This animal study (n=500) investigated the neural circuitry underlying the antidepressant efficacy of ketamine (10 -; 25mg/kg) in rodents and found that it blocks the activity of the lateral habenula, a network that normally inhibits reward processing, whose inhibition is in turn unblocked via ketamine.
Introduction
The N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine has attracted enormous interest in mental health research owing to its rapid antidepressant actions, but its mechanism of action has remained elusive.Methods/Results: Here we show that blockade of NMDAR-dependent bursting activity in the ‘anti-reward center’, the lateral habenula (LHb), mediates the rapid antidepressant actions of ketamine in rat and mouse models of depression. LHb neurons show a significant increase in burst activity and theta-band synchronization in depressive-like animals, which is reversed by ketamine. Burst-evoking photostimulation of LHb drives behavioural despair and anhedonia. Pharmacology and modelling experiments reveal that LHb bursting requires both NMDARs and low-voltage-sensitive T-type calcium channels (T-VSCCs). Furthermore, local blockade of NMDAR or T-VSCCs in the LHb is sufficient to induce rapid antidepressant effects.
Discussion
Our results suggest a simple model whereby ketamine quickly elevates mood by blocking NMDAR-dependent bursting activity of LHb neurons to disinhibit downstream monoaminergic reward centres, and provide a framework for developing new rapid-acting antidepressants.
Yang and colleagues used complementary rodent models, neurophysiology, pharmacology, optogenetics and biophysical modelling to test whether lateral habenula (LHb) burst firing underlies depression-like behaviour and the rapid antidepressant actions of ketamine. Male congenitally learned helpless (cLH) rats and C57BL/6 mice subjected to chronic restraint stress (CRS) served as depression-like models. Animals were group- or pair-housed under a 12-h light–dark cycle with ad libitum food and water; all procedures were approved by the institutional animal care committee. Behavioural assays included the forced swim test (FST), sucrose preference test (SPT), open field test (OFT) and a real-time place aversion (RTPA) paradigm. Systemic drug dosing was intraperitoneal (ketamine 25 mg/kg for rats, 10 mg/kg for mice; ethosuximide 200 mg/kg in mice), and local LHb infusions were performed through bilateral guide cannulae (1 μl per side) delivering ketamine (25 μg), AP5, NBQX or mibefradil at specified concentrations. Timing of behavioural testing after infusion varied by drug (for example, 1 h after ketamine or mibefradil, 0.5 h after AP5 or NBQX). LC-MS/MS was used to measure local ketamine concentrations in habenular tissue following systemic or local administration. For electrophysiology, the investigators prepared coronal or sagittal LHb brain slices (300–350 μm) for whole-cell current- and voltage-clamp recordings. Spontaneous activity was recorded at I = 0 pA to classify neurons as silent, tonic-firing or burst-firing; pharmacological manipulations in slices tested ketamine, AP5, NBQX, mibefradil, ZD7288, TTX, NMDA, AMPA and fluoxetine. In vivo multi-tetrode recordings and LFPs were obtained from freely behaving mice using an eight-tetrode microdrive implanted in the LHb; spike sorting and burst detection criteria were defined from slice data (bursts begin with maximal inter-spike interval 20 ms and end with maximal inter-spike interval 100 ms). Optogenetic manipulations used AAV vectors to express eNpHR3.0 (for rebound bursts) or oChIEF (for high-frequency stimulation) targeted to LHb neurons; optetrode recordings confirmed stimulation effects in vivo. Finally, the team constructed a reduced single-compartment biophysical model to explore interactions between NMDARs, T-type voltage-sensitive calcium channels (T-VSCCs) and membrane potential in generating LHb bursts.
