Disrupted basolateral amygdala circuits supports negative valence bias in depressive states

Abstract

Negative bias is an essential characteristic of depressive episodes leading patients to attribute more negative valence to environmental cues. This negative bias affects all levels of information processing including emotional response, attention and memory, leading to the development and maintenance of depressive symptoms. In this context, pleasant stimuli become less attractive and unpleasant ones more aversive, yet the related neural circuits underlying this bias remain largely unknown. By studying a mice model for depression chronically receiving corticosterone (CORT), we showed a negative bias in valence attribution to olfactory stimuli that responds to antidepressant drug. This result paralleled the alterations in odor value assignment we observed in bipolar depressed patients. Given the crucial role of amygdala in valence coding and its strong link with depression, we hypothesized that basolateral amygdala (BLA) circuits alterations might support negative shift associated with depressive states. Contrary to humans, where limits in spatial resolution of imaging tools impair easy amygdala segmentation, recently unravelled specific BLA circuits implicated in negative and positive valence attribution could be studied in mice. Combining CTB and rabies-based tracing with ex vivo measurements of neuronal activity, we demonstrated that negative valence bias is supported by disrupted activity of specific BLA circuits during depressive states. Chronic CORT administration induced decreased recruitment of BLA-to-NAc neurons preferentially involved in positive valence encoding, while increasing recruitment of BLA-to-CeA neurons preferentially involved in negative valence encoding. Importantly, this dysfunction was dampened by chemogenetic hyperactivation of BLA-to-NAc neurons. Moreover, altered BLA activity correlated with durable presynaptic connectivity changes coming from the paraventricular nucleus of the thalamus, recently demonstrated as orchestrating valence assignment in the amygdala. Together, our findings suggest that specific BLA circuits alterations might support negative bias in depressive states and provide new avenues for translational research to understand the mechanisms underlying depression and treatment efficacy.

Fig. 1. Negative olfactory valence bias in depressed patients and CORT-model of depression in mice.

A Depressed bipolar patients (n = 23) classified less odors as pleasant ones than control subjects (n = 11) and remitted bipolar patients (n = 25 ; **p < 0.01), and more as unpleasant ones compared to remitted bipolar depresssed patients and control subjects (*p < 0.05) (Group: F(2,56) = 0.00, p > 0.999, Valence: F(2,112) = 30.1, p < 0.001, Interaction: F(4,112) = 4.15, p = 0.004). B–D The number of classified neutral odors did not change with depression severity (B). Bipolar depressive patients with higher depression severity (i.e. higher MADRS score) classified less odors as pleasant (C), and more odors as unpleasant (D). E Mice received vehicle (grey, n = 18) or chronic CORT (red, n = 17) to model depression. F CORT mice presented anxiety-like phenotypes in the open field (OF, t(33) = 2.26, p = 0.030) and light and dark box (LDb, t(33) = 2.62, p = 0.013), and depressive-like behaviors in the splash test (ST, U = 59, p = 0.001) but not in the tail suspension test (TST, U = 149, p = 0.909). G Global emotionality score is increased in CORT mice (t(32) = 4.23). H Scheme of the olfactory preference test using neutral (grey), appetitive (pink) and aversive (blue) odors. I Representative mouse tracks colored by the density of position points. J CORT mice explored less ♀ urine, TMA and TMT than Veh controls (Group: F(1,20) = 10.63, p = 0.004; Odor: F(3.60) = 163.50, p < 0.001; Interaction: F(3.60) = 1.54, p = 0.214). K Principal Component Analysis on the 23 behavioral parameters evaluated in 87 mice from both CORT (red) and Veh (grey) mice. L The olfactory preference test (OP) standed for 86% of contributions to PC1 (blue bar parts). The OF (32%) and TST (28%) contributed mostly to PC2. M PC1, PC2 and PC2:PC3 significantly predicted the Veh/CORT status. N Mice received chronic CORT to model depression (grey, n = 7), or CORT for 4 weeks then fluoxetine in addition to CORT for the following weeks (green, n = 14). O The reduction of the global emotionality score shows an improvement of the depressed- and anxious-like phenotype in responsive mice (U = 11.78, p = 0.0005). Fluoxetine-responsive mice are defined by an emotionality score lower than −0.5. P CORT + FLX-Responsive (n = 5) mice explored more peanut oil and ♀ urine, but less TMA, than CORT (n = 7) and CORT + FLX-Non Responsive (n = 9) groups (Group: F(2,19) = 9.629, p = 0.0013; Odor: F(2.192) = 86.61, p < 0.001; Interaction: F(6.57) = 4.11, p = 0.0017). ^#0.05 ≤ p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Bars are mean ± sem.

