Instantaneous antidepressant effect of lateral habenula deep brain stimulation in rats studied with functional magnetic resonance imaging

The available treatments for depression have substantial limitations, including low response rates and substantial lag time before a response is achieved. We applied deep brain stimulation (DBS) to the lateral habenula (LHb) of two rat models of depression (Wistar Kyoto rats and lipopolysaccharide-treated rats) and observed an immediate (within seconds to minutes) alleviation of depressive-like symptoms with a high response rate. Simultaneous functional magnetic resonance imaging (fMRI) conducted on the same sets of depressive rats used in behavioral tests revealed DBS-induced activation of multiple regions in afferent and efferent circuitry of the LHb. The activation levels of brain regions connected to the medial LHb (M-LHb) was correlated with the extent of behavioral improvements. Rats with more medial stimulation sites in the LHb exhibited greater antidepressant effects than those with more lateral stimulation sites. These results indicated that the antidromic activation of the limbic system and orthodromic activation of the monoaminergic systems connected to the M-LHb played a critical role in the rapid antidepressant effects of LHb-DBS. This study indicates that M-LHb-DBS might act as a valuable, rapid-acting antidepressant therapeutic strategy for treatment-resistant depression and demonstrates the potential of using fMRI activation of specific brain regions as biomarkers to predict and evaluate antidepressant efficacy.

lipopolysaccharide-treated rats) and observed an immediate (within seconds to minutes) 23 alleviation of depressive-like symptoms with a high response rate. Simultaneous functional 24 magnetic resonance imaging (fMRI) conducted on the same sets of depressive rats used in

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LHb-DBS instantaneously reduces depressive symptomatology 95 Bipolar GF microelectrodes were implanted in the right LHb of WKY and LPS-treated rats that 96 showed depressive symptomatology (Fig. 1b-d). The WKY rat is characterized as an animal  Electrode tip placements within the LHb for DBS were verified for each subject by T2-weighted 123 rapid acquisition with relaxation enhancement (RARE) anatomical MRI images acquired 124 immediately after implantation (Fig. 1c). The negligible artifact induced by the GF electrodes did implanted electrodes. Electrode tip localization within the LHb was also confirmed by 128 hematoxylin and eosin (H&E) staining at the end of the study which showed consistent results as 129 those from MRI scans (Fig. 1d). Unless otherwise specified, rats with GF electrodes successfully   (Fig. 2b). The increases in locomotor activity were also reflected by the 166 increased total distance travelled (in the entire field) as well as the increased average speed in the 167 center area (Supplementary Fig. S1). An example of the locomotor activity of an LPS-treated rat  174 Overall, WKY and LPS-treated depressive rats showed a high response rate to LHb-DBS. Of 175 nineteen WKY and LPS-treated rats, sixteen (~84.2%) showed an increase in sugar preference 176 greater than 50%, thirteen (~68.4%) showed an increase in average speed greater than 5-fold, and 177 14 (~73.7%) exhibited at least one entry into the center area in the OFT compared to no entries 178 before DBS. Of ten WKY rats, six had a decrease in the duration of immobility in the FST 179 greater than 50%. In addition, DBS of same parameters delivered to electrodes implanted outside  . 219 The small-to-absent artifact produced by the GF electrodes enabled fMRI scanning of all brain  The time courses of the BOLD signals in several anatomical regions of interest (ROIs) were 238 calculated and averaged from all WKY and LPS-treated rats. We observed clear BOLD signal 239 changes time-locked to the stimulation pulse blocks (Fig. 3d). Of all of the regions examined in 240 WKY rats, the mPFC showed the largest percent changes in BOLD signals (4.87±0.42%, 241 mean±SEM, n = 30 scans from 10 rats), slightly higher than those in the DBS target, the LHb 242 (4.77±0.38%, mean±SEM, n = 30 scans from 10 rats). For LPS-treated rats, the LHb exhibited 243 11 the largest changes in BOLD signals (7.02±0.35%, mean±SEM, n=27 scans from 9 rats), and the 244 mPFC exhibited the second largest changes (4.79±0.28%, mean±SEM, n=27 scans from 9 rats).   Table S1). The activation of the IPN/VTA showed a relatively weak correlation 280 with the increase in average speed, but no correlation was observed between that and the number 281 of entries into the center or distance travelled in the center (Fig. 4b, Supplementary Table S1).

