Figures and data in Dorsal hippocampus mediates light–tone associations in male mice | eLife

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Figure 1 with 3 supplements

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Simultaneous associations between light and tone are required for sensory preconditioning responding.

(A) Schematic representation of the _LTSPC task in males (two training sessions) with a representation of paired, unpaired, and no-shock experimental groups. The temporal dynamics of freezing represented in bins of 10 s across time of experiment of the Probe Test 1 (tone) (B on the left) and Probe Test 2 (light) (C on the left). The percentage of time spent freezing during OFF and ON periods of the Probe Test 1 (tone) (B on the right) and Probe Test 2 (light) (C on the right). *Significant p-value (<0.05) after false discovery rate (FDR). GLM: generalized linear model fitted to gamma distribution with planned comparisons. KW: Kruskal–Wallis across experimental groups: paired, unpaired, no-shock. * p<0,05; ** p<0,01 (between OFF and ON periods); # p<0,05; ## p<0,01, ###, p<0,001. See statistical details in Supplementary file 1A.

Figure 1—figure supplement 1

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New automatized tool to measure freezing responses.

We developed a new automated tool to assess freezing behavior using a fear conditioning protocol (A). This novel tool was based on pose estimation using DeepLabCut and a customized Python script, which considers freezing when the Euclidean distance is lower than 0,02 cm per pair of frames for videos with 25 fps, resulting in a speed lower than 0,5 cm/s. To validate this tool, we correlate the freezing measures during all phases (upper) (B) and probe test (lower) (C) on males (left) and on females (right) with ezTrack (a software dedicated to the automatized freezing quantification) and our own manual counting performed by an expert in this type of behaviors. See statistical details in Supplementary file 1B.

Figure 1—figure supplement 2

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Simultaneous associations between light and tone to develop a sensory preconditioning protocol in females.

(A) Schematic representation of the _LTSPC task in females (one training session) with a representation of paired, unpaired, and no-shock experimental groups. The temporal dynamics of freezing represented in bins of 10 s across time of experiment of the Probe Test 1 (tone) (B on the left) and Probe Test 2 (light) (C on the left). The percentage of time spent freezing during OFF and ON periods of the Probe Test 1 (tone) (B on the right) and Probe Test 2 (light) (C on the right). (D) Independent experiment to test fear generalization observed in the female, we extended the interval between cue presentations during preconditioning (Unpaired) or by removing the light during the conditioning phase (no light). *Significant p-value (<0.05) after false discovery rate (FDR). GLM: generalized linear model fitted to gamma distribution with planned comparisons. ** p<0,01;*** p<0,001; (between OFF and ON periods). See statistical details in Supplementary file 1B.

Figure 1—figure supplement 3

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Spearman correlations of mediated (tone) and direct (light) learning for the protocol of sensory preconditioning for males (A) and females (B).

Panel A shows a significant positive correlation in males (ρ = 0.70, p = 0.011), whereas panel B shows no significant correlation in females (ρ = 0.02, p = 0.920).

Figure 2 with 3 supplements

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Hippocampal cell activity during _LTSPC.

(A) Schematic representation of _LTSPC task while fiber photometry recordings of RCaMP (calcium sensor in synapsin-positive neurons) and Cre-dependent GCaMP (calcium sensor in PV-positive interneurons) in dHPC and vHPC of PV^cre mice. (B) dHPC modulation during preconditioning of _LTSPC: on the left upper panel z scores of $\frac{Δ f}{f}$ (where f represents fluorescence) of GCaMP PV-positive interneurons on dHPC (green), left bottom z scores of $\frac{Δ f}{f}$ of RCaMP in neurons of dHPC (red), right upper number of events with a $\frac{Δ f}{f} > 2$ of GCaMP PV-positive interneurons on dHPC, and right bottom number of trials with a $\frac{Δ f}{f} > 2$ of Rcamp in neurons of dHPC. (C) vHPC modulation during _LTSPC: on left upper panel z scores of $\frac{Δ f}{f}$ of GCaMP PV-positive interneurons of vHPC (green), left bottom z scores of $\frac{Δ f}{f}$ of RCaMP in neurons of vHPC (red), right upper number of trials with a $\frac{Δ f}{f} > 2$ of GCaMP in PV-positive interneurons of vHPC, and right bottom number of trials with a $\frac{Δ f}{f} > 2$ of RCaMP in neurons of vHPC. (D) maximal value of $\frac{Δ f}{f}$ in the first 1-s window after pairings compared with baseline, on the left (dHPC) and on the right (vHPC). * p-<0.05; ** p<0,01. See statistical details in Supplementary file 1A.

