Dorsolateral septum somatostatin interneurons gate mobility to calibrate context-specific behavioral fear responses

Abstract

Adaptive fear responses to external threats rely upon efficient relay of computations underlying contextual encoding to subcortical circuits. Brain-wide analysis of highly coactivated ensembles following contextual fear discrimination identified the dorsolateral septum (DLS) as a relay of the dentate gyrus-CA3 circuit. Retrograde monosynaptic tracing and electrophysiological whole-cell recordings demonstrated that DLS somatostatin-expressing interneurons (SST-INs) receive direct CA3 inputs. Longitudinal in vivo calcium imaging of DLS SST-INs in awake, behaving mice identified a stable population of footshock-responsive SST-INs during contextual conditioning whose activity tracked and predicted non-freezing epochs during subsequent recall in the training context but not in a similar, neutral context or open field. Optogenetic attenuation or stimulation of DLS SST-INs bidirectionally modulated conditioned fear responses and recruited proximal and distal subcortical targets. Together, these observations suggest a role for a potentially hard-wired DLS SST-IN subpopulation as arbiters of mobility that calibrate context-appropriate behavioral fear responses.

Figure 1.. Contextual fear discrimination is associated with activation of a DG-CA3-DLS network.

a-b) Schematic representation of CFCDL procedure in which mice were trained to discriminate between a footshock delivered in context A and a safe context B (group AB) or A’ (group AA’). c) Freezing behavior in AB and AA’ groups over 5 blocks of training. Means ± SEM; n= 5, 5 mice per group, mixed factor two-way ANOVA (repeated measure over time): group AB: time F _{(4, 32)} = 13.83, P < 0.001; context F _{(1, 8)} = 20.83, P < 0.01; interaction F _{(4, 32)} = 3.676, P < 0.01; group AA’: time F _{(4, 32)} = 47.71, P < 0.001; context F _{(1, 8)} = 0.04855, NS; interaction F _{(4, 32)} = 5.043, P < 0.01. *p < 0.05, context A versus context B or A’. d) Freezing behavior during final exposure to the safe context. Means ± SEM; n= 5, 5 mice per group, unpaired Student two-tailed T-test, ***p < 0.001, context B versus A’. e) c-Fos immunohistochemistry (yellow arrowheads) in mice previously exposed to context B or A’ as well as a naive control group. Representative images for 5 independent animals per group. Scale bar: 50 μm. f) Schematic depicting brain-wide c-Fos analysis in AB and AA’ mice as compared to naive controls. c-Fos immunoreactivity unchanged (blue), B and A’ different from naive (grey), and B and A’ different from each other (red). Arrows indicate c-Fos changes in relation to freezing behavior. g) Inter-regional correlations for brain-wide c-Fos immunoreactivity using within-and between-subject design in context A’ and B. h) Schematic representation of the most robust inter-regional correlations (data are corrected for multiple comparisons). Note the robust positive correlations between CA3 and DLS but not CA1. See methods and statistics detailed in Supplementary Table 1 and Table 2.

Figure 2.. DLS SST-INs receive direct monosynaptic inputs from CA3

a) SST-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 3 independent animals. Scale bar: 50 μm. Means ± SEM; n= 3 mice per group. b) Presynaptic partners were identified in the MS/DBN, dorsal subiculum, CA1, dorsal and ventral CA3. Representative images for 3 independent animals. Scale bar: 100 μm. Means ± SEM; n= 3 mice per group. c) Example recordings of blue light-evoked monosynaptic inputs onto DLS neurons in acute slices obtained from adult SST-Cre::Ai14 bigenic mice. Clear yellow arrowheads indicate tdTomato-labeled SST INs; solid yellow arrowhead, dye fills of the recorded cell. Representative images for 4 independent animals. Scale bar: 30 μm. Traces show synaptic currents evoked in both SST-INs (top) and non-SST INs (bottom) and the 10 ms light pulse is indicated by a blue box. Individual trials are shown in gray and averages in red and black. d) Average amplitude and latency for both cell types. Means ± SEM; n= 11, 6 cells per group, unpaired Student two-tailed T-test. e) Firing properties of SST and non-SST INs tested by current injection. f) Example recordings of miniature synaptic currents in SST neurons (red) and non-SST cells (black). g) Amplitude and frequency for mEPSCs in SST and non-SST INs. Means ± SEM; n= 6, 5 cells per group, Student two-tailed T-test. All statistics detailed in Supplementary Table 1.

Figure 3.. A stable subpopulation of SST-INs is recruited during fear conditioning and predicts freezing behavior during recall

