Dorsal raphe serotonergic neurons mediate depression in a mouse model of loss of control over stress

doi:10.21203/rs.3.rs-1645735/v1

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Dorsal raphe serotonergic neurons mediate depression in a mouse model of loss of control over stress

https://doi.org/10.21203/rs.3.rs-1645735/v1

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Our previous study found that, in a triadic experiment, helplessness exhibited in mice in the loss of control over stress (LOC) group in response to aversive events was more prominent than that seen in the yoked (Yoked) group of mice. However, the mechanisms responsible for such phenomena are poorly understood. Increasing evidence has demonstrated that 5-hydroxytryptamine (5-HT) plays a critical role in emotional behaviours. Therefore, we hypothesized that the serotonergic system might mediate depression induced by the loss of control over stress. Our results showed that the activity level of 5-HT neurons of mice in the LOC group was significantly higher than that of mice in the Yoked and Control groups. Additionally, optogenetic inhibition of 5-HT neuron projections from the dorsal raphe nucleus (DRN) to the ventral hippocampus (VH) significantly reduced escape latency and freezing time, improved escape defect behaviour of mice in the LOC/Yoked group, and downregulated the expression of DRN 5-HT_1A autoreceptor (DRN 5-HT_1AR) of mice in the LOC group. These results provide compelling evidence that the sensitization of dorsal raphe 5-HT neurons mediates depression in a mouse model of loss of control over stress.

Depression

Loss of control

5-HT

5-HT1A autoreceptor

DRN

Stress resulting from loss, defeat, threat, and humiliation can influence the onset or course of depression [1, 2]. Previous studies demonstrated that severe or chronic stress coupled with a lack of predictability or perceived lack of control over stress could induce depression [3]. We recently reported that the loss of existing control over mild foot shock (LOC) could produce more significant depression-like behaviours than an unchanged continuous lack of control over an identical foot shock in a novel triadic design, where the LOC paradigm could mimic learned depression in humans or rodents induced by the loss of control over aversive life events [4]. However, the mechanisms underlying this phenomenon are unknown and deserve further study. 5-HT mediates the regulation of emotional behaviours [5]. As the target of selective serotonin reuptake–inhibitor (SSRI) drugs in depression treatment, the 5-HT system is logically a source of candidate genes underlying depression [6]. Several animal models of anxiety and depression-like behaviours have exhibited a reduction in the firing of 5-HT neurons [7]. Genetic depletion of 5-HT neuronal vesicular neurotransmission has been shown to alleviate anxiety [8]. 5-HT deficiency in the brain also increases vulnerability to psychological stress and decreases the antidepressant effects of fluoxetine following stress exposure [9]. However, hyperactivity and sensitization of DRN 5-HT neurons are critical for the development of behavioural consequences in response to uncontrollable stress. Excessive activation of DRN 5-HT neurons during uncontrolled electric shock can trigger electric shock-avoidance behaviour [10]. Both the inescapable electric shock and the evasion of electric shock increase 5-HT release in the DRN [11]. The above suggests that stressors with different intensities and controllability can cause opposite reactions in the firing of DRN 5-HT neurons.

5-HT neuronal projections in the brain originate from the DRN. Most 5-HT neurons in both the DRN and the median raphe nuclei (MRN) projecting to the forebrain are involved in emotional behaviours [12, 13]. Activation of 5-HT neurons in the MRN was correlated with enhanced anxiety-like behaviour in mice, whereas activation of 5-HT neurons in the DRN had no effect on anxiety-like behaviour [14]. At the same time, optogenetic activation of DRN 5-HT neurons also impairs reward-associated behaviours [15]. The antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and nonlearned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels [16]. Furthermore, selective activation of the medial prefrontal cortex (mPFC) 5-HT-positive neuronal projections to the DRN increases swimming behaviour in the forced-swim test (FST) [17]. These findings suggest that 5-HT projections from the DRN to the cortex play an important role in regulating depressive behaviour.

