Lateral Septum Somatostatin Neurons are Activated by Diverse Stressors

Abstract

The lateral septum (LS) is a forebrain structure that has been implicated in a wide range of behavioral and physiological responses to stress. However, the specific populations of neurons in the LS that mediate stress responses remain incompletely understood. Here, we show that neurons in the dorsal lateral septum (LSd) that express the somatostatin gene (hereafter, LSd^Sst neurons) are activated by diverse stressors. Retrograde tracing from LSd^Sst neurons revealed that these neurons are directly innervated by neurons in the locus coeruleus (LC), the primary source of norepinephrine well-known to mediate diverse stress-related functions in the brain. Consistently, we found that norepinephrine increased excitatory synaptic transmission onto LSd^Sst neurons, suggesting the functional connectivity between LSd^Sst neurons and LC noradrenergic neurons. However, optogenetic stimulation of LSd^Sst neurons did not affect stress-related behaviors or autonomic functions, likely owing to the functional heterogeneity within this population. Together, our findings show that LSd^Sst neurons are activated by diverse stressors and suggest that norepinephrine released from the LC may modulate the activity of LSd^Sst neurons under stressful circumstances.

Keywords: Lateral septum; Locus coeruleus; Norepinephrine; Somatostatin; Stress.

Fig. 1

LSd^Sst neurons are activated in response to diverse stressors. (A) Schematics showing Cre-dependent expression of GCaMP6 and fiberoptic cannula implantation for fiber photometry recordings from LSd^Sst neurons. Representative confocal images show LSd- restricted expression. Scale bar, 200 µm. (B) Schematic of the fiber photometry system. (C) Velocity of mice was only weakly correlated with the activity of LSd^Sst neurons (n=7, Pearson’s r=0.244, p=0.08). (D) Spatial calcium activity map of a representative mouse showing the higher activity of LSd^Sst neurons in the center zone of the open field arena. Average normalized calcium responses are color-coded for each pixel of spatial location. (E) Average calcium activity of LSd^Sst neurons in the center and the periphery (n=7, p=0.03). (F) Peri-event plot showing increases in the activity of LSd^Sst neurons as mice approach and enter the center zone. (G) Representative calcium response heatmap showing increased activity of LSd^Sst neurons in the open arms of the elevated plus maze. (H) Average calcium activity of LSd^Sst neurons for open arms (Open) was higher than for closed arms (Closed) (n=5, p=0.008). (I) Peri-event plot showing increases in the activity of LSd^Sst neurons as mice approach and enter open arms. (J) Representative traces showing increased activity of LSd^Sst neurons during the baselines and physical restraint. (K) Calcium transient amplitudes of LSd^Sst neurons were increased by restraint (n=5, one-way repeated measures ANOVA interaction, F(2,8)=19.65, p=0.0008). HC, homecage; Res, restraint. (L) Mice were given a series of electric footshocks. (M) Average calcium transients around the shock show time-locked responses of LSd^Sst neurons (n=5). Shaded box, footshock. (N) Average normalized calcium responses of both LSd^Sst neurons to shock delivery (Shock, 0~2 s from the shock onset) were larger than activity level during rest of the session (Base) (n=5, p=0.008). Data are represented as mean±s.e.m. Asterisks indicate significance levels for comparisons in each panel using Bonferroni post-tests following one-way repeated measures ANOVA or Wilcoxon rank-sum test (*p<0.05, **p<0.01).

