METHOD AND PHARMACEUTICAL COMPOSITION FOR TREATING DEPRESSION

Abstract

A method for treating a subject with depression characterized by having an increased burst firing in neurons of a lateral habenula in the subject is provided. The method includes a step of administering to the subject a pharmaceutical composition capable of inhibiting the burst firing in the lateral habenula of the subject. The pharmaceutical composition includes one or more active pharmaceutical agents, which can suppress the burst firing in the lateral habenula of the subject and can include at least one of an N-methyl-D-aspartate receptor (NMDAR) inhibitor or a T-type calcium channel inhibitor. The pharmaceutical composition can be in a formulation allowing for local administration to the lateral habenula of the subject, or can be in a formulation configured for systemic administration to the subject. A method for testing a test substance for an antidepressive effect is also provided.

Claims

1. A method for treating a depression in a subject, wherein the depression is characterized by having an increased burst firing in neurons of a lateral habenula (LHb) of the subject, the method comprising: administering to the subject a pharmaceutical composition capable of inhibiting the burst firing in the LHb of the subject.

2. The method of claim 1, wherein the administering to the subject a pharmaceutical composition capable of inhibiting the burst firing in the LHb of the subject comprises: administering the pharmaceutical composition locally to the LHb of the subject.

3. The method of claim 2, wherein the pharmaceutical composition comprises at least one of an N-methyl-D-aspartate receptor (NMDAR) inhibitor, or a T-type calcium channel inhibitor.

4. The method of claim 1, wherein the administering to the subject a pharmaceutical composition capable of inhibiting the burst firing in the lateral habenula of the subject comprises: administering the pharmaceutical composition systemically to the subject.

5. The method of claim 4, wherein the pharmaceutical composition comprises at least one of an NMDAR inhibitor capable of crossing a blood-brain barrier or a T-type calcium channel inhibitor capable of crossing the blood-brain barrier.

6. A pharmaceutical composition for treating a depression having an increased burst firing in neurons of a lateral habenula (LHb) in a subject, comprising one or more active pharmaceutical agents capable of inhibiting the burst firing in the lateral habenula of the subject.

7. The pharmaceutical composition of claim 6, wherein the one or more active pharmaceutical agents comprise at least one of an N-methyl-D-aspartate receptor (NMDAR) inhibitor or a T-type calcium channel inhibitor.

8. The pharmaceutical composition of claim 7, wherein each of the one or more active pharmaceutical agents is capable of crossing a blood-brain barrier of the subject such that the pharmaceutical composition is in a formulation allowing for systemic administration to the subject.

9. The pharmaceutical composition of claim 8, wherein the one or more active pharmaceutical agents comprise an N-methyl-D-aspartate receptor (NMDAR) inhibitor and a T-type calcium channel inhibitor.

10. The pharmaceutical composition of claim 9, wherein: the NMDAR inhibitor comprises ketamine; and the T-type calcium channel inhibitor comprises at least one of ethosuximide or methsuximide.

11. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in a formulation configured for local administration to the lateral habenula of the subject.

12. The pharmaceutical composition of claim 11, wherein the one or more active pharmaceutical agents comprise a combination of an NMDAR inhibitor and a T-type calcium channel inhibitor.

13. The pharmaceutical composition of claim 12, wherein: the NMDAR inhibitor comprises at least one of ketamine or APS; and the T-type calcium channel inhibitor comprises at least one of ethosuximide or mibefradil.

14. A method for testing a test substance for an antidepressive effect, comprising: providing a testing model characterized by electrophysiologically having an increased burst firing in lateral habenula (LHb) neurons contained therein; administering the test substance to the testing model; examining an electrophysiological activity of the LHb neurons in the testing model; and determining, if there is a reduction of the burst firing, that the test substance is an antidepressant candidate having the antidepressive effect.

15. The method of claim 14, wherein the testing model is an LHb in vitro model or an LHb animal model.

16. The method of claim 15, wherein the testing model is an LHb in vitro model, and the LHb neurons in the LHb in vitro model show spontaneous burst firing or are induced to show an increased burst firing.

17. The method of claim 16, wherein: the LHb neurons are induced to show an increased burst firing by hyperpolarization current injection; or the LHb neurons overexpress an opsin, and are configured to inducibly show an increased burst firing upon eliciting by a enabling light signal corresponding to the opsin.

18. The method of claim 17, wherein the opsin comprises at least one of: an inhibitory opsin eNpHR3.0, wherein the corresponding enabling light signal comprises yellow light pulses; or an excitatory opsin oChief, wherein the enabling light signal comprises high frequecy blue light pulses.

19. The method of claim 15, wherein the testing model is an LHb animal model engineered to overexpress an opsin in the LHb neurons, wherein the LHb neurons are configured to inducibly show an increased burst firing upon eliciting by an enabling light signal to the LHb neurons.

20. The method of claim 14, wherein the examining an electrophysiological activity of the LHb neurons in the testing model comprises: performing, if the testing model is an LHb in vitro model, a whole cell patch-clamp recording in the LHb neurons in the LHb in vitro model; or performing, if the testing model is an LHb animal model, an in vivo multi-tetrode recording in the LHb of the animal model.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0111] FIGS. 1A-1I show local blockade of NMDARs in LHb is sufficient to elicit rapid and sustained antidepressant effects. FIG. 1A, Illustration of bilateral implantation of cannulae in LHb of cLH rats. CTX: cortex; HPC: hippocampus. Bottom: white dashed lines indicate location of habenula. FIGS. 1B-1G, Acute antidepressant effects of local bilateral infusion of ketamine (25 g, 1 ul each side, 1 h, B-D), AP5 (40 nmol, 1 ul each side, 0.5 h, E-G) in LHb in forced swim test (FST, C, F) and sucrose preference test (SPT, D, G). Infusion sites of drugs are verified by CTB (B, E, see Methods). FIGS. 1H-1I, Sustained antidepressant effects of local bilateral infusion of ketamine (25 ug, 1 ul each side, 14 d) in LHb in forced swim test (FST, H) and sucrose preference test (SPT, I). Data are means.e.m. **P<0.01, ***P<0.005, n.s. not significant.