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Local blockade of LHb NMDARs produced rapid antidepressant-like effects. Bilateral infusion of ketamine into the LHb of cLH rats (25 μg per side, 1 μl) reduced immobility in the FST and increased sucrose preference within 1 h; effects were dose-dependent. LC-MS/MS showed that this local infusion produced a habenular ketamine concentration of 5.2 ± 1.3 μM, comparable in order of magnitude to levels after systemic ketamine (11.0 ± 1.7 μM after 25 mg/kg). Local infusion of the NMDAR antagonist AP5 into LHb similarly reduced immobility and increased sucrose preference, whereas local AMPAR blockade with NBQX did not produce significant antidepressant effects. Neither ketamine nor AP5 altered general locomotion in the OFT. Electrophysiological recordings revealed increased LHb bursting in depression models. In LHb slices from cLH rats the proportion of bursting neurons rose from 7% in controls to 23% in cLH animals (P = 0.003), and the percentage of spikes occurring within bursts increased from 7% to 43%. Inter-spike interval (ISI) histograms showed an extra peak around 14 ms (~71 Hz) in cLH rats. CRS mice displayed analogous changes (increased percentage of bursting cells, increased spikes in bursting mode and an extra ISI peak near 20 ms). In vivo multi-tetrode recordings from LHb of CRS mice showed elevated burst activity and a higher mean firing rate relative to controls. Systemic ketamine (10 mg/kg, i.p.) given 1 h before recording markedly suppressed LHb bursting (P < 0.0001), reduced overall firing rate, and shifted ISI distributions toward control levels. Network synchrony in LHb was altered in CRS mice and normalised by ketamine. Spike-triggered averages of local field potentials revealed a dominant ~7 Hz theta-band peak and increased spike-field coherence in CRS mice (SFC in theta band, P = 0.022), both of which were reversed to control levels 1 h after systemic ketamine. Mechanistically, LHb bursting required NMDAR activation and T-VSCC function. Bath application of ketamine to slices (100 μM) eliminated spontaneous bursts within seconds without changing resting membrane potential or miniature EPSCs; lower concentrations (10 μM, 1 μM) also blocked bursts with longer latency. AP5 (100 μM) likewise abolished bursts, while full AMPAR blockade with NBQX (10 μM) only moderately reduced burst probability. NMDA perfusion in Mg2+-free ACSF induced bursting in previously silent neurons, an effect blocked by ketamine. Ramp hyperpolarisation protocols converted the majority of LHb neurons to high-frequency bursting (evoked in 90% of rat and 93% of mouse neurons), with intra-burst frequency positively correlated with hyperpolarisation; spontaneous bursting peaked around -56 to -60 mV. T-VSCC currents were detected in LHb neurons, and mibefradil (10 μM) reduced burst probability and plateau potential amplitude without altering RMPs; ZD7288 produced a smaller effect. A minimal biophysical model reproduced voltage dependence and knockout-like effects of blocking NMDARs or T-VSCCs. Pharmacological blockade of T-VSCCs had rapid antidepressant-like behavioural effects. Systemic ethosuximide in CRS mice and bilateral infusion of mibefradil into LHb of cLH rats both produced rapid reductions in FST immobility and increases in sucrose preference without changing total distance in the OFT. Finally, optogenetic experiments showed that driving LHb rebound bursts induced aversion and depression-like behaviours: 1 Hz, 100 ms yellow-light activation of eNpHR3.0 to evoke rebound bursts produced real-time place aversion, increased immobility in the FST (P = 0.0049) and decreased sucrose preference, whereas a stimulation protocol that increased overall spike rate without producing burst patterns (5 Hz oChIEF stimulation) did not induce depression-like phenotypes. Importantly, ketamine reversed the behavioural effects driven by optogenetically evoked rebound bursts.
