Ventral hippocampal afferents to the nucleus accumbens regulate susceptibility to depression methods
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LTD attenuation of vHIP-NAc synaptic transmission is pro-resilient
To determine whether decreasing glutamatergic synaptic transmission at vHIP-NAc synapses could promote resilience in previously defeated mice, we injected AAV5-CaMKIIa-ChR2-EYFP or AAV5-CaMKIIa-EYFP into vHIP and 6 weeks later exposed mice to CSDS. Optic fibres targeting medial NAc were then surgically implanted, and optogenetic low frequency stimulation (LFS; 473 nm, 10 min, 1 Hz, 4 ms pulse width) of ChR2-infected terminals in NAc was used to induce long-term depression (LTD) of MSN EPSCs. One week after surgery, we delivered LFS to reduce vHIP-NAc synaptic transmission 45 min before social interaction testing ( ); LTD-like responses were electrophysiologically validated by optogenetic terminal stimulation of inputs in brain slices and in vivo (; ) as previously described. vHIP ChR2-infected mice spent more time interacting wit...
Methods
Male 6-8 week-old C57BL/6 J mice, and 6-month-old CD1 retired breeders, were maintained on a 12-h light-dark cycle (lights on at 0700 hours) at 22-25 °C with ad libitum access to food and water. C57 mice were housed 5 per cage except following defeat experiments and after fiber implantation surgery at which point mice were singly housed. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Icahn School of Medicine at Mount Sinai. All behavioural testing occurred during the animals' light cycle. Experimenter was blinded to experimental group and order of testing was counterbalanced during behavioural experiments and across days for electrophysiology experiments. For in vivo LTD experiments ( ), animals were randomized within cages before surgery. For acute stimulation experiments ( ), g...
Immunohistochemistry
Forty-eight hours after CSDS (see below), mice were anaesthetized with chloral hydrate and transcardially perfused with 0.1M PBS followed by 4% paraformaldehyde in PBS. Brains were postfixed overnight and then cryopreserved in 30% sucrose. Brains were sectioned on a freezing microtome at 35 µm. Sections were rinsed three times in PBS and blocked in 4% normal donkey serum (NDS) with 0.5% Triton-X in PBS (PBST) for 2 h at room temperature. Sections were then incubated in primary antibodies overnight at 4 °C (chicken anti-GFP, #AB13970, Abcam, 1:500 and rabbit anti-Egr1, #4154 S, Cell Signaling, 1:1,000) in PBST. Sections were then washed three times in PBS and incubated with secondary antibodies (donkey anti-rabbit Cy3, donkey anti-chicken Alexa Fluor 488; 1:500, Jackson ImmunoResearch) for 2 h at room temperature. Sections were washed three times in PB...
Stereotaxic surgery and viral vectors
Adenoassociated virus (AAV) vectors expressing channelrhodopsin-2 (ChR2) fused with enhanced yellow fluorescent protein (EYFP) under the control of the CaMKIIa promoter (AAV5-CaMKIIa-ChR2-EYFP) were used for optical activation of glutamatergic afferent terminals of NAc (mPFC, vHIP and AMY). CaMKIIa expression is specific to excitatory glutamatergic neurons, and the use of this promoter in viral vectors has been shown to target such neurons. AAV5-CaMKIIa-ChR2-EYFP and AAV5-CaMKIIa-EYFP were purchased from the University of North Carolina Vector Core. To label neurons projecting to NAc, retrograding AAV2/5-CaMKIIa-EYFP purchased from University of Pennsylvania Viral Core was infused into NAc. (Note that AAV2/5 represents a different serotype than the AAV5 vector, even though the UPenn Core labels it as 'AAV5.') For ster...
In vitro optogenetic patch-clamp electrophysiology
All recordings were conducted blind to the experimental conditions 2-28 days after CSDS. Whole-cell recordings were obtained from NAc MSNs in acute brain slices from mice that had been stereotaxically injected with AAV5-CaMKIIa-ChR2-EYFP into mPFC, vHIP or AMY. Eight to 12 weeks after viral surgery, mice were anaesthetized with isofluorane and perfused 1 min with ice-cold aCSF (128 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 4, 10 mM D-glucose, 24 mM NaHCO 3, 2 mM CaCl 2 and 2 mM MgCl 2; oxygenated with 95% O 2 and 5% CO 2, pH 7.4, 295-305 mOsm). Acute brain slices (200 µm) containing NAc shell were cut using a microslicer (DTK-1000, Ted Pella) in cold sucrose aCSF (254 mM sucrose, 3 mM KCl, 1.25 mM NaH 2 PO 4, 10 mM D-glucose, 24 mM NaHCO 3, 2 mM CaCl2 and 2̴...