[0112] FIGS. 2A-2N show bursting activity is enhanced in rat and mouse model of depression, which is reversed by ketamine. FIG. 2A, Whole cell patch recordings sites across different subregions of LHb. FIGS. 2B-2D, Representative traces showing spontaneous activity of three LHb neuron types, namely silent (FIG. 2B), tonic (FIG. 2C), and burst (FIG. 2D) firing types. Grey shaded areas shown at the right with enlarged time scales to illustrate shape of spike pattern. Red traces are responses of same neurons after TTX treatment. FIGS. 2E-2F, Scattered plots (FIG. 2E) and cumulative curves (FIG. 2F) denoting the mean and distribution of resting membrane potentials (RMPs). FIGS. 2G-2H, Intra burst frequency (FIG. 2G) but not inter burst frequency (FIG. 2H) reversely correlated with RMPs. FIGS. 2I-2N, Bursting neurons are significantly increased in cLH rat model (FIGS. 2I-2K) and chronic restraint stress (CRS) mouse model (FIGS. 2L-2N) of depression, which is reversed by ketamine. FIGS. 2I and 2L, Pie charts illustrating the percentage abundance of the three types of LHb neurons in SD and cLH rats (FIG. 2I), or control and CRS mice (FIG. 2L). FIGS. 2J and 2M, Bar graph illustrating the percentage of burst- and tonic-type spikes in all spikes recorded. FIGS. 2K and 2N, Histogram of inter-spike intervals (ISI, ms) distribution. Data are means.e.m. *P<0.05, **P<0.01, ****P<0.0001.

[0113] FIGS. 3A-3G show ketamine suppresses enhanced LHb bursting activity and theta-band synchronization in vivo in chronic restraint mice. FIG. 3A, Recording sites of each tetrode track in LHb of CRS and control mice. FIG. 3B, Example traces (left) and averaged spike waveform (right) of recorded neurons from LHb of control (top), CRS (middle) and the same CRS unit after ketamine injection (bottom). Bursts (red shades) are identified by ISI method (see Methods). FIGS. 3C-3D, Percent of spikes in bursting mode and number of bursts per minute of neurons recorded from control, CRS mice (FIG. 3C), and the same unit in CRS mice 1 h before and after ketamine injection (FIG. 3D). FIG. 3E, Cumulative distribution of ISI from control, CRS mice, and CRS mice treated with ketamine. Dashed lines indicate the 50% percentile of ISI (control: 143 ms; CRS: 33 ms, CRS+ketamine: 121 ms). FIG. 3F, Spike-triggered averages (STAs) of neurons recorded from control, CRS, and CRS mice after ketamine injection. Note the distance between the neighboring troughs is around 140 ms (corresponding to 7 Hz) in CRS mice. FIG. 3G, Spike-field coherence (SFC) of neurons recorded from control, CRS, and CRS mice after ketamine injection. Left: SFC of each unit; Middle: average SFC; Right: percent SFC in theta band (4-10 Hz). Data are means.e.m. *P<0.05, ***P<0.001, ****P<0.0001.

[0114] FIGS. 4A-4J show LHb bursting requires activation of NMDARs. FIG. 4A, Example traces showing evoked EPSCs when the cells are held at 80 mV. NMDAR-EPSCs are isolated by application of picrotoxin and NBQX in Mg.sup.2+ free ACSF, and confirmed by AP5 blockade. FIG. 4B, Amplitudes of NMDAR-EPSCs under different voltages (EPSCs are recorded under 0 Mg.sup.2+, picrotoxin and NBQX); the isolated NMDAR-EPSCs is completely blocked by AP5. FIGS. 4C-4H, Example traces (left) and statistics (right, sampled within 1 min after drug application) showing effects of ketamine (FIGS. 4C-4D), AP5 (FIGS. 4E-4F) or NBQX (FIGS. 4G-4H) on spontaneous bursts in LHb. Spikes in bursting mode are marked in blue. Spikes in tonic-firing mode are marked in black. FIG. 4I, Example trace of an originally silent LHb neurons induced to bursts by NMDA perfusion and returned to silence after ketamine application. Note that NMDA induces both large EPSP (green shaded) and bursting discharges. FIG. 4J, Summary of NMDA perfusion and ketamine effect on bursting. Data are means.e.m., *P<0.05, **P<0.01, ***P<0.001.

[0115] FIGS. 5A-5J show LHb bursting requires membrane hyperpolarization and T-VSCCs.