5-HT_1A autoreceptors are mainly located on cell bodies (DRN or MRN), and 5-HT_1A receptors are mainly located in the axonal terminal (ventral or dorsal hippocampus) [18, 19]. Activation of either autoreceptor elicits a decrease in the extracellular concentration of 5-HT in terminal regions [20, 21], indicating that their regulation of 5-HT neuronal activity is inhibitory in nature. At the same time, the somatodendritic 5-HT_1A receptor in the DRN directly controls 5-HT cell firing and release and governs the overall 5-HT environment in the brain [22]; in addition, the activity of 5-HT_1A autoreceptors may influence the actions of other 5-HT receptors [23]. A genetic polymorphism that leads to an increase in 5-HT_1A autoreceptor expression is associated with increased vulnerability to depression in humans [24]. Thus, 5-HT_1AR likely plays a vital role in depression. Presynaptically decreasing the number or density of 5-HT_1AR in the DRN or hippocampus can increase anxiety-like behaviour [25]. In addition, studies have shown that uncontrollable stress can lead to functional desensitization of 5-HT_1A autoreceptors and enhance the activity of 5-HT neurons in the DRN [26]. The downregulation of 5-HT_1A R produced by inescapable electric shock was shown to inhibit passivity/anxiety behaviour [3]. These results suggest that 5-HT_1A autoreceptors play an important role in the regulation of stress-induced behavioural responses.

Here, we sought to probe the role of 5-HT neurons in the DRN in depression-like behaviours generated by the loss of control over stress. The data showed that after the two stress procedures, the firing of 5-HT neuronal activity was reduced in yoked mice but increased in LOC mice, and the 5-HT_1A R expression of mice in the DRN in the LOC group was significantly decreased compared with that of mice in the yoked group and control group. These results suggested that DRN 5-HT_1AR mediates the effect unpredictable shock has on mice by the selective activation of 5-HT neurons in the DRN. Excessive activation of 5-HT neurons leads to depression-like behaviour.

2.1. Animals

All animal procedures were carried out in accordance with the guidelines for the care and use of Laboratory Animals issued by the USA National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Shaanxi Normal University. C57BL/6 mice were purchased from Xi’an Jiaotong University. SERT-Cre mice were gifted by the National Institute of Biological Sciences of Minmin Luo Laboratory. Mice were acclimated for one week before use, housed in groups with ad libitum access to food and water, and maintained at room temperature (23 ± 1°C) and under a 12 hours light/dark cycle.

2.2. Operant model of learned helplessness in mice

Loss of control over shocks was performed as previously described with minimal modifications [4]. The experiments were carried out in operant response test chambers (30 × 24 × 30 cm, MED-QT0640, Med Associates Inc., St. Albans, VT, USA), equipped with house lights, a ventilator fan, and two nose-poke detectors (left and right) located 2 cm above the metal grid floor. One of the nose-poke detectors was randomly designated to be “active” to terminate the foot shock when the mice poked the hole. A LED light was illuminated as a cue during the shock, and nose poke data were collected automatically by the equipped software. The mice were randomly assigned to the following 3 groups: control with non-shocks (Control), loss of control over stress (LOC), yoked to LOC (Yoked). For mice assigned to the LOC group, the procedure of foot shocks was illustrated in Fig. 1A. During acclimation (day 1), mice were allowed to freely explore the operant chambers for over 100 min without foot shock delivery. During the period of escapable shocks (day 2, day 3, and day 4), an individual mouse was placed in the operant chamber again. A shock of 0.15 mA was delivered to the mouse, and at the same time, a LED light above the left nose poker was turned on. Nose poking on the left but not the right nose-poker terminated the shock and turned off the LED light, and the mouse was then given a 1.5 s tone and a pseudorandom period of non-shock ranging from 30 s to 60 s. This is defined as one trial. One experimental block included 10 continuous trials and a break of 10 min. On each day of the escapable shocks, 5 blocks were delivered, and the total time of a session was 100 min approximately. During the period of inescapable shocks (day 5, day 6, and day 7), the previous left nose-poker was changed to inactive, meaning that mice could no longer turn off the shock by poking at the left nose-poker. On each day of the inescapable shocks, mice were given 5 blocks of shocks, and the total time of each session was 100 min too. Each block consisted of a shock of 0.15 mA over 10 min and a break of 10 min. For mice assigned to the Yoked group, mice received exactly the same duration, intensity, and pattern of shock controlled by their LOC group partners; however, their behavioral responses had no effect on the shock during the whole period (day 2–7). The mice assigned to the Control group were placed in the operant chambers for 100 min each day for continuous 7 days but did not receive any shock. The numbers of nose-poking and/or the shock time were automatically recorded during the experiment. The apparatuses were thoroughly cleaned with 30% ethanol after each experiment.

