What is the functional relevance of GABAB receptor dependent hyperexcitability for the emergence of depressive like symptoms

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GABA B receptors trafficking

Newly synthesized cell-surface receptors are processed and passed through distinct membrane compartments before reaching the plasma membrane, including the endoplasmic reticulum (ER), cis-Golgi network, and trans-Golgi network. Different check points regulate GABABR transport to the surface membrane. Notably an Arginine-rich endoplasmic reticulum retention signal is present within the C-terminal tail of the GABAB1 subunit (Calver et al., 2001; Margeta-Mitrovic et al., 2000; Pagano et al., 2001) as well as an di-lysine motif that inhibits its transport from trans-Golgi to the cell surface (Restituito et al., 2005). Heterodimerization with GABAB2 seems to induce a conformational change that masks retention signals promoting its exit from the endoplasmic reticulum and the GABAB1 transport to the cell surface (Gassmann et al., 2005; Pagano et al., 2001). Once at the plasma membrane GABABRs undergo constitutive clathrin and dynamin-dependent endocytosis (Grampp et al., 2007, 2008; Pooler et al., 2009; Vargas et al., 2008; Wilkins et al., 2008). After internalization, GABABRs are transferred to fast and slow recycling endosomes, as well as lysosomes. The none-degraded receptors can then recycle back to the membrane (Grampp et al., 2008). Blocking vesicle fusion to the plasma membrane by monensin induced degradation of 50% of the receptors by redirecting them to lysosomes, suggesting a stable equilibrium between internalization and recycling (for review see Benke, 2010). In the following paragraph, I will give several examples of how this process could be regulated determining the surface expression of GABABRs.

