Ketamine blocks bursting in the lateral habenula to rapidly relieve depression

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.

Abstract

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.”

Authors: Yan Yang, Yihui Cui, Kangning Sang, Yiyan Dong, Zheyi Ni, Shuangshuang Ma & Hailan Hu

Summary

The NMDAR antagonist ketamine elicits fast and sustained antidepressant effects in humans and animal models of depression. It also has a fast metabolic turnover rate.

The LHb is implicated in the coding of negative emotion and the pathophysiology of major depression. It hosts primarily glutamatergic neurons, but inhibits the brain’s reward centres through a relay at the RMTg.

Blockade of LHb NMDARs is antidepressant

Congenitally learned helpless rats showed an antidepressant response to systemic injection of ketamine in the forced swim test. Local infusion of ketamine into the LHb of cLH rats quickly rescued the depression-like behaviours, including behavioural despair and anhedonia, one hour after infusion.

Local bilateral infusion of the specific NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) into the LHb reduced immobile time in the FST and increased sucrose preference in the SPT, similar to the effects of ketamine, but did not change general locomotion.

Increased LHb burst firing in depression

We performed whole-cell patch-clamp on neurons in LHb coronal slices and recorded spontaneous neuronal activity under current clamp at resting conditions. We found that LHb neurons were intrinsically active and fell into three categories: silent, tonic-firing and burst-firing.

Although basic membrane properties were indistinguishable between neurons from cLH and wild-type rats, the percentage of bursting neurons increased significantly from 7% in wild-type controls to 23% in cLH rats.

Using a second animal model of depression, we found that bursting is enhanced in LHb neurons from mice with chronic restraint stress. Ketamine reduced bursting back to control levels in both animals.

In vivo multi-tetrode recording in the LHb of freely behaving mice showed that neurons from CRS mice showed a notable increase in bursting activity but not tonic firing, compared with neurons from naive control mice. Ketamine significantly suppressed bursting activity and overall firing rate.

We tested whether LHb network synchronization was altered in CRS mice. The results showed that there emerged a dominant frequency of 7 Hz in the power spectra of spike-triggered averages of local field potentials, indicating that spikes tended to phase-lock with LFPs in the theta-band range.

LHb bursts require NMDARs

Ketamine completely blocked burst activity in LHb neurons, and a specific NMDAR antagonist, AP5, also stopped burst activity in LHb neurons, but with a longer delay. NBQX and fluoxetine did not affect burst activity at this rapid time scale.

LHb bursts depend on RMP and T-VSCCs

We found that changing the RMPs of LHb neurons could alter the pattern of spiking activity. The number of spikes in each burst was normally distributed from 80 mV to 40 mV and peaked at 90 mV.

Local blockade of NMDARs in LHb is sufficient to elicit rapid antidepressant effects, as demonstrated by the effects of bilateral implantation of cannulae in the LHb of cLH rats and the effects of intra-LHb infusion of ketamine, AP5, and NBQX.

Voltage-dependent transition of firing mode occurred in spontaneously bursting LHb neurons, and 100% of originally tonic-firing neurons were transformed to burst-firing mode by delivering a depolarizing current injection.

A pacemaker channel, T-VSCC, was detected in rat and mouse LHb neurons. The specific T-VSCC blocker mibefradil and an antagonist of another pacemaker channel, HCN, were effective in reducing bursts, but not RMPs.

Although AMPAR blockade reduced LHb burst probability only slightly, increasing AMPAR current or application of AMPA onto LHb neurons increased burst frequency.

The above results predict that drugs that block T-VSCCs may also have rapid antidepressant effects. Indeed, 2-ethyl-2-methylsuccinimide and mibefradil cause rapid antidepressant effects in both the FST and the SPT.

Ketamine suppresses enhanced LHb bursting activity and theta-band synchronization in animal models of depression. Ketamine reduces burst activity in the LHb by reducing the abundance of burst-firing neurons and increasing the percentage of burst- and tonic-type spikes in all spikes recorded. Neurons recorded from control and CRS mice had a higher rate of spikes in bursting mode and a lower rate of bursts per minute than neurons recorded from CRS mice after ketamine injection.

LHb bursts drive depression

We used optogenetic tools to drive burst activity in the LHb, but could not induce depression-like behaviours with this protocol.

We devised a protocol involving a transient (100-ms) hyperpolarization ramp current injection, which induced NMDAR- and T-VSCC-dependent rebound bursts in LHb brain slices with 100% success. We then used an inhibitory opsin, eNpHR3.0, to drive rebound bursts in the LHb. NMDA perfusion and ketamine affected bursts differently, with P 0.05, P 0.01, P 0.001 and NS, not significant, respectively.

