| Background Major Depressive Disorder(MDD)affects millions and thousands of people worldwide and causes severe socioeconomic burden annually.Despite numerous medications for the disease,between one-and two-thirds of patients are unresponsive to the primary pharmacotherapy.In addition,up to 13% of depressed patients never experience sustained remissionafter 5 years’ treatment.These patients are considered to have treatment-resistant depression(TRD),and are often referred for deep brain stimulation(DBS)treatment which has shown significant therapeutic benefits for patients with otherwise treatment-resistant movement(such as Parkinson’s Disease)and affective disorders.Despite its long history of use,however,the therapeutic mechanism underlying DBS remains uncertain.The lateral habenula(LHb)is an epithalamic structure connected to the basal ganglia and limbic system.It participates in cortically driven functions such as the evaluation of motivational and reward values,and the anticipation of motivational events.The LHb has been a hypothetical DBS target that has gained validity in clinical studies.O ne recent animal study demonstrated increased blood monoamine levels after LHb-DBS,which led to the hypothesis that LHb-DBS reversed depressive behaviors by elevating monoamine secretion.However,the hypothesis is controversial with the successful application of DBS for TRD not responsive to monoamine transporter inhibitor(e.g.,fluoxetine),which increase monoamine levels.The result suggests that the hypothesis is insufficient to explain all the phenomenahave been observed.In fact,the patient described above had a history of poor outcomes following monoamine-elevating antidepressant use prior to receiving LHb-DBS.Therefore,non-monoaminergic mechanisms are likely to be involved in reversing depressive symptoms through LHb-DBS.DBS is reported to exhibit a rapid antidepressant effect,unlike the monoamine transporter inhibitors.The rapid antidepressant effects are highly related to the phosphorylation of the mammalian target of rapamycin(m TOR),which contributes to an increase in local dendritic translation of proteins that are essential for dendritic spine synthesis and synaptic remodeling.Thus,activation of the m TOR pathway often induced synaptic potentiation and neuroplasticity.Previous study indicated that synaptic potentiation was a potential mechanism underlying DBS,and has been shown to cause symptomatic improvements.Additionally,biochemical changes such as brain-derived neurotrophic factor(BDNF)secretion and long-term synaptic changes like long-term potentiation(LTP)have been identified during DBS.Notably,the fast antidepressant effects of ketamine are blocked by L-type voltage-dependent calcium channel(L-VDCC)antagonists such as nifedipine;L-VDCC channels are also necessary for activation of m TOR signaling.Thus,we hypothesized that synaptic potentiation related to m TOR activation,and mediated by L-VDCCs,generates the antidepressant effects of LHb-DBS.Aim To study this hypothesis,we used a rodent model of depression to analyze changes in depressive-like behaviors and m TO R activity,and in vitro hippocampal electrophysiology to analyze synaptic potentiation and electrical function,after exposure to LHb-DBS.Research methods 1.To determine the effects of LH training and LHb-DBS a.Learned helplessness(LH)paradigm Male Sprague-Dawley rats(Sun Yat-san University Laboratory Animal Center,4– 7 weeks of age)were trained for the LH experiments.The paradigm contained two sessions.The “training session” consisted of 120 inescapable,uncontrollableelectric foot shocks at 0.8 m A over 40 min in the shocking chambers,with random shock durations and inter shock times ranging from 5 s to 15 s.Total shock duration was 20 min.The “testing session” was conducted 24 h after training.LH was evaluated by an escape task,during which a non-shocking chamber was added next to the shock chamber.This session consisted of 15 escapable shocks with 0.8 m A intensity and 24 s inter-trial intervals.Each shock lasted up to 60 s,but could be terminated by escaping to the non-shocking chamber.Out of the 15 trials,more than 10 escape failures was defined as “learned helplessness”(LH);less than 5 failures as “non-learned helplessness”(NLH).The rats were housed under a 12-h light-dark cycle(light on from 7 a.m.to 7 p.m.)in stable conditions with food and water ad libitum.All animal studies and experimental procedures were approved by the Animal Care and Use Committee of the Institute of Neuroscience,C hinese Academy of Sciences.The sucrose preference test and Forced swim test will be used to identify the efficiency of LH training b.Deep brain stimulation and drug