GABAB receptor regulation

Desensitization following prolonged exposure to the agonist is a common feature of G-protein coupled receptors (GPCRs) down-regulation, reducing protein levels at the surface to prevent overstimulation. Usually, desensitization involves phosphorylation by the G protein-coupled receptor kinases (GRK) recruiting arrestin, dynamin, and clathrin, leading to internalization of the receptor downregulating signaling at the membrane (Gainetdinov et al., 2004). However, GABABRs do not seem to follow this common regulatory pathway. Instead, prolonged exposure to GABABR agonist, baclofen leads to decreased phosphorylation level (at the serine residue (S)892 of the GABAB2) promoting degradation of the receptor; an effect that is attenuated by increasing PKA activity (Fairfax et al., 2004). Consistently, PKA phosphorylation of GABAB2 S892 has been shown to decrease the desensitization rate through potential stabilization of the receptor at the membrane and promotes GABABR signaling (Couve et al., 2002). Instead, protein kinase C (PKC)-dependent desensitization decreased coupling of the receptor to the G protein following prolonged receptor activation. This mechanism involved phosphorylation of GABAB1 subunit by PKC and subsequent N-ethylmaleimide-sensitive fusion (NSF) protein dissociation from GABABRs both necessary to reduce G protein activation (Pontier et al., 2006).
Apart from the agonist-dependent desensitization, sustained application of glutamate or N-methyl-D-aspartic acid (NMDA) has also been described to regulate GABABR function. This regulatory pathway increases GABABR targeting to lysosomes and its subsequent degradation, resulting in a decrease in receptor expression at the cell surface (Guetg et al., 2010; Kantamneni et al., 2014; Maier et al., 2010; Terunuma et al., 2010a; Vargas et al., 2008). Mechanistically, prolonged NMDARs activation triggers phosphorylation of GABAB1 at S867 by the CaMKII promoting the degradation of the receptor (Guetg et al., 2010). Moreover, in cortical and hippocampal cultured neurons, the balance between GABAB2-S783 phosphorylation and dephosphorylation by the AMP-activated protein kinase (AMPK) and the protein phosphatase type 2 (PP2A) respectively, governs post-endocytic sorting of GABABRs. Indeed, phosphorylation of S783 on GABAB2 subunit by the AMP-activated protein kinase (AMPK) stabilizes the complex at the membrane and decreases desensitization (Kuramoto et al., 2007; Terunuma et al., 2010a) whereas dephosphorylation of S783 by PP2A following prolonged NMDA exposure targets the receptors for lysosomal degradation (Terunuma et al., 2010a). Interestingly, electrophysiological studies in brain slice also reported PP2A-dependent down-regulation of GABABRs involving a retention within the internal compartments in a pathophysiological context (Hearing et al., 2013; Padgett et al., 2012).
Importantly, GABABR signaling depends on several other proteins interacting with the receptor or their effectors (GIRK/VGCC), such as the members of the potassium channel tetradimerization Domain containing proteins (KCTD), the regulator of G protein signaling (RGS), the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP), the Sorting nexin 27 (SNX27), among many others (Labouèbe et al., 2007; Munoz and Slesinger, 2014a; Sauter et al., 2005; Schwenk et al., 2010). Indeed interacting proteins not only regulate their targeting to specific compartments but are also needed for synthesis modulation; their intracellular signaling, for their cross-linkage to neuronal cytoskeleton, membrane assembly as well as for their allosteric activation/inactivation (for review see see (Benke, 2013; Bettler and Fakler, 2017; Couve et al., 2004; Lujan and Ciruela, 2012; Luján et al., 2014; Lüscher and Slesinger, 2010; Padgett and Slesinger, 2010; Pinard et al., 2010; Terunuma et al., 2010b). As example, RGS proteins enhance the GTPase activity of Gα subunits, accelerating the deactivation of G protein signaling (Doupnik, 2015; Sjögren, 2011). Consistently, genetically silencing RGS2 has been reported to lead to a higher GABABR-GIRK coupling efficiency in the VTA (Labouèbe et al., 2007). One other example is the four K+ channel tetramerization domain-containing (KCTD) proteins. KCTDs have been identified as auxiliary subunits of the GABABRs. By forming tetramers and binding to the C-terminal tail of GABAB2, KCTDs stabilize GABABR at the cell surface (Ivankova et al., 2013), increase agonist potency, accelerate onset and promote desensitization of the GABABRs (Fritzius et al., 2017; Schwenk et al., 2010).
Additionally, the modulation of GABABR function also rely on regulation of its effectors such as GIRK channels. For example, a proteomic study reveals that Girk3 subunit interacts with the SNX27, a protein implicated in the trafficking of an array of neuronal signaling proteins between the endosome and the plasma membrane (Balana et al., 2011; Lauffer et al., 2010;
Lunn et al., 2007; Temkin et al., 2011). Functionally, genetical ablation of the SNX27 in the VTA DA neurons has been described to reduce the GABABR -GIRK signaling (Munoz and Slesinger, 2014b).
Overall, these studies suggest an important role of GABABRs in dampening neuronal activity, presynaptically modulating neurotransmitter release via inactivation of VGCCs but also through modulation of postsynaptic GIRK channels that control neuronal excitability.
LHb hyperactivity has been related to the expression of depressive like symptoms (Li et al., 2011, 2013; Seo et al., 2017). Previous studies mainly focused on the role of fast glutamatergic and GABAergic transmission onto LHb in a rodent model of depression (Li et al., 2011, 2013; Shabel et al., 2014). However, considering the critical role of GABABRs in controlling neuronal activity, whether GABABRs dysregulation within LHb could contribute to the establishment of a depressive phenotype has still to be investigated. Importantly, GABABRs signaling have been described in pathological states including addiction, anxiety and mood disorder (for review Bowery, 2006; Cryan and Slattery, 2010; Lüscher and Slesinger, 2010). Regarding depression, the role of the GABABRs is still under debate (Cryan and Slattery, 2010; Lujan and Ciruela, 2012). Indeed, an unidirectional relationship between GABABR signaling and a precise depressive like behavioral outcome has not been provided. One possible explanation is that the different results so far obtained are not taking into account the topographical localization (pre and post-synaptic) or region specificity of the receptors. In this thesis, I will present two sets of data investigating the plasticity of GABABRs in the LHb as an early cellular process following exposure to stress that is not only crucial for neuronal output but also for behavioral traits of mood disorders.

