Decision letter: Instantaneous antidepressant effect of lateral habenula deep brain stimulation in rats studied with functional MRI

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 MRI (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) were 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. Editor's evaluation This important paper is a real tour de force that combines functional MRI, behaviour, and brain stimulation to characterise the effect of stimulation of the lateral habenula in a rodent model for depression. The results are compelling and provide additional information potentially relevant to both surgical targeting and mechanism of action for this deep brain stimulation target. https://doi.org/10.7554/eLife.84693.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Major depressive disorder (MDD), also known as depression, is estimated to affect approximately 300 million individuals worldwide and is a leading cause of disability according to the World Health Organization (World Health Organization, 2017). Almost 30% of MDD patients fail to respond to one or more adequate antidepressants and thus exhibit treatment-resistant depression (TRD; Conway et al., 2017). TRD is associated with increased morbidity and healthcare costs as well as reduced life quality and work productivity, all of which significantly contribute to the overall burden of MDD (Mrazek et al., 2014). Because of the limited effectiveness of available psychological and pharmacological treatments for chronic TRD, various nonpharmacological interventions, including repetitive transcranial magnetic stimulation (rTMS), transcranial direct current stimulation, vagus nerve stimulation, epidural cortical stimulation, electroconvulsive therapy (ECT) and deep brain stimulation (DBS), have been explored as therapeutic options for TRD (Wong and Licinio, 2001; Shelton et al., 2010; Cusin and Dougherty, 2012; Dandekar et al., 2018). However, these available treatment choices often have low response rates, multiple (often intolerable) side effects, and substantial lag times before a response is achieved. Lag time is especially dangerous and undesirable because rapid antidepressant effects are critical for patients with suicidal ideation, who account for 15% of TRD patients. DBS emerged in 2005 and developed into a promising strategy for the management of TRD (Mayberg et al., 2005). Clinical and preclinical studies have shown that DBS targeting various brain regions, including the subcallosal cingulate, the ventral capsule/ventral striatum, medial forebrain bundle, nucleus accumbens (NAc), and lateral habenular nucleus (LHb), can induce remission of depressive symptoms (Anderson et al., 2012; Dandekar et al., 2018; Zhou et al., 2018). However, the response rates and effectiveness have varied widely among studies. In addition, most studies have focused on the long-term effects of DBS, on the order of days to weeks; few have investigated the short-term, rapid antidepressant effects (Wang et al., 2020; Scangos et al., 2021). The mechanisms underlying the therapeutic response to DBS remain unclear. Accumulating evidence indicates that the LHb, which innervates multiple brain regions and directly influences the serotonergic, noradrenergic, and dopaminergic brain systems, exhibits hyperactivity in depressed states (Hikosaka, 2010; Hu et al., 2020). Recently, LHb-DBS has attracted intense interest for the treatment of TRD. Electrical stimulation of the LHb improved depressive-like behavior in rat models of TRD (Meng et al., 2011; Lim et al., 2015; Tchenio et al., 2017; Jakobs et al., 2019). A recent case report observed both short- and long-term improvements in depression, anxiety, and sleep in one human patient after administering high-frequency DBS to the left LHb (Wang et al., 2020), suggesting that LHb-DBS may represent a potential form of rapid-acting antidepressant therapy. However, due to the small sample sizes and open label design of relevant studies, the efficacy of this rapid-acting antidepressant therapy and its underlying mechanism remains elusive. In this study, we observed an immediate (within seconds to minutes) alleviation of depressive-like symptoms with a high-response rate under DBS targeting the LHb (LHb-DBS) in two rat models of depression, Wistar Kyoto (WKY) and lipopolysaccharide (LPS)-treated rats. The remission of depressive symptoms manifested as a significantly increased sucrose preference, decreased immobility time in the forced-swim test, and increased locomotor activity, including increased activity in the center area of an open arena. Simultaneous functional MRI (fMRI) was conducted on the same sets of depressive rats used for behavioral tests under the