Yang and colleagues interpret their findings as support for a model in which a depression-like state depends on NMDAR- and T-VSCC-dependent bursting of LHb neurons, and ketamine’s rapid antidepressant action arises largely from blockade of this bursting. They note that burst firing can produce stronger downstream inhibition—via the rostromedial tegmental nucleus or local inhibitory circuits in VTA and DRN—thereby suppressing dopaminergic and serotonergic reward circuits; blocking LHb bursts would therefore release this inhibitory brake and rapidly elevate activity in monoaminergic centres. The investigators contrast the rapid electrophysiological effects they observed with the unresolved mechanisms underlying ketamine’s longer-lasting actions, which they acknowledge may involve BDNF upregulation or synaptogenesis. They report that T-VSCC currents were not increased in depression models, but that resting membrane potentials of LHb neurons were hyperpolarized by approximately 5–6 mV in depressed animals; given that hyperpolarisation facilitates de-inactivation of T-VSCCs and entry into burst mode, the authors suggest that mechanisms which lower LHb RMPs may drive pathological bursting. They further indicate that an accompanying paper implicates increased astrocytic Kir4.1 and enhanced potassium buffering as a candidate for this RMP shift. Key limitations and uncertainties are acknowledged: the work explains how ketamine can act rapidly but does not resolve the basis for its sustained antidepressant effects. The authors also emphasise that LHb burst generation reflects intrinsic properties modulated by synaptic inputs, implying multiple points at which future interventions might act. Overall, they propose LHb bursting as a mechanistic framework for rapid antidepressant action and a potential target for novel fast-acting therapies.
Animals. Male cLH rats (8-16 weeks of age) and age-matched male Sprague Dawley rats (SLAC Laboratory Animal Co.) were used. The cLH rats were screened using the learned helpless testfor breeding as previously described. Male adult (8-16 weeks of age) C57BL/6 mice (SLAC) were used for establishing the chronic restraint stress (CRS) depression model. Rats were group-housed two per cage and mice were housed four per cage under a 12-h light-dark cycle (light on from 7 a.m. to 7 p.m.) with free access to food and water ad libitum. All animal studies and experimental procedures were approved by the Animal Care and Use Committee of the animal facility at Zhejiang University. Systemic drug delivery for antidepressant. All drugs were dissolved in 0.9% saline, and administered intraperitoneally (i.p.). Concentrations were as follows: ketamine (Gutian Pharma Co.) 25 mg/kg for ratsand 10 mg/kg for mice; ethosuximide (2-ethyl-2-methylsuccinimide: a T-VSCC blocker, which can cross the blood-brain barrier, Selleck) 200 mg/kg for mice. One hour after drug delivery, animals were used for behavioural or in vivo electrophysiology studies, or killed for in vitro electrophysiology studies. Cannula infusion experiment. A 26-gauge double guide cannulae (centre-to-centre distance 1.4 mm, Plastics One) was placed at a 2° angle to the coronal plane (without the 2° rotation, we found it difficult to hit both sides of the LHb) and inserted bilaterally into the LHb (AP, -3.7 mm from bregma; ML, ± 0.7 mm; DV, -4.05 mm from the brain surface) of cLH rats. A 33-gauge double dummy cannulae (Plastic One), secured with a dust cap, was inserted into the guide cannula to prevent clogging during the recovery period. After rats had recovered for at least 7 days, drugs were microinjected with a 33-gauge double injector cannulae, which has a 0.6-mm extension beyond the tip of the guide cannula, while cLH rats were anaesthetized with isoflurane on an anaesthetic machine. The extensions were manually sharpened before insertion. Ketamine (25 μ g/μ l, IC 50 = 5.35 μ M 38 ), AP5 (40 nmol/μ l, IC 50 = 30 μ M 39 ), NBQX (1 or 5 nmol/μ l, IC 50 = 0.15 μ M 40 ) or mibefradil (10 mol/μ l, IC 50 = 2.7 μ M 41 ) were dissolved in 0.9% saline. Before the local drug infusion, tip-sharpened 33-gauge double injector cannulae were inserted into the guide cannulae to ensure clear passage and then pulled out. One microlitre of drug was infused (0.1 μ l/min) into each side through another set of tip-sharpened 33-gauge double injector cannulae, which were connected to the microsyringe. The injector cannulae were left in place for an additional 10 min to minimize