ORIGINALRESEARCH 
published:12December2018 doi:10.3389/fnmol.2018.00456 
Edited by: 
BoldizsarCzeh, University ofPécs,Hungary 
Reviewed by: 
NashatAbumaria, FudanUniversity,China Vivian B.Neis, FederalUniversity ofSantaCatarina, Brazil MiraJakovcevski, Max-Planck-Institut fPsychiatrie, Germany 
*Correspondence: 
BartłomiejPochwat pochwat@if-pan.krakow.pl GabrielNowak nowak@if-pan.krakow.pl 
Received: 31July2018 Accepted: 26 November2018 Published: 12 December2018 
Citation: 
PochwatB,SzewczykB,KotarskaK, Rafało-Uli´
nskaA,SiwiecM,SowaJE, TokarskiK,Siwek A, BouronA, FriedlandK andNowakG(2018) HyperforinPotentiates Antidepressant-Like Activity of Lanicemine inMice. Front.Mol. Neurosci.11:456. doi:10.3389/fnmol.2018.00456 
HyperforinPotentiates Antidepressant-LikeActivity of LanicemineinMice 
BartłomiejPochwat1*,BernadetaSzewczyk1,KatarzynaKotarska1,AnnaRafało-Uli´, 
nska1 MarcinSiwiec2, JoannaE.Sowa2,KrzysztofTokarski2,AgataSiwek3,AlexandreBouron4, KristinaFriedland5 andGabrielNowak1,3* 
1Laboratory ofNeurobiology ofTraceElements,Department ofNeurobiology, InstituteofPharmacology,PolishAcademy of Sciences,Krakow,Poland, 2Department ofPhysiology, InstituteofPharmacology,PolishAcademy ofSciences,Krakow, Poland, 3Department ofPharmacobiology,Faculty ofPharmacy,JagiellonianUniversityMedicalCollege,Krakow,Poland, 4UniversitéGrenobleAlpes,CNRS,CEA,BIG-LCBM,Grenoble,France, 5Pharmacology andToxicology, Instituteof Pharmacy andBiochemistry,JohannesGutenbergUniversity Mainz,Mainz,Germany 
N-methyl-D-aspartate receptor (NMDAR) modulators induce rapid and sustained antidepressant like-activity in rodents through a molecular mechanism of action that involves the activation of Ca2+ dependent signaling pathways. Moreover, ketamine, a globalNMDARantagonistis apotent, novel, andatypicaldrugthathasbeen successfully used to treat major depressive disorder (MDD). However, because ketamine evokes unwanted side effects, alternative strategies have been developed for the treatment of depression. The objective of the present study was to determine the antidepressant effects of either a single dose of hyperforin or lanicemine vs. their combined effects in mice. Hyperforin modulates intracellular Ca2+ levels by activating Ca2+ -conducting non-selective canonical transient receptor potential 6 channel (TRPC6) channels. Lanicemine, on the other hand, blocks NMDARs and regulates Ca2+ dependent processes. To evaluate the antidepressant-like activity of hyperforin and lanicemine, a set of in vivo (behavioral) and in vitro methods (western blotting, Ca2+ imaging studies, electrophysiological, and radioligandbinding assays) was employed.Combined administration of hyperforin and lanicemine evoked long-lasting antidepressant-like effects in both naive and chronic corticosterone-treated mice while also enhancing the expression of the synapsin I, GluA1 subunit, and brain derived neurotrophic factor (BDNF) proteins in the frontal cortex. In Ca2+ imaging studies, lanicemine enhanced Ca2+ infuxinducedbyhyperforin.Moreover,compound such asMK-2206(Aktkinase inhibitor) inhibited the antidepressant-like activity of hyperforin in the tail suspension test (TST). Hyperforin reversed disturbances induced by MK-801 in the novel object recognition(NOR)testandhadno effects onNMDAcurrentsandbindingtoNMDAR.Our results suggest that co-administration ofhyperforin andlanicemineinduceslong-lasting antidepressant effects in mice and that both substances may have different molecular targets. 
Keywords:depression,NMDA -receptor,hyperforin,lanicemine,TRPC6,ketamine 
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INTRODUCTION 
Chronic intake, delayed onset of action, drug resistance, and numerous side eects of current antidepressants have forced researchers to look for new and safer drugs with rapid onset and longer acting times (Rosenblat et al., 2015; Sanacora and Schatzberg, 2015). Results obtained in recent years have been encouraging, especially with ketamine, a global NMDAR antagonist.The antidepressant-like activity ofketaminehasbeen showninmany preclinical studies(Maeng etal.,2008; Li etal., 2010, 2011; Autry et al., 2011; Gideons et al., 2014; Miller et al., 2014). A single non-anesthetic dose of ketamine reversed the symptoms of MDD (Berman et al., 2000). The antidepressant eects of ketamine were also observed in patients who suered fromtreatment-resistantdepression or suicidalideations(Zarate et al., 2006a; DiazGranados et al., 2010; Murrough et al., 2013; Price et al., 2014). Despite successful clinical trials with ketamine, its use on a large scale is fraught with diyculties andis controversial.Ketamine caninduce unwanted side eects such as psychotomimetic symptoms and cognitive disturbances (Rajagopalet al.,2016). 