LHb-DBS (Figure 1a). The use of MRI-compatible graphene fiber (GF) electrodes to deliver the DBS pulses enabled complete and unbiased activation pattern mapping across the rat brain by fMRI. LHb-DBS activated multiple regions in afferent and efferent circuitry of the LHb, including those in limbic, serotonergic and dopaminergic systems. The activation levels of brain regions connected to the medial LHb (M-LHb) was correlated with the extent of behavioral improvements. Rats with DBS sites more medial in the LHb exhibited greater antidepressant effects than those with more lateral DBS sites. These results indicate that the antidromic activation of the limbic system and orthodromic activation of the monoaminergic systems connected to the M-LHb play critical roles regarding the instant antidepressant response to LHb-DBS. Our work indicates that DBS of the M-LHb might represent a valuable rapid-acting antidepressant therapy for treating TRD and that fMRI activation of specific brain regions may serve as a biomarker for predicting and evaluating antidepressant efficacy. Figure 1 Download asset Open asset Lateral habenula (LHb)-deep brain stimulation (DBS) to rat models of depression studied by functional MRI (fMRI). (a) Schematics showing the immediate antidepressant effect of LHb-DBS and fMRI studies in rats. (b) A schematic brain section showing the placement of a graphene fiber (GF) bipolar electrode in the LHb of a rat. (c) A representative coronal section from the T2-weighted MRI scan of a rat brain with a GF bipolar microelectrode implanted in the LHb. (d) An image of a hematoxylin and eosin (H&E) stained brain section with the GF electrode implanted in the LHb from the same rat shown in c. Scale bar, 500 μm. Results LHb-DBS instantaneously reduces depressive symptomatology Bipolar GF microelectrodes were implanted in the right LHb of WKY and LPS-treated rats that showed depressive symptomatology (Figure 1b–d). The WKY rat is characterized as an animal model of endogenous depression and exhibits a set of behavioral abnormalities that emulate many symptoms observed in depressive patients, such as increased emotionality and reactivity to stress as well as considerable resistance to classic antidepressants (Will et al., 2003; Aleksandrova et al., 2019; Planchez et al., 2019). As shown in Figure 2a, compared to normal Sprague‒Dawley (SD, Control) rats, depressive WKY rats exhibited a significantly lower sucrose preference in the sucrose preference test (SPT, 0.49±0.01 versus 0.84±0.02, mean ± SEM, n=10, same for below) and spent more time immobile in the forced swim test (FST, 193±17 s versus 88±14 s). Lower sucrose preference and longer durations of immobility in the FST indicate anhedonia and despair-like behavior, respectively. In the open field test (OFT), which is used to assess anxiety-related behaviors, these WKY rats exhibited decreased locomotor activity, including significantly reduced average speed and increased durations of immobility (Figure 2a). In addition, these WKY rats exhibited almost no entries into the center of the open field and spent no time in that area (Figure 2a). These results confirmed the presence of depressive symptomatology in WKY rats. Figure 2 with 2 supplements see all Download asset Open asset Lateral habenula (LHb)-deep brain stimulation (DBS) immediately alleviates depressive-like symptoms in Wistar Kyoto (WKY) and lipopolysaccharide (LPS)-treated rats (models of depression). Quantification of the behavioral responses of WKY (a) and LPS-treated (b) rats to LHb-DBS (DBS), including the sucrose preference index, the duration of immobility in the forced swim test (FST), average speed in entire field in the open field test (OFT), total duration of immobility in the OFT, number of entries into the center area of the OFT, time spent in the center area of the OFT, and distance traveled in the center area of the OFT. For WKY rats, the behavioral performances of normal Sprague‒Dawley (SD) rats (Control) and WKY rats before DBS (Pre) were included for comparison. For LPS-treated rats, the behavioral performances of these rats before LPS injection (Baseline) and before DBS (Pre) were included for comparison. Data from the same animals are connected with black lines. Data are represented as the mean ± SEM (n=10 animals for WKY rats, n=9 animals for LPS-treated rats). The dots indicate data from each individual subject. To compare scores on the sucrose preference test (SPT), FST, and performance in total area of the OFT in WKY rats between the Pre and DBS groups, a two-tailed paired t test was used. A two-tailed unpaired t test was used for comparisons between the Control and Pre groups as well as between the Control and DBS