Chemogenetic inhibition of vHPC-LS disrupts social novelty preference

(A) A schematic showing AAV5-hSyn-DIO-hM4D(Gi)-mCherry injection in the vHPC and retroAAV-Cre injection in the LS. (B) Example histology showing hM4Di-mCherry expression in vHPC-LS neurons. (C) After being pair housed for 72 hours with a sex-matched and age-matched conspecific for familiarization, mice are run through the Social Discrimination Task (SDT). In the task, mice can freely explore an arena containing two encaged conspecifics, one novel and one familiar. (D) Control mice expressing the mCherry-only virus in vHPC-LS neurons were injected with either saline (left) or CNO (right) prior to being run on the SDT. Control mice, regardless of treatment group, preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific (Two-factor ANOVA with drug condition (saline or CNO) and conspecific identity (novel or familiar) as factors; interaction: p = 0.638, main effect of conspecific identity: p = 6.4E-06, post hoc Sidak multiple comparison tests; Saline: p = 1.1E-04, CNO: p = 1.9E-05; mCherry: n = 19 mice). (E) Discrimination scores show that control mice under both saline and CNO conditions preferentially spend more time investigating the novel conspecific relative to familiar conspecific (One sample t-test, Saline: p = 0.0103, CNO: p = 0.0013, mCherry: n = 19 mice). (F, G) Chemogenetic inhibition of vHPC-LS neurons with CNO disrupted the preference of mice for novel conspecific in the SDT. In contrast, hM4Di mice expressing mice exhibited a strong preference for the novel conspecific over the familiar conspecific when mice were administered saline prior to being run on the SDT (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 8.4E-04, main effect of conspecific identity: p = 0.003; post hoc Sidak multiple comparison tests; Saline: p = 2E-05, CNO: p = 0.971; discrimination score: one sample t-test, Saline: p = 0.0009, CNO: p = 0.9222; hM4Di: n = 20 mice). (H, I) Control mice expressing the mCherry-only virus in vHPC-LS neurons were injected with either saline (left) or CNO (right) prior to being run on a food discrimination task. Control mice preferentially spent more time in the proximity of familiar food regardless of the drug treatment group (Two-factor ANOVA with food identity (novel or familiar) and drug condition as factors; interaction: p = 0.254, main effect of food identity: p = 0.003, post hoc Sidak multiple comparison tests; Saline: p = 0.0323, CNO: p = 0.0009; discrimination score: one sample t-test, Saline: p = 0.0134, CNO: p = 0.008; mCherry: n = 18 mice). (J, K) Importantly, inhibition of hM4Di expressing vHPC-LS neurons in the presence of CNO had no effect on the ability of mice to preferentially investigate the familiar food relative to the novel food. Both CNO and saline-injected hM4Di animals preferentially spent more time in the proximity of the familiar food (Two-factor ANOVA with food identity and drug condition as factors; interaction: p = 0.508, main effect of food preference: p = 3.2E-05, post hoc Sidak multiple comparison tests; Saline: p = 1.7E-04, CNO: p = 0.03; discrimination score: one sample t-test, Saline: p = 0.0016, CNO: p = 0.0419; hM4Di: n = 16 mice). (L, N) We observed that both saline and CNO injected control (L) and hM4Di (N) mice spent similar amounts of time in the center of the open field arena (paired t-test, mCherry: p = 0.3167, hM4Di: p = 0.1837; mCherry: n = 19 mice, hM4Di=20 mice). (M, O) In both mCherry (M) and hM4Di (O) mice, saline and CNO injections did not affect the velocity (pixel/second) of the animals (paired t-test, mCherry: p = 0.2694, hM4Di: p = 0.7886; mCherry: n = 19 mice, hM4Di: n = 20 mice).

vHPC-LS neuron inhibition increases investigation of a mouse paired with inhibition