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The activation intensity of the DRN showed the strongest correlation with the increase in the 283 average speed (r = 0.92, P = 2.3 e -8 ) among all the brain regions and was also significantly 284 correlated with the number of entries into the center (r = 0.60, P = 0.007) and the distance 285 travelled in the center (r = 0.55, P = 0.01) (Fig. 4b, Supplementary Fig. S5). Notably, except for 286 the SLEA, all the brain regions with activation levels correlated with the above three indicators    (Hu et al., 2020). Based on the T2-weighted anatomical 307 images, we delineated the electrode tip location of each rat and summarized them in Fig. 5a. T2-308 14 weighted MRI images of all the WKY rats and LPS-treated rats used in this study are included in 309 Supplementary Fig. S6. 310 The rats with DBS sites in the M-LHb and close to the medial habenula (MHb), as indicated by 311 the red dots in Fig. 5a, showed greater improvements in average speed than the rats with more LHb and far from MHb, with the difference approaching the borderline of significance (Fig. 5b).  its long-term effect, which requires days to weeks of stimulation before a response is achieved 367 (Schlaepfer et al., 2014). The rapid-acting antidepressant effect of DBS has rarely been reported 368 (Wang et al., 2020; Scangos et al., 2021). 369 In our studies, we found that of nineteen WKY and LPS-treated rats, sixteen (~84.2%) showed 370 an increase in sucrose preference greater than 50%, thirteen (~68.4%) showed an increase in 371 average speed greater than 5-fold, and 14 (~73.7%) had at least one entry into the center area in 372 the OFT. Of ten WKY rats, six had a decrease in the duration of immobility in the FST greater 373 than 50%. More importantly, the depressive behavior was alleviated immediately upon delivery  (Yang et al., 2018). We believe that  (Abler et al., 2007). 415 We suspect that the activation of the SLEA by LHb-DBS was mediated by the activation of in the motor cortex for either type of rat. It was reported that the suppression of motor action in 429 depressive rats arises from the inhibition of dopaminergic and serotonergic neurons in the VTA, 430 substantia nigra pars compacta (SNc) and raphe nuclei due to hyperactivity of the LHb 431 (Hikosaka, 2010). The absence of motor cortex activation by LHb-DBS suggests that the 432 improvement of the locomotor activity was not due to the direct activation of the motor circuit.  (Hu et al., 2020). The strong correlation between the activation level and    The FST was performed as previously reported (Porsolt et al., 1977;Slattery and Cryan, 2012). 510 A transparent acrylic cylinder (20 cm diameter, 50 cm height) was filled with water (23±2 ℃ 511 temperature) for forced swimming. The water depth was set to prevent animals from touching 512 the bottom with their tails or hind limbs. The day before the test, animals were habituated to the 513 swimming apparatus for 15 min. About 24 hrs after the habituation, rats were subjected to a 15-514 min FST test session, which was divided into 'pre' (0-5 min), 'during' (5-10 min), and 'after'   The rat was subjected to a 15-min OFT test session, which was divided into 'pre' (0-5 min), 527 'during' (5-10 min), and 'after' (10-15 min) LHb-DBS, with locomotor performance recorded.

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The LHb-DBS was applied in the 'during' (5-10 min) session. The data upon DBS presented in 529 this work was either from the 'during' or 'after' session. This selection was remained consistent followed by a bolus injection of dexmedetomidine (0.022 mg kg -1 ). During MRI scanning, 538 isoflurane (0.5%) delivered via a nose cone combined with continuous infusion of 539 dexmedetomidine (0.015 mg kg −1 hr −1 ) was used to maintain anesthesia (Brynildsen et al., 2017). 540 Animal temperature, respiration, and blood oxygen saturation were all monitored and within 541 normal ranges (Model 1025, SA Instruments, USA). Body temperature was maintained at 542 37±0.5 °C using a circulated hot water bed and a hot air blower. The fMRI scans were acquired on anesthetized rats for 210 s (210 repetitions), during which 551 stimulation was applied in a 60 s-OFF/ 30 s-ON/ 120 s-OFF cycle, with the following parameters: 552 bipolar square-wave current with an amplitude of 600 μA, frequency of 130 Hz, and pulse width 553 of 90 μs. A higher current amplitude at 600 μA was used in DBS-fMRI studies to circumvent the 554 issue of low DBS-fMRI sensitivity at low stimulus amplitude. This generated BOLD response 555 patterns that were qualitatively similar, yet more robust than 300 μA. The EPI scans were 556 repeated three times per rat for within subject averaging. 557 fMRI data analysis. The fMRI Data analysis was performed using a custom-written code 558 developed using Matlab (R2018a, MathWorks, USA) and SPM12 (http://www.fil.ion.ucl.ac.uk/).