groups. Because the data on variables from the center area of the OFT were not normally distributed in WKY rats, Wilcoxon’s matched pairs signed-rank test was used to compare the Pre and DBS groups, and the Mann‒Whitney U test was used to compare the Control and Pre groups as well as the Control and DBS groups. Data from the SPT, FST, and total area of the OFT in LPS-treated rats were compared by one-way repeated-measures ANOVA tests with Tukey post hoc tests. Because the data from the center area of the OFT of LPS-treated rats was nonnormally distributed, Friedman’s tests were used for comparison. n.s.: not significant, *p<0.05, **p<0.01, and ***p<0.001. The last panels in (a) and (b) show examples of the locomotor activity of a WKY rat and an LPS-treated rat before (Pre, black line, 5 min) and during (DBS, blue line, 5 min) LHb-DBS with graphene fiber (GF) bipolar electrodes. The area outlined by the red square was defined as the center area, accounting for one-ninth of the total area size. See Figure 2—figure supplements 1 and 2 for additional details. Results from detailed statistical tests are summarized in Supplementary file 1. An intraperitoneal injection of LPS is known to increase the levels of inflammatory factors, resulting in depression-like behavior (Cui et al., 2018; Planchez et al., 2019; Zhao et al., 2020a). In our experiments, LPS-treated Wistar rats exhibited a significant reduction in sucrose preference and locomotor activity, including decreased average speed and increased immobility time in the OFT (Figure 2b, n=9 rats). The numbers of entries, time spent within, and total distance traveled in the center area of the OFT were also significantly reduced (Figure 2b, n=9 rats). No significant change was observed in the immobility time in the FST (Figure 2b, n=9 rats). This finding is consistent with previous observations that LPS administration at lower doses did not alter behavioral performance in the FST (Tonelli et al., 2008; Pitychoutis et al., 2009). Taken together, these results indicate the successful establishment of an inflammatory model of depression, given the clear anhedonia and anxiety-like behaviors. Electrode tip placements within the LHb for DBS were verified for each subject by T2-weighted rapid acquisition with relaxation enhancement (RARE) anatomical MRI acquired immediately after implantation (Figure 1c). The negligible artifact induced by the GF electrodes did not obscure the LHb and allowed accurate identification of the electrode tip positions in MRI scans. These advantages enabled simple and precise in vivo verification of the placement of the implanted electrodes. Electrode tip localization within the LHb was also confirmed by hematoxylin and eosin (H&E) staining at the end of the study which showed consistent results as those from MRI scans (Figure 1d). Unless otherwise specified, rats with GF electrodes successfully implanted into the LHb were selected and used in the behavioral tests and DBS-fMRI experiments. High-frequency stimulation consisting of 130 Hz constant-current pulses with an amplitude of 300 μA and a duration of 90 μs (biphasic and symmetric) was applied to the GF bipolar microelectrodes implanted in rat models of depression. After 15 min of LHb-DBS, the sucrose preference index (SPI) of the WKY rats increased from 0.49±0.01 to 0.84±0.04 (mean ± SEM, n=10, same for below; Figure 2a). This immediate improvement in sucrose preference indicates the rapid and significant reversal of anhedonia in these WKY rats by LHb-DBS. The despair-like symptoms were also instantly alleviated upon the administration of LHb-DBS, as indicated by the decreased immobility time in the FST from 193±17 s to 85±12 s (Figure 2a). In the OFT, LHb-DBS significantly increased the average speed from 0.58±0.07 cm s–1 to 4.27±0.26 cm s–1 and decreased the duration of immobility from 260.97±3.24 s to 144.85±4.49 s. In addition, upon LHb-DBS, these rats made more entries into (0 versus 6.50±1.96), spent more time in (0 s versus 32.00±14.33 s), and traveled a longer distance (0 cm versus 118.08±35.01 cm) in the center area. An example of the locomotor activity of a WKY rat before (black line, 5 min) and during (blue line, 5 min) LHb-DBS is shown in Figure 2a. Together, these results indicated that LHb-DBS significantly alleviated depressive symptomatology in WKY rats. More importantly, this antidepressant effect occurred immediately or within a few minutes after the administration of LHb-DBS, indicating a short lag time to response. The immediate antidepressant effect of LHb-DBS was also observed in LPS-treated depressive rats. Upon administration of LHb-DBS, LPS-treated depressive rats showed a significant increase in sucrose preference