHb infusions were performed 30 min before FC training. Cued FC was tested 7 days later. During training, rats freely explored the cage for a baseline period of 180 s that was followed by 4 tone-shock pairings (17 s of tone followed by 3 s, 0.6 mA shock) interspaced by 70 s. During memory recall, tone was presented for 60 s after a pre-tone period of 180 s. B Left panel: freezing over time. Dashed line delimits tone presentation. Right panel: average freezing for pre- and tone periods. Freezing during pre-tone period was low and not different between vehicle and muscimol groups (pre-tone freezing: 0.79 ± 0.55% for muscimol group vs. 10.97 ± 5.51% for vehicle group; t₍₂₈₎ = 1.469, p = 0.2828). In contrast, during tone presentation, a highly significant reduction in freezing was observed in the muscimol group (t₍₂₈₎ = 5.396, p < 0.0001, n_vehicle = 8, n_muscimol = 9). Additional statistics can be found in the Statistics details section.

Fig. 3. FC formed under inactivation of the LHb could be retrieved in context + tone conditions.

A Experiment diagram: bilateral intra LHb infusions of vehicle/muscimol were performed 30 min before training. Animals were trained in cued FC. Memory was evaluated 7 or 21 days later in the same context used for training (Context A). The tone was presented for 60 s after a pre-tone period of 180 s. B Freezing during recall session 7 days after training. Left panel: freezing over time. Dashed line delimits tone presentation. Right panel: average freezing for pre- and tone periods. Freezing to the context was higher in vehicle group (t₍₄₆₎ = 2.613, p = 0.0474). Tone elicited a robust freezing in the muscimol group equivalent to the vehicle group (t₍₄₆₎ = 0.899, p = 0.8460, n_vehicle = 13, n_muscimol = 13). Additional statistics can be found in the Statistics details section of Supplementary Material. C Freezing during recall session 21 days after FC training. Left panel: freezing over time. Dashed line delimits tone presentation. Right panel: average freezing for pre- and tone periods. 21 days after FC training, freezing in context + tone conditions in the muscimol group was lower than in the vehicle group (t₍₂₆₎ = 2.683, p = 0.0491) indicating a reduced temporal stability of memory elicited by the presentation of the cue in the training context. n_vehicle = 8, n_muscimol = 8. Additional statistics can be found in the Statistics details section of Supplementary Material.

Fig. 4. Optogenetic inactivation of the LHb during cue and US, impaired cued but not contextual FC.

A Experiment diagram: Top-Left: animals were bilaterally transfected with AAV-ArchT-GFP or AAV-GFP in the LHb and implanted with optic fibers above the LHb 4 weeks before training. Top-Right: during training, optogenetic light stimulation was delivered starting with the tone and stopping 5 s after the shock (tone and shock presentations were as previously described for cued FC). Bottom: diagram of training and memory tests. Cued memory was tested 7 days after training in Context B. The same animals were re-tested the following day in the training context to evaluate contextual FC memory and tone freezing in context + tone condition. B Microphotographs of the AAV-ArchT-GFP infection at ~3.6 mm posterior to Bregma (top, middle, bottom: DAPI, GFP, and merge respectively). Dashed white lines in the middle panel delimitates brain structures. * indicates the optic fiber tract. MHb: medial habenula, sm: stria medullaris, 3V: third ventricle. Scale bars: 1 mm. Freezing over time during tone test at day 7 (C) and during context + tone test at day 8 (D). Dashed line delimits tone presentation. E Average freezing on tone test, and context + tone test sessions. During tone test ArchT group displayed lower levels of freezing to the tone than GFP group (t₍₆₆₎ = 3.410, p = 0.0078). The following day, during context + tone session, freezing levels of the ArchT group to both the context and the tone were equivalent to those of the GFP group (t₍₆₆₎ = 0.178, p = 1.0000 and t₍₆₆₎ = 1.145, p = 0.8742 respectively; n_GFP = 9, n_ArchT = 10). Additional statistics can be found in the Statistics details section of Supplementary Material.

Fig. 5. Optogenetic excitation of the LHb during complete FC training impairs contextual and cued FC.

A Experiment diagram: Top-Left: animals were bilaterally infected with AAV-oChIEF-tdTomato in the LHb and implanted with optic fibers above the LHb 4 weeks before training. Top-Right: during whole-training session the LHb was stimulated with 5 ms light pulses at 20 Hz to disrupt endogenous neuronal activity. Bottom: diagram of training and tests. Cued memory was tested 7 days after training in Context B. The same animals were re-tested the following day in the training context to evaluate contextual FC memory and tone freezing in a context + tone session. B Microphotographs of the AAV-oChIEF-tdTomato infection at approximately 3.3 mm posterior to bregma (top, middle, bottom: DAPI, tdTomato fluorescence, and merge respectively). Dashed white lines in the middle panel delimitates brain structures. * indicates the optic fiber tract. MHb: medial habenula, sm: stria medullaris, 3V: third ventricle. Scale bars: 1 mm. Freezing over time during cued test at day 7 (C) and during context + tone test at day 8 (D). Dashed line delimited area indicates tone presentation. E Average freezing of tone test and context + tone test sessions. Disruption of endogenous LHb activity during training by optogenetic stimulation-induced deficits in cued (tone freezing during Tone Test: control vs oChIEF + light: t₍₄₂₎ = 4.454, p = 0.0004; tone freezing during Context + Tone test: control vs oChIEF + light: t₍₄₂₎ = 2.995, p = 0.0317 respectively), and contextual memories (pre-tone freezing during context + tone test: control vs oChIEF + light: t₍₄₂₎ = 3.874, p = 0.0026). n_Control = 7, n_{oChIEF + light} = 6. Additional statistics can be found in the Statistics details section of Supplementary Material.