Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability

The lateral habenula encodes aversive stimuli contributing to negative emotional states during drug withdrawal. Here we report that morphine withdrawal in mice leads to microglia adaptations and diminishes glutamatergic transmission onto raphe-projecting lateral habenula neurons. Chemogenetic inhibition of this circuit promotes morphine withdrawal-like social deficits. Morphine withdrawal-driven synaptic plasticity and reduced sociability require tumor necrosis factor-α (TNF-α) release and neuronal TNF receptor 1 activation. Hence, habenular cytokines control synaptic and behavioral adaptations during drug withdrawal. The authors show that decreased synaptic efficacy onto raphe-projecting lateral habenula neurons supports opiate withdrawal-induced sociability deficits, and they demonstrate a role for TNF-α signaling in this process.

125 Similarly, LHb dysfunction contributes to the negative states emerging in 126 addiction, although its implications for sociability remains poorly addressed.
135 Next, we recapitulated MORwd-driven reduction in social preference in 136 C57Bl6 mice . We then prepared slices 137 from MORwd mice showing low or high sociability scores, and found that 138 Med LHb AMPAR:NMDAR ratios positively correlated with the social score ( Fig.   139 3h). This indicates that reduced synaptic strength in the LHb predicts opiate-140 WD-driven sociability deficits.
141 Notably, microglia and TNFα signaling also contributes to social behaviors 12 .

145
We found that MORwd-driven TNFα release requires neuronal TNF-R1 to 146 reduce AMPAR transmission onto raphe-projecting, medially-located, LHb 147 neurons. This ultimately gates MORwd-driven social impairment, a negative 148 symptom typical of opiate withdrawal. 149 150 151 Together with sociability deficits, MORwd also leads to anxiety and 152 hyperalgesia 1 . Since the contribution of LHb on these two behavioral aspects 153 remains elusive we cannot rule out that MORwd-driven habenular plasticity is 154 specific for withdrawal-mediated sociability defects.

184
In conclusion, our data support the participation of cytokine-mediated 185 plasticity for opiate-evoked negative symptoms, a mechanism by which LHb 186 ultimately contributes to the addiction spiral.

Microglia analysis 506
Mice were anhestetized and perfused with cold 4% paraformaldehyde (PFA) 507 in PBS (phosphate-buffered saline). The brains were extracted, post-fixed in 508 4% PFA in PBS, and incubated in 30% sucrose in PBS until they sank. 30 µm 509 slices were cut at the cryostat, and stored in PBS containing 0.02% NaN3 for 510 future analysis. Brain sections were permeabilized at room temperature (RT) 511 in 0.5% Triton X-100 (Sigma) for 1 hr RT, followed by 1 hr RT blocking in 2% 512 BSA 0.5% Triton X-100 and overnight incubation with primary antibody (Iba1 513 1:1000, Wako Chemicals, Cat. No. 019-19741 and CD68 1:400, Bio-Rad Cat. 514 No. MCA1957 8 ) at 4°C. Upon washing, sections were incubated 2 hr RT with 515 Alexa-fluorophore-conjugated secondary antibodies (Invitrogen), and 516 counterstained with DAPI (Invitrogen). 517 Confocal microscopy was performed with a TCS-SP5 (Leica) Laser Scanning 518 System, by using a 20X dry objective and images were processed and 519 analyzed by Fiji Software or Imaris Software (Bitplane, Switzerland), as 520 appropriate. Imaris was used for 3D rendering of confocal images for 521 quantification of volumes. 522 For density analysis, for each acquisition, the DAPI channel was max-523 projected and the medial and lateral portions of the lateral habenula were 524 manually drawn as region of interest. Then, stacks ranging from 15 to 20µm in 525 thickness, with z-step size of 1µm, were processed as follows: Iba1 and DAPI 526 channels were thresholded in Fiji and multiplied to each other for each stack, 527 with the image calculator function. The resulting thresholded stack was max-528 projected and the microglia nuclei were counted with Analyze Particle 529

function. 530
For cell soma size and Iba1 intensity, each acquisition was max-projected and 531 the contour of cell somata in the medial portion of the lateral habenula were 532 manually drawn based on the Iba1 immunoreactivity, and analyzed per size in 533 µm 2 and intensity. 534 3D imaging analysis was performed by Imaris applying recorded algorithms 535 (fixed thresholds for signal intensity) to all the images of the same experiment, 536 in order to produce unbiased signal quantification. In each experiment, one 537 brain slice per animal (n=4) per each group was acquired. The microglial cell 538 volume and the volume of phagocytic structures were reconstructed based on 539 the absolute intensity of Iba1 and CD68 signals, respectively. The volume of 540 CD68 was then normalized for the Iba1 volume, to take in account the cell 541 Varese, Italy) divided into three chambers. The walls of the center chamber 547 had doors to allow free access to all compartments. The luminosity was 548 around 10 lux. Thirty minutes after naloxone injection each mouse was placed 549 in the arena for a habituation period of 10 min and was allowed to freely 550 explore the whole empty arena. The social preference test was performed 551 immediately after the end of the habituation: two enclosures with vertical bars 552 were placed in the middle of the two lateral compartments, while the central 553 chamber remained empty. One enclosure was empty (serving as an 554 inanimate object) whereas the other contained a social stimulus (unfamiliar 555 juvenile mouse 25 ± 1 days old). The enclosures allowed visual, auditory, 556 olfactory and tactile contact between the experimental mice and the social 557 stimuli mice. The juvenile mice in the enclosures were habituated to the 558 apparatus and the enclosures for 3 days before the experiment and each one 559 of them served as a social stimulus for no more than 2 experimental mice (at 560 least 6 weeks old). The test lasted 10 minutes where experimental mice were 561 allowed to freely explore the apparatus and the enclosures. The position of 562 the empty and juvenile-containing enclosures alternated and was 563 counterbalanced for each trial to avoid any bias effects. Every session was 564 video-tracked and recorded using Ethovision XT (Noldus, Wageningen, the 565 Netherlands) or AnyMaze (Stoelting, Ireland), which provided an automated 566 recording of the entries and time spent in the compartments, the distance 567 moved and the velocity. The time spent in each chamber was assessed and 568 then used to determine the preference score for the social compartment as 569 compared to the object compartment (social/(social + object)). The arena was 570 cleaned with 1% acetic acid solution and dried between trials. 571 572 Analysis and statistics. Animals were randomly assigned to experimental 573 groups. Compiled data are always reported and represented as whisker box 574 plots (whisker top/bottom represent 90/10 th percentile, box top/bottom 575 represent 75/25 th percentile and median) or mean ± SEM, with single data 576 points plotted (single cell for electrophysiology and single animal for 577 behavioral experiments). Animals or data points were not excluded unless 578 stated and normality test was applied. Data collection and analysis were not 579 performed blind to the conditions of the experiments. When applicable, 580 statistical tests were paired or unpaired t-test and one-way or two-way 581 ANOVA. Significance for correlations was obtained applying Pearson's 582 estimates. Testing was always performed two-tailed with α = 0.05. More 583 information on the methods and analysis can be found in the Life Science 584 Reporting Summary. 585 586 587

Data availability statement 588
The data sets generated during and/or analyzed during the current study are 589 available from the corresponding author on reasonable request.