and increased locomotor activity, including behavior in the center area of the OFT. Specifically, the SPI increased from 0.48±0.03 to 0.81±0.03 (mean ± SEM, n=9 rats, same for below). In the OFT, the average speed increased from 1.21±0.27 cm s–1 to 5.31±0.58 cm s–1, the duration of immobility decreased from 231.21±7.14 s to 126.91±9.81 s, the number of entries into the center area increased from 0 to 1.33±0.44, the time spent in the center increased from 0 s to 2.24±0.79 s, and the total distance traveled in the center increased from 0 cm to 26.25±9.99 cm (Figure 2b). The increases in locomotor activity were also reflected by the increased total distance traveled (in the entire field) as well as the increased average speed in the center area (Figure 2—figure supplement 1). An example of the locomotor activity of an LPS-treated rat before (black line, 5 min) and during (blue line, 5 min) LHb-DBS is shown in Figure 2b. Due to the lack of behavioral despair in LPS-treated rats, we did not observe any decrease in the duration of immobility in the FST in these rats upon the administration of LHb-DBS. Instead, we observed an increased duration of immobility. This increase is consistent with the time effect of the FST reported in a previous minute-by-minute analysis of the FST (Pitychoutis et al., 2009; Mezadri et al., 2011; Costa et al., 2013). Overall, WKY and LPS-treated depressive rats showed a high-response rate to LHb-DBS. Of 19 WKY and LPS-treated rats, 16 (~84.2%) showed an increase in sugar preference greater than 50%, 13 (~68.4%) showed an increase in average speed greater than fivefold, and 14 (~73.7%) exhibited at least one entry into the center area in the OFT compared to no entries before DBS. Of 10 WKY rats, 6 had a decrease in the duration of immobility in the FST greater than 50%. In addition, DBS of same parameters delivered to electrodes implanted outside the LHb failed to exert antidepressant effects (Figure 2—figure supplement 2a). fMRI studies of LHb-DBS The fMRI scans were performed during LHb-DBS on the same sets of WKY and LPS-treated rat models of depression under anesthesia. The stimulation pulses were the same as those used for behavioral tests, except that a higher pulse amplitude of 600 μA was used to achieve a more robust effect and circumvent the issue of low DBS-fMRI sensitivity at low stimulus amplitude (Albaugh et al., 2016). The 600 μA stimulation generated blood-oxygenation-level-dependent (BOLD) signal patterns qualitatively similar to but more robust than stimulation at 300 μA. Notably, the high-charge injection capacity of the GF electrodes allowed for the delivery of the 600 μA pulses without polarizing the electrode beyond the potentials for water reduction or oxidation with a small electrode size of only ~75 μm, thus ensuring both safety and stimulation resolution (Zhao et al., 2020b). The small-to-absent artifact produced by the GF electrodes enabled fMRI scanning of all brain regions, thus resulting in full and unbiased activation pattern mapping under LHb-DBS in rat models. Robust positive BOLD responses were evoked ipsilaterally in multiple regions along the direct afferent and efferent circuitry of the LHb as well as several regions outside the direct circuity and the DBS target (the LHb). Representative BOLD activation maps of a WKY rat and a LPS-treated rat are shown in Figure 3b and c, and individual activation maps from all the WKY rats and LPS-treated rats used in this study are shown in Figure 3—figure supplements 1 and 2. The activation patterns were similar in all rats, although individual rats differed in the intensity of BOLD activation. The activated brain regions with afferent connections to the LHb included the septum, diagonal band nucleus (DBN), lateral preoptic area (LPO), lateral hypothalamic area (LHA), and medial prefrontal cortex (mPFC). The ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) are two activated regions with efferent connections from the LHb. Since the interpeduncular nucleus (IPN) is close to the VTA and it is difficult to accurately distinguish these two areas in fMRI studies, we labeled this region as IPN/VTA. In addition, we observed the activation of the sublenticular extended amygdala (SLEA), cingulate cortex (Cg), and retrosplenial cortex (RS), which are not directly connected to the LHb. Despite DBS-induced improvements in locomotor activity, no significant BOLD activation in the motor cortex was observed under LHb-DBS. Figure 3 with 2 supplements see all Download asset Open asset Functional blood-oxygenation-level-dependent (BOLD) activation evoked by lateral habenula (LHb)-deep brain stimulation (DBS) with graphene fiber (GF) stimulating electrodes in rat