Distinct Dorsal and Ventral Hippocampal CA3 Outputs Govern Contextual Fear Discrimination

Abstract

Considerable work emphasizes a role for hippocampal circuits in governing contextual fear discrimination. However, the intra- and extrahippocampal pathways that route contextual information to cortical and subcortical circuits to guide adaptive behavioral responses are poorly understood. Using terminal-specific optogenetic silencing in a contextual fear discrimination learning paradigm, we identify opposing roles for dorsal CA3-CA1 (dCA3-dCA1) projections and dorsal CA3-dorsolateral septum (dCA3-DLS) projections in calibrating fear responses to certain and ambiguous contextual threats, respectively. Ventral CA3-DLS (vCA3-DLS) projections suppress fear responses in both certain and ambiguous contexts, whereas ventral CA3-CA1 (vCA3-vCA1) projections promote fear responses in both these contexts. Lastly, using retrograde monosynaptic tracing, ex vivo electrophysiological recordings, and optogenetics, we identify a sparse population of DLS parvalbumin (PV) neurons as putative relays of dCA3-DLS projections to diverse subcortical circuits. Taken together, these studies illuminate how distinct dCA3 and vCA3 outputs calibrate contextual fear discrimination.

Keywords: contextual fear discrimination; dorsal hippocampus; dorsolateral septum; hippocampus; lateral septum; memory; ventral hippocampus.

Figure 1.. Topographical Organization of Excitatory CA3 Projections in DLS

(A and B) Forward mapping of CA3 excitatory terminals in DLS obtained from the progeny of G32–4 bred with Ai27 (tdTomato) or Ai35 (GFP). In these mouse lines, ChR2-tdTomato or Arch-GFP is restricted mainly to CA3 (bottom panels). Note the abundance of terminals in DLS (top panels). (C and D) Local injection of CaMKII-ChR2-eYFP in dorsal or ventral CA3 of C57B6/J mice leads to restricted expression of eYFP in the medial or lateral part of DLS, respectively. Asterisks denote the abundance of terminals in dorsal and ventral CA1, respectively. (E) Dorsal CA3 inhibitory neurons do not innervate DLS, as evidenced by selective infection of GAD2-Cre mice with DIO-ChR2-eYFP. Asterisk denotes the presence of terminals in the medial septum. Scale bar: 100 μm.

Figure 2.. Dorsal CA3 Excitatory Projections to CA1 Instruct Fear Responses to Certain but Not Ambiguous Contextual Threats

(A and B) Schematic of the behavioral timeline. (C) Schematic illustrating dorsal CA3 infection with CaMKII-eNpHR3.0 and fiber optic implantation on top of dorsal CA1 in C57B6/J mice. (D–G) Silencing dorsal CA3 terminals in dorsal CA1 has no effect on locomotor behavior and innate anxiety in OF (D and E), EPM (F), and NSF (G). (H and I) Silencing dorsal CA3 terminals in dorsal CA1 decreases freezing behavior in context A but not B (H), which results in a decrease in fear discrimination ratio on block 7 (I). Data (means ± SEM; n = 14, 15 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test and log-rank (Mantel Cox) test. *p < 0.05, eNpHR3.0 versus eYFP; #p < 0.05, block 7 versus block 6. Statistics detailed in Table S1. (J) Schematic representation of the effect of light silencing dorsal CA3 terminals in CA1 on brain-wide c-Fos expression 60 min following exposure to context A (day 21). Regions highlighted in red denote a significant effect of eNpHR3.0, and arrows indicate the direction of the effect. See also Figures S1, S3, and S4.