spread of the drug along the injection track. The FST or SPT was performed 1 h after the injection of ketamine or mibefradil and 0.5 h after the injection of AP5 or NBQX. To verify the drug infusion sites, rats were injected with 1 μ l CTB-488 to each side of the LHb after all behavioural tests. For immunostaining, rats were then euthanized 30 min after CTB injection and processed as described. Brain slices were counterstained with Hoechst before mounting on the slides. Fluorescent image acquisition was performed with an Olympus VS120 virtual microscopy slide scanning system. Only data from rats with correctly sited injections were used. LC-MS/MS measurement of ketamine concentration. Male cLH rats were anaesthetized by isoflurane and subsequently decapitated 1 h after drug administration (i.p. or LHb cannulae infusion). Habenular or ventral hippocampal tissues (about 4 mg) were immediately dissected from the brain with Brain Matrix (RWD). The tissues were rinsed with PBS and immediately frozen in liquid nitrogen and stored at -80 °C until analysis. After samples were thawed, 100 μ l solution (ddH2O: acetonitrile = 9:1, v/v) and steel balls were added to homogenize samples for 2 min at 60 Hz with a tissue grinder. Ten microlitres of fluvoxamine solution (0.5 μ g/ml) was then added for vortex mixing, followed by the addition of 100 μ l supersaturated sodium carbonate. After vortex mixing for 10 s, the samples were extracted by liquid-liquid extraction with 800 μ l methyl tert-butyl ether under continuous shaking for 2 min at room temperature. After centrifugation for 10 min at 13,000 r.p.m. at 4 °C, 640 μ l of the upper layer was removed into a centrifugal concentrator (CentriVap, LABCONCO) to spin at 1,725 r.p.m. and 30 °C for 45 min, then vortexed with 40 μ l solution (ddH 2 O: acetonitrile = 1:1, v/v). After centrifugation for 5 min at 13,000 r.p.m. and 4 °C, the upper layer was injected into the chromatographic system. The concentrations of (R,S)-ketamine in habenular and hippocampal tissue were determined by achiral LC-MS/MS following a previously described methodwith slight modifications. The analysis was accomplished using an Agilent Extend-C18 column (3.0 mm × 100 mm ID, 1.8 μ m; Varian). The mobile phase consisted of 0.1% methanoic acid buffer (pH 2.67) as component A and acetonitrile as component B at a flow rate of 0.3 ml/min, temporized at 10 °C (injection volume: 7 μ l). A linear gradient was run as follows: 0-0.2 min, 10% B; 0.2-1.5 min, from 10% B increased to 90% B; 1.5-3.0 min, 90% B; 3.0-3.1 min, from 90% B decreased to 10% B; 3.1-4.0 min, 10% B. Ten microlitres of (R,S)-ketamine of different concentrations (0.1, 1, 5, 10, 50, 100, 200 ng/ml) was mixed with 90 μ l brain tissue to establish the standard calibration curve. The quantification of (R,S)-ketamine was accomplished by calculating area ratios using fluvoxamine (10 μ l of 0.5 μ g/ml solution loaded) as the internal standard. The MS/MS analysis was performed using a triple quadruple mass spectrometer model AB SCIEX 4000 plus (AB Company). The data were acquired and analysed using MutiQuant 3.0.2. Positive electrospray ionization data were acquired using multiple reaction monitoring (MRM) using the following transitions for (R,S)-ketamine studies: 238 → 125. Viral vectors. AAV2/9-CaMKIIa-eNpHR3.0-eYFP (titre: 7.45 × 10 12 vector genome (v.g.)/ml, dilution: 1:5, 0.1 μ l unilateral into LHb, Taitool Bioscience), AAV2/9-Ubi-eGFP (titre: 2.5 × 10 13 v.g./ml, 1:30, 0.1 μ l unilateral into LHb, University of Massachusetts, Guangping Gao Laboratory), and AAV2/9-hSyn-oChIEF-tdTomato (titre: 6.29 × 10 12 v.g./ml, 1:5, 0.1 μ l unilateral into LHb, Obio Technology) were aliquoted and stored at -80 °C until use. Behavioural assays. All behavioural assays were performed on animals 12-16 weeks old. Most behaviours were performed during the light phase except for the sucrose preference test, which was performed during the dark phase to maximize the consumption of solution, and the open field test for CRS mice. Behavioural analysis was performed blinded to experimental conditions. Chronic restraint stress (CRS). Mice were subjected to chronic-restraint stress by placement in 50-ml conical tubes with holes for air flow for 2-3 h per day for 14 consecutive days. Open field test (OFT). Animals were placed in the centre of an arena (40 cm × 40 cm × 40.5 cm for mice and 100 cm × 100 cm × 50 cm for rats) in a room with dim light for 10 min. A video camera positioned directly