Figure 2—figure supplement 1

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Fiber photometry recordings of RCaMP (calcium sensor in synapsin-positive neurons) and Cre-dependent GCaMP (calcium sensor in PV-positive interneurons) in dHPC and vHPC of PVcre mice.

Photometry recording during each preconditioning trial (A), each light presentation during conditioning (B), and each shock presentation during conditioning (C). (A–C) Maximal value of $\frac{Δ f}{f}$ in the first 1-s window after stimulus compared with baseline, on the upper (GCaMP) and on the lower (RCaMP) in dHPC and vHPC. * p<0.05. See statistical details in Supplementary file 1B.

Figure 2—figure supplement 2

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Hippocampal cell activity at the onset of the light in conditioning sessions.

(A) On the top, schematic representation of fiber photometry recordings with RCAMP1 and GCAMP8 in dHPV and vHPC of PV-Cre mice at the onset of the conditioned light (i.e., after pairing between light and electric footshock) during the conditioning phase. (B) dHPC modulation during preconditioning of _LTSPC: on left upper panel, z scores of $\frac{Δ f}{f}$ (where f represents fluorescence) of GCAMP8 PV-positive interneurons on dHPC (green); left bottom panel, z scores of $\frac{Δ f}{f}$ of RCAMP1 neurons in dHPC (red); right upper panel, number of events with a $\frac{Δ f}{f} > 2$ of GCAMP8 PV-positive interneurons in dHPC, and right bottom panel, number of trials with a $\frac{Δ f}{f} > 2$ of RCAMP1 neurons in dHPC. (C) vHPC modulation during _LTSPC: on left upper panel, z scores of $\frac{Δ f}{f}$ of GCAMP8 PV-positive interneurons in vHPC (green); left bottom panel, z scores of $\frac{Δ f}{f}$ of RCAMP1 neurons in vHPC (red); right upper panel, number of trials with a $\frac{Δ f}{f} > 2$ of GCAMP8 PV-positive interneurons in vHPC, and right bottom panel, number of trials with a $\frac{Δ f}{f} > 2$ of RCAMP1 neurons in vHPC. (D) maximal value of $\frac{Δ f}{f}$ in the first second window after pairings compared with baseline, on left (dorsal hippocampus, dHPC) and on right (ventral hippocampus, vHPC). * p<0.05; ** p≤0,01. Statistical details in Supplementary file 1B.

Figure 2—figure supplement 3

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Hippocampal cell activity at the electric footshock onset.

