Early Life Stress Impairs VTA Coordination of BLA Network and Behavioral States

Abstract

Motivated behaviors, such as social interactions, are governed by the interplay between mesocorticolimbic structures, such as the ventral tegmental area (VTA), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC). Adverse childhood experiences and early life stress (ELS) can impact these networks and behaviors, which is associated with increased risk for psychiatric illnesses. While it is known that the VTA projects to both the BLA and mPFC, the influence of these inputs on local network activity which govern behavioral states-and whether ELS impacts VTA-mediated network communication-remains unknown. Our study demonstrates that VTA inputs influence BLA oscillations and entrainment of mPFC activity in mice and that ELS weakens the ability of the VTA to coordinate BLA network states, while also impairing dopaminergic signaling between VTA and BLA. Optogenetic stimulation of VTA_BLA terminals decreased social interaction in ELS mice, which can be recapitulated in control mice by inhibiting VTA→BLA communication. These data suggest that ELS impacts social reward via the VTA→BLA dopamine network.

Keywords: basolateral amygdala; dopamine; early life stress; oscillations; ventral tegmental area.

Figure 1.

VTA_BLA photostimulation DAT-Cre mice drives network activity. inhibits ability for VTA_BLA to drive network activity. A, Schematic of sagittal view shows at P70, in DAT-Cre mice (N = 7), AAV-Syn-ChrimsonR-tDT was injected into VTA, with an optrode placed into BLA while a grounding screw was placed over frontal cortex (FC). B, Summary of LFP power ratio from BLA between pulsed stimulation and baseline periods (baseline = 0) where power was measured at each stimulation frequency for three, 2 min trials. Asterisks within bars indicate significant change from baseline. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Figure 2.

VTA_BLA robustly drives BLA and mPFC network activity. Representative traces and STFT transformations for a CNT animal during a (A) 5 Hz, (B) 10 Hz, (C) 20 Hz, and (D) 40 Hz trial. Top, Stimulated frequency LFP trace atop filtered (2–100 Hz) LFP traces (gray) from BLA (left) and mPFC (right). Middle, zoomed in portions from above traces show five periods before, during, and after 10 mW 640 nm photoexcitation. Bottom, STFT transformation from respective traces; horizontal white lines indicate onset/offset of 640 nm laser. E, Grouped bar plot, organized by stimulation order, shows LFP power ratio between pulsed stimulation and baseline periods where power was measured at each stimulation frequency for CNT (blue, n = 7) animals. F, Same as E with the exception that analyses performed on ELS (red, n = 8) animals. Spectrograms were not bandpass filtered but the power around stimulation frequencies were retrieved for the relative power plot in subpanels E and F.

Figure 3.

ELS inhibits ability for VTA_BLA to drive network activity. A, Experimental timeline for ELS cohort. Timeline is identical for CNT cohort excluding maternal separation (D1–21). B, Left, Schematic of sagittal view shows at P70, AAV-Syn-ChrRimsonR-tDT was injected into VTA, with an optrode and electrode placed into BLA and mPFC, respectively. Right, Representative fluorescence images of -tdT expression in the BLA. C, Top, Representative 20 Hz LFP trace (colored) overlaid on raw LFP trace (2–100 Hz; gray) from BLA (left) and mPFC (right). Middle, Zoomed in portions from above traces show five periods before, during, and after 10 mW 640 nm photoexcitation. Bottom, STFT transformation from respective traces; vertical dashed white lines indicate onset/offset of 640 nm laser. Spectrogram was not bandpass filtered but the power around stimulation frequencies were retrieved for the relative power plot for D. D, Summary of LFP power ratio from BLA (left) and mPFC (right) between pulsed stimulation and baseline periods (baseline = 0) where power was measured at each stimulation frequency for CNT (blue, n = 7) and ELS (red, n = 8) conditions. Asterisks within bars indicate significant change from baseline. Asterisks atop horizontal bars indicate difference between CNT and ELS. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Figure 4.

ELS minimizes VTA_BLA→mPFC functional network connectivity. A, Representative LFP traces from BLA and mPFC before (top) and during (bottom) 40 Hz VTA_BLA photoexcitation for a CNT mouse. B, Top, Representative summary bar plots for animal in A detailing VTA-evoked BLA→mPFC coherence at each stimulation frequency. Bottom, Phase histogram for baseline (gray) and 40 Hz stimulation (red) in A. C, D, Same analyses seen in A and B for a representative ELS mouse. Summary of LFP coherence (PLV, E; and iPLV, F) ratio between pulsed stimulation and baseline periods where BLA→mPFC coherence is measured at each stimulation frequency for CNT (blue, n = 7) and ELS (red, n = 8) conditions. Dashed horizontal line indicates baseline response. Asterisks within bars indicate significant change from baseline. Asterisks atop horizontal bars indicate difference between CNT and ELS. ^#p ∼ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Figure 5.