Therefore, other NMDAR antagonists have been tested. Unfortunately, clinical trials with low trapping NMDAR antagonists such as lanicemine (Sanacora et al., 2014, 2017) and otherglobalNMDAR antagonistslike memantine(Zarate et al., 2006b) have given inconsistent results and do not show ketamine-like antidepressant potentials. Memantine and lanicemine are also not as eective as ketamine in preclinical studies (Gideons et al., 2014; Qu Y. et al., 2017). However, the data from Preskorn et al. (2008) indicate that NMDA blockade with traxoprodil, a selective antagonist of the GluN2B subunit of the NMDA receptor also produces a robust antidepressant eect. These observations were used to formulate alternative hypotheses that NMDAR blockade could not be the primary molecular mechanism of ketamine action (Zanos et al., 2016, 2017; Collingridge et al., 2017). Moreover, individual enantiomers of ketamine have been shown to activate dierent intracellular signaling pathways with the induction of dierent profles of antidepressant­like activity in mice (Yang et al., 2017, 2018b). Despite all these controversies and doubts surrounding ketamine’s primary mode of action, it has been shown that its antidepressant activity in animals is causally dependent on the enhanced processes of neuroplasticity induced in brain regions like the prefrontal cortex (PFC) and hippocampus (Hp) (Li et al., 2010, 2011; Ardalan et al., 2017). At the molecular level, these processes are dependent on the interplay between glutamate receptors, Ca2+ channels, intracellular Ca2+ levels, Ca2+­dependent proteins like Akt, ERK, mTOR, and neurotrophins such asBDNF(Duman andVoleti,2012;Duman et al.,2016; Workman et al., 2018). The role of Ca2+ homeostasis in the antidepressant activity of ketamine was recently reported by Yang et al. (2018a). They showed that the local blockade of NMDAR by ketamine or blockade of the low voltage-sensitive T-type calcium channels(T-VSCC) by mibefradilinthelateral habenulainduced antidepressant-like eectsin rats(Yang et al., 2018a). 
BecauseCa2+ isthefocalpointto allthe processes mentioned above, we have focused our study on hyperforin, a Ca2+­modulator. Hyperforin is the natural and biologically active compound extracted from Hypericum perforatum (St John’s Wort)(Cervoetal.,2002).BothSt John’sWortandhyperforin attenuated symptoms of mild to moderate depression in several clinical trials and displayed antidepressant-like activity in preclinical studies (Zanoli, 2004). Hyperforin also restored cognitive abilitiesin rats subjectedto chronic stress(Liu et al., 2015). 
From a molecular perspective, hyperforin has a multi­directionalmechanismofaction.Itblocksconductanceofligand-gated(GABA,NMDA, andAMPA receptors)(Chatterjee et al., 1999;Kumar etal.,2006)and voltage-gated channels(Ca2+,K+ , andNa+)(Chatterjeeetal.,1999;Fisunov etal.,2000).Incontrast to blockade of ion transport through the plasma membrane, hyperforin can generate inward Ca2+ currents. In vitro studies have shownthathyperforinincreasesintracellularCa2+ levelsby activating TRPC6 or by releasing Ca2+ from the mitochondria (Leuner et al., 2007;Tu et al., 2009, 2010). Thus, hyperforin is a potent modulator ofintracellularCa2+ levels.Heiser etal.(2013) showed thathyperforinincreased the activity ofRAS/MEK/ERK and PI3K/Akt or CAMKIV intracellular signaling pathways in PC12 cells andhippocampalCA1 neurons. Gibon et al.(2013) also showed that hyperforin enhanced the expression of TrkB (BDNFreceptor), c-AMP responsebinding-protein(CREB) and thephosphorylatedformofCREB(p-CREB)in primary cortical neuronsincludingTrkBinthe cortex of adult mice. 
Ithasbeen shownthatin vitro(Heiser etal.,2013),hyperforin activates Ca2+-dependent signaling pathways involved in neuroplasticity akin to ketamine and NMDAR antagonists, which areknownto activateCa2+-dependent signalingpathways in vivo(Duman andVoleti,2012).In addition,Qu etal. recently described a functional relationship between NMDAR and TRPC6. They showed that TRPC6 expression is regulated by NMDARin vitro(QuZ. etal.,2017).Basedonthis, we askedthe following question: “Can a single dose of hyperforin potentiate antidepressant-like activity a singledose ofNMDAR antagonists in mice andwhatisthe mechanism of action?” 
To answerthisquestion we selectedtwoNMDARantagonists: lanicemine and MK-801. In the frst phase of the study, we determined whether there was an interaction between NMDAR and hyperforin usingTST in naive mice. Secondly, we determinedthe eects ofthe combinedadministration of a single dose of hyperforin and lanicemine in mice exposed to chronic corticosteronetreatment. 
Because hyperforin’s potential to improve cognitive activity has been described elsewhere (Klusa et al., 2001; Liu et al., 2015), in the next part of our study we evaluated hyperforin’s potential to attenuate cognitive defcits induced by MK-801 in the NOR test. This is a very important area of research, because ketamine and other NMDAR antagonists can induce cognitivedefcits(Rajagopal et al.,2016)which are also present in MDD (Lam et al., 2014), Next, we determined whether hyperforin’s antidepressant-like activity wasdependent on select Ca2+signaling pathways and the glutamate system. The last phase of the study was devoted to determining the potential 