Experimental subjects and stress paradigms

For the first study, 4- to 7-week-old wild-type male C57Bl/6J mice (Janvier laboratories, France) were used in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals and of the ethics committee Charles Darwin #5 of the University Pierre et Marie Curie Mice were housed in groups of 4–6 per cage with water and food ad libitum. Mice were randomly allocated to experimental groups.
In the second study, 4-9 weeks wild-type male and female C57Bl/6J were used. All procedures were used in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (Committee Charles Darwin #5, University Pierre et Marie Curie, Pairs). Part of the current study was carried out in the Department of Fundamental Neuroscience (Lausanne, Switzerland). Pregnant dams C57Bl/6J were received at the gestational stage E13-18 (Janvier laboratories, France). Mothers were housed two per cages with access of food and water at libitum. After birth, pups of either sex remained untouched until postnatal day (P) 7. At P7, litters were randomly divided in 2 groups. Control group were weaned at P22/23 and housed in groups of 3-5 mice per cage with water and food ad libitum. Maternally separated (MS) mice were weaned at P17 and housed in groups of 3-5 mice per cage. Control and MS mice were housed separately.

Foot shock paradigm (FS)

The inescapable-shock procedure was previously described (Stamatakis and Stuber, 2012). Briefly, we placed mice into standard mouse behavioral chambers (Imetronics) equipped with a metal grid floor. We let them habituate to the new environment for 5 min. In a 20-min session animals received either 19 or 0 unpredictable foot shocks (1 mA, 500 ms) with an intershock interval of 30, 60 or 90 s. We anesthetized mice for patch-clamp electrophysiology 1 h, 24 h, 7 d, 14 d or 30 d after the session ended.

Learned-helplessness model (LHp)

The procedure consisted of two sessions of inescapable foot shocks (one session per day; 360 foot shocks per session; 0.3 mA; shock duration between 1 and 3 s; and random intershock intervals) followed 24 h after the last session by a test session to assess the LHp. The testing was performed in a shuttle box (13 × 18 × 30 cm) equipped with a grid floor and a door separating the two compartments. The test consisted of 30 trials of escapable foot shocks. Each trial started with a 5-s-long light stimulus followed by a 10 s shock (0.1–0.3 mA). The intertrial interval was 30 s. When the mouse shuttled in the other compartments during the light cue, the avoidance was scored. When it shuttled during the shock, the escape latency was measured. When the mouse was unable to escape, the failure was scored. The shock terminated any time that the animal shuttled in the other compartment. Out of the 30 trials, more than 15 failures were defined as an LH. Only LHp mice were behaviorally and electrophysiologically tested.

Maternal separation paradigm

The maternal separation group consisted of pups removed from their litter and isolated in small compartments for 6 hours per day (light phase 8:19h) repeated from P7 to P15 and followed by an early weaning at P17. During the separation, animals were maintained in heating plate and water was provided, maintaining constant temperature and humidity. The control group consisted of mice from independent litters, which were not manipulated until the regular weaning at P21 except during cage changing. During cage changing some old bedding and nest were transferred into the new cage in order to limit novelty stress. Behavior testing and recording where performed 2 to 5 weeks after the stress protocol.

Behavioral tests

All behavioral tests were conducted during the light phase (8:00–19:00), 1 or 7 d after the shock procedure. Animals were tested only for a single behavioral paradigm, and operators were blinded to the experimental group during the scoring.
Locomotor activity. To assess the locomotor activity we tested mice in an open-field arena. Mice were placed in the center of a plastic box (50 cm × 50 cm × 45 cm) in a room with dim light. We let them to explore the arena for 5 m and then we acquired the video tracks. During the 15-min session, animal behavior was videotaped and subsequently analyzed (Anymaze, Ireland).
Forced-swim test. The forced-swim test was conducted in normal light conditions. Mice were placed in a cylinder of water (temperature of 23–25 °C; 14 cm in diameter, 27 cm in height for mice) for 6 min. The depth of water was set to prevent animals from touching the bottom with their hind limbs. Animal behavior was video-tracked from the top (Viewpoint, France). The latency to the first immobility event and the immobility time of each animal spent during the test were counted online by two independent observers in a blinded manner. Immobility was defined as floating or remaining motionless, which means absence of all movement except for the motions required to maintain the head above water.
The tail suspension test The tail suspension test was performed with mice being suspended by their tails with adhesive tape for a single session of 6 min. Immobility time of each animal was scored online by the experimenter. Mice were considered immobile only when they suspended passively and motionless Sucrose preference test. For the sucrose preference test, mice were single-housed and habituated with two bottles of 1% sucrose for 2 d. At day 3 (test day) mice were exposed to two bottles filled with either 1% sucrose or water for 24 h. The sucrose preference was defined as the ratio of the consumption of sucrose solution versus total intake (sucrose + water) during the test day and expressed as a percentage.
The shuttle box test The shuttle box (13 × 18 × 30 cm) was equipped with an electrified grid floor and a door separating the two compartments. The test session consisted of 30 trials of escapable foot-shocks (10 sec at 0.1–0.3 mA) separated by an interval of 30s. The shock terminated any time that the animal shuttled in the other compartment. Failure is defined as the absence of shuttling to the other compartment within the 10 sec shock delivery.