models of depression. The same sets of rats were used as those in the behavioral tests. (a) Definition of regions of interest (ROIs) for different brain regions. The numbers below slices denote the relative distance from bregma (in mm). The same set of distance numbers applies to the slices in b and c. (b and c) Representative BOLD maps of a Wistar Kyoto (WKY) rat (b) and a lipopolysaccharide (LPS)-treated rat (c). The BOLD activation maps are overlaid onto averaged anatomical images. The color bar denotes t-score values obtained by GLM analyses, with a significance threshold of corrected p<0.001. The vertical white lines in a–c indicate the graphene fiber (GF) bipolar electrode. (d) BOLD signal time series at anatomically defined ROIs evoked by LHb-DBS in WKY (blue) and LPS-treated (red) rats. The stimulation epoch is indicated by the gray-shaded band. The solid lines show the average signal, and the shaded regions represent the SEM, n=30 scans from 10 WKY, n=27 scans from 9 LPS-treated rats. DBN, diagonal band nucleus; LPO, lateral preoptic area; LHA, lateral hypothalamic area; IPN, interpeduncular nucleus; VTA, ventral tegmental area; DRN, dorsal raphe nucleus; mPFC, medial prefrontal cortex; Cg, cingulate cortex; RS, retrosplenial cortex; LHb, lateral habenula; SLEA, sublenticular extended amygdala. See Figure 3—figure supplements 1 and 2 for additional details. The time courses of the BOLD signals in several anatomical regions of interest (ROIs) were calculated and averaged from all WKY and LPS-treated rats. We observed clear BOLD signal changes time-locked to the stimulation pulse blocks (Figure 3d). Of all of the regions examined in WKY rats, the mPFC showed the largest percent changes in BOLD signals (4.87 ± 0.42%, mean ± SEM, n=30 scans from 10 rats), slightly higher than those in the DBS target, the LHb (4.77 ± 0.38%, mean ± SEM, n=30 scans from 10 rats). For LPS-treated rats, the LHb exhibited the largest changes in BOLD signals (7.02 ± 0.35%, mean ± SEM, n=27 scans from 9 rats), and the mPFC exhibited the second largest changes (4.79 ± 0.28%, mean ± SEM, n=27 scans from 9 rats). The limbic areas, including the septum, DBN, LPO, and LHA, exhibited an overall lower BOLD response than the cortical regions, including the mPFC, Cg, and RS. The IPN/VTA showed activation approximating that of the cortical regions, and the activity level of the DRN was close to that of limbic regions. A characteristic “double peak” in the BOLD signal was observed in specific regions, including the Cg, mPFC, and DRN, possibly due to the recruitment of two distinct circuitries or a delayed neurotransmission effect (Van Den Berge et al., 2017). The BOLD signals of those depressive rats with electrodes implanted outside the LHb showed distinctly different patterns from those with accurate electrode implantation into the LHb. Some rats showed almost no activation across the entire brain, and others showed activation only in the target LHb, Cg, and mPFC (Figure 2—figure supplement 2b). Correlation between antidepressant efficacy and fMRI responses To shed light on the mechanism underlying the immediate antidepressant effect of LHb-DBS, we conducted Pearson correlation analysis on the behavioral improvement and the BOLD activation levels of various brain regions in individual rats. The increase in average speed in the OFT, as characterized by the ratio of speed with and without DBS, the number of entries into the center of the open field, and the distance traveled in the center were significantly correlated with the beta values of BOLD signals in the septum, DBN, LPO, LHA, DRN, and SLEA (p<0.05, Figure 4a and b, Figure 4—figure supplement 1) but not correlated with those in the Cg, RS, mPFC, and LHb (Figure 4—source data 1). The activation of the IPN/VTA showed a relatively weak correlation with the increase in average speed, but no correlation was observed between that and the number of entries into the center or distance traveled in the center (Figure 4a and b, Figure 4—source data 1). The activation intensity of the DRN showed the strongest correlation with the increase in the average speed (r=0.92, p=2.3 e–8) among all the brain regions and was also significantly correlated with the number of entries into the center (r=0.60, p=0.007) and the distance traveled in the center (r=0.55, p=0.01; Figure 4a and b, Figure 4—figure supplement 1). Notably, except for the SLEA, all the brain regions with activation levels correlated with the above three indicators of the OFT have direct afferent or efferent connections to the LHb (Figure 4c). No correlation was observed between the BOLD activation levels and sucrose preference or the duration of immobility in the FST. Figure 4 