[0116] FIG. 5A, Representative trace of a LHb neuron transformed from bursting- to tonic-firing mode with a ramp-like current injection, showing bursting at more hyperpolarized potential and tonic firing at more depolarized membrane potential. Spikes in bursting and tonic-firing mode are marked in blue and black respectively. FIG. 5B, Percentage of LHb neurons that can be induced into bursting mode with a hyperpolarizing current injection during the current ramp. Number in the bar indicates cell number. FIGS. 5C-5E, Correlations of membrane potential versus inner burst frequency (FIG. 5C), burst duration (FIG. 5D) and inner burst spike number (FIG. 5E) generated by current ramps. FIGS. 5F and 5G, Example trace (left) and statistics (right) of a spontaneously tonic-firing neuron transformed to burst-firing mode by hyperpolarization (FIG. 5F), and a spontaneously bursting neuron transformed to tonic firing by depolarization (FIG. 5G). FIGS. 5H-5I, Example traces (left) and statistics (right, sampled within 1 min after drug application) showing effects of T-VSCC blocker miberfradil (FIG. 5H) or HCN blocker ZD7288 (FIG. 5I) on spontaneous bursts in LHb. FIG. 5J, An example trace summarizing the ionic components and channel mechanisms involved in LHb bursting. Activation of T-VSCCs removes the Mg blockade of NMDARs. The opening of these two channels synergistically drive membrane potential toward the threshold for a burst of APs. After the quick inactivation of T-VSCCs and NMDARs, the return of RMP back to below 55 mV de-inactivates T-VSCCs, which initiates another cycle of burst. Data are mean s.e.m., *P<0.05, **P<0.01, n.s. not significant.

[0117] FIGS. 6A-6C show local bilateral infusion of mibefradil in LHb exerts a rapid antidepressant effect in FST (FIG. 6B) and SPT (FIG. 6C). Infusion sites are verified by CTB (FIG. 6A). Data are means.e.m., *P<0.005, **P<0.01, n.s. not significant.

[0118] FIGS. 7A-7H show eNpHR-induced rebound burst drives behavioral aversion and depressive-like symptoms. FIG. 7A, Construct of AAV2/9-eNpHR3.0 (top), example site of viral injection and optic fiber implantation (middle), and illustration of optrode recording (bottom). FIGS. 7B-7C, Representative traces showing rebound bursts reliably elicited by pulsed yellow light in LHb brain slices in vitro (FIG. 7B) and in vivo from mice infected with AAV2/9-eNpHR3.0. Spikes in bursting and tonic-firing mode are marked in blue and black respectively. Percentage of successfully induced burst is shown on right. FIG. 7D, Raster plots (top) and post-stimulus time histogram (bottom) of an example LHb neuron responding to 100 ms yellow light stimulation from in vivo optrode recording. FIG. 7E, Distribution of intra burst frequencies and intra burst spike numbers of eNpHR3.0-driven rebound bursts (left) are comparable to those in CRS mice (right). Means are represented by the black crosses. FIG. 7F, Real-time place aversion (RTPA) induced by eNpHR3.0-driven bursts. Left: representative heat maps of RTPA; Right: quantitative aversion score (see Methods). FIGS. 7G-7H, Depressive-like behaviors in FST (FIG. 7G) and SPT (FIG. 7H) induced by eNpHR3.0-driven bursts. Data are represented as means.e.m., **P<0.01.

[0119] FIGS. 8A-8C show stimulation yielded the same overall firing rate as the rebound burst protocol do not cause depressive-like phenotypes. FIG. 8A, Representative trace showing LHb neurons following a 5 Hz tonic blue light stimulation protocol in LHb brain slices infected with AAV2/9-oCHIEF. Percentage of responsive neurons shown on the right. FIGS. 8B-8C, 5 Hz photostimulation of mice expressing oChIEF does not change locomotion in OPT (FIG. 8B), and does not induce depressive phenotypes in FST (FIG. 8C).

[0120] FIG. 9 shows antidepressant-like effect of low dose ketamine and ethosuximide co-treatment in mice. Low dose ketamine (2.5 mg/kg) or ethosuximide (ETH, 100 mg/kg) is ineffective in the mouse FST. Co-treatment with subeffective doses of both drugs has an antidepressant-like effect.

[0121] FIGS. 10A-10E. Pharmacological manipulations of hyperpolarization- triggered rebound bursts in LHb. FIGS. 10A-10B, Example traces (left) and statistics (right) showing effects of ketamine (FIG. 10A) or AP5 (FIG. 10B) on rebound burst. Current injection steps are illustrated under the bottom of the trace. FIG. 10C, Example traces (left) and statistics (right) showing effects of T-VSCC blocker miberfradil on rebound bursts. FIGS. 10D-10E, Example traces (left) and statistics (right) showing effects of combined application of mibefradil and AP5 (FIG. 10D) or mibrfradil and ketamine (FIG. 10E) on rebound bursts. Data are means.e.m., ****P<0.0001.

DETAILED DESCRIPTION

[0122] The technical details and benefits of the invention provided in the present disclosure are further described in the following examples, which are intended to illustrate the inventions and not to limit the scope of the present disclosure.

Example 1. Materials and Methods

[0123] Animals. Male cLH rats (8-16 weeks of age) and age-matched male Sprague Dawley rats (SLAC Laboratory Animal Co., Shanghai) were used. The cLH rats were screened by learned helpless test for breeding as previously described (Schulz et al., 2010). 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/cage and mice were four/cage respectively 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.

[0124] Viral vectors. AAV2/9-CaMKII-eNpHR3.0-eYFP (titer: 7.4510.sup.12 v.g./ml, dilution: 1:5, 0.1 l unilateral into LHb, Taitool Bioscience, China), AAV2/9-Ubi-eGFP (titer: 2.510.sup.13 v.g./ml, 1:30, 0.1 l each side of LHb, University of Massachusetts, Guangping Gao Lab, USA), AAV2/9-hSyn-oChIEF-tdTomato (titer: 6.2910.sup.12 v.g./ml, 1:5, 0.1 l unilateral into LHb, Obio Technology, Shanghai, Corp., Ltd) were aliquoted and stored at 80 C. until use.