(A) A schematic showing retroAAV-Cre-mCherry injection in the LS and AAV5-Ef1a-DIO eNpHR 3.0-EYFP injection in the vHPC. Optical ferrules were implanted in the vHPC to inhibit NpHR expressing vHPC-LS neurons. (B) Example histology showing vHPC-LS neurons labeled with NpHR (expressing both EYFP and mCherry) and ferrule placement. (C) As with the chemogenetic condition, mice were pair housed for 72 hours with a sex-matched, and age-matched conspecific for familiarization, then are run through the SDT. In the task, mice can freely explore an arena containing two encaged conspecifics, one novel and one familiar. vHPC-LS neurons were inhibited (532 nm; 6 mW; constant light stimulation) in the proximity of one of the two conspecifics. (D) Control mice expressing EGFP had light stimulation paired with either a novel (N-ON) or a familiar conspecific (F-ON). We also had a stimulation-free condition (OFF). Control mice, regardless of stimulation group, preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific (Two-factor ANOVA with light condition (on or off) and conspecific identity as factors; interaction: p = 0.898, main effect of conspecific identity: p = 2.8E-05; post hoc Sidak multiple comparison tests; OFF: p = 0.032; N-ON: p = 0.012; F-ON: p = 0.032; EGFP: n = 20 mice). (E) Discrimination scores show that control mice in all light stimulation conditions preferentially spend more time investigating the novel conspecific relative to familiar conspecific (One sample t-test, OFF: p = 0.008, N-ON: p = 0.009, F-ON: p = 0.028; EGFP: n = 20 mice). (F, G) Mice expressing NpHR had light stimulation paired with a novel (N-ON) or familiar conspecific (F-ON), in addition to a stimulation-free condition (OFF). NpHR mice preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific, except when stimulation was paired with a familiar conspecific (Two-factor ANOVA with light condition and conspecific identity as factors; interaction: p = 0.024, main effect of conspecific identity p = 0.006, post hoc Sidak multiple comparison tests; OFF: p = 0.006, N-ON: p = 0.037, F-ON: p = 0.949, discrimination score: one sample t-test, OFF: p = 0.002, N-ON: p = 0.19, F-ON: p = 0.719; NpHR: n = 15 mice). (H, I) EGFP mice were then run through the SDT, but with two novel conspecifics to look at the impact of stimulation on novelty preference. Mice were run in either light off (OFF) condition or stimulated when in the proximity of one of two novel animals (N’-ON). Control mice, regardless of stimulation group, spent an equivalent amount of time in the proximity of each novel conspecific (Two-factor ANOVA with light condition and conspecific identity as factors; interaction: p = 0.006, no main effect of conspecific identity: p = 0.608; post hoc Sidak multiple comparison tests; OFF: p = 0.376, N-ON: p = 0.075; discrimination scores: one sample t-test, OFF: p = 0.535, N-ON: p = 0.128; EGFP: n = 20 mice) (J, K) NpHR mice were then run through the novel SDT in the light off or N’-ON condition. NpHR mice exhibited a preference for the novel conspecific paired with vHPC-LS inhibition (Two-factor ANOVA with light condition and conspecific identity as factors, interaction: p = 0.040, main effect of conspecific identity: p = 0.005, post hoc Sidak multiple comparison tests; N-OFF: p = 0.961, N-ON: p = 0.001; discrimination scores: one sample t-test, OFF: p = 0.796, N’-ON: p = 0.029; NpHR: n = 20 mice) (L, M) Control mice, regardless of stimulation group, showed an equal preference for both novel objects (Two-factor ANOVA with light condition and object identity (N or N’) as factors; interaction: p = 0.878, no main effect of object identity: p = 0.220, post hoc Sidak multiple comparison tests; N-OFF: p = 0.704, N-ON: p = 0.556; discrimination score: one sample t-test, OFF: p = 0.697, N-ON: p = 0.422; EGFP: n = 14 mice) (N, O) vHPC-LS inhibition had no effect on object preference even when one of the two novel objects were paired with stimulation. (Two-factor ANOVA with light condition and object identity as factors; interaction: p = 0.871, no main effect of object identity: p = 0.505, post hoc Sidak multiple comparison tests; N-OFF: p = 0.853, N-ON: p = 0.968; discrimination score: one sample t-test, OFF: p = 0.720, N-ON: p = 0.905; NpHR: n = 11 mice) (P, R) We observed that, regardless of light condition, control (P) and NpHR (R) mice spent similar amounts of time in the center of the open field arena (one-way ANOVA, EGFP: p = 0.094, 20 mice; NpHR: p = 0.254, 15 mice). (Q, S) We observed that light conditions had no effect on the speed (pix/sec) of control (Q) and NpHR (S) mice (One-way ANOVA, EGFP: p = 0.259, 20 mice; NpHR: p = 0.775, 15 mice). Green bars denote light ON condition and gray bars denote light OFF condition.