with 1 supplement see all Download asset Open asset Correlation between functional MRI (fMRI) responses and behavioral improvement. Scatter plots of the regional beta values of blood-oxygenation-level-dependent (BOLD) responses and performance indicators on the open field test (OFT) from all the Wistar Kyoto (WKY) and lipopolysaccharide (LPS)-treated rats upon administration of lateral habenula (LHb)-deep brain stimulation (DBS), including (a) change of average speed, defined as the ratio of average speed with DBS (DBS) and without DBS (non-DBS), and (b) number of entries into the center. The Pearson’s correlation coefficient r between behavioral improvement in the OFT and beta values of BOLD responses across rats were calculated for each regions of interest (ROI). (c) A schematic showing the placement of the stimulating electrode in LHb and summary of the activated brain areas. Arrows to and from LHb indicate afferent and efferent connections, respectively. See Figure 4—figure supplement 1 and Figure 4—source data 1 for additional details. Figure 4—source data 1 Correlation analysis results between functional MRI (fMRI) responses in some brain areas and behavioral improvement. The Pearson’s correlation coefficient r between beta values of blood-oxygenation-level-dependent (BOLD) responses and performance indicators of open field test (OFT) across rats was calculated for the cingulate cortex (Cg), retrosplenial cortex (RS), medial prefrontal cortex (mPFC), lateral habenula (LHb), and interpeduncular nucleus (IPN)/ventral tegmental area (VTA), including change of average speed, defined as the ratio of average speed with deep brain stimulation (DBS) and without DBS (non-DBS), number of entries into the center and distance traveled in center. https://cdn.elifesciences.org/articles/84693/elife-84693-fig4-data1-v1.docx Download elife-84693-fig4-data1-v1.docx Antidepressant efficacy depended on DBS position The excellent MRI compatibility of the GF electrodes facilitated exact determination of the locations of electrode tips within the LHb in vivo, thus allowing us to correlate the antidepressant efficacy with the stimulating positions relative to the anatomical substructures of the target nuclei. On the basis of differential afferent and efferent connections, the LHb can be divided into the M-LHb and the lateral LHb (L-LHb; Hu et al., 2020). Based on the T2-weighted anatomical images, we delineated the electrode tip location of each rat and summarized them in Figure 5a. T2-weighted MRI images of all the WKY rats and LPS-treated rats used in this study are included in Figure 5—figure supplement 1. Figure 5 with 2 supplements see all Download asset Open asset Stimulation position dependence of the antidepressant efficacy and blood-oxygenation-level-dependent (BOLD) responses. (a) A schematic diagram showing the positions of the graphene fiber (GF) electrode tips within the habenula from all the Wistar Kyoto (WKY) and lipopolysaccharide (LPS)-treated depressive rats. Each dot represents the electrode tip position in a depressive rat. Labels with “W” indicate WKY rats and those with “L” indicate LPS-treated rats. According to the positions in the medial-lateral direction, the depressive rats were divided into three groups, with red dots corresponding to the group with stimulation positions more medial, black dots corresponding to the group with stimulation positions in the middle, and blue dots corresponding to the group with stimulation positions more lateral in lateral habenula (LHb). The electrode tip position was delineated from the T2-weighted MRI images of each rat. The dashed line indicates the boundary between the medial LHb (M-LHb) and the lateral LHb (L-LHb). (b) Performance indicators in the open field test (OFT) and beta values of BOLD responses in several regions of interest (ROIs) from the three groups of rats with different stimulation locations. The change of average speed is defined as the ratio of average speed with deep brain stimulation (DBS) and without DBS (non-DBS). The number of entries into the center, time spent in the center and distance traveled in the center area of the OFT correspond to the behavioral performance with DBS. *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA tests with Tukey post hoc analysis for change of average speed, Kruskall–Wallis ANOVA for indicators in the center of the OFT, one-way ANOVA tests with Tukey post hoc analysis for beta values of BOLD responses. Red, black, and blue histogram bars represent the three groups marked with red, black, and blue dots in (a). The dots represent the data of each individual rat. The error bars indicate the SEM. (c) BOLD activation maps averaged from the three groups o