[0125] Cannula infusion experiment. A 26-gauge double guide cannulae (center-to-center distance 1.4 mm, Plastics One) was placed with a 2-degree angle with coronal plane (without the 2 degree rotation, we found it difficult to hit both sides of LHb) and inserted bilaterally into the LHb (AP, 3.7 mm from bregma; ML, 0.7 mm; DV, 4.1 mm from the brain surface) of cLH rats. A 33-gauge double dummy cannulae (Plastic One), secured with a dust cap, was inserted into guide cannula to prevent clogging during recover period. After rats were 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 anesthetic machine. The extensions were manually sharpened before insertion.

[0126] Ketamine (25 g/l), AP5 (40 nmol/l, IC50=30 M), NBQX (1 nmol/l, IC50=0.15 M) or mibefradil (10 nmol/l) were dissolved in 0.9% saline respectively. Ketamine was purchased from Gutian Pharma Co., Fujian, and stored at room temperature. Before the drug local infusion, tip-sharpened 33-gauge double injector cannulae were inserted into the guide cannulae to assure clear passage and then pulled out. 1 l of drug was infused (0.1 l/min) into each side through another 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. FST or SPT was performed 1 h after the injection of ketamine or mibefradil, 0.5 h after the injection of AP5 or NBQX. To verify the drug infusion sites, rats were injected with 1 l CTB-488 to each side of LHb after all behavioral 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 correct injections were used.

[0127] LHb brain slice preparation. Animals (P45-70 rats and P65-75 mice) were anesthetized with isoflurane and 10% chloral hydrate, and then perfused with 20 ml ice-cold ACSF (oxygenated with 95% O.sub.2+5% CO.sub.2) containing (mM): 125 NaCl, 2.5 KCl, 25 NaHCO.sub.3, 1.25 NaH2PO4, 1 MgCl.sub.2 and 25 Glucose, with 1 mM pyruvate added. The brain was removed as quickly as possible after decapitation and put into chilled and oxygenated ACSF. Coronal (for most of experiments if not specified) or sagittal slices containing habenular (350 m- and 300 m-thickness for rats and mice, respectively) were sectioned in cold ACSF by a Leica2000 vibratome and then transferred to ASCF at 32 C. for incubation and recovery. ACSF was continuously gassed with 95% O.sub.2 and 5% CO.sub.2. Slices were allowed to recover for at least 1 hour before recording. For cLH rats, since a very high percentage (90%) of cLH offsprings are learned helpless, we did not perform LH test before taking them for brain slice recording. For CRS mice, both CRS and their wild-type controls went through FST test before brain slice recording. We then used the CRS animals which showed high immobility scores (immobile time >140 s) and control mice which showed low immobility (immobile time <110 s) in FST for slice recording.

[0128] In vitro Electrophysiological recording. For LHb neuron recordings, currents were measured under whole-cell patch clamp using pipettes with a typical resistance of 5-6 M filled with internal solution containing (mM): 10.sup.5 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. The external ACSF solution contained (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO.sub.3, 1.25 NaH.sub.2PO.sub.4, 1 MgCl.sub.2, 2 CaCl.sub.2 and 25 Glucose. Cells were visualized with infrared optics on an upright microscope (BX51WI, Olympus). A MultiClamp 700B amplifier and pCLAMP10 software were used for electrophysiology (Axon Instruments). The series resistance and capacitance was compensated automatically after stable Giga seal were formed. Recordings were typically performed between 3-6 min after break-in. The spontaneous neuronal activity was recorded under current-clamp (I=0 pA).

[0129] LHb neurons display three modes of spontaneous activity at resting conditions. Silent cells showed no spike activities during recording. Tonic cells spontaneous generated tonic trains of action potentials at frequency between 0.1-10 Hz, rarely up to 10-20 Hz. Burst-firing cells spontaneously generate clusters of spikes with an initially high but progressively declining intra-burst firing frequency in each burst. For bursting neurons, 99% (from n=50 bursting neurons) of their spikes occurred within bursts.

[0130] Evoked-NMDAR-EPSC was recorded under voltage clamp from 50 mV to 80 mV in a modified extracellular ACSF solution with 0 Mg.sup.2+. Evoked T-type VSCC current was recorded under voltage clamp starting from a holding potential of 50 mV before increased to conditioning potential (100 mV) for is preceding the command steps (5 mV, 0.1 Hz/step increment). LHb T-VSCC currents were obtained by subtraction of recorded traces in presence or absence of mibefradil.

[0131] In vivo electrophysiology. For in vivo recording experiments, a custom-made microdrive array consisting of 8 tetrodes (impedance 250-500 K, California fine wire) was implanted in LHb (AP, 1.72 mm; ML, 0.46 mm; DV, 2.44 mm from the brain surface) of Ctrl or CRS mice. Stainless steel wires were attached to two screws on the skull as ground. The microdrive was secured to the skull with dental cement. After recovery for 2 weeks, mice were allowed to adapt to the recording headstage 10 min per day for 2-3 days. Spontaneous spiking activity (digitized at 40 kHz, band-pass filtered between 300-6000 Hz) and LFP (digitized at 1 kHz sampling rate, low-pass filtered up to 250 Hz) were recorded simultaneously for 30 min during the still period of the mice in their home cages with a gain of 5000. A single channel without detectable unit was assigned as a reference electrode. The tetrodes were lowered insteps of 70 m after each recording session followed with at least 2 day-recovery. For CRS mice, data were recorded for 30 min before and 1 h after ketamine treatment (10 mg/kg, i.p.). If mice received a second ketamine injection, at least a 2-week interval was introduced before the next recording session. All procedures were performed during the light phase. The positions of the electrodes were verified by electrolytic lesions (30 A, 10-15 s) at the end of all the experiments.