On the top, schematic representation of the fiber photometry recordings with RCAMP1 and GCAMP8 on dorsal or ventral hippocampus of PV-Cre mice at the onset of the electric footshock during the conditioning phase. (B) dHPC modulation during preconditioning of _LTSPC: on left upper panel, z scores of $\frac{Δ f}{f}$ (where f represents fluorescence) of GCAMP8 PV-positive interneurons on dHPC (green); left bottom panel, z scores of $\frac{Δ f}{f}$ of RCAMP1 neurons in dHPC (red); right upper panel, number of events with a $\frac{Δ f}{f} > 2$ of GCAMP8 PV-positive interneurons in dHPC, and right bottom panel, number of trials with a $\frac{Δ f}{f} > 2$ of RCAMP1 neurons in dHPC. (C) vHPC modulation during _LTSPC: on left upper panel, z scores of $\frac{Δ f}{f}$ of GCAMP8 PV-positive interneurons in vHPC (green); left bottom panel, z scores of $\frac{Δ f}{f}$ of RCAMP1 neurons in vHPC (red); right upper panel, number of trials with a $\frac{Δ f}{f} > 2$ of GCAMP8 PV-positive interneurons in vHPC, and right bottom panel, number of trials with a $\frac{Δ f}{f} > 2$ of RCAMP1 neurons in vHPC. (D) maximal value of $\frac{Δ f}{f}$ in the first second window after pairings compared with baseline, on left (dorsal hippocampus, dHPC) and on right (ventral hippocampus, vHPC). * p<0.05; ** p<0,01. Statistical details in Supplementary file 1B.

Figure 3

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CaMKII neurons during preconditioning on dHPC and vHPC.

(A) Maximal value of $\frac{Δ f}{f}$ in the first 1-s window after stimulus compared with baseline, for each stimulus during preconditioning session, on the left (dHPC) and on the right (vHPC). (B) dHPC modulation during preconditioning of _LTSPC: on left z scores of $\frac{Δ f}{f}$ (where f represents fluorescence) of GCaMP in CaMKII-positive neurons on dHPC (green), on right number of events with a $\frac{Δ f}{f} > 2$ of GCaMP CaMKII-positive neurons on dHPC. (C) vHPC modulation during preconditioning of _LTSPC: on left z scores of $\frac{Δ f}{f}$ (where f represents fluorescence) of GCaMP in CaMKII-positive neurons on vHPC (green), on right number of events with a $\frac{Δ f}{f} > 2$ of GCaMP CaMKII-positive neurons on vHPC. (D) Maximal value of $\frac{Δ f}{f}$ in the first 1-s window after stimulus compared with baseline, for the average of the six pairings stimuli during preconditioning session, on the left (dHPC) and on the right (vHPC).(E) Photometry recording during each preconditioning trial. * p<0.05. Statistical details in Supplementary file 1A.

Figure 4 with 3 supplements

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Chemogenetic modulation of dorsal and ventral hippocampus during _LTSPC.

(A) Schematic representation of _LTSPC task combined with chemogenetic approaches, where an inhibitory DREADD was infused in dHPC (representative image on the left) and vHPC (representative image on the right). During the preconditioning (DPC) or the Probe Test (DPT), a DREADD agonist (J60) was injected intraperitoneally (controls were injected with the agonist in both phases) (diagram of DREADD agonist administration on the center). The temporal dynamics of freezing represented in bins of 10 s across time of experiment of the Probe Test 1 (tone) of dHPC (B on the left) and vHPC (C on the left) and Probe Test 2 (light) of dHPC (D on the left) and vHPC (E on the left). The percentage of time spent in freezing during OFF and ON periods (left) of the Probe Test 1 (tone) of animals infused in dHPC (B on the right) and vHPC (C on the right). The percentage of time spent in freezing during OFF and ON periods (left) of the Probe Test 2 (light) of animals infused in dHPC (D on the right) and vHPC (E on the right). Controls are labeleld as Constrols_d (on dHPC) and Controls_V (on vHPC). The preconditioning groups are labeled as DPCd (on dHPC) and DPCv (on vHPC). The Probe Test groups are labeled as DPTd (on dHPC) and DPTv (on vHPC). *Significant p-value (<0.05) after false discovery rate (FDR). GLM: generalized linear model fitted to gamma distribution with planned comparisons. ** p<0,01; *** p<0,001. See statistical details in Supplementary file 1A.

Figure 4—figure supplement 1

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Additional controls for chemogenetics experiments.