CSDS and social interaction
All experiments utilized an established CSDS protocol to induce depressive-like behaviours in mice. C57BL/6 J mice were subjected to 10 daily, 5-min defeats by a novel CD1 aggressor mouse and were then housed across a plexiglass divider to allow for sensory contact. Control mice were housed in cages separated from other control mice by a plexiglass divider and were rotated to a different cage daily. Resilient and susceptible mice were identified by their respective preference for, or avoidance of, interaction with a novel mouse after 10 days of defeat in a social interaction test. In vivo optogenetic experiments used the entire population of defeated mice and did not specifically select for resilient or susceptible to facilitate the detection of bidirectional shifts in social interaction behaviour.
Statistics
No statistical methods were used to predetermine sample sizes but sample sizes are consistent with those reported in previous publications from our laboratory and sufficient for the types of experiments. Variance was similar between groups being statistically compared and data were normally distributed supporting the use of parametric statistics. Some animals or data points were lost in the course of social defeat, due to cage-flooding post-surgery, loss of fiber implant or equipment failure during behavioural testing. Grubb's test was used to identify and remove significant outliers from qPCR data. qPCR and paired-pulse data were analysed by one-way ANOVA and social interaction data were analysed by two-way repeated measures ANOVA. In all instances, Bonferroni post-hoc tests were used to resolve significant main effects or interactions. In one-way designs all pairwise comparisons wer...
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Evidence quotes (7)
To determine whether decreasing glutamatergic synaptic transmission at vHIP-NAc synapses could promote resilience in previously defeated mice, we injected AAV5-CaMKIIa-ChR2-EYFP or AAV5-CaMKIIa-EYFP into vHIP and 6 weeks later exposed mice to CSDS. Optic fibres targeting medial NAc were then surgically implanted, and optogenetic low frequency stimulation (LFS; 473 nm, 10 min, 1 Hz, 4 ms pulse width) of ChR2-infected terminals in NAc was used to induce long-term depression (LTD) of MSN EPSCs. One week after surgery, we delivered LFS to reduce vHIP-NAc synaptic transmission 45 min before social interaction testing ( ); LTD-like responses were electrophysiologically validated by optogenetic terminal stimulation of inputs in brain slices and in vivo (; ) as previously described. vHIP ChR2-infected mice spent more time interacting with a target mouse in comparison with EYFP-infected controls ( ) and similar to what is normally observed in resilient mice indicating a pro-resilient effect. The same manipulations did not affect social interaction in mice injected with AAV5-CaMKIIa-ChR2-EYFP in mPFC or AMY ( ). vHIP, mPF...
Male 6-8 week-old C57BL/6 J mice, and 6-month-old CD1 retired breeders, were maintained on a 12-h light-dark cycle (lights on at 0700 hours) at 22-25 °C with ad libitum access to food and water. C57 mice were housed 5 per cage except following defeat experiments and after fiber implantation surgery at which point mice were singly housed. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Icahn School of Medicine at Mount Sinai. All behavioural testing occurred during the animals' light cycle. Experimenter was blinded to experimental group and order of testing was counterbalanced during behavioural experiments and across days for electrophysiology experiments. For in vivo LTD experiments ( ), animals were randomized within cages before surgery. For acute stimulation experiments ( ), groups were matched on post-defeat social interaction scores to generate two groups with equivalent means and variance.
Forty-eight hours after CSDS (see below), mice were anaesthetized with chloral hydrate and transcardially perfused with 0.1M PBS followed by 4% paraformaldehyde in PBS. Brains were postfixed overnight and then cryopreserved in 30% sucrose. Brains were sectioned on a freezing microtome at 35 µm. Sections were rinsed three times in PBS and blocked in 4% normal donkey serum (NDS) with 0.5% Triton-X in PBS (PBST) for 2 h at room temperature. Sections were then incubated in primary antibodies overnight at 4 °C (chicken anti-GFP, #AB13970, Abcam, 1:500 and rabbit anti-Egr1, #4154 S, Cell Signaling, 1:1,000) in PBST. Sections were then washed three times in PBS and incubated with secondary antibodies (donkey anti-rabbit Cy3, donkey anti-chicken Alexa Fluor 488; 1:500, Jackson ImmunoResearch) for 2 h at room temperature. Sections were washed three times in PBS. Hoechst stain (Invitrogen) was added to the final wash for 25 min. Sections were washed one final time before mounting and coverslipping with ProLong Gold (Invitrogen).