[0132] Spike sorting. All waveforms recorded from each tetrode were imported in Offline Sorter V3 (Plexon Inc.). Single units were manually identified by threshold crossing and principal component analysis (PCA). Spikes with inter-spike interval (ISI) less than the refractory period (1.4 ms) were excluded. Cross correlograms were plotted to ensure that no cell was discriminated more than once on overlapping tetrodes. Only units with signal to noise ratio larger than 2 were used.

[0133] Behavioral assays. All behavioral assays were performed on animals 12-16 weeks old, in the light cycle (7:00-19:00) except for the sucrose preference test, which was performed during dark phase to maximize the consumption of solutions. Behavioral analysis was performed blindly.

[0134] 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 (23-24 C.) and swam for 6 min under normal light. Animal behaviors were videotaped from the side. The immobile time during the last 4 min test was counted offline by an observer blind of the animal treatments. 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 turned on as previously described, 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 behavior, the cable length was adjusted to allow the cable just touch the water surface.

[0135] 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. Total consumption of each fluid was measured and sucrose preference was defined as the ratio of sucrose consumption divided by total consumptions of water and sucrose. For optogenetic manipulations, mice were gently placed in a white arena containing normal bedding and allowed to freely move in the arena. During the 90-min test, light was delivered during 30-60 min. Laser intensity during eNpHR3.0 stimulation was adjusted to 10 mW since LHb neurons could not follow 16 mW stimulation for longer than 10 min. Sucrose preference scores were measured for every 30 min. Only animals that had a >30% baseline sucrose preference during the first 30 min session would proceed to the next session. Otherwise they would be tested later on a different day. Behavioral analyses and experiments were performed blindly.

[0136] 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 hours per day for 14 consecutive days. ROC curve (Receiver Operating Characteristic curve) of immobile time in forced-swim test was used to assess the successful rate of CRS on depressive-like behaviors. The nearest point to (0, 100) on ROC curve was selected as successful rate according to GraphPad Statistics Guide.

[0137] Real-time place aversion. Mice were placed in a white open chamber (52 cm26 cm23 cm) consisting of two chambers, and allowed to freely move between chambers for 20 minutes to assess their baseline place preference. During the following 20-min test, we assigned a stimulated side with a counterbalanced manner. Laser stimulation was turned on as previously described, as soon as mice entered the stimulated side and terminated once mice crossed to the non-stimulated side. A video camera positioned above the chamber recorded each trial. Mouse locations and velocity were tracked and analyzed using Any-maze Software (Stoelting Co.). Avoidance score=(Time in stimulated sideTime in non-stimulated side).sub.test(Time in stimulated sideTime in non-stimulated side).sub.baseline

[0138] Open field test (OFT). Animals were placed in the center of an arena (40 cm40 cm 40.5 cm for mice; and 100 cm100 cm50 cm for rats) in a room with dim light for 6 min. A video camera positioned directly above the arena was used to track the movement of each animal (Any-maze, Stoelting, US). For optogenetic manipulations, mice were allowed to freely move throughout the arena for 9 min, with laser stimulation occurring during the middle 3 min epoch (eNpHR3.0: 589 nm, 1 Hz, 16 mW, 100 ms pulses; oChIEF: 473 nm, 100 Hz, 25 mW, either 5 ms pulses, 5 pulses/s for the pulsed 100 Hz protocol or 5 Hz, 5 ms pulses for the 5 Hz protocol).

[0139] Statistical analysis. Required sample sizes were estimated based on our past experience performing similar experiments. Mice were randomly assigned to treatment groups. Analysis were performed in a manner blinded to treatment assignments in all behavioral experiments. Statistical analyses were performed using GraphPad Prism software v6. By pre-established criteria, values were excluded from the analyses if the viral injection or drug delivering sites were out of LHb. All statistical tests were two-tailed, and significance was assigned at P<0.05. Normality and equal variances between group samples were assessed using the D'Agostino & Pearson omnibus normality test and Brown-Forsythe tests respectively. When normality and equal variance between sample groups was achieved, one-way ANOVAs (followed by Bonferroni's multiple comparisons test), or t test were used. Where normality or equal variance of samples failed, Kruskal-Wallis one-way ANOVAs (followed by Dunn's correction), Mann-Whitney U test, or Wilcoxon matched-pairs signed rank test were performed. Linear regression test, Chi-square test, Fisher's exact test or two-way ANOVAs (followed by Bonferroni's multiple comparisons test) was used in appropriate situations.

Example 2. Local Blockade of NMDARs in LHb is Sufficient to be Rapid Antidepressant

[0140] The antidepressant effect of ketamine was tested on a well-accepted animal model of depression, the congenitally learned helpless (cLH) rats. It was tested whether ketamine may exert its antidepressant effect through LHb by performing bilateral infusion of ketamine in the LHb through dual guide cannulae (FIG. 1A). Local infusion of ketamine (25 ug, 1 ul each side) in the LHb of cLH rats was sufficient to quickly rescue the depressive-like behaviors, including the behavioral despair as measured by the immobility time in the FST (FIG. 1C) and the anhedonia as measured by the sucrose preference test (SPT FIG. 1D) 1 hr after infusion. To determine whether NMDAR inhibition is the main mechanism underlying the antidepressant effects of ketamine, a specific NMDAR antagonist AP5 (40 nmol, 1 ul, each side) was locally infused in the LHb, and found that AP5 efficiently reduced the immobile time in the FST (FIG. 1F), as well as increased hedonic behaviors in the SPT (FIG. 1G), similarly as ketamine. Indeed, infusion of ketamine (25 ug, 1 ul each side) into LHb of cLH rats induced sustained antidepressant effects for 14 days after injection (FIGS. 1H and 1I). All these results suggest that LHb local infusion of NMDAR antagonist can induce rapid and sustained antidepressant effect.