2.3 Shuttle box test (SBT)

As described previously [4], escape behavior was observed in shuttle boxes for one day (Shuttle box test 2, SBT2). At the beginning of each session, mice were placed into the shuttle boxes and given 10 min for habituation, during which they were allowed to explore the box. Mice then received two foot shocks with an intensity of 0.15 mA through both sides of the grid floor. The foot shocks were terminated when the mice successfully crossed over to the opposite side of the shuttle box (fixed ratio 1, FR-1). Following the second FR-1 trial, the mice were allowed to move freely in the shuttle box for 20 min to monitor for freezing behavior. After three additional FR1 trials, mice were given 25 fixed-ratio 2 (FR-2) trials with the same intensity (0.15 mA) of shocks in which they had to cross through the shuttle box door twice in order to shut down the foot shock. The maximum length of foot shock was 60 s with a rest interval of 30 s. A single test session lasted approximately 1 h. To observe the escape deficit for a longer period of time, we carried out a 5-day test in the shuttle box (Shuttle box test 1, SBT1). As done in a previous study [4], mice were given 2 min to acclimate to the boxes before the study started. Each trial started with turning on a light for 5 s, followed by a 5-second foot shock (0.15 mA) with a rest interval of 30 seconds. After each session, the apparatus was thoroughly cleaned with 30% ethanol.

2.4 Open field test (OFT)

The open field box consists of a square black box (50 × 50 × 40 cm) made of an acrylic board with an illuminated center area. The study was performed 24 hours after the last exposure to LOC/Yoked/Control conditions, with mice being habituated in the testing room for 1 h before starting the test. Then, each mouse was removed from its home cage, placed in the middle of the field box, and allowed to explore freely for 10-min. The EthoVision-XT (AccuScan, Columbus, OH, USA) was used to monitor and record the activity of the mice. After each session, the apparatus was thoroughly cleaned with 30% ethanol.

2.5 Elevated plus maze (EPM)

After the open field testing, mice were moved back to their home cage for 2 h before starting the elevated plus maze test. The elevated plus maze, which was raised 50 cm above the floor, consisted of two enclosed arms (70 × 7 × 14 cm) and two open arms (70 × 7 cm) that form a plus (+) shape. The mice were placed in the intersection of the four arms of the elevated plus maze and faced a closed arm. A camera was used to record the behaviors of the mice for 5 min. The time spent and entries made on the open and closed arms were measured. The maze was cleaned with 30% ethanol between trials.

2.6 Sucrose preference test (SPT)

This was performed as reported in previous studies [4]. The mice were transferred individually to a chamber (22 × 17 × 19 cm) equipped with two contact lickometers connected to two bottles filled with regular water for a 2-h apparatus adaptation period. The mice’s contact with the lickometer was monitored and recorded. All mice were moved back to their home cage and had ad libitum access to water and food and then were deprived of water and food for 12 hours. After deprivation, each mouse was individually placed in a chamber of the SPT apparatus again and given two bottles containing 1% sucrose solution and regular water, respectively. The number of licks of normal water and sucrose solution was recorded for 2 hours. The preference index was calculated as the ratio of the number of licks of the sucrose solution to the total number of licks.

2.7 Forced swimming test (FST)

This was performed as previously reported [4]. After the SPT, the mice were habituated in their home cages for 2 h prior to FST. The behaviors during FST were monitored and recorded by a video camera mounted on the side of the cylinder. The videos were analyzed by the Ethovision XT animal software. The water was changed after every session to avoid influencing the next mice. Immobility behavior is defined as the lack of any movement except for those necessary to keep the nose above water. The first minute was the adaptation stage, and the last 5 minutes were defined as the test stage.