Drug/intervention

LB-100 treatment: Mice were injected with LB-100 (1.5 mg/kg; i.p.; Lixte Inc.) or saline 6–8 h after the FS (2 h was used for biochemical assays). LHp animals received LB-100 24 h after the test day. A set of mice (aged 5 weeks), were single-housed for 3 d and were then treated with LB-100 i.p. (1.5 mg/kg/d) for 7 d. Body weight and food pellet and water intake were monitored every 2 d. Three days after the last injections, the mice were tested for their locomotor activity Dreadd(Gi) experiment : Behavioral experiments in DREADD-injected animals were performed three weeks after viral infusion. For the shuttle Box, the tail suspension test, and the locomotor activity all the groups (YFP or DREADDi injected animals) were injected 15 minutes with CNO i.p.(1mg kg-1). For sucrose preference experiments, all groups were injected with CNO i.p. (1mg kg-1) every 3h for the extent of the preference session (24h) to maintain a constant DREADD-mediated inhibition.
Deep brain stimulation MS mice for DBS experiments were first preselected on the basis of their failure rate in the Shuttle box test (A cutoff of 12 failures was used for the preselection). 50 mice were tested, and 17 of these animals met the criteria. Standard surgical procedures were used to implant bipolar concentric electrodes unilaterally into the LHb (coordinates −1.45 mm AP, ± 0.45 mm ML and −3.1 mm DV). After 5 days recovery from surgery, DBS or no (Sham) stimulation was applied for 1h (seven stimulus trains of 130 Hz, separated by 40 ms intervals; 150 µA intensity) prior testing each mouse in the shuttle box test.
CochR expression For optogenetic experiments rAAV2.1-hSyn-CoChr-eGFP (University of North Carolina, US) was infused in the entopeduncular nucleus. Recordings were performed 3 weeks after surgery. The injection sites were carefully examined and only animals with correct injections were kept for behavioral and electrophysiological analysis.
DBS electrodes implantation Electrodes (Bilaney, UK) were unilaterally implanted using similar procedures and coordinates in the LHb. DBS electrodes were chronically implanted using a Superbond resin cements (Sun medical, Japan).
Retrolabelling of VTA and RMTg projecting neurons For the experiment analyzing the output specificity of I-Baclofen, mice were bilaterally injected with a mixture of herpes simplex virus (McGovern Institute, US) expressing enhanced GFP and red retrobeads (Lumafluor, US) into the RMTg or the VTA. Recordings from fluorescent LHb neurons were performed ±12 days following the surgery, and injection site were verified using the retrobeads labelling.