LS-VTA neurons receive dense monosynaptic input from the hippocampus

(A) A schematic of the viral strategy to determine if LS neurons project to the VTA. The schematic shows AAV5-CAG-flex-EGFP injection into the LS and retroAAV-Cre-mCherry injection into the VTA. (B) Example histology showing Retro-Cre-mCherry targeting in the VTA (mCherry). (C) Histology showing LS neurons (GFP) that project to the VTA. (D) A schematic of the viral tracing strategy for investigating monosynaptic inputs into LS-VTA neurons using a modified rabies tracing method (E, F) Example histology showing starter cells expressing both GFP and mCherry in LS. (G) Coronal section showing all detected mCherry labeled input neurons to LS-VTA neurons along the anterior-posterior axis of the hippocampus (−22.5, −3.0, and −3.5 mm from bregma, left to right). Green dots represent inputs to LS-VTA neurons from the hippocampal formation. Purple: Isocortex; Pink: Hypothalamus; Orange: Thalamus; Yellow: Midbrain. (H) Proportion of total neurons from hippocampal formation (HPF), Isocortex (IC), Hypothalamus (HY), and Thalamus (TH). A significantly larger proportion of HPF neurons send input to LS-VTA neurons compared to the HY and TH (One-way ANOVA: p = 0.0107, Post hoc multiple comparison test; HPF vs IC: p = 0.1579, HPF vs HY: p = 0.0206, HPF vs TH: p = 0.0056). (I) Breakdown of proportion of total neurons from subsections of the hippocampal formation. Amongst the HPF, CA1 and CA3 regions send more inputs to LS-VTA neurons compared to other subregions. (One-way ANOVA: p = 0.0096, Post hoc multiple comparison test; CA1 vs DG: p = 0.0411, CA1 vs SUBd: p = 0.0511, CA3 vs DG: p = 0.0134, CA3 vs SUBd: p = 0.0166, CA3 vs SUBv: p = 0.0325). (J,K) Distribution of CA1 (J) and CA3 (K) onto LS-VTA neurons along the dorsoventral axis of the hippocampus. More vCA1 neurons project to LS-VTA neurons compared to dCA1 (J, paired t-test: p = 0.0496). A comparable proportion of dCA3 and vCA3 neurons project to LS-VTA (K, paired t-test: p = 0.7736). n = 3 mice.

LS-VTA neurons play a role in social discrimination and food discrimination

(A) A schematic of AAV5-hSyn-DIO-hM4D(Gi)-mCherry injection in the LS and retro AAV-Cre injection in the VTA (B) Example histology showing hM4Di-mCherry expression in LS-VTA neurons. (C) Mice are pair housed for 72 hours with a sex matched, and age matched conspecific for familiarization, mice are run through the Social Discrimination Task (SDT). In the task, mice are allowed to freely explore an arena containing two encaged conspecifics, one novel and one familiar. (D, E) Control mice expressing the mCherry only virus in LS-VTA neurons were injected with either saline (left) or CNO (right) prior to being run on the SDT. Control mice, regardless of treatment group, preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 0.307, main effect of conspecific identity p = 6.8E-04, post hoc Sidak multiple comparison tests; Saline: p = 2.2E-04, CNO: p = 0.003; discrimination score: one sample t-test, Saline: p = 0.045, CNO: p = 0.032, n = 11 mice). (F, G) Chemogenetic inhibition of LS-VTA neurons with CNO disrupted the preference of mice for novel conspecific in the SDT. In contrast, hM4Di mice expressing mice exhibited a strong preference for the novel conspecific over the familiar conspecific when mice when administered saline prior to being run on the SDT (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 5.1E-04, post hoc Sidak multiple comparison tests; Saline: p = 0.001, CNO: p = 0.091; discrimination score: one sample t-test, Saline: p = 0.012, CNO: p = 0.208, n = 10 mice). (H, I) Control mice expressing the mCherry-only virus in LS-VTA neurons were injected with either saline (left) or CNO (right) prior to being run on a food discrimination task. Control mice preferentially spent more time in the proximity of the familiar food regardless of the drug treatment group (Two-factor ANOVA with drug condition and food identity as factors; interaction: p = 0.941, main effect of food preference: p = 2.7E-05, post hoc Sidak multiple comparison tests; Saline: p = 0.002, CNO: p = 0.001; discrimination score: one sample t-test, Saline: p = 0.007, CNO: p = 0.055, n = 10 mice) (J, K) Interestingly, there is a trend towards disrupted food preference in the hM4Di animals when administered CNO. This was not observed when the animals received saline injections and saline injected animals preferred the familiar food (Two-factor ANOVA with drug condition and food identity as factors; interaction: p = 0.076, post hoc Sidak multiple comparison tests; Saline: p = 0.07, CNO: p = 0.81; discrimination score: one sample t-test, Saline: p = 0.065 CNO: p = 0.863, n = 9 mice). (L, N) CNO administration did not affect the time spent in the center of the open field arena in both mCherry and hM4Di mice (paired t-test, mCherry: p = 0.2196; hM4Di: p = 0.2182; mCherry n = 8 mice, hM4Di n = 7 mice). (M, O) Velocity (pixel/seconds) of Control and hM4D mice were unaltered by CNO administration (paired t-test, mCherry: p = 0.0733, hM4Di: p = 0.7193; mCherry n = 8 mice, hM4Di n = 7 mice).