Antagonists of T-VSCCs block LHb bursts and cause rapid antidepression. Systemic ethosuximide injection or local bilateral infusion of mibefradil into the LHb causes antidepression 1 h after treatment.

We tested whether rebound bursts in the LHb could acutely drive aversion and depression-like symptoms in freely behaving mice. The results indicate that it is the pattern of burst firing rather than a mere increase in spikes that is important for the induction of depression-like behaviours.

Discussion

We have provided multiple lines of evidence to support the idea that ketamine can act rapidly by targeting bursting in the LHb. This burst firing can reduce synaptic transmission failure, enhance the signal-to-noise ratio, facilitate synaptic plasticity or promote neuropeptide release.

The LHb bursting activity was not changed in animal models of depression, but the RMPs were.

eNpHR3.0-induced rebound bursting drives behavioural aversion and depression-like symptoms in LHb brain slices, which are reversible by ketamine. The aversion and depression-like symptoms are induced by pulsed yellow light stimulation, and the rebound bursts are reliably elicited by LHb neurons.

Hyperpolarization of RMPs can convert silent or tonic-firing neurons to bursting mode, and increased levels of astrocytic Kir4.1 and potassium buffering may be responsible for this process.

received 18 May 2017; accepted 9 January 2018.

Ketamine’s antidepressant effects are mediated by NMDA receptors, which are found in the prefrontal cortex and ventral tegmental area. Ketamine’s antidepressant effects are independent of dopaminergic neurons or the NMDA receptor. The basal ganglia circuit for evaluating action outcomes is activated by serotonin and excitatory, aversive, and suppressed by lateral habenula inputs. Morris, Smith, K. A., Cowen, P. J., Friston, K. J. & Dolan, R. J. reported that activity in the habenula and dorsal raphé nuclei were correlated following tryptophan depletion, and Shumake, J. and Edwards, E. reported that the habenula and ventral tegmental area showed opposite metabolic changes.

Tye, K. M., Hu, H., Fields, H. L., Baxter, M. G., Saper, C. B. & Holland, P. C. studied how dopamine neurons encode and express depression-related behaviour. Tian, J., Uchida, N., Zhou, L., Chang, S. Y., Kim, U., Weiss, T. & Veh, R. W., studied the habenula in rat brain slices and found that multiple mechanisms underlie dopamine prediction errors. T-type Ca2+ channels in normal and abnormal brain functions, astrocyte Kir4.1 in the lateral habenula drives neuronal bursts in depression, and bursts are a unit of neural information.

Supplementary Information is available in the online version of the paper.

We thank L.-S. Yu, J. Pan, S.-M. Duan, X.-H. Zhang, X.-W. Chen, Y.-S. Shu, X. Ju, C. Lohmann and W. Yang for their support and comments on the manuscript.

Author contributions include H.H., Y.C., Y.Y., S.M., K.S., Y.D., K.N. and Z.N. who designed the study, performed the experiments, and wrote the manuscript.

Author information is available at www.nature.com/reprints. The authors declare competing financial interests, which are available in the online version of the paper.

MethOdS

Male cLH rats and age-matched male Sprague Dawley rats were used for breeding and the chronic restraint stress (CRS) depression model was established using male adult C57BL/6 mice.

Systemic drug delivery for antidepressant was performed using ketamine and ethosuximide. One hour after drug delivery, animals were used for behavioural or in vivo electrophysiology studies.

A 26-gauge double guide cannulae was placed at a 2° angle to the coronal plane and inserted bilaterally into the LHb of cLH rats. A 33-gauge double injector cannulae was inserted after the rats had recovered for at least 7 days.

Ketamine, AP5, NBQX or mibefradil were dissolved in 0.9% saline and infused into the LHb of rats. Then, the injection sites were verified with CTB-488 and brain slices were counterstained with Hoechst before mounting on the slides.

Rats were anaesthetized by isoflurane, decapitated 1 h after drug administration, and their brain tissues were immediately dissected. The tissues were homogenized, dissolved in acetonitrile, and extracted with methyl tert-butyl ether.

(R,S)-ketamine concentrations in habenular and hippocampal tissue were determined by achiral LC – MS/MS following a previously described method6,42 with slight modifications. The analysis was performed using an Agilent Extend-C18 column and a triple quadruple mass spectrometer model AB SCIEX 4000 plus.

Viral vectors AAV2/9-CaMKIIa-eNpHR3.0-eYFP, AAV2/9-Ubi-eGFP, and AAV2/9-hSynoChIEF-tdTomato were aliquoted and stored at 80 °C until use.