Example 3. Three Types of LHb Neurons, the Silent, Tonic and Burst Firing Types

[0141] To investigate the activity pattern of the LHb neurons, whole cell patch clamp is performed in the LHb coronal slices, and recorded spontaneous neuronal activity under current clamp at resting conditions (I=0 pA). It was found that LHb neurons were intrinsically active and fell into three categories, namely, the silent, tonic- and burst-firing types (FIGS. 2A-2D). The three classes of neurons were distributed among different sub-nuclei of the LHb with no clear subregion enrichment (FIG. 2A).

[0142] The resting membrane potentials (RMPs) of LHb neurons were on average more depolarized than those in the hippocampus or cortex (FIGS. 2E and 2F). Notably, the bursting neurons have significantly more hyperpolarized RMPs compared with the silent and tonic firing neurons (Silent: 47.81.3 mV, tonic: 45.50.8 mV, bursting: 610.9 mV, FIGS. 2E and 2F).

[0143] To test the potential contribution of bursting activity in the hyperactive state of the LHb under depression, the spike patterns of LHb neurons from the cLH or wild type SD rats were compared. While the percentage of bursting neurons was significantly increased from 7% (n =8/121) in the SD controls to 23% (n=21/102) in the cLH rats (FIG. 2I). The percentage of spikes in the bursting mode was also increased from 7% in SD to 43% in the cLH rats (FIG. 2J). It was then analyzed the inter-spike intervals (ISI), which represent the duration of the silent periods between two neighboring single spikes. A typical bursting cell shows a bimodal distribution of ISIs since it is composed of relatively large inter-burst intervals and small intra-burst intervals. In contrast, tonic firing cells show a more homogenous Poisson's distribution of ISIs. The ISIs of LHb neurons in SD rats were mostly normally distributed between 50 to 150 ms (n=24 neurons, FIG. 2K). In contrast, ISIs of LHb neurons from cLH rats exhibited a clear bimodal distribution with an extra sharp and condensed cluster of high frequency events centered around 14 ms (corresponding to 71 Hz), indicating a significant weight increase of burst firings (n=24 neurons, FIG. 2K).

[0144] To test if enhanced bursting is universal in depression, we used a second animal model of depression, mice with chronic restraint stress (CRS). Patch clamp recording of LHb neurons in these mice revealed similar phenomena, namely, percentage of bursting cells and percentage of spikes in bursting were both dramatically increased (FIGS. 2L and 2M). ISIs of LHb neurons in CRS mice also displayed bimodal distribution and an extra peak at 20 ms (FIG. 2N).

Example 4. Ketamine Suppresses Enhanced LHb Bursting Activity and Theta-Band Synchronization In Vivo in Chronic Restraint Mice

[0145] To test whether burst also occurs in vivo in the LHb and whether it is bidirectionally modulated by depression state and ketamine, in vivo multi-tetrode recording was performed in the LHb of freely behaving mice (FIGS. 3A and 3B). Unlike in in vitro slice conditions where LHb neurons spike with either tonic or bursting mode, spike patterns of LHb neurons recorded in vivo switched between tonic and burst firing modes (FIG. 3B). LHb neurons from CRS mice showed a notable increase in bursting activity (FIG. 3C) but not tonic firing, compared with control nave mice. Injection of ketamine at the antidepressant dosage (10 mg/kg, i.p., 1 h prior to recording) significantly suppressed the LHb bursting activity (FIG. 3D). The cumulative frequency distributions of ISI, which were clearly different between CRS and control mice, were significantly shifted toward control level by ketamine (FIG. 3E).

[0146] Burst firing was known to increase network synchronization. It was thus tested whether LHb network synchronization was altered in the CRS animals. It was first calculated the spike-triggered averages (STAs) of local field potential (LFP), which revealed oscillatory synchronization between spikes and LFP. In the control mice, the distribution of the power spectra of STAs was relatively flat (FIG. 3F), indicating a lack of synchronization. In the CRS mice, there emerged a dominant frequency of 7 Hz in the power spectra of STAs (FIG. 3F), indicating that spikes tended to phase-lock with LFP in the theta-band range (4-10 Hz). Consistently, CRS mice showed significantly higher spike-field coherence (SFC, reflecting normalized power spectra of STAs) in the theta band range compared with control mice (FIG. 3G). These changes in LHb network synchronization in CRS mice as reflected in STA and SFC were reversed to control level 1 hr after systemic injection of ketamine (10 mg/kg, i.p., FIG. 3G).

[0147] Together with the in vitro slice experiments, these in vivo results in freely behaving animals provide strong evidences that LHb bursting is pathologically enhanced in depression, which can be efficiently alleviated by ketamine.

Example 5. Bursts in LHb Directly Require Activation of NMDAR

[0148] Given that NMDAR-mediated calcium influx plays a pivotal role in burst generation in several brain regions, and in light of the data that systematic injection of ketamine suppressed bursting activity (FIGS. 2A-2N and 3A-3G), it was tested if NMDARs are directly required for the bursting activity in the LHb. First to confirm that LHb expresses functional NMDARs, NMDAR-dependent excitatory post-synaptic potentials (NMDAR-EPSCs) in sagittal LHb slices was recorded by stimulating the input stria medullaris (SM) fiber in the presence of AMPA receptor (AMPAR) blocker NBQX and GABA receptor (GABAR) blocker picrotoxin and 0 Mg.sup.2+, and isolated characteristic NMDAR-currents, which can be abolished by AP5 (FIGS. 4A and 4B). Next, ketamine (100 M) was bath applied onto spontaneously bursting neurons recorded in the LHb brain slices, and found that ketamine almost completely eliminated spontaneous bursts (FIGS. 4C and 4D). As illustrated in FIG. 4C, within seconds after bath application of ketamine, a burst-firing LHb neuron was converted to tonic-firing mode. Similarly, bath application of a specific NMDAR antagonist AP5 (100 M) also stopped burst-firing (FIGS. 4E and 4F). Interestingly, consistent with the behavioral effects from cannular infusion, blockade of AMPAR with NBQX (10 M) reduced bursts, but to a much smaller extent than NMDAR blockade (FIGS. 4G and 4H).