Adenoassociated virus (AAV) vectors expressing channelrhodopsin-2 (ChR2) fused with enhanced yellow fluorescent protein (EYFP) under the control of the CaMKIIa promoter (AAV5-CaMKIIa-ChR2-EYFP) were used for optical activation of glutamatergic afferent terminals of NAc (mPFC, vHIP and AMY). CaMKIIa expression is specific to excitatory glutamatergic neurons, and the use of this promoter in viral vectors has been shown to target such neurons. AAV5-CaMKIIa-ChR2-EYFP and AAV5-CaMKIIa-EYFP were purchased from the University of North Carolina Vector Core. To label neurons projecting to NAc, retrograding AAV2/5-CaMKIIa-EYFP purchased from University of Pennsylvania Viral Core was infused into NAc. (Note that AAV2/5 represents a different serotype than the AAV5 vector, even though the UPenn Core labels it as 'AAV5.') For stereotaxic surgeries, mice were anaesthetised with a mixture of ketamine (100 mg kg -1 ) and xylazine (10 mg kg -1 ) and positioned in a small-animal stereotaxic instrument (Kopf Instruments). The skull surface was exposed and 33-gauge syringe needles (Hamilton) were...
All recordings were conducted blind to the experimental conditions 2-28 days after CSDS. Whole-cell recordings were obtained from NAc MSNs in acute brain slices from mice that had been stereotaxically injected with AAV5-CaMKIIa-ChR2-EYFP into mPFC, vHIP or AMY. Eight to 12 weeks after viral surgery, mice were anaesthetized with isofluorane and perfused 1 min with ice-cold aCSF (128 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 4, 10 mM D-glucose, 24 mM NaHCO 3, 2 mM CaCl 2 and 2 mM MgCl 2; oxygenated with 95% O 2 and 5% CO 2, pH 7.4, 295-305 mOsm). Acute brain slices (200 µm) containing NAc shell were cut using a microslicer (DTK-1000, Ted Pella) in cold sucrose aCSF (254 mM sucrose, 3 mM KCl, 1.25 mM NaH 2 PO 4, 10 mM D-glucose, 24 mM NaHCO 3, 2 mM CaCl2 and 2 mM MgCl 2; oxygenated with 95% O 2 and 5% CO 2, pH 7.4, 295-305 mOsm). Slices were incubated in aCSF for 1 h at 32 °C and then returned to room temperature for a minimum of 1 h before recording. Patch pipettes (3-5 mΩ) for whole-cell voltage-clamp re...
All experiments utilized an established CSDS protocol to induce depressive-like behaviours in mice. C57BL/6 J mice were subjected to 10 daily, 5-min defeats by a novel CD1 aggressor mouse and were then housed across a plexiglass divider to allow for sensory contact. Control mice were housed in cages separated from other control mice by a plexiglass divider and were rotated to a different cage daily. Resilient and susceptible mice were identified by their respective preference for, or avoidance of, interaction with a novel mouse after 10 days of defeat in a social interaction test. In vivo optogenetic experiments used the entire population of defeated mice and did not specifically select for resilient or susceptible to facilitate the detection of bidirectional shifts in social interaction behaviour.
No statistical methods were used to predetermine sample sizes but sample sizes are consistent with those reported in previous publications from our laboratory and sufficient for the types of experiments. Variance was similar between groups being statistically compared and data were normally distributed supporting the use of parametric statistics. Some animals or data points were lost in the course of social defeat, due to cage-flooding post-surgery, loss of fiber implant or equipment failure during behavioural testing. Grubb's test was used to identify and remove significant outliers from qPCR data. qPCR and paired-pulse data were analysed by one-way ANOVA and social interaction data were analysed by two-way repeated measures ANOVA. In all instances, Bonferroni post-hoc tests were used to resolve significant main effects or interactions. In one-way designs all pairwise comparisons were tested and in two-way repeated measures designs the effect of the between subjects variable was compared at each level of the within subjects variable. Two group comparisons (open field and forced swim test) were analysed by unpaired two-tailed Student's t -tests. Significance was set at P <0.05 f...
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