Percentage change of the Off versus On periods to the control groups during mediated (A) and direct (B) of dHPC and mediated (C) and direct (D) of vHPC. The control groups are constituted by: CS (animals injected in HPC with AAV-CamkII-mCherry with saline (i.p.) during preconditioning and probe test), CJ60 (animals injected in HPC with AAV-CamkII-mCherry with J60 (i.p.) during preconditioning and probe test), and DS (animals injected in HPC with AAV-CamkII-hM4Di with J60 (i.p.) during Preconditioning and probe test). (E) Frequency of calcium transients in CaMKII-positive neurons in the dHPC in C57BL6J mice in basal and J60 conditions. (F) Frequency of calcium transients in CaMKII-positive neurons in the vHPC in C57BL6J mice in basal and J60 conditions. (G) During the conditioning phase, the DREADD agonist J60 was injected intraperitoneally and the percentage of time spent in freezing during OFF and ON periods of the Probe Test 1 (mediated learning) and Probe Test 2 (direct learning) is shown. * p<0,05; ** p<0.01; *** p<0,001. Statistical details in Supplementary file 1B.

Figure 4—figure supplement 2

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Inhibition of PV interneurons in dorsal hippocampus during light–tone associations did not impact mediated learning.

(A) Schematic representation of the chemogenetic approach in PV-Cre mice, where a Cre-dependent inhibitory DREADD was infused in dHPC (representative image on right). Frequency of calcium transients in (B) dHPC and (C) vHPC in PV-positive mice in basal and under J60 conditions. (D) During the preconditioning phase, the DREADD agonist J60 was injected intraperitoneally and the percentage of time spent in freezing during OFF and ON periods of the Probe Test 1 (mediated learning) and Probe Test 2 (direct learning) is shown. * p<0.05; ** p<0.01. Statistical details in Supplementary file 1B.

Figure 4—figure supplement 3

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Temporal dynamics of the conditioning phase of all sensory preconditioning experiments.

(A) Freezing percentage across the conditioning phase composed of two sessions (Training 1 and Training 2) of the protocol of sensory preconditioning on males with the respective control groups (Paired, Unpaired, and No-Shock). (B) Freezing percentage across the conditioning phase composed of one session (Training 1) of the protocol of sensory preconditioning on females with the respective control groups (Paired, Unpaired, and No-Shock). (C) Freezing percentage across the conditioning phase composed of two sessions (Training 1 and Training 2) during chemogenetic modulation of dHPC on preconditioning (DPCd) and on probe test (DPTc) compared with controls (Controls_d) during sensory preconditioning on males. (D) Freezing percentage across the conditioning phase composed of two sessions (Training 1 and Training 2) during chemogenetic modulation of vHPC on preconditioning (DPCv) and on probe test (DPTv) compared with controls (Controls_v) during sensory preconditioning on males. (E) Freezing percentage across the conditioning phase composed of two sessions (Training 1 and Training 2) during chemogenetic modulation of CaMKII-positive neurons on dHPC during conditioning phase (J60) compared saline-treated animals during sensory preconditioning on males. In yellow bars, the periods of light on, and in red dashed lines, the periods of 2-s shock stimulus.

Additional files

MDAR checklist: https://cdn.elifesciences.org/articles/105863/elife-105863-mdarchecklist1-v1.docx
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Supplementary file 1 Statistical tables for main figures and figure supplements.: https://cdn.elifesciences.org/articles/105863/elife-105863-supp1-v1.docx
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Source data 1 Raw data for main figures.: https://cdn.elifesciences.org/articles/105863/elife-105863-data1-v1.xlsx
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Source data 2 Raw data for figure supplements.: https://cdn.elifesciences.org/articles/105863/elife-105863-data2-v1.xlsx
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Julia S Pinho
Carla Ramon-Duaso
Irene Manzanares-Sierra
Arnau Busquets-Garcia

(2025)

Dorsal hippocampus mediates light–tone associations in male mice

eLife 14:RP105863.

https://doi.org/10.7554/eLife.105863.3

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