2.8 In vivo electrophysiology

This was performed as previously described [27]. At 24–48 hours after the last exposure to the LOC/Yoked/Control conditions, mice were removed from their home cage and anesthetized with 25% urethane (Sigma-Aldrich, 1.25 g/kg, i.p.). After fixation of the head in the stereotaxic frame (Neurostar/Germany), the skull was exposed to reveal bregma. The eyes were kept moist using erythromycin eye ointment, and body temperature was maintained at 37 ± 0.5℃ via an adjustable heating pad. The coordinates for the DRN were obtained from bregma (-4.7 mm anteroposterior (AP), 0 mm midlateral (ML), and − 2.7 to -3.2 mm dorsoventral (DV)), and a cranial window was created above the target. Glass electrode (2.0 mm o.d. × 1.16 mm i.d; Harvard Apparatus, UK; 10–40 Mohm) filled with 2M NaCl was penetrated slowly using a micromanipulator (Sutter Instruments DP-311). Signals were amplified and filtered (0.3–5 kHz). Data were acquired via a CED Data Acquisition & Analysis System, sampled at 10 kHz, and stored by Spike 2 software (Cambridge Electronic Design, UK) for offline analysis. Once a neuron with a stable baseline firing rate was extracellularly recorded for at least 5 min, juxtacellular labeling was performed. After labeling, animals were left for at least 1 h before transcardial perfusion with phosphate-buffered saline (PBS) solution followed by 4% paraformaldehyde (PFA). Brains were removed and processed for 5-HT immunohistochemistry later. DRN 5-HT neurons were identified as described previously [28]. Briefly, the criteria include: (i) neurons with a slow (0.1–3.5 spikes/s) and regular spontaneous firing and action potentials of 1–2 ms duration, 0.5-2 mV amplitude as well as a prominent positive deflection, followed by a negative or negative/positive transient; and (ii) the location of the recorded neurons in the DRN was histologically confirmed [29]. Original oscilloscope traces of the typical 5-HT neuron in the DRN are shown in Fig. 4A. Baseline spike trains (5 min) were analyzed to obtain the average firing rate and rhythmic changes.

2.9 Western Blot Analysis

Protein levels were measured using western blot analysis, with β-actin used to ensure equal loading of the samples, as seen in previously published studies [30] and in the Supporting Information Material and Methods 1. Primary antibody dilutions used were as follows; anti-serotonin-1A autoreceptors( 5-HT_1AR) (#ab-85615, 1 : 1000; Abcam), β-actin (#ab-7817, 1 : 10000; Abcam).

2.10 Cannula implantation and drug administration

This was performed according to previously published studies [30]. Solutions containing 5-HT_1AR-selective agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT ) (2 µg, 0.5 µl), 5-HT_1AR-selective antagonist WAY100635 (N-h2-[4-(2-methoxy- phenyl) -1-piperazinyl]ethylj -N-(2-pyridinyl) (150 µg, 0.5 µl) (You et al., 2015) or 0.9% NaCl (0.5µL) was microinjected bilaterally into the DRN using a syringe pump (HARVARD, US) connected to the implanted cannula.

2.11 Virus injections, optic fiber implantation, and optogenetic manipulation of DRN neurons

The specific operation steps of the experiment are as follows: ①SERT-Cre mice and the littermates were anesthetized with isoflurane and fixed in the stereotaxic frame (Neurostar / Germany); ②After craniotomy, a microinjector was inserted into DRN (AP: -4.6 mm, ML: 0 mm, DV: 2.7 mm)/VH (AP: -2.0 mm, ML༚1.5 mm, DV༚1.3 mm), Using a microsyringe pump, 200 nl of rAAV-Ef1α-DIO-eNpHR3.0-EYFP/Retro rAAV- EF1a-DIO-eNpHR3.0-EYFP or rAAV- Ef1α-DIO-EYFP/rAAV-EF1a-DIO -eNpHR3.0-EYFP purchased from the Wuhan Secret Brain Science and Technology Co., Ltd, were bilaterally injected into the DRN of mice at 20 nl/min, with the microinjector kept in place for 10 minutes after the injections were finished and then slowly withdrawn; ③The mice were moved back to their home cages and were allowed three weeks to recover before optic fiber implantation. For optical implantation, 24 hours after the modeling paradigm, an optical fiber in a ceramic ferrule was inserted into the DRN/VH through craniotomy, the ceramic ferrule was supported with dental acrylic, and the mice were allowed to recover for one week before random grouping; ④The mice received were placed in shuttle boxes to investigate escape deficit, one pulse of yellow light generated by a diode-pumped solid-state laser were given in the 2nd and 4th block to inhibit DRN/VH to DRN 5-HT neuronal projections through the optical fiber (593 nm wavelength, 300s pulse duration, 20 mW output power; fiber diameter = 200 mm, NA = 0.37). Light stimulation was immediately terminated when the 3rd and 5th blocks began.

2.12 Whole cell recording

Sert-eNpHR3.0 mice were deeply anaesthetized with Urethane and intracardially perfused with 5ml ice-cold oxygenated perfusion solution. The methods of slice preparation, whole-cell patch recording, and photo-stimulation are similar to those described elsewhere [15].