Figure 3.. Dorsal CA3 Excitatory Projections to DLS Instruct Fear Responses to Ambiguous Contextual Threats

(A and B) Schematic of the behavioral timeline. (C) Schematic illustrating dorsal CA3 infection with CaMKII-eNpHR3.0 and fiber optic implantation on top of anterior DLS in C57B6/J mice. (D–G) Silencing dorsal CA3 terminals in anterior DLS has no effect on locomotor behavior and innate anxiety in OF (D and E), EPM (F), and NSF (G). (H and I) Silencing dorsal CA3 terminals in anterior DLS decreases freezing behavior in context B but not A (H), which results in an increase in fear discrimination ratio on block 7 (I). Data (means ± SEM; n = 8,8 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test and log-rank (Mantel Cox) test. *p < 0.05, eNpHR3.0 versus eYFP; #p < 0.05, block 7 versus block 6. Statistics detailed in Table S1. (J) Schematic representation of the effect of light silencing dorsal CA3 terminals in DLS on brain-wide c-Fos expression 30 min following exposure to context B (day 21). Regions highlighted in red denote a significant effect of eNpHR3.0, and arrows indicate the direction of the effect. (K and L) Schematic of the behavioral timeline. (M–O) Silencing dorsal CA3 terminals in anterior DLS increases freezing behavior in context A but not B early on training and (M), and decreases freezing behavior in context B but not A later on training (O) following repeated training in context A (N). Data (means ± SEM; n = 8,10 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test. *p < 0.05, eNpHR3.0versus eYFP. Statistics detailed in Table S1. See also Figures S1, S2, S3, S5, and S7.

Figure 4.. Ventral CA3 Excitatory Projections to CA1 and DLS Exert Opposing Roles in Gating Contextual Fear Responses

(A and B) Schematic of the behavioral timeline. (C) Schematic illustrating ventral CA3 infection with CaMKII-eNpHR3.0 and fiber optic implantation on top of ventral CA1 in C57B6/J mice. (D–G) Silencing ventral CA3 terminals in ventral CA1 has no effect on locomotor behavior and innate anxiety in OF (D and E) and EPM (F) while decreasing the latency to feed in the familiar environment in NSF (G). (H and I) Silencing ventral CA3 terminals in ventral CA1 decreases freezing behavior in context A and B (H) without altering fear discrimination ratio on block 7 (I). (J) Schematic illustrating ventral CA3 infection with CaMKII-eNpHR3.0 and fiber optic implantation on top of anterior DLS in C57B6/J mice. (K–N) Silencing ventral CA3 terminals in DLS did not affect locomotor behavior and innate anxiety in OF (K and L) and EPM (M) while increasing the latency to feed in the familiar environment in NSF (N). (O and P) Silencing ventral CA3 terminals in anterior DLS increases freezing behavior in context A and B (O), without altering fear discrimination ratio on block 7 (P). Data (means ± SEM; n = 8,8 per group and n = 14,24 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test and log-rank (Mantel Cox) test. #p < 0.05, eNpHR3.0 versus eYFP. Statistics detailed in Table S1. (Q) Schematic representation of the effect of light silencing vCA3 terminals in aDLS on brain-wide c-Fos expression 30 min following exposure to context B (day 21). Regions highlighted in red denote a significant effect of eNpHR3.0, and arrows indicate the direction of the effect. See also Figures S1, S6, and S7.