treatments The bipolar concentric stainless steel electrodes(insulated,8 mm in length,0.8 mm tip separation)were unilaterally implanted into the LHb(coordinates [mm]:-3.7 AP,+0.7 ML,-5.4 DV)with a catheter placed into the lateral ventricle(coordinates [mm]:-1.5 AP,±2.0 ML,-3.5 DV).After the 2 week recovery period,all rats in the stimulated group were given four periods of DBS(130 Hz trains separated by 40 ms intervals;150 μA).Each period lasted for one hour and was separated by 24 h(Li B et al,2011).In the drug treatment group,20 μM nifedipine(Abcam,100 m M stored in DMSO)dissolved in artificial cerebrospinal fluid(ACSF)was injected intracerebroventricularly once per day for 4 days before the stimulation procedure;the same volume of vehicle was injected as a control.The electrodes remained implanted in the LHb,but DBS was not provided in the Sham-DBS group.The sucrose preference test and Forced swim test will be used to identify the efficiency of LHb-DBS.2.Western analysisThirty minutes after DBS,the rats were sacrificed for Western blotting.The hippocampus was homogenized by sonication in ice cold lysis solution containing phosphatase and protease inhibitors(PPI,Sigma,Saint Louis,MO)and sample buffer(Laemmli,Sigma).The homogenate was boiled and loaded into 4–12% Bis-Tris gel.After electrophoresis,the gel was transferred onto polyvinylidene difluoride membranes and blocked with 5% nonfat milk powder in TBS containing 0.05% Tween,and probed with antibodies against phosphorylated m TOR(1:2000;Cell Signaling Technology #2971)and GAPDH(1:4000;Beyotime #AG019),at 4°C overnight.After rinses in TBS-Tween,the membrane was incubated for 1 h at room temperature in horseradish peroxidase-conjugated goat anti-rabbit Ig G(1:20000,Abcam #ab97051).The immunoblot was developed with western EC L substrate(BIO-RAD#170-5060).Antibodies against m TOR(1:2000;Cell Signaling Technology #2972)were used to probe the expression of m TO R in another gel.Blot quantitation was performed in NIH Image J software,and densitometry values were normalized with respect to the values obtained with antibody against GAPDH.3.Electrophysiology The newly trained LH rats were sacrificed for brain dissection in ice-cold artificial cerebrospinal fluid(124 m M Na C l,3 m M KCl,1.25 m M Na H2PO4,1.5 m M Mg SO4,2.5 m M Ca C l2,26 m M Na HCO3,and 10 m M glucose)bubbled with 95% O2/5% CO2.Brain slices(400 μm)were cut on a vibratome and kept in a holding chamber at 33 °C at the interface of ACSF and humidified 95% O2/5% CO2 for >1 h.The slices were then transferred to a submersion-type recording chamber and perfused with ACSF at 27±1 °C(flow rate = 1–2 ml min-1).Extracellular recordings of field population excitatory postsynaptic potentials(f EPSPs)were obtained from the dendritic layer and pyramidal cell layer of the CA1 region of the hippocampus,using separate electrodes filled with ACSF(2-3 MΩ)and a 200 b amplifier(Axon,USA).Schaffer collateral-commissural fibers were electrically stimulated with 0.1-0.2 ms constant current paired pulses at a 30 s interval with a bipolar electrode(TM33CCINS,WPI,USA).The intensities for each recording were adjusted to produce an evoked response that was 50-60 % of the maximum EPSP.The average of field EPSPs during an initial control period of the recording was used to determine the initial baseline response(represented as 100% in subsequent figures).To replicate DBS in vitro,another electrode was placed in the slice,directly into the LHb.After establishing a stable baseline,a control input-output(IO)curve was obtained;a second IO curve was obtained after simulating LHb-DBS.Nifedipine(20 μM)was added to the perfusion medium during the stimulation phase to analyze the potential role of L-VDCCs.Hippocampal C A1 pyramidal neurons visualized under a Zeiss microscope were patch-clamped at 27±1 °C with the 200 b amplifier during the simulated LHb-DBS.The pipette resistance was in the range of 3-4 MΩ.For spike current recordings,neurons were clamped in ACSF at-65 m V.Result and discussionThis study demonstrated that LHb-DBS can reverse the depressive-like behavior,likely by synaptic potentiation and enhanced neruoplasticity.It also showed that the antidepressant effect required participation of L-VDCCs.The data hypothesized that LHb-DBS could directly activate peri-electrode astrocytes in addition to neurons,which could subsequently signal further hippocampal astrocytes via Ca2+ waves to increase the astrocyte-released glutamate.The increased glutamate could induce the depolarization needed for L-VDCC-regulated Ca2+ influx to activate phosphorylation of m TOR,and thus improve neuroplasticity. |
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