LS neurons make monosynaptic inputs onto dopaminergic neurons in the VTA

(A) Schematic of the viral intersectional strategy for tracing monosynaptic inputs onto dopaminergic neurons in the VTA. A Cre-dependent helper virus (AAV1-synP-FLEX.splitTVA.EGFP.B19G) was first injected into the VTA of Th-Cre+ mice. After allowing 3 weeks for viral expression, a mCherry labeled, delta G deleted rabies virus was also injected into the VTA (RVdG-mCherry). (B) Representative image showing the injection site in the VTA. In green are VTADA neurons labeled by the helper virus. In red are neurons labeled by the rabies virus. Starter cells that are labeled with both the helper and rabies virus are yellow in this image. High resolution (20x) images of the inset showing expression of the TVA-deltaG helper virus in green and the RVdG-mCherry in red (middle panels). Filled white arrowheads point to starter cells, they are double labeled and appear yellow (right panel). DAPI stain is in blue (left panel). (C) Coronal section showing all detected mCherry labeled LS input neurons to VTADA neurons along the anterior-posterior axis of the lateral septum. Each blue dot represents an individual LS neuron that projects to VTADA neurons. (D-F) A normalized density plot along the medio-lateral (D; M-L), anterior-posterior (E; A-P) and dorsal-ventral axis (F; D-V) showing LS neurons that project to dopamine neurons in the VTA. Bin width: 0.1 mm (M-L), 0.2 mm (A-P), 0.2 mm (D-V). (G) LS neurons that project onto dopamine neurons in the VTA separated into caudal (LSc), rostral (LSr) and ventral subdivisions (LSv) of the LS. The rostral subdivision of the LS projected most strongly to the VTA (One-way ANOVA: p = 5E-06, post hoc Tukey test; LSr vs LSv: p = 1.2E-05, LSr vs LSv: p = 6E-06, LSc vs LSv: p = 0.26, n = 3 mice).

Chemogenetic inhibition of vHPC-LS disrupts social novelty preference in both male and female mice

(A) Control male mice expressing the mCherry-only virus in vHPC-LS neurons were injected with either saline (left) or CNO (right) prior to being run on the SDT. Control mice, regardless of treatment group, preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 0.480, main effect of conspecific identity: p = 7.84E-04, post hoc Sidak multiple comparison tests; Saline: p = 0.011, CNO: p = 0.001; mCherry: n = 12 mice). (B) Discrimination scores show that control mice under CNO condition preferentially spend more time investigating the novel conspecific relative to familiar conspecific (One sample t-test, saline: CNO: p = 0.008, mCherry: n = 12 mice). Preference strongly trends towards novel preferring in saline injected control mice (One sample t-test, Saline: p = 0.082, mCherry: n = 12 mice). (C, D) Chemogenetic inhibition of vHPC-LS neurons with CNO disrupted the preference of male mice for novel conspecific in the SDT. In contrast, hM4Di mice expressing mice injected with saline exhibited a strong preference for the novel conspecific over the familiar conspecific (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 6.28E-04; post hoc Sidak multiple comparison tests; Saline: p = 0.001, CNO: p = 0.058; discrimination score: one sample t-test, Saline: p = 0.03, CNO: p = 0.059; hM4Di: n = 9 mice). (E) Control female mice expressing the mCherry-only virus in vHPC-LS neurons were injected with either saline (left) or CNO (right) prior to being run on the SDT. Control female mice, regardless of treatment group, preferentially spent more time in the proximity of the novel conspecific relative to the familiar conspecific (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 0.984, but main effect of conspecific identity: p = 0.003, post hoc Sidak multiple comparison tests; Saline: p = 0.008, CNO: p = 0.008; mCherry: n = 7 mice). (B) Discrimination scores show that in control female mice under both CNO and saline conditions there is strong trend towards preferentially spending more time near a novel same-sex conspecific relative to familiar conspecific (One sample t-test, Saline: p = 0.076, CNO: p = 0.053, mCherry: n = 7 mice). (C, D) Chemogenetic inhibition of vHPC-LS neurons with CNO disrupted the preference of female mice for novel conspecific in the SDT. In contrast, hM4Di mice expressing mice injected with saline exhibited a strong preference for the novel conspecific over the familiar conspecific (Two-factor ANOVA with drug condition and conspecific identity as factors; interaction: p = 0.162, but main effect of conspecific identity: p = 0.006, post hoc Sidak multiple comparison tests; Saline: p = 0.004, CNO: p = 0.212; discrimination score: one sample t-test, Saline: p = 0.018, CNO: p = 0.301; hM4Di: n = 11 mice).

Histological reconstruction of optical fiber tip placements

(A) Reconstruction of optical fiber tip placements in the vHPC in mice expressing NpHR (dark green) or EGFP (light green) in vHPC-LS neurons.