[0149] To further verify the causal link between NMDAR activity and LHb bursting, we perfused NMDA (20 M) onto the LHb brain slice in the presence of AMPAR blocker NBQX and GABAR blocker picrotoxin and 0 Mg.sup.2+ to activate NMDARs. NMDA application induced strong bursting activity in 10 out of 13 originally silent LHb neurons (FIGS. 4I and 4J). Again this bursting activity was blocked by additional bath application of 100 M ketamine (FIG. 4J). FIG. 4I shows a representative example of such neurons, exhibiting dramatically enhanced EPSPs and burst firing quickly after perfusion of NMDA, and then transforming to tonic followed by silent mode after wash-in of ketamine.

Example 6. Bursts in LHb also Depend on Hyperpolarization and Synergistic Activation of T-VSCCs

[0150] Given the correlation between the RMPs and firing mode of LHb neurons, we next tested whether changing RMPs can alter the pattern of spiking activity in LHb. By applying a transient ramp-like current injection enabling RMPs to change progressively from around 80 to 40 mV (FIG. 5A), we found that in 90% of rat and 93% of mouse LHb neurons, the hyperpolarization current injection was able to evoke high frequency bursts of Aps (FIG. 5B). Similar as found in the spontaneous bursting neurons (FIG. 2G), the intra-burst frequencies of the ramp-evoked rebound bursts were positively correlated with the hyperpolarization level of membrane potential (FIG. 5C). The duration of bursts tended to decrease with more hyperpolarization (FIG. 5D). Consequently, the number of spikes in each burst, which is the product of the intra-burst frequency and burst duration, were normally distributed from 80 mV to 40 mV and peaked at 5660 mV (FIG. 5E), close to the average RMPs observed in spontaneous bursting LHb neurons (FIG. 2E). As current ramped into more depolarized potentials, burst firings transformed into tonic firings of single Aps, whose frequency increased with the level of depolarization (FIG. 5A).

[0151] This voltage-dependent transition of firing mode also occurred in spontaneously-spiking LHb neurons. By delivering a hyperpolarizing current injection (1020 pA), 75% of originally tonic-firing neurons could be transformed to burst-firing mode (FIG. 5F). Vice versa, with a depolarizing current injection (1020 pA), 100% of originally bursting neurons could be transformed to tonic firing mode (FIG. 5G). These results indicated that, within the very same LHb neuron, the activity pattern can be transformed from tonic to burst firing, or vice versa, depending on the membrane potential.

[0152] Since NMDAR alone does not explain the voltage-dependence of LHb bursts, we searched for more ion channels involved. The T-type Voltage Sensitive Calcium Channels (T-VSCCs, including Cav3.1, 3.2, 3.3) are known to have pacemaker activity and are expressed in LHb neurons. Unlike other types of voltage sensitive calcium channels, T-VSCCs are inactivated quickly after opening at depolarized membrane potentials, but can be de-inactivated to initiate burst firings when the membrane potential is hyperpolarized for longer than 100 ms. Bath application of mibefradil (10 M), onto the LHb brain slices effectively decreased the bursting probability and reduced the amplitude of plateau potential of spontaneous bursts (FIG. 5H). ZD7288(50 M), an antagonist of another pacemaker channel (hyperpolarization-activated cyclic nucleotide-gated (HCN) channel), had a significant but much smaller effect on bursts than mibefradil (P=0.018, FIG. 5I).

[0153] To understand how NMDARs and T-VSCCs work synergistically to mediate LHb burst firing, we constructed a minimal biophysical model incorporating these two channels (FIG. 5J). Burst generation was successfully modeled and depended critically on the ionic currents of T-VSCC (I.sub.T) and NMDAR (I.sub.NMDA): hyperpolarization of neurons to membrane potentials negative to 55 mV slowly de-inactivates T-VSCC. I.sub.T continues to grow as the de-inactivated T-VSCCs increase, leading to a transient Ca plateau potential. The Ca plateau helps remove the magnesium blockade of NMDARs while T-VSCC inactivates rapidly during the depolarization. After the Ca.sup.2+ plateau reaches approximately 45 mV, I.sub.NMDA dominants the driving force to further depolarize RMP to the threshold for Na spike generation. The falling back to RMP below 55 mV again de-inactivates I.sub.T and results in the intrinsic propensity of LHb neurons to generate the next cycle of burst (FIG. 5J).

Example 7. Local LHb Blockade of T-VSCCs is Rapidly Anti-Depressive

[0154] The above results predict that drugs blocking T-VSCCs may be also antidepressant. To test that, we performed bilateral infusion of a selective T-VSCC blocker, mibefradil (10 nmol, 1 ul each side), in the LHb of cLH rats through dual guide cannulae (FIG. 6A). mibefradil infusion quickly rescued the depressive-like behaviors, including the immobility in the FST (FIG. 6B) and the anhedonia in the SPT (FIG. 6C) 1 hr after infusion.