ELS reduces ELS reduces VTA_BLA DA response. A, Schematic of sagittal and coronal views show AAV9-Syn-ChrRimsonR-tDT or AAV9-hSyn-eGFP was injected into VTA, AAV9-hSyn-GRAB_DA2m injected into BLA, with GRIN lens implanted above BLA. B, Representative traces show dopamine (DA) response across a recording window consisting of 15–5 s trials of 20 mW red (620 ± 30 nm) 40 Hz photoexcitation indicated by horizontal black bars; eGFP, gray; CNT, blue; ELS, red. C, Grouped bar plot shows trial-averaged normalized DA response for each second of 5 sec stimulation for eGFP (n = 4), CNT (n = 4), and ELS (n = 5) animals. Dashed horizontal line indicates baseline response. D, Summary plot shows session-averaged normalized DA response for YFP, CNT, and ELS animals. *p < 0.05. Error bars represent SEM.

Figure 6.

VTA_BLA activation in ELS reduces social interaction. A, Schematic of three-chamber social interaction task (3CST) during Toy-Toy sessions for habituation (ChrR-Off, top) and testing (ChrR-On, bottom) trials. B, Representative heatmaps during 3CST show location for a CNT (left) and ELS (right) animal during habituation and testing trials, BI and BII, respectively. Heat, Min-Max scaled duration across trial. BIII shows subtraction plots, with heat indicating the difference in time spent at each location between testing and habituation trials. C, D, Same as A and B except interaction objects are a toy and novel, female conspecific (Toy-Female). E, Grouped bar plots show interaction preference scores for CNT (n = 7) and ELS (n = 8) animals across habituation and testing trials for Toy-Toy (left) and Toy-Female (right) sessions; value of 1 is equivalent time spent in stimulation and opposite side. Asterisks centered over trial: one side of chamber is significantly preferred. Asterisks centered across trials: preference scores during testing trials significantly differed from habituation trials. F, Grouped bar plot shows the effect of ELS on locomotion activity in 3CST, displayed as time (seconds) spent in each chamber during each animal (control, N = 7; ELS, N = 8) habituation trial. Mixed ANOVA (position × lifegroup) excluding center position revealed no significant effect (p > 0.05). G, Grouped bar plot shows normalized preference scores from E for CNT and ELS animals across Toy-Toy and Toy-Female sessions; value of 1: equivalent preference score between habituation and testing trials. Preference score is the percentage of time the mouse spends in the stimulation zone versus both sides of the area; a value >1 suggests a preference for the stimulation side. Normalized preference score compares the PS during testing with the habituation trial; a value >1 indicates increased stimulation-side preference compared with preference during habituation. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.

Figure 7.

VTA_BLA inhibition mimics social deficit observed following ELS + VTA_BLA excitation. A, Top, Schematic of coronal slice showing AAV8-hsyn-GFP-Jaws was injected into VTA with optrode implanted into BLA. Bottom, Schematic of inhibitory mechanism of red-shifted Jaws acting on anion-gated membrane following 640 nm photostimulation. B, Schematic of 3CST during Toy-Toy sessions for habituation (Jaws-Off, Top) and testing (Jaws-On, Bottom) trials. C, Schematic showing same experimental set-up as in B with the exception being interaction objects are a toy and novel, female conspecific (Toy-Female). D, Grouped bar plots show interaction preference scores for Toy-Toy and Toy-Female sessions habituation and testing; value of 1 is equivalent time spent in stimulation and opposite side. Significance stars centered over trial: one side of chamber is significantly preferred. Significance stars centered across trials: preference scores during testing trials significantly differed from habituation trials. E, Grouped bar plot shows normalized preference scores from D for CNT + Jaws animals (n = 5) as well as CNT + ChrR and ELS + ChrR animals (from Fig. 6) across Toy-Toy and Toy-Female sessions; value of 1: equivalent preference score between habituation and testing trials. Preference score is the percentage of time the mouse spends in the stimulation zone versus both sides of the area; a value >1 suggests a preference for the stimulation side. Normalized preference score compares the PS during testing with the habituation trial; a value >1 indicates increased stimulation-side preference compared with preference during habituation. *p < 0.05. **p < 0.01.