Fig. 2. Altered BLA circuits activity after chronic CORT administration.

A, B Retrograde fluorescent CTB647 and CTB555 dyes were injected in the NAc (green) and CeA (orange) respectively. Odors were presented to trigger the immediate-early gene c-Fos expression in CORT (red) and Veh (grey) mice. C Representative image of BLA c-Fos expression colocalized with CTB647 and/or CTB555. Scale bars, 100 µm (left) and 20 µm (right). D–H Quantification of c-Fos+ (D), c-Fos + /CTB647 + E), c-Fos + /CTB555+ (F) and c-Fos + /CTB647 + /CTB555+ cell number (G) in the BLA. BLA c-Fos+ cell density was similar between groups (D, t[81] = 0.93, p = 0.356, Veh n = 45; CORT n = 38). BLA c-Fos + /CTB647+ cell density decreased in CORT mice (E, U = 356, *p = 0.032, Veh n = 37, CORT n = 28), whereas c-Fos + /CTB555+ cell density increased (F, U = 328, **p = 0.006, Veh n = 40, CORT n = 27). BLA c-Fos + /CTB647 + /CTB555+ cell density tended to decrease in CORT mice (G, U = 144.5, p = 0.070, Veh n = 29, CORT n = 15) (H) Distribution of CTB647 and/or CTB555 colocalization among the total number of c-Fos+ cells in the BLA (χ²(3) = 340.0, ***p < 0.001). Bars are mean ± sem.

Fig. 3. CORT treatment changes synaptic connectivity of BLA neurons.

A Mice received vehicle (grey, n = 10) or chronic CORT (red, n = 9) to model depression. B AAVr-Pgk-Cre in the NAc and AAV-hSyn-Flex-G-TVA-GFP and EnvA-RVAG-mCherryin the BLA were injected to trace monosynaptic input to BLA-to-NAc projecting cells (in green, n = 10). C Same than A except that AAVr-Pgk-Cre were injected in the CeA to labelled BLA-to-CeA cells (in orange, n = 9). D, E Distribution of BLA-to-NAc (D, in green) or BLA-to-CeA (E, in orange), starters cells (in yellow), inputs cells (in pink) and unidentifed cells (in gray) among the total number of c-Fos+ cells in the BLA (D: χ²(3) = 35.09, p < 0.0001; E: χ²(3) = 39.58, p < 0.0001). F, G Map of the differences in inputs arriving to BLA-to-NAc (F) and BLA-to-CeA (G) projecting neurons in CORT-treated compared to Vehicle group. Only regions showing a tendency or significant difference are colour-coded (for full data see Fig. S6H, I). H–I Representative images (right) and percentage of inputs neurons (left) to BLA-to-NAc (H) and BLA-to-CeA (I) arriving from subregions of PVT posterior/anterior or BLA/LA respectively, in both CORT and Veh groups (relative to the total) (H, BLA-to-NAc: Group: F(1, 8) = 37.69, p = 0.0003; Region: F(1, 8) = 185.5, p < 0,0001; Interaction: F([1, 8) = 37.69; p = 0.0003; (I), BLA-to-CeA: Group: F(1, 7) = 12.08, p = 0.0103; Region: F([1, 7) = 39.03, p = 0,0004; Interaction: F(1, 7) = 6.489, p = 0.0382). Scale bar = 100 μm (H) and 20 μm (I). MO somatomotor areas, AUD auditory areas, AI agranular insular area, TEA temporal association areas, ECT ectorhinal area, PIR piriform area, COA cortical amygdalar area, PAA piriform-amygdalar area, TR postpiriform transition area, LA lateral amygdala, BLA basolateral amygdala, BMA basomedial amygdala, PA posterior amygdala, EP endopiriform nucleus, ENT retrohippocampal region, MEA medial amygdala, CEA Central amygdala, PAL pallidum, TH thalamus; HY hypothalamus. Bars are mean ± sem.