Electrophysiology

Animals were anesthetized with ketamine and xylazine (50 mg/kg and 10 mg/kg, respectively; i.p.; Sigma-Aldrich, France). Analysis was performed in a non-blinded fashion.
Preparation LHb-containing brain slices was done in bubbled ice-cold 95% O2/5% CO2-equilibrated solution containing: 110 mM choline chloride; 25 mM glucose; 25 mM NaHCO3; 7 mM MgCl2; 11.6 mM ascorbic acid; 3.1 mM sodium pyruvate; 2,5 mM KCl; 1.25 mM NaH2PO4; 0.5 mM CaCl2. 250 μm thick sagittal slices (or coronal 2nd study), were stored at room temperature in 95% O2/5% CO2–equilibrated artificial cerebrospinal fluid (ACSF) containing: 124 mM NaCl; 26.2 mM NaHCO3; 11 mM glucose; 2.5 mM KCl; 2.5 mM CaCl2; 1.3 mM MgCl2; 1 mM NaH2PO4. Recordings (flow rate of 2.5 ml/min) were made under an Olympus-BX51 microscope (Olympus, France) at 31 °C. Currents were amplified, filtered at 5 kHz and digitized at 20 kHz. Access resistance was monitored by a step of −4 mV (0.1 Hz). Experiments were discarded if the access resistance increased more than 20%. Animals were anesthetized with ketamine and xylazine (50 mg/kg and 10 mg/kg, respectively; i.p.; Sigma-Aldrich, France). Analysis was performed in a non-blinded fashion.
Internal solution The internal solution used to examine GABAB and/or GIRK currents and neuronal excitability contained: 140 mM potassium gluconate, 4 mM NaCl, 2 mM MgCl2, 1.1 mM EGTA, 5 mM HEPES, 2 mM Na2ATP, 5 mM sodium creatine phosphate, and 0.6 mM Na3GTP (pH 7.3 with KOH). The liquid junction potential was ~12 mV. When we measured the synaptic inhibitory or excitatory release, the internal solution contained: 130 mM CsCl; 4 mM NaCl; 2 mM MgCl2; 1.1 mM EGTA; 5 mM HEPES; 2 mM Na2ATP; 5 mM sodium creatine phosphate; 0.6 mM Na3GTP; and 0.1 mM spermine. The liquid junction potential was −3 mV.
Whole-cell voltage clamp recordings were achieved to measure GABAB-GIRK currents in presence of bicuculline (10 μM), NBQX (20 μM) and AP5 (50 μM). For agonist-induced currents, changes in holding currents in response to bath application of baclofen were measured (at −50 mV every 5-10 s). The plotted values correspond to the difference between the baseline and the plateau (for the baclofen and ML297 experiments) or the difference between the plateau and the value of holding current after barium (for the I-GTP-γS). GABAB-GIRK currents were confirmed by antagonism with 10 μM of CGP54626. When stated, 100 μM of GTP-γS was added to the internal solution in place of Na3GTP. Plateau currents were then reversed by 1 mM Barium application, a selective inhibitor of K+ channels. Changes in holding currents in response to GIRK agonist were measured (at −50 mV every 5-10 s) by bath application of ML-297 (50 µM), a Selective GIRK1/2 channel activator then reversed by 1 mM Barium application. Synaptic GABAB slow IPSCs were optically evoked by trains of 10 pulses delivered at 20 Hz through a 470 nm LED. The fast GABA amplitude correspond to the amplitude of the first pic of the train, the slow GABA current instead were measured after picrotoxin bath application, and correspond to the I-max. Miniature excitatory postsynaptic currents (mEPSCs) were recorded in voltage-clamp mode at −60 mV in presence of bicuculline (10 μM), AP5 (50 μM) and tetrodotoxin (TTX, 1 μM). Miniature inhibitory postsynaptic currents (mIPSCs) were recorded (–60 mV) in presence of NBQX (20 μM) AP5 (50 μM) and TTX (1 μM). EPSCs were evoked through an ACSF-filled monopolar glass electrode placed in the LHb. For the experiments in which high-frequency stimulation trains were used to determine presynaptic release probability (5 pulses at 20 Hz), QX314 (5 mM) was included in the internal solution to prevent the generation of sodium spikes.
Current-clamp experiments were performed using a series of current steps (from −80 pA to 100 pA or when the cell reached a depolarization block) injected to induce action potentials (10-pA injection current per step, duration of 500 ms). Cells were maintained at -55mV throughout the experiment. When testing changes in tonic firing, cells were depolarized to obtain stable firing activity in current clamp mode.

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Analysis and drugs.

All drugs were obtained from Abcam (Cambridge, UK) and Hello Bio, and Tocris (Bristol, UK) and dissolved in water, except for TTX (citric acid 1%), ML297 and CNO (DMSO). Online/offline analysis were performed using IGOR-6 (Wavemetrics, US) and Prism (Graphpad, US). Data analysis for in vivo electrophysiology was performed off-line using Spike2 (CED, UK) software. Sample size required for the experiment was empirically tested by running pilots experiments in the laboratory. While behavioral experiments were run in a single-to-triple trial, electrophysiological experiments were replicated at least 5 times. Experiments were replicated in the laboratory at least twice. Animals were randomly assigned to experimental groups. Data distribution was assumed to be normal, and single data points are always plotted. Compiled data are expressed as mean ± s.e.m. All groups were tested with Grubbs exclusion test (limit set at 0.05) to determine outliers. Significance was set at p < 0.05 using Student’s t-test two-sided, Kolmogorov-smirnov test, one-way, two-way or three-way Anova with multiple comparison when applicable.