All behavioural assays were performed on animals 12 – 16 weeks old. Behavioural analysis was performed blinded to experimental conditions.

Open field test (OFT) was performed with mice and rats in a room with dim light for 10 min. Laser stimulation occurred during the middle 3-min epoch.

Forced swim test was conducted with mice and rats in a cylinder of water (23 – 25 °C) and videotaped from the side. Optogenetic manipulations were performed immediately after mice were placed in the water and lasted for 6 min.

Mice were single housed, habituated with two bottles of water and two bottles of 2% sucrose, and then exposed to one bottle of 2% sucrose and one bottle of water for 2 h in the dark phase. Their sucrose preference was measured.

Mice were placed in a white open chamber consisting of two chambers, and a laser stimulation was delivered as soon as mice entered the stimulated side and terminated once mice crossed to the non-stimulated side.

Optogenetic light delivery and protocols were used to stimulate LHb neurons with a 589-nm yellow light laser at 1 Hz, 100 ms pulse, or a 473-nm blue light laser at 20 Hz, tonic 5-ms pulses, or 5 Hz, tonic 5-ms pulses.

Animals were anaesthetized with isoflurane and 10% chloral hydrate, then perfused with ice-cold ACSF containing 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 1 MgCl2 and 25 glucose, with 1 mM pyruvate added. Brain slices were sectioned and allowed to recover for 1 h before recording.

LHb neuron recordings were performed using whole-cell patch-clamp with pipettes filled with internal solution containing 105 K-gluconate, 30 KCl, 4 Mg-ATP, 0.3 Na-GTP, 0.3 EGTA, 10 HEPES and 10 Na-phosphocreatine, with pH set to 7.35.

LHb neurons show three modes of spontaneous activity at resting conditions: silent cells, tonic cells, and bursting cells. Burst-firing neurons show a bimodal distribution of inter-spike intervals, while tonic-firing neurons show a more homogenous Poisson distribution of ISIs.

Evoked NMDAR EPSCs and T-type VSCC currents were recorded in sagittal LHb slices by stimulating the input stria medullaris fibre in a modified extracellular ACSF solution48 with the AMPAR blocker NBQX, GABAR blocker picrotoxin and 0 Mg2+.

Ketamine, AP5, NBQX, mibefradil, ZD7288, TTX, picrotoxin, NMDA, AMPA and fluoxetine were used for electrophysiology. Baselines were recorded for at least 3 min before drugs were perfused.

Ketamine (100 M) was tested on mEPSCs under voltage clamp in the presence of TTX (1 M) and picrotoxin (100 M) in ACSF. Data were analysed by Mini Analysis Program (Synaptosoft) with an amplitude threshold of 5 pA.

For in vivo electrophysiology experiments, a custom-made microdrive array consisting of eight tetrodes was implanted into the LHb of control or CRS mice. Spontaneous spiking activity and LFP were recorded simultaneously for 30 min during the still period of the mice in their home cages.

Spike sorting was performed on all waveforms recorded from each tetrode. Single units were identified by threshold crossing and principal component analysis.

Data analysis was conducted by Neuroexplorer4 (Plexon Inc.) and MATLAB. In vivo bursts were defined as clusters of spikes beginning with a maximal inter-spike interval of 20 ms and ending with a maximal inter-spike interval of 100 ms.

We filtered the local field potentials using a fourth-order Butterworth filter, calculated spike-triggered averages (STAs), and quantified STAs using spike-field coherence (SFC). SFC in the theta band was calculated as the average SFC in the theta frequency domain.

Optetrode recording was applied to mice injected with AAV2/9-CaMKIIa-eNpHR3.0-eYFP into the LHb. 100 ms light pulses at 1 Hz were delivered to evoke rebound bursting.

Mice were deeply anaesthetized and placed in a stereotactic frame. Virus was injected into the LHb one side of the brain using a pulled glass capillary with a pressure microinjector, and a 200-m fibre-optic cannula was placed 300 m above the centre of viral injection site.

Statistical analysis was performed using GraphPad Prism software v6, and mice were randomly assigned to treatment groups. Data were analyzed using one-way ANOVA (followed by Bonferroni’s multiple comparisons test), t-test, linear regression test, Chi-square test, Fisher’s exact test or two-way ANOVA (followed by Bonferroni’s multiple comparisons test).

Ketamine elicits sustained antidepressant-like activity via a serotonin-dependent mechanism, memantine and ethosuximide exert differential effects on efficacy of competitive and non-competitive antagonists, and Ro 40-5967 blocks differently T-type and L-type Ca++ channels.