2.13 Immunofluorescence

Sert-eNpHR3.0 or Sert-EYFP mice were deeply anesthetized with Urethane and intracardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4). The methods of slice preparation, fluorescence staining, and imaging analysis are similar to those described elsewhere [15]. The 16 µm coronal brain slices were cut with a freezing microtome (Leica CM1950, Nussloch, Germany). The slices were blocked with phosphate- buffered saline (PBS, containing 10% normal goat serum and 2% Triton X-100) for 1 h on a shaker at room temperature (22 ± 1°C) and incubated overnight at 4°C with the primary antibody (Anti-TPH2 1:200, #ab121013, US). After rinsing with PBS, the slices were incubated again for 1 h at room temperature with the secondary antibody (CY3 1:400, #ab6949, USA). Fluorescent signals were collected using a confocal microscope (SP8 SR, Leica).

2.14 Data Analysis and Statistics

Statistical analysis/figures were performed with SPSS 20.0/GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA) by one-way analysis of variance (ANOVA) or two-way ANOVA with Bonferroni’s post hoc test. The numbers of nose pokes were analyzed by an unpaired two-tailed Student’s t-test, respectively. One-way analysis of variance (ANOVA) with a Turkey post hoc test was used to evaluate the escape latency. In vivo recording data were analyzed by one-way ANOVA followed by a Tukey test. The change in the percentage of bursting cells were analyzed by the χ² test. AUC was analyzed using a two-way repeated measure ANOVA. The comparison between pre- and post-treatment was analyzed with a paired two-tailed Student’s t-test. The value of p < 0.05 was considered to be that of statistical significance, and data are expressed as mean ± SEM.

3.1 Mice in the LOC group acquired and then lost control over foot shocks

Before the stress procedure, no significant differences among mice were found in the number of pokes to the active and inactive nose pokes on Day 1. The LOC group was first exposed to escapable foot shocks for 3 days and then to inescapable foot shocks for 3 days. The yoked group received identical foot shocks for 6 days (Fig. 1D-F). After the LOC group experienced negative reinforcement learning over three consecutive days (Days 2–4), the escape latency decreased rapidly across the five stimulation blocks on Day 4 (F_{(1, 63)} = 268.658, p < 0.001, Fig. 1D-F). Nose-poke towards the active side was significantly increased compared with that of the inactive side on Days 3–4 (Day 3: F_{(1, 63)} = 191.891, p < 0.001; Day 4: F_{(1, 63)} = 388.579, p < 0.001, Fig. 1D-F). No difference in the numbers of active nose pokes was found in the Yoked group (Fig. 1G-K), indicating that only mice in the LOC group acquired control over the stressor. While the foot shock was no longer able to be terminated by poking the previously active poker on Days 5–7, the number of active nose pokes was still significantly larger than that of inactive nose pokes in the LOC group on Days 5–6 (Day 4: F_{(1, 63)} = 274.843, p < 0.001; Day 5: F_{(1, 63)} = 8.927, p < 0.01). However, there was no significant difference between the number of active nose pokes and inactive nose pokes in the LOC group on Day 7 (F_{(1, 63)} = 0.89, p = 0.349; Fig. 1E), indicating extinction of the learned escape behaviours in the LOC group. There was no difference between the numbers of the two nose pokers in the Yoked group on Days 5–6 (Fig. 1G-K). The above results suggested that mice in the LOC group suffered from both psychological and physical stress (foot shock), while mice in the Yoked group only experienced physical stress.

3.2 Mice with a loss of control over shocks exhibit more significant escape deficits and more anxiety, anhedonia, and despair-like behaviours

As performed in our previous study [4], mice receiving the above-described foot shock treatments over a period of 7 days were randomly divided into three groups: the LOC group (n = 9), the Yoked group (n = 9), and the Control group (n = 9). On Day 8, all groups received SBT1 and SBT2 to assess escape behaviours (Fig. 2A-B), during which significant differences were found in the mean escape latency (F_(2,195) = 131.1, p < 0.001) and the number of failures (F_(2,195) = 72.31, p < 0.001) in SBT1 among the above groups. Figure 2D and 2F show significant differences in mean escape latency (F_(2,120) = 15.72, p < 0.001) and freezing time in the chamber (F _{(2, 24)} = 4.302, p < 0.05) among the 3 groups in the SBT2. In both SBT1 and SBT2, mice in the LOC group exhibited a more serious escape deficit than the other two groups. Because each pair of mice from the LOC and Yoked groups received identical shocks, the significant differences in mean escape latency, number of failures, and freezing time between the two groups were caused by the loss of control over shocks (p < 0.01) in the LOC group. Similarly, the mice in the Yoked group also displayed increased freezing time and mean escape latency (p < 0.001) as well as an increased number of failures (p < 0.001) compared with those in the Control group, which suggests the stressful effects of shock.