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The preprocessing included the following steps. (1) EPI images were converted from Bruker 560 format to NIFTI format (nominal voxel size was enlarged 10-fold to facilitate image processing 561 in SPM). (2) After image format conversion, the rat brain was extracted manually from each 562 image using ITK-SNAP (http://www.itksnap.org/). (3) The images of each scan were corrected 563 for slice timing and realigned to the first volume for that scan. (4) For spatial normalization, the 564 realigned EPI images were co-registered to the rat's own T2 anatomical images, which were 565 normalized to a rat brain template. (5) Spatial smoothing was applied with a 0.8-mm full width at 566 half maximum Gaussian kernel. After preprocessing, statistical analysis was conducted across 567 subjects using a general linear model with reference to the stimulation paradigm, and a modified 568 rodent hemodynamic response function was used (Chen et al., 2020). Standard 1st-level analysis 569 was performed for each EPI scan of each animal, and the corresponding t-statistic maps of each 570 subject were calculated, with false discovery rate (FDR)-corrected set at p < 0.001 and cluster 571 size set at > 10 voxels. close to the VTA and it is difficult to accurately distinguish these two areas in fMRI studies, we 579 labeled this region as IPN/VTA. For each scan, the time series was converted to relative BOLD 580 response ΔS(t)/S0, where ΔS(t) was generated by subtracting the mean of pre-stimulation period 581 (S0) of that scan. The BOLD signal time courses were calculated for each ROI. Moreover, the 582 mean statistics beta values of these ROIs were also calculated for each subject. 583 H&E staining. Electrode tip localization within LHb was confirmed by H&E staining at the end 584 of the study. Rats were anesthetized and transcardially perfused with 100 -150 mL PBS, 585 followed by 200 -300 mL 4% paraformaldehyde (PFA) in PBS. The brain was taken out with 586 the implants removed from the skull. After being immersed in 4% PFA solution for 24 hours at 587 room temperature for the purpose of post-fixation, the brain was immersed successively in 10% 588 (w/v), 20% (w/v) and 30% (w/v) sucrose solution for dehydration until it settled to the bottom of 589 the solution. The brain was segmented coronally to remove extra brain tissue, leaving out ~3 mm 590 thick section which included the entire habenula. The tissue sample was cryoprotected using the 591 optimal cutting temperature (OCT) compound (Tissue-Tek, USA) and frozen at -80 °C to stiffen 592 the OCT compound. Frozen tissues were sliced coronally into 7 -10 μm thick sections using a 593 cryostat machine (Leica CM3050 S, Germany), mounted on glass slides and stored at -20 °C.

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The frozen brain coronal slices were put at room temperature for 15 mins and then rinsed with 595 PBS for 5 mins to remove OCT compound. The cell nuclei were stained with haematoxylin 596 solution (#BA4097, Baso, China) for 1-2 mins and rinsed with water. Then the slices were put 597 into 1% hydrochloric acid ethanol solution for differentiation for 1 -5 s and rinsed with water.

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The differentiation was followed by a 50 ℃ water rinse for 10 -20 s to stop the action of the acid 599 alcohol. The color of nucleus was checked using microscope. The cytoplasm was stained with 600 eosin solution (#BA4022, Baso, China) for 1 -2 mins and rinsed with water at room temperature.

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The slices were then dehydrated using graded alcohol in ascending order as 70%, 90% and 95%