Context and objectives of the study I: GABAB receptors trigger

LHb hyperactivity, an early marker of depressive like symptoms

Major depressive disorder is one of the leading causes of disability worldwide, that afflict nowadays more than 300 million of people. Yet, more than 30% of the patients do not respond adequately to the currently available treatment. One explanation remains the poor understanding of the etiology of the disease. Depression is characterized by alteration of motivational behavior such as anhedonia and deficit in coping with aversive stimuli. In this context, LHb have gained a particular attention. Indeed the literature presented so far, highlight a pivotal role of the LHb in regulating monoaminergic activity and motivated behavior. Particularly, LHb neurons encode aversive stimuli by phasically increasing their firing rate and activation of LHb terminals onto midbrain neurons induces avoidance behavior in mice. Moreover, previous studies provide evidence that LHb hyperactivity could underlie certain symptoms of depression. However, insights in the cellular and molecular mechanism underlying this hyperexcitability and their causal link with the emergence of depressive like symptoms remain poor. In this study, we aimed to understand early cellular modifications occurring in the LHb after a stressful experience, one of the main triggers to engage depressive behaviors in animals and humans. Neuronal activity is tightly control by the balance of excitatory and inhibitory transmission. We focused in the GABABR signaling which (1) play an important role in the dampening of the neuronal activity in other brain areas and (2) present modification of expression in neuropsychiatric disorder such as addiction and depression both in humans and animal model of depression rodents. Yet, there is no functional evidence of a role of GABABRs in controlling LHb neurons functions and its involvement in encoding negative state. Here we reported that, aversive experience, such as foot-shock exposure, induces LHb neuronal hyperactivity and depressive-like symptoms. This occurs along with an increase in PP2A activity together with a GABABR-GIRK internalization leading to rapid and persistent weakening of GABABR-GIRK currents. The inhibition of protein phosphatase-2A (PP2A), a regulator of GABABR-GIRK surface expression, restores stress-induced GABABR-GIRK reduction and neuronal hyperxcitability. Furthermore, PP2A inhibition ameliorates depressive symptoms after FS and in a LHp of depression. These data establish causality between GABABR-GIRK plasticity LHb yperexcitability and the emergence of depressive like symptoms opening a new window toward new target in the treatment of mood disorder.
The lateral habenula (LHb) encodes aversive signals and its aberrant activity contributes to depressive-like symptoms. However, a limited understanding of the cellular mechanisms underlying LHb hyperactivity has precluded the development of pharmacological strategies to ameliorate depressive phenotypes. Here, we report that aversive experience in mice, such as foot-shock exposure (FsE), induces LHb neuronal hyperactivity and depressive-like symptoms. This occurs along with increased protein-phosphatase-2A (PP2A) activity, a known regulator of GABAB receptor (GABABR) and G-protein-gated inwardly rectifying potassium (GIRK) channel surface expression. Accordingly, FsE triggers GABAB1 and GIRK2 internalization leading to rapid and persistent weakening of GABAB-activated GIRK-mediated (GABAB-GIRK) currents. Pharmacological inhibition of PP2A restores both GABAB-GIRK function and neuronal excitability. As a consequence, PP2A inhibition ameliorates depressive- like symptoms after FsE and in a learned helplessness model of depression. Thus, GABAB- GIRK plasticity in the LHb represents a cellular substrate for aversive experience. Furthermore, its reversal by PP2A inhibition may provide a novel therapeutic approach to alleviate depressive symptoms in disorders characterized by LHb hyperactivity.
Unpredictable aversive stimuli trigger rapid avoidance responses and, if persistent, contribute to the emergence of depressive-like symptoms in both animals and humans1. The lateral habenula (LHb) bridges forebrain and midbrain nuclei and encodes aversive stimuli2. Analysis of fMRI data in depressed humans3 and metabolic activity in rodent models of depression4 (e.g. learned helplessness5) suggests that LHb hyperactivity may contribute to depressive-like symptoms6,7-9. However, the early cellular adaptations and precise molecular targets responsible for LHb hyperexcitability remain elusive.
Modifications in GABAB receptor (GABAB-R) expression and polymorphisms of the GIRK genes contribute to depressive symptoms in humans and rodents. This provides substantial evidence for the involvement of the GABAB-mediated G-protein-gated inwardly rectifying potassium channels (GABAB-GIRK) signaling in the etiology of mood disorders10,11.
Furthermore, pharmacological and genetic-based observations support the idea that adaptations in GABAB-GIRK signaling may represent a viable target to ameliorate depressive-like symptoms12,13,14. Although these findings suggest that GABAB-GIRK function plays a role in mood disorders, they fail to provide a precise anatomical substrate in which modifications of GABAB-GIRK occur, and how they may ultimately contribute to depressive symptoms.
Here, we provide evidence that GABAB-GIRK signaling in the LHb is involved in the expression of depressive-like symptoms in mice. Unpredictable foot-shock exposure (FsE) triggered a reduction in GABAB-GIRK signaling, increased PP2A activity (a known regulator of membrane GABAB -GIRK complexes15) and neuronal hyperexcitability in the LHb, promoting depressive-like behaviors. We found that local GIRK overexpression or pharmacological inhibition of PP2A rescue these FsE-driven cellular modifications. As a consequence, these interventions ameliorated behavioral phenotypes including despair, anhedonia and learned helplessness, which model distinct aspects of depression5,16,17. These data establish causality between GABAB-GIRK plasticity and LHb hyperexcitability, offering a viable rescue strategy to reverse cellular adaptations and behavioral traits of depression.