The open field test and elevated plus maze were used to evaluate whether this stress can induce anxiety-like behaviours after 8 days of modelling in the triadic groups. Mice in the LOC group spent more time in the middle of the open field than those in the Yoked or the Control group, while the Yoked mice spent more time in that area than mice in the Control group (Fig. 2E). However, the overall activities in the open-field test were not significantly different among the three groups (Fig. 3E). Figure 3F shows that mice in both the LOC group and the Yoked group spent less time in the open arms of the elevated plus maze than mice in the Control group, but mice in the LOC group spent less time in the open arms than mice in the Yoked group. These results demonstrated that 7 days of mild electric foot shocks in the LOC and Yoked groups could cause anxiety-like behaviour even though it did not affect locomotion significantly, and psychological stress from a lack of control exacerbated the mice’ anxiety induced by physical stress from shocks.

To investigate whether psychological stress plays a role in depression-like behaviours, we performed sucrose preference and forced swimming tests to evaluate depression-like behaviours in the triadic groups after 8 days of modelling. As shown in Fig. 3G, the LOC mice had significantly decreased sucrose consumption compared to both Yoked and Control mice, while no significant difference was found between the Yoked and the Control mice. Figure 3K also shows that the immobile time of the LOC mice during the forced swimming test was significantly longer than that of the Yoked or the Control groups. The behaviours suggested that the LOC mice may have experienced the phenomenon of anhedonia and despair, which are the core depression-like symptoms, caused by psychological stress.

3.3. Loss of control over shocks downregulated the expression of 5-HT_1AR proteins in the DRN

It has been reported that uncontrollable stressors produce desensitization of 5-HT_1AR in the DRN, which is then responsible for the experience of uncontrollable stress [26]. Here, we investigated whether the loss of control over shocks could affect 5-HT_1A R protein expression in the DRN. As shown in Fig. 3A, to investigate the role of the DRN 5-HT_1AR, 8-OH-DPAT, which is an agonist of 5-HT_1AR, was administered locally in the DRNs of mice in two groups each of control/Yoked/LOC through implanted cannulas 20 min before the SBT1 test on Day 4. We found that activating 5-HT_1AR in the DRN reduced the mean escape latencies (F_{(5, 305)} = 42.29, p < 0.001; Fig. 3B) and the number of failures (F_{(5, 300)} = 42.64, p < 0.001; Fig. 3C) in the LOC group, suggesting that the loss of control over shocks produces desensitization of DRN 5-HT_1AR, which mediates the behavioural consequences of the LOC group.

3.4. Mice with depression-like behaviours show enhanced activity of DRN serotoninergic neurons

Next, we examined the activation of serotonergic neurons in the DRN of LOC mice. At 24 h after the mice were exposed to the triadic model over 8 days, we first performed the shuttle box test and then recorded the spontaneous activity of DRN serotoninergic neurons in anaesthetized mice. As shown in Fig. 4, the average firing rate of mice in the LOC group was significantly increased compared to that of mice in both the Yoked group (p < 0.001, Fig. 4E) and the Control group (p < 0.001, Fig. 4E). Additionally, the average firing rate of mice in the Yoked group was significantly higher than that of mice in the Control group (p < 0.01, Fig. 4E). The interspike interval of spontaneous firing was also significantly shorter in the LOC group than in either the Yoked group or the Control group (Fig. 4F). These data suggested that the escape deficit induced by the loss of control over shock is directly related to an increase in DRN serotoninergic neuron firing.

3.5. Optogenetic inhibition of DRN 5-HT neurons alleviates the escape deficit of mice with depression-like behaviours

Previous studies have demonstrated that 5-HT neuronal signalling mediates the pathophysiology and therapeutics of major depressive disorders [31]. To address whether inhibition of DRN serotonergic neurons improves depression-like behaviours in LOC mice, we performed optogenetic manipulation to inhibit DRN 5-HT neurons in SERT-Cre mice. Following stereotaxic infusion of the Cre-dependent adeno-associated virus rAAV-Ef1α-DIO

-eNpHR3.0-EYFP into the DRN of SERT-Cre transgenic mice, we recorded the firing rate of raphe serotonergic neurons in the acute coronal brain slices of eNpHR3.0-expressing SERT-cre mice. We found that the firing frequency was decreased significantly by inhibition of eNpHR3.0-expressing neurons (Fig. 5C). Subsequently, we investigated behavioural changes through an optical fibre implanted in the DRN of eNpHR3.0-expressing mice. In vivo optogenetic stimulation with yellow light decreased the mean escape latencies and the freezing time of the eNpHR3.0-expressing mice in shuttle box test 2 (Figs. 5D-L).