Hasan, M. et al. determined ketamine and its metabolites by LC-MS/MS in human serum, urine and fecal samples, and Kim, K. S. & Han, P. L. optimized chronic stress paradigms using anxiety- and depression-like behavioral parameters, and Powell, T. R. Molecular and cellular approaches for diversifying and extending optogenetics are described, as well as improved channelrhodopsin variants with improved properties and kinetics. The transient, low-threshold current in rat thalamocortical relay neurones controls habenula output and is modified by antidepressant treatment. mGluR-LTD regulates synaptic output in the lateral habenula, and calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-d-aspartate in subthalamic neurons in vitro. Rat nucleus reticularis thalami Ih current, guinea pig substantia nigra pars compacta NMDA receptors, serotonin and fluoxetine regulated excitability of prefrontal cortical fast-spiking interneurons and pyramidal neurons, large-scale neural ensemble recording in the brains of freely behaving mice, and human memory strength.

Extended Data Figure 1 shows the antidepressant response to systemic ketamine injection in FST 1 h after treatment in cLH rats, as well as the OFT results for CRS mice 1 h after intraperitoneal injection of saline or ethosuximide.

Extended Data Figure 2 | I-V relationship, input resistance and burst duration are not changed in animal models of depression. The duration of bursts is longer in behaving animals in vivo than in brain slices.

In vivo recordings of LHb neurons show that burst duration (the time interval between the first and last spike within the same burst) does not differ between cLH and wild-type rats.

Chronic restraint stress induces depression-like phenotypes and increased burst firing, which can be reversed by ketamine. CRS mice have increased burst firing in the LHb, and ketamine reverses this increase. The percentage of burst- and tonic-type spikes in all spikes recorded is i, and the distribution of inter-spike intervals (ISIs) is j.

Ketamine suppresses LHb bursting activity in vivo by reducing the ISIs between consecutive spikes and by reducing the mean of total and tonic firing rates, intra- and inter-burst intervals, and ISI distribution.

Inter-burst frequencies and number of spikes per burst were measured in vivo in control mice, CRS mice, and CRS mice after ketamine injection. The distance between the neighbouring troughs was around 140 ms (corresponding to 7 Hz) in CRS mice.

Drug effects on NMDAR currents, RMPs and miniature EPSCs of LHb neurons are shown in Figure 5. Ketamine, AP5, NBQX and mibefradil do not affect RMPs of LHb neurons.

ZD7288 causes a small but significant hyperpolarization of LHb neurons. The hyperpolarization was induced by perfusion of ketamine, and was characterized by an increase in mEPSC amplitude and mEPSC inter-events interval and average frequency.

Extended Data Figure 6 shows that LHb bursts are voltage dependent and that pharmacological manipulation of hyperpolarization-triggered rebound bursts in LHb results in increased burst frequency, burst duration and intra-burst spike number.

LHb neurons show T-VSCC currents and RMPs in animal models of depression. The T-VSCC currents are obtained by subtraction of recorded traces without mibefradil from those with mibefradil (10 M).

No difference in LHb T-VSCC currents was detected between wild-type and cLH rats or control and CRS mice. However, neuronal RMPs were more hyperpolarized in cLH rats and CRS mice than in controls.

Extended Data Figure 8 shows a single compartment model of an LHb neuron incorporating T-VSCCs and NMDAR channels. The model shows that the bursting probability was evaluated across ten independent trials with simulated synaptic inputs.

NBQX or AMPA treatment decreased spontaneous bursts in the LHb model neuron, and the Ca2+ plateau potential helped remove the magnesium blockade of NMDARs. INMDA dominates the driving force to further depolarize RMP to the threshold for Na spike generation.

The effects of AMPA or picrotoxin on spontaneous burst frequency in the LHb are shown in Figure 9.

LHb neurons infected with AAV2/9-oChIEF can follow only the first of five 100-Hz pulsed blue light stimulations and do not show depressive phenotypes. LHb neurons infected with AAV2/9- oChIEF did not respond to 5-Hz tonic blue light stimulation or to 20-Hz tonic photostimulation, and did not change locomotion in the OFT or induce depressive phenotypes in the FST or SPT.

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This study used male cLH rats and age-matched male Sprague Dawley rats to analyze burst and synchronization data. Male adult C57BL/6 mice were used to establish the chronic restraint stress (CRS) depression model.

Study details

Compounds studied
Ketamine

Topics studied
Neuroscience

Study characteristics
Animal Study

Participants
500

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