Depressive-like symptoms and cellular modifications in the LHb

Although LHb neuronal firing contributes to depressive-like states18, whether cellular modifications occur in the LHb after an aversive experience that ultimately contributes to depressive symptoms remains unknown. In humans uncontrollable and unpredicted stressful events lead to negative emotional feelings and behaviour similar to those found in depression19. The inescapable shock represents a paradigm that recapitulates symptoms of depression in a variety of animals20. Here, we examined the behavioral consequences of aversive experience by exposing C57/BL6J mice to inescapable and unpredictable foot-shocks21. Control mice were exposed to the same behavioral chamber in the absence of any foot-shocks. Re-exposure of mice to the shock -associated context 24 hours after the protocol led to a high level of freezing behavior (Fig. 1a), typical of fear memory22, without modifying the animals’ locomotor activity (Supplementary Fig. 1a). Seven days after FsE, animal behavior analyzed in the forced swim test paradigm (FST)23 revealed a reduced latency to first immobility and increased total immobility, indicative of a depressive-like state (Fig. 1b). As hyperexcitability in the LHb contributes to depressive-like phenotypes in rodents7,24,25, we hypothesized that the establishment of FsE-driven depressive-like traits relies, at least in part, on modifications in LHb function. In acute brain slices prepared 1 hour after FsE, we found that the spontaneous firing of LHb neurons recorded in cell-attached configuration was higher in slices from FsE mice compared to control ( Supplementary Fig. 1b). Furthermore, in whole-cell mode, LHb neurons in the FsE group exhibited a higher number of action potentials evoked by current injections compared to control (Figs. 1c and d) . Together, these data suggest that FsE produces behavioral responses reminiscent of depressive-like states as well as LHb neuronal hyperexcitability.

GABAB-GIRK signaling in the LHb

GABAB-Rs exert control over neuronal excitability via the activation of hyperpolarizing actions of GIRK channels26. To address whether GABAB-Rs and/or GIRKs represent a cellular substrate in the LHb underlying neuronal hyperexcitability and the emergence of depressive -like behaviors, we first assessed GABAB-GIRK signaling in the LHb of naïve mice. A saturating dose of the GABAB-R agonist baclofen (100 µM) evoked an outward current (I-Baclofen) reversed by the GABAB-R antagonist CGP54626 (10 µM; Supplementary Figs. 2a and b). I -Baclofen was dose-dependent, occurred along with a decrease in input resistance, and was reduced in presence of barium (Ba 2+, 1 mM, Supplementary Figs. 2a–e), consistent with the activation of GIRK channels. In support of this conclusion, reverse-transcription PCR in LHb revealed the expression of GIRK1-2-3-4 subunits (Supplementary Fig. 2f) 26. Furthermore, GABAB-GIRK signaling controls baseline LHb neuronal activity as bath application of the antagonist CGP54626 led to inward currents and increased firing (Supplementary Figs. 2g and h). These results indicate that GABAB-GIRK signaling provide a relevant inhibitory control over LHb neuronal activity.