above the arena was used to track the movement of each animal (Any-maze, Stoelting). For optogenetic manipulations, mice were allowed to move freely throughout the arena for 9 min, with laser stimulation occurring during the middle 3-min epoch. Forced swim test (FST). Animals were individually placed in a cylinder (12 cm diameter, 25 cm height for mice; 20 cm diameter, 50 cm height for rats) of water (23-25 °C) and swam for 6 min under normal light. Water depth was set to prevent animals from touching the bottom with their tails or hind limbs. Animal behaviours were videotaped from the side. The immobile time during the last 4-min test was counted offline by an observer blinded to animal treatment. Immobile time was defined as time when animals remained floating or motionless with only movements necessary for keeping balance in the water. For rats, an additional pre-test was conducted 24 h before the test, during which rats were individually placed in a cylinder of water with conditions described above for 15 min. For optogenetic manipulations, laser stimulation was delivered immediately after mice were placed in the water and lasted for 6 min. In order to minimize the impact of the optogenetic cable on swimming behaviour, the cable length was adjusted to allow the cable to just touch the water surface. Sucrose preference test (SPT). Animals were single housed and habituated with two bottles of water for 2 days, followed by two bottles of 2% sucrose for 2 days. Animals were then water deprived for 24 h and then exposed to one bottle of 2% sucrose and one bottle of water for 2 h in the dark phase. Bottle positions were switched after 1 h (for 2 h test). Total consumption of each fluid was measured and sucrose preference was defined as the average sucrose consumption ratio during the first and second hours. Sucrose consumption ratio was calculated by dividing the total consumption of sucrose by the total consumption of both water and sucrose. For optogenetic manipulations, mice were gently placed in a white arena containing normal bedding and allowed to move freely in the arena. During the 90-min test, light was delivered during 30-60 min. Sucrose preference scores were measured for every 30 min. Only animals that had a baseline sucrose preference greater than 30% during the first 30-min session proceeded to the next session. Otherwise they were tested later on a different day.
We have provided multiple lines of evidence to support the idea that a depression-like state depends critically on a bursting mode of firing in the LHb, and that the rapid antidepressant effects of ketamine are induced largely by targeting this burst firing. Burst firing can reduce synaptic transmission failure, enhance the signal-to-noise ratio, facilitate synaptic plasticity or promote neuropeptide release. Thus, enhanced bursting in the LHb may provide a stronger output than tonic firing onto the downstream inhibitory RMTg or onto inhibitory interneurons within the VTA or DRN, to suppress the activity of dopaminergic, serotinergic or glutamatergic neurons in the reward centres of the brain. By blocking NMDAR-dependent LHb bursts, ketamine can then release this inhibition brake onto the reward centres to elicit its rapid antidepressant effects. Although our data addresses how ketamine can act so rapidly, the mechanism for the long-term effects of ketamine remains unclear; it might involve upregulation of brain-derived neurotrophic factor (BDNF)or synaptogenesis. Given the importance of LHb bursting activity for the depressive state, it is plausible that entry into this mode should be carefully controlled. We found no significant changes in T-VSCC currents in the LHb of animal models of depression (Extended Data Fig.). Instead, the RMPs of LHb neurons in these models were more Article reSeArcH hyperpolarized (by 5-6 mV) than those of control animals (Extended Data Fig.). Considering that hyperpolarization can convert silent or tonic-firing neurons to bursting mode (Extended Data Fig.), we hypothesize that a molecular mechanism that lowers the RMPs of LHb neurons to the level at which T-VSCCs can be de-inactivated may account for the increase of LHb bursting in depression-like states. In the accompanying paper, we show that increased levels of astrocytic Kir4.1 and potassium buffering may be responsible for the hyperpolarization of RMPs and for the increased bursting of LHb neurons in depression.
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