3.6. Optogenetic inhibition of 5-HT neurons in the VH improves the escape deficit of mice with depression-like behaviours

The DRN contains a large number of 5-HT neurons that project to the VTA, nucleus accumbens (NAc), and lateral hypothalamus (LH) [32]. Previous reports showed that chronic stress could negatively regulate adult hippocampal neurogenesis [33]. Furthermore, serotonergic fibres labelled with YFP immunoreactivity were observed throughout the hippocampal region [34]. To verify whether DRN-ventral hippocampus (VH) 5-HT neuronal projections exist and whether they play a role in the depression-like behaviour induced by a loss of control over shocks, we stereotaxically injected retro rAAV-EF1a-DIO-eNpHR3.0-EYFP) into the VH. In addition, as shown in Fig. 6A, Fig. 6B and C, continuous photostimulation at the 5-HT terminals during the 2nd training and the 4th training (590 nm, 5 mW) reduced the mean escape latencies (F_(1,8) = 7.947, p = 0.023, p < 0.05) and the freezing time (Figs. 6B-C) in the modelling experiments. These data suggest that DRN-VH serotonergic activation plays a pivotal role in depression-like behaviour induced by the loss of control over shocks.

In the present study, we investigated the role of 5-HT neurons in depression-like behaviours induced by a loss of control over shocks. We found that the LOC mice that first received controllable foot shocks and then received uncontrollable foot shocks exhibited depression-like behaviours, as seen in our previous reports [4]. DRN 5-HT_1AR was also downregulated in the LOC group. The basal firing activities of DRN 5-HT neurons were increased in both the LOC and Yoked groups; however, the former exhibited a more significant enhancement compared to the latter. Optogenetic inhibition of DRN 5-HT neurons or 5-HT neuronal projection to the VH from the DRN alleviated depression-like behaviours in LOC mice.

Chronic stressful life events are risk factors for depression, the pathophysiology of which is strongly associated with impairments in 5-HT neurotransmission [7]. 5-HT directly or indirectly regulates various stress-induced depressions, and thus, it is an important physiological basis for the formation of depression [35]. 5-HT_1AR is an inhibitory G-protein coupled receptor expressed both in 5-HT neurons (as an autoreceptor) where 5-HT tone is regulated through feedback inhibition [36] and in target areas receiving 5-HT innervation (as a heteroreceptor) [37,38]. 5-HT_1A R can interact with 5-HT neurotransmitters released by itself or by nearby 5-HT neurons to inhibit the activity of 5-HT neurons in the DRN and negatively regulate the level of 5-HT neurotransmitters [39]. The manipulations prevented the behavioural changes caused by uncontrollable stress by eliminating the uncontrollable stress-induced impairment of 5-HT_1AR inhibition. Voluntary exercise could block the behavioural effects of inescapable shock (IS) by increasing DRN 5-HT_1AR mRNA [40]. The inhibition of 5-HT_1AR function is a part of the causal network that mediates the behavioural effects of IS. 5-HT_1AR is critically involved in regulating mood and anxiety levels. Exposure to chronic stress could inhibit postsynaptic 5-HT_1AR function in limbic areas [41]. The functional desensitization of DRN 5-HT_1AR after IS can help explain the relationship between DRN 5-HT_1AR alterations and both anxiety- and depression-related psychopathologies. The binding of 5-HT_1AR in the raphe was reduced in patients with depression, social anxiety disorder, and panic disorder [42,43, 44 45]. In this work, we found that the expression of DRN 5-HT_1AR was downregulated significantly in LOC mice, similar to previous studies, suggesting that 5-HT_1AR plays a crucial role in the behaviours of LOC mice, especially escape deficits and freezing behaviours.

Prolonged stress produces neural adaptations in the midbrain serotonin system, influencing emotional and behavioural disorders. DRN 5-HT neurons may play complex and perhaps contradictory roles in the different types of depression-like behaviours produced by various stressors. For example, unpredictable mild stress reduces the firing activities of 5-HT neurons in the DRN. In contrast, the uncontrollable stress increases their firing activities [10]. In our study, spontaneous activity of DRN 5-HT neurons was significantly enhanced in the LOC group compared to the Yoked group, and optogenetic inhibition of DRN 5-HT neurons relieved the depression-like behaviours induced by the loss of control over shocks. Our data suggest that the enhanced basal DRN 5-HT neuronal activity, which could be modulated by decreased 5-HT_1AR expression, may contribute to the depression-like behaviours elicited by the loss of control over shocks and cause psychological stress-related behaviour deficits.