Example 8. NpHR-Induced Rebound Burst Drives Behavioral Aversion and Depressive-Like Symptoms

[0155] Based on the observation that a hyperpolarization ramp current could induce burst firing in the LHb (FIGS. 5A and 5B), a protocol employing a transient (100 ms) hyperpolarization current injection was devised, which induced rebound bursts in the LHb brain slices with 100% success rate. We thus used an inhibitory opsin, eNpHR3.0 (an enhanced variant of halorhodopsin) to drive rebound bursts in the LHb (FIG. 7A). 1 Hz, 100 ms of 589 nm yellow light pulses reliably elicited robust rebound bursts in in vitro slice recording with a high intra-burst frequency and 90% success rate (FIG. 7B), as well as in in vivo as revealed by optrode recording (FIGS. 7C and 7D). The intra-burst frequency and intra-burst number of spikes produced by this rebound burst protocol were comparable to those detected in depressed CRS mice (FIG. 7E).

[0156] It was then tested whether the rebound bursts in the LHb could acutely drive aversion and depressive-like symptoms in freely behaving mice. In the real-time place aversion (RTPA) assay, 1 Hz yellow light photostimulation significantly reduced the time spent in the light-paired chamber in mice injected with AAV-eNpHR3.0 but not those with AAV-eGFP (FIG. 7F). Furthermore, 1 Hz yellow light photostimulation significantly increased the immobility (FIG. 7G) and decreased sucrose preference (FIG. 7H) in the eNpHR3.0 group.

[0157] To prove that it is the bursting firing mode but not the general increase in firing rate that is important for the induction of depressive-like behaviors, we applied a stimulation protocol (5 Hz on AAV-oChIEF injected mice) that yields the same overall firing rate as the rebound burst protocol (FIG. 8A). This did not cause depressive-like phenotypes (FIGS. 8B and 8C).

[0158] Collectively, these results indicated that bursting activity in the LHb can acutely drive depressive-like state.

Example 9. Co-Administration of Subeffective Antidepressant Doses of NMDAR Antagonist and T-VSCC Antagonist has an Antidepressant-Like Effect

[0159] Low dose ketamine (one of the NMDAR antagonists, 2.5 mg/kg) or ethosuximide (ETH, one of the T-VSCC antagonists, 100 mg/kg) is ineffective in the forced swim test in C56BL/6 mice 1 hour after drug administration (i.p.) (FIG. 9). In co-administration of 2.5 mg/kg of ketamine and 100 mg/kg ethosuximide, as subeffective antidepressant doses of either drug, the immobility time was reduced and latency to immobility was increased compared to control group (FIG. 9). Hence, Co-administration of subeffective antidepressant doses of ketamine and ethosuximide could an antidepressant-like effect.

[0160] These above in vivo experiments suggest that administering an NMDA receptor inhibitor at a dosage lower than its working dosage in combination with administering a T-VSCC receptor inhibitor at a dosage lower than working dosage, could be effective in generating a significant anti-depression effect.

Example 10. Pharmacological Manipulations of Hyperpolarization-Triggered Rebound Bursts in LHb

[0161] To further test the effect of NMDAR inhibitors combined with T-VSCC inhibitors on the induction of burst firing, the effects on inducing burst firing by NMDAR inhibitors and T-VSCC inhibitors alone or in combination were tested.

[0162] Based on the observation that a hyperpolarization ramp current could induce burst firing in the LHb, a protocol was devised employing a transient (100 ms) hyperpolarization current injection, which induced rebound bursts in the LHb brain slices with 100% success rate (FIGS. 10A-10E). As seen, 100 pA hyperpolarization current was injected into the lateral habenular neurons for 100 ms, which made the neurons hyperpolarized, and the hyperpolarization state induced burst firings of the lateral habenular neurons.

[0163] When the lateral habenular slices were perfused with ketamine (100 M, FIG. 10A), AP5 (100 M, FIG. 10B) or mibefradil (10 M, FIG. 10C), the probability of neuron hyperpolarization-induced burst was partially reduced to 0.19, 0.12 and 0.05, respectively. When AP5 (100 M) was administered in combination with mibefradil (10 M, FIG. 10D) or ketamine (100 M) was administered in combination with mibefradil (10 M, FIG. 10E), the probability of neuron hyperpolarization- induced burst in both tests were further reduced to almost zero, which means a complete blocking of the hyperpolarization-induced burst.

[0164] Thus in other words, the hyperpolarization-induced rebound bursts can be partially inhibited by ketamine (100 M, FIG. 10A), AP5 (100 M, FIG. 10B), or mibefradil (10 M, FIG. 10C), but can be almost fully blocked by mibefradil (10 M) in combination with ketamine (100 M, FIG. 10E) or AP5 (100 M, FIG. 10D), indicating that the combination of NMDAR antagonists and T-VSCC antagonists has a strong synergy in modulating the hyperpolarization-induced rebound bursts.

[0165] Collectively, these above in vitro experiments demonstrate that a combination of a low dose of an NMDA receptor inhibitor that is lower than the effective dose when administered alone and a low dose of a T-VSCC receptor inhibitor that is also lower than the effective dose when administered alone can produce a much more pronounced and significant antidepressant effect. It is also noteworthy that since the lower than effective doses of the NMDA receptor inhibitor and the T-VSCC receptor inhibitor are administered, there is potentially another benefit for a reduced side effect for both drugs.

[0166] Unless otherwise indicated, the practice of the present disclosure will employ common technologies of organic chemistry, polymer chemistry, biotechnology, and the like. It is apparently that in addition to the above description and examples than as specifically described, the present disclosure can also be achieved in other ways. Other aspects within the scope of the disclosure and improvement of the present disclosure will be apparent to the ordinary skilled in the art. According to the teachings of the present disclosure, many modifications and variations are possible, and therefore it is within the scope of the present disclosure.

[0167] Unless otherwise indicated herein, the temperature unit degrees refers to Celsius degrees, namely C.

[0168] All references that have been referred to in the present application are incorporated by reference in their entirety.

REFERENCES

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