FsE-driven plasticity of GABAB-GIRK in the LHb

To test the role of GABAB-GIRK signaling in FsE-induced LHb hyperexcitability, we assessed I-Baclofen 1 hour after FsE. I-Baclofen in slices from FsE mice was reduced compared to controls throughout the LHb (Fig. 1e; Supplementary Fig. 3a). The FsE-evoked reduction in I-Baclofen persisted up to 14 days and returned to control values by 30 days after the FsE (Fig. 1e). I -Baclofen in the presence of Ba2+ was comparable between groups ( Supplementary Fig. 3b), indicating that solely the GABAB-GIRK component is diminished after aversive experience. To also probe GIRK channel function we constitutively activated GIRKs via G / – dependent mechanisms through the infusion of GTP-γS27. Intracellular dialysis of GTP-γS (100 µM) led to an outward current sensitive to extracellular Ba2+, indicative of GIRK channel activation (Fig. 1f). LHb neurons from slices of FsE mice showed reduced GTP-γS-induced currents (Fig. 1f), suggesting that FsE weakens GABAB-GIRK signaling in the LHb.
FsE pairs a painful stimulus with a negative experience. We therefore tested whether aversive conditions independent of painful stimuli also modify GABAB-GIRK signaling in LHb28,29. The predator odor stress paradigm involves an aversive natural stimulus, as opposed to foot-shock. Similarly to FsE mice, I-Baclofen was depressed in animals exposed to the predator odor ( Supplementary Fig. 3c). Furthermore, 1 hour of restraint stress, classically employed to cause depressive-like states in rodents, also reduced I-Baclofen in LHb neurons (Supplementary Fig. 3d) 28,29.
To test whether FsE also modifies fast synaptic neurotransmission, we recorded miniature excitatory-inhibitory postsynaptic currents (mEPSCs- mIPSCs) after the FsE ( Supplementary Figs. 4a and b). Quantal excitatory and inhibitory synaptic transmission remained unaffected in frequency and amplitude (Supplementary Figs. 4a and b). No modifications were found of excitatory synaptic strength or AMPA-R subunit composition, as AMPA-NMDA ratios and rectification indices remained unaffected (Supplementary Fig. 4c). We next evoked EPSCs and IPSCs by high-frequency extracellular stimulation30, which yields responses comparable between control and FsE mice (Supplementary Fig. 4d). This indicates that fast synaptic transmission onto LHb neurons, recorded 1 hour after FsE remained unaffected.

Table of contents :

I. A brief history of depression
II. Treatment of major depressive disorder
III. Studying depression in laboratory animals
A. Learned helplessness model
B. The chronic mild stress
C. Social defeat
D. Maternal separation
Neural circuits of depression
Emerging role of LHb in depression
I. Lateral habenula as a node to control midbrain regions
II. Lateral habenula function in processing negative information
III. Lateral habenula modifications in depression
I. GABAB receptor structure and function
II. GABA B receptors trafficking
III. GABAB receptor regulation
Methodological section
Experimental subjects and stress paradigms
Behavioral tests
Drug/intervention
Surgery
Electrophysiology
Analysis and drugs.
I. Stress-driven mechanisms underlying GABAB receptor plasticity in the LHb
II. Induction mechanism of GABAB receptor plasticity following different kind of stress
III. Circuit specificity of GABAB receptor regulation in the LHb
IV. What is the functional relevance of GABAB receptor dependent hyperexcitability for the emergence of depressive like symptoms
V. Targeting LHb hyperexcitability to treat depression?
A. Caveat of DBS intervention:
B. PP2A inhibitors as potential antidepressant drugs
C. Consequences of LHb hyperexcitability on downstream circuitries
Conclusion

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