DRN 5-HT neurons have extensive projections in the forebrain and are considered to be the main source of 5-HT in the hippocampus [46]. The injection of 5-HT in the hippocampus exerted anti-stress depression effects [47]. However, very few studies have focused on the contribution of the DRN–VH pathway to stress and stress-related psychiatric disorders, including anxiety and depression. In the present work, we found 5-HT neuronal projections from the DRN to the VH. In addition, optogenetic inhibition of the 5-HT neuronal terminal in the VH reduced the average escape latency and freezing time in LOC mice, suggesting that DRN-VH 5-HT neuronal projections play a critical role in the depression-like behaviours elicited by the loss of control over shocks. In future studies, we will continue to address the mechanisms by which DRN-VH projections regulate the depression-like behaviours induced by the loss of control over shocks.

Acknowledgments

We thank Dr. Luo Minmin of School of Life Sciences, Peking University for providing SERT-Cre mice.

Author Contribution Conceptualization:

MR. Methodology:YFL, LY, QHZ, NRL and ZDY Validation: WR, YFL, CLW and HFQ. Formal analysis: WR, YFL, and HFQ. Investigation: YFL, LY, WR and HFQ. Resources: YFL, QHZ, WR and HFQ. Writing, original draft: YFL, WR and HFQ. Writing, review and editing: YFL, LY, QHZ, WR and HFQ. Supervision: YFL. Funding acquisition: YFL, WR and HFQ. All authors read and approved the fnal manuscript.

Funding This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82004489), the Shaanxi Provincial Department of Education Foundation funding project (Grant No. 20JK0592), Shaanxi Provincial Department of Science and Technology (Grant No. 2022JQ-949) and the Fundamental Research Funds for Shaanxi University of Chinese Medicine (Grant No. 123890026).

Data Availability All data generated during this study are available from the corresponding author on request.

Code Availability Not applicable.

Declarations

Ethics Approval All the experimental procedures were approved by the Medicine Animal Care and Use Committee of Shaanxi Normal University and conformed to the Guide for the National Institutional Animal Care and the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals and their suffering.

Consent to Participate Not applicable.

Consent for Publication Not applicable.

Conflict of Interest The authors declare no competing interests.

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Dorsal raphe serotonergic neurons mediate depression in a mouse model of loss of control over stress

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Animals

2.2. Operant model of learned helplessness in mice

2.3 Shuttle box test (SBT)

2.4 Open field test (OFT)

2.5 Elevated plus maze (EPM)

2.6 Sucrose preference test (SPT)

2.7 Forced swimming test (FST)

2.8 In vivo electrophysiology

2.9 Western Blot Analysis

2.10 Cannula implantation and drug administration

3. Results

3.1 Mice in the LOC group acquired and then lost control over foot shocks

3.3. Loss of control over shocks downregulated the expression of 5-HT_1AR proteins in the DRN

3.4. Mice with depression-like behaviours show enhanced activity of DRN serotoninergic neurons

3.5. Optogenetic inhibition of DRN 5-HT neurons alleviates the escape deficit of mice with depression-like behaviours

Discussion

Declarations

References

Status:

Version 1

Dorsal raphe serotonergic neurons mediate depression in a mouse model of loss of control over stress

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Animals

2.2. Operant model of learned helplessness in mice

2.3 Shuttle box test (SBT)

2.4 Open field test (OFT)

2.5 Elevated plus maze (EPM)

2.6 Sucrose preference test (SPT)

2.7 Forced swimming test (FST)

2.8 In vivo electrophysiology

2.9 Western Blot Analysis

2.10 Cannula implantation and drug administration

3. Results

3.1 Mice in the LOC group acquired and then lost control over foot shocks

3.3. Loss of control over shocks downregulated the expression of 5-HT1AR proteins in the DRN

3.4. Mice with depression-like behaviours show enhanced activity of DRN serotoninergic neurons

3.5. Optogenetic inhibition of DRN 5-HT neurons alleviates the escape deficit of mice with depression-like behaviours

Discussion

Declarations

References

Status:

Version 1

3.3. Loss of control over shocks downregulated the expression of 5-HT_1AR proteins in the DRN