Fig. 4. Chemogenetic activation of BLA circuits distinctly impact CORT phenotype.

A To mimic chronic CORT phenotype were injected AAVr-Pgk-Cre in the CeA and AAV-hSyn-DIO-hM3Dq-mCherry (orange, n = 9, or AAV-hSyn-DIO-mCherry for the controls, grey, n = 11) in the BLA to activate BLA-to-CeA cells (B, C) CNO intra-peritoneal (i.p.) injection in the hM3Dq mice had no effect on global emotionality score (t(20) = 0.49, p = 0.626). D Representative mouse tracks in the olfactory preference test. E Chemogenetic activation of BLA-to-CeA cells did not modify olfactory valence compared to mCherry controls (Group: F(1,18) = 0.10, p = 0.752; Odor: F(3,54) = 75.02, p < 0.001; Interaction: F(3,54) = 1.16, p = 0.336). F Representative images of BLA c-Fos expression mCherry (left) and hM3Dq mice (right). G Percentage of c-Fos expression among mCherry+ cells after CNO injection (mCherry n = 5, hM3Dq n = 5; t(8) = 8.31, ***p < 0.001). Total number of mCherry+ cell number analysed dot not diffet between groups (t(8) = 0.37, p = 0.722). H AAVr-Pgk-Cre in the NAc and AAV-hSyn-DIO-hM3Dq-mCherry (green, n = 10 or AAV-hSyn-DIO-mCherry for the controls, grey, n = 12) in the BLA were injected to activate BLA-to-NAc cells. I All mice were treated with CORT. J CNO intra-peritoneal (i.p.) injection in the hM3Dq mice reduce the global emotionality score (t(20) = 3.40). K Representative mouse tracks in the olfactory preference test. L CORT-hM3Dq mice explored more peanut oil and ♀ urine relative to CORT-mCherry controls, but not aversive odors (Group: F(1.20) = 6.56, p = 0.019; Odor: F(3, 60) = 11.2, p < 0.001; Interaction: F(3, 60) = 7.31, p < 0.001). M Representative images and magnifications of BLA c-Fos expression CORT-mCherry (left) and CORT-hM3Dq mice (right). N Percentage of c-Fos expression among mCherry+ cells after CNO injection (CORT-mCherry n = 12, CORT-hM3Dq n = 7; t(6.135) = 5.51). Total number of mCherry+ cell number analysed dot not diffet between groups (U = 36, p = 0.650). **p < 0.01, ***p < 0.001. Scale bar, 125 µm. Bars are mean ± sem.

Fig. 5. Summary scheme of the results.

Depressive state in both BD patients and CORT-induced model for depression induce a negative olfactory bias (right) respect to healthy state (left). Patients present decreased number of odors rated as pleasant associated with increased number of rated unpleasant odors respect to control subject, while CORT-treated mice exhibit decreased approach behavior towards appetitive odors and increased avoidance behaviour towards aversive odors respect to control group. The chronic CORT administration elevates the activity of BLA projecting neurons to the CeA, which preferentially encode negative valence, and reduces the activity of BLA projecting neurons to the NAc, preferentially encoding positive valence.