Fig. 2

LSd^Sst neurons are connected with regions implicated in stress response. (A) AAV vector expressing cytosol-filling mGFP and terminal bouton-labeling mRuby-fused synaptophysin was injected into the LSd of the Sst^{cre /+} mice for anterograde tracing (n=3). Summary of the tracing experiment is shown. (B) Example image of the injection site, showing the expression of both mRuby and mGFP. (C) Representative images of the identified projection targets. Green, mGFP; black, mRuby. MnPO, median preoptic area; AH, anterior hypothalamus; SI, substantia innominata; HDB, horizontal limb of the diagonal band of Broca; MCPO, magnocellular preoptic nucleus; PH, posterior hypothalamus; VTA, ventral tegmental area; SuMm, medial supramammillary nucleus; SuMl, lateral supramammillary nucleus; MM, medial mammillary nucleus. Scale bars, 500 μm (C), 50 μm (B, insets). (D) AAV vectors Cre-dependently expressing rabies G protein and TVA receptor, and G-deficient EnvA-pseudotyped RV carrying eGFP were injected into the LSd of the Sst^{cre /+} mice for tracing monosynaptic inputs of LSd^Sst neurons (n=3). Summary of the retrograde tracing is shown. (E) Representative confocal image of the injection site, showing the expression of mCherry fused to TVA (red) and eGFP (green). (F) Representative images of monosynaptically connected upstream neurons of LSd^Sst neurons. MS, medial septum; VDB, nucleus of the vertical limb of the diagonal band; HPC, hippocampus; aca, anterior commissure; VP, ventral pallidum; LPO, lateral preoptic area; LH, lateral hypothalamus; DMH, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus; TU, tuberal nucleus; 3V, third ventricle; PAG, periaqueductal gray; LC, locus coeruleus.

Fig. 3

NE increases spontaneous excitatory synaptic transmission onto LSd^Sst neurons. (A) Top, schematic of Cre-dependent expression of eYFP and patch-clamp whole-cell recording from LSd^Sst neurons. Bottom, representative differential interference contrast (DIC) and fluorescence images of a LSd^Sst neuron expressing eYFP during recording. (B~D) Bath application of 10 μM NE increased mEPSC amplitude without affecting mEPSC frequency. Representative traces (B) and group data for mEPSC amplitude (n=9, p=0.039) (C) and frequency (n=9, p=0.570) (D). (E~G) Application of 100 nM corticosterone did not affect either mEPSC amplitude (n=7, p=0.297) (F) or frequency (n=7, p=0.938) (G). Scale bar, 10 pA, 1 s. Data are represented as mean±s.e.m. Asterisks indicate significance levels for comparisons using Wilcoxon rank-sum test (*p<0.05).

Fig. 4

Optogenetic stimulation of LSd^Sst neurons does not affect stress-related behaviors or autonomic functions. (A) Schematic of patch-clamp recording from ChR2-expressing LSd^Sst neurons. The LS area was illuminated with blue light for optogenetic stimulations. Right, example images of a LSd^Sst neuron expressing ChR2-eYFP during recording. DIC, differential interference contrast. (B) Representative traces showing the responses of LSd^Sst neurons upon the delivery of light pulse trains in 15 Hz or 30 Hz. (C) 15 Hz blue laser-stimulation evokes action potentials (AP) with high fidelity, whereas 30 Hz stimulation shows 50~60% of success rate in inducing action potentials (n=4 cells from 2 animals). (D) Schematics showing Cre-dependent expression of ChR2 and fiberoptic cannula implantation for bilateral optogenetic stimulations of LSd^Sst. Representative confocal image shows LSd-restricted expression of ChR2. Scale bar, 200 µm. (E~K) Optogenetic stimulation of LSd^Sst neurons did not affect time spent in open arms of the elevated plus maze (n=11 ChR2, n=12 Ctrl, p=0.339) (E), time spent in the center (n=11 ChR2, n=11 Ctrl, two-way repeated measures ANOVA interaction, F(1,20)=0.097, p=0.759) (F) or velocity (n=11 ChR2, n=11 Ctrl, two-way repeated measures ANOVA interaction, F(1,20)=7.787, p=0.011) (G) in an open field arena, struggling time in the tail suspension test (n=4 ChR2, n=7 Ctrl, two-way repeated measures ANOVA interaction, F(2,18)=0.778, p=0.474) (H), nor real-time place preference (n=11 ChR2, n=12 Ctrl, p=0.347) (I). Dashed line indicates 50% preference. This manipulation also did not affect heart rate (n=12, one-way repeated measures ANOVA interaction, F(2,22)=0.199, p=0.821) (J) and respiratory rate (n=12, one-way repeated measures ANOVA interaction, F(2,22)=1.114, p=0.339) (K), Data are represented as mean±s.e.m. Asterisks indicate significance levels for comparisons in each panel using Bonferroni post-tests following two-way repeated measures ANOVA or Wilcoxon rank-sum test.