a) GFP Immunohistochemistry (GCaMP6m) and tdTomato expression in SST-Cre::Ai14 bigenic mice. Nuclei are counterstained with DAPI. Scale bar: 200 μm. Red horizontal bar indicates field of view. b) Field of view for in vivo GCaMP6m signal restricted to SST-INs in DLS. Representative image for 8 independent animals. Yellow arrowheads indicate representative cells with observable variations of fluorescence across time (maximum projection). Scale bar: 100 μm. c) Timeline of longitudinal calcium imaging experiments. d) Example of calcium transients detected with CNMF-E from 7 different cells during exposure to context A and context B during freezing (grey bars) and non-freezing epochs (representative of 8 independent animals). Note the limited number of transients during freezing epochs. Scale bar: x axis:10 sec, y axis:5 sd. e) Heat maps indicate the percent time (% total) spent in the different zones of the OF and EPM on two consecutive sessions. f) Heat maps indicate the calcium transient frequency (Hz) for 121 cells detected with CNMF-E per zone in the OF and EPM on two consecutive sessions. g) Raster plot of calcium transient frequency (Hz) detected with CNMF-E (average of session 1 and 2) in OF and EPM during freezing and non-freezing epochs in context A and B. Sorting of 121 cells based on their responsiveness to the first 3 footshocks administered during CFC1 (black arrow). h) Average calcium transient frequency (Hz) for the overall population (n=121 cells) identified with CNMF-E in the OF, EPM, context A and context B (average of two consecutive sessions). Means ± SEM and data distribution represented as violin plots; n= 121 cells, one-way ANOVA (repeated measures over time) followed by Tukey’s multiple comparisons post-hoc test. *p < 0.05. i) Sorting of the 121 cells detected with CNMF-E based on calcium transients observed in response to footshocks delivered during CFC1 and CFC2. Cells that never responded to footshocks (grey), cells that responded to 1, 2 or 3 footshocks during CFC1 (blue) and CFC2 (green). Cells that responded to footshocks during both CFC1 and CFC2 (red). j-k) Probability density function (PDF) for overlap between SST^FSH cells (22/121) and SST^non-FSH cells (64/121) detected with CNMF-E across CFC1 and CFC2 with a truncated null distribution l) Average calcium transient frequency (Hz) for SST^FSH cells and SST^non-FSH cells during freezing (red) and no freezing (blue) events in context A and context B. Means ± SEM and data distribution represented as violin plots; n= 22,64 cells, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test across no freezing events, *p < 0.05, FSH+ cells in A versus all groups. m) Calcium transient frequency analysis for behavioral state transitions for SST^FSH cells, SST^non-FSH cells or a randomly assigned cell population of equivalent size to that of SST^FSH cells. Behavioral state transitions consist in the onset (Off-On) and offset (On-Off) of freezing bouts (grey area) in context A or B as well as immobility bouts in the open field (grey area). Means ± SEM; n= 22, 64, 100 cells per group, mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post-hoc test across contexts, *p < 0.05, Ca2+ frequency in A versus all groups. n-o) Decoder analysis across mice based on calcium transient frequency local minima (n) and local maxima (o) predicting behavioral state transitions (freezing or immobility) in context A (blue), context B (red) or OF (green) using 1, 2 or 3 SST^FSH cells or SST^non-FSH cells per mouse. Means ± SEM; n= 6,8 mice per group, mixed factor two-way ANOVA (repeated measure over time) as well as one sample Student two-tailed T-test, *p < 0.05, data point versus chance. All statistics detailed in Supplementary Table 1.

Figure 4.. Optogenetic silencing of DLS SST-INs increases contextual fear responses.

a-b) Schematic of behavioral testing timeline. c) Schematic illustrating infection of SST DLS-INs with DIO-eNpHR3.0 and fiber optic implantation on top of DLS in SST-Cre mice. d-g) Silencing SST cell bodies in DLS (constant illumination) has no effect on locomotor behavior and innate anxiety in OF (d-e) and EPM (f) and novelty suppressed feeding (NSF) (g). Means ± SEM; n= 7, 7 mice per group, mixed factor two-way ANOVA. h) Silencing SST cell bodies (constant illumination) increases freezing behavior in context A and B on block training 7. Means ± SEM; n= 7, 7 mice per group, mixed factor two-way ANOVA (repeated measure over time) followed by Bonferroni’s multiple comparisons post-hoc test. i) Silencing SST cell bodies (constant illumination) did not alter fear discrimination ratio. Means ± SEM; n= 7, 7 mice per group, mixed factor two-way ANOVA (repeated measure over time). j) Schematic representation of the effect of light silencing of SST cell bodies 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 DIO-eNpHR3.0 and arrows indicate the direction of the effect. k) Detailed c-Fos immunostaining quantifications. Brain regions highlighted with red boxes indicate a significant effect of DIO-eNpHR3.0. Means ± SEM; n= 4, 5, 5 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. *p < 0.05, DIO-EYFP or DIO-eNpHR3.0 versus controls, #p < 0.05, DIO-EYFP versus DIO-eNpHR3.0. All statistics detailed in Supplementary Table 1.

Figure 5.. Optogenetic stimulation of DLS SST-INs attenuates anxiety and contextual fear responses.

a-b) Schematic of behavioral testing timeline. c) Schematic illustrating infection of SST DLS-INs with DIO-ChR2 and fiber optic implantation on top of DLS in SST-Cre mice. d-g) Stimulating SST cell bodies in DLS (15 Hz) increases the time spent exploring the open arms in the EPM (f) but has no effect on locomotor behavior and innate anxiety in OF (d-e) and novelty suppressed feeding (NSF) (g). Means ± SEM; n= 7, 6 mice per group, mixed factor two-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test. *p < 0.05, DIO-EYFP versus DIO-ChR2 (f). h) Stimulating SST cell bodies (15 Hz) decreases freezing behavior in context A and B on block training 7. Means ± SEM; n= 7, 6 mice per group, mixed factor two-way ANOVA (repeated measure over time). i) Stimulating SST cell bodies (15 Hz) did not alter fear discrimination ratio. Means ± SEM; n= 7, 6 mice per group, mixed factor two-way ANOVA (repeated measure over time). j) Schematic representation of the effect of light stimulation of SST cell bodies 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 DIO-ChR2 and arrows indicate the direction of the effect. k) Detailed c-Fos immunostaining quantifications. Brain regions highlighted with red boxes indicate a significant effect of DIO-ChR2. Means ± SEM; n= 4, 5, 5 mice per group, one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test. *p < 0.05, DIO-EYFP or DIO-ChR2 versus controls, #p < 0.05, DIO-EYFP versus DIO-ChR2. All statistics detailed in Supplementary Table 1.