Figure 5.. Characterization of DLS PV Neurons

(A) Mapping of tdTomato-expressing cells in the medial and lateral part of DLS in the progeny of PV-Cre mice bred with Ai14 mice. Scale bar: 100 μm. Representative images for 3 independent animals. Scale bar: 100 μm. (B) Quantifications of tdTomato-expressing cells in DLS along the rostrocaudal and mediolateral axis. Data (means ± SEM; n = 3 mice per group) were analyzed using paired two-tailed Student t test. *p < 0.05, medial versus lateral. (C) Immunohistochemistry for PV and endogenous expression of tdTomato in the DLS of GAD2-Cre::Ai14, SST-Cre::Ai14 and PV-Cre::Ai14. Note the lack of overlap between PV and tdTomato in GAD2-Cre::Ai14 and SST-Cre:Ai14 mice. Representative images for 3 independent animals (single experiment). Scale bar: 25 μm. (D) Quantifications of the overlap of tdTomato-expressing cells with PV immunpositive cells in the cingulate cortex, DLS, MS, nucleus accumbens shell and core, and caudate putamen medial and lateral of PV-Cre::Ai14. Data (means ± SEM; n = 3 mice per group). (E) PV-Cre::TVA bigenic mice were injected with helper virus (AAV8-EF1a-FLEX-HB) followed by pseudotyped G-deleted rabies virus (EnvA-SADΔG-mCherry) in the DLS. Yellow arrowheads denote starter cells, which are positive for both GFP (helper) and mCherry (rabies). Representative images for 2 independent animals. Scale bar: 50 μm. Means ± SEM; n = 2 mice per group. (F) Presynaptic partners were identified in the MS/DBN, dorsal subiculum, dorsal CA1, and dorsal and ventral CA3. Representative images for 2 independent animals. Scale bar: 100 μm. Means ± SEM; n = 2 mice per group. (H–K) CA3 provides powerful synaptic input to PV neurons in DLS. (H) Acute slices obtained from 6-week-old PV-Cre::Ai14 bigenic mice injected with CaMKII-ChR2 into CA3 were used for ex vivo slice electrophysiology. Solid yellow arrowhead indicates tdTomato-labeled PV neuron. Representative image for 4 independent animals. Scale bar: 10 μm. (I) Light-evoked short latency (2 ± 0.7ms) excitatory postsynaptic currents (eEPSCs) were detected in PV and non-PV neurons following CA3 terminal activation. A light-evoked delayed (46 ± 5 ms) inhibitory current (eIPSC) was observed in a subset of non-PV neurons. (J) Example recordings of blue-light-evoked inputs onto DLS neurons. Traces show synaptic currents (EPSCs: bottom, IPSCs: top) evoked in both PV- and non-PV-expressing neurons (red and black, respectively), and the 5-ms light pulse is indicated by a blue box. (K) Average amplitude for both cell types. Data (means ± SEM; n = 6, 4 animals per group) were analyzed using unpaired Student two-tailed t test (detailed in Table S1). (L) Immunohistochemistry for eYFP in the DLS, DBN, NAcc, BNST, LH, PVN, and SUM of PV-Cre mice injected with AAV5-DIO-ChR2-eYFP. Representative images for 3 independent animals (single experiment). Scale bar: 100 μm.

Figure 6.. Optogenetic Silencing of DLS PV Neurons Increases Contextual Fear Discrimination

(A and B) Schematic of the behavioral timeline. (C) Schematic illustrating infection of DLS PV neurons with DIO-eNpHR3.0 and fiber optic implantation on top of DLS in PV-Cre mice. (D–G) Silencing DLS PV neurons has no effect on locomotor behavior and innate anxiety in OF (D and E), EPM (F), and NSF (G). (H and I) Silencing DLS PV neurons decreases freezing behavior in context B but not A (H), which results in an increase in fear discrimination ratio on block 7 (I). Data (means ± SEM; n = 8,8 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test and log-rank (Mantel Cox) test. *p < 0.05, eNpHR3.0 versus eYFP. Statistics detailed in Table S1. (J) Schematic representation of the effect of light silencing PV neurons in DLS on brain-wide c-Fos expression 60 min following exposure to context B (day 21). Regions highlighted in red denote a significant effect of eNpHR3.0, and arrows indicate the direction of the effect. See also Figures S1 and S8.

Figure 7.. Optogenetic Stimulation of DLS PV Neurons Decreases Contextual Fear Discrimination

(A and B) Schematic of the behavioral timeline. (C) Schematic illustrating infection of DLS PV neurons with DIO-ChR2 and fiber optic implantation on top of DLS in PV-Cre mice. (D–G) Stimulating DLS PV neurons has no effect on locomotor behavior and innate anxiety in OF (D and E) and NSF (G) but induces a strong anxiolytic effect in EPM (F). (H and I) Stimulating DLS PV neurons alters freezing behavior in both context A and B (H), which results in decrease in fear discrimination ratio on block 7 (I). Data (means ± SEM; n = 8, 6 mice per group) were analyzed using mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post hoc test and log-rank (Mantel Cox) test. *p < 0.05, ChR2 versus eYFP. Statistics detailed in Table S1. (J) Schematic representation of the effect of light stimulating PV neurons in DLS on brain-wide c-Fos expression 60 min following exposure to context B (day 21). Regions highlighted in red denote a significant effect of ChR2, and arrows indicate the direction of the effect. See also Figures S1, S2, and S8.