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biochemical and electrophysiological mechanisms induced by hyperforin andlanicemine. 
MATERIALSANDMETHODS 


Animals 
Male andfemale adult(9–10 weeks,23–25g) C57BL/6J mice (Charles River)wereusedintheexperiments. Micewerehoused under a natural12hlight/dark cyclein a room with controlled temperature with ad libitum access to water and food. All behavioral experiments were performed between 9a.m. and 2 
p.m. All studies were performed according to the guidelines of the European Community Council (Directive 86/609/EEC) and were approved by the Ethical Committee of the Institute of Pharmacology. 

Compounds andTreatment 
All the compounds/drugs used in the study were obtained commercially excepthyperforin(sodium salt), which was agift from Dr. Wilmar Schwabe GmbH & Co(Karlshrue Germany). Doses of compounds were chosen based on results from our preliminary studies (lanicemine and hyperforin) or from literature(MK-801, fuoxetine,NMDA).Pertinentinformation onthe various compoundsis presentedin Table1. 

CannulaeImplantation 
Forintracerebroventricular(i.c.v.)injection of compounds, mice were anesthetized withketamine(100 mg/kgi.p.) and xylazine (10 mg/kg i.p.; Biowet, Poland) and stereotaxically, bilaterally implantedwith theguide cannulae(8mm);(coordinates relative tobregma:1mmlateral,0.2mm posteriorand3,7mm ventral). After 14 days of recovery, mice were subjected to injection and behavioral studies. Compounds were applied to each ventricle for1min,followedby a1mindiusiontime. Micereceived one injection eachthroughboth ventricles(1 µLper ventricle)using aninfusion cannula(9mm) 15minbefore i.p. hyperforin(2.5 mg/kg) administration.1h afterhyperforintreatmentTST was conducted. 

ChronicExposure toCorticosterone 
MaleC57BL/6Jmice(10 weeks old) whichincluded controls and Cort-treated(Cort) groups werehoused6 per cage. TheCort group received corticosterone(25µg/ml) in drinking water for the frst4 weeks.Corticosterone(SigmaAldrich) wasdissolved in concentratedethanoland addedtothedrinking watertogive a fnal concentration of0.5%.Controls, only received0.5% ethanol in drinking water. All bottles were wrapped in foil and replaced every 3 days. In the 5th week, corticosterone concentration was gradually reduced as follows: Cortgroup received 12.5µg/ml of corticosterone in the frst 3 days followed by 6.25µg/ml in the next3days. 
Animals were Cort free during the next 7 days, after which behavioral tests were carried out. All behavioral tests were performed onthe same mice.Administration ofCortindrinking waterhasbeendescribedinseveral papers(Gourley et al.,2008; Miller et al.,2014;Zanos et al.,2016). 

ForcedSwimTest 
The Forced swim test (FST) was conducted as previously described (Szewczyk et al., 2010). Mice were put individually in aglass cylinder(10cmdiameter,25cmhigh) flled with water (23–25.C) to the height of 10cm for 6min. Following that, immobility time was measured in the last 4min of the experiment. Mice that remained foating passively werejudged tobeimmobile. 

TailSuspensionTest 
TST wasconducted as previously described(Steruetal.,1985). Briefy, eachmouse wasindividually suspendedbythetail, using adhesivetape(2cmfromthetail tip)gluedtoasolidfat surface. Immobility time was measuredfor6min.A mouse wasjudged to be immobile when it was hanging passively and completely motionless. 

LocomotorActivity 
Evaluation of the specifcity of the eects observed in the FST andTST was assessedby locomotor activity(Table2). Plexiglas locomotor activity chambers(40 × 20 × 15cm) in a20-station photo-beam activity system(ColumbusInstruments,Opto-M3­activity meter) were used to measure locomotor activity. Each mouse was placedindividuallyinthe chamberfor a6min session duringwhichthetotal number of ambulations was measured. 

SplashTest 
ASplash test was conducted asdescribedby Yalcin et al.(2008). Briefy, 10% sucrose solution was sprayed on the back of each mouse. The viscous nature of the solution induces enhanced groomingbehavior. Each sessionlasted5min withgrooming behavior measuredaccordingly. 

NovelObjectRecognitionTest 
Thistest wasconducted as previouslydescribed(Wozniak et al., 2016). In brief, training, habituation and test sessions took place in a black plastic rectangular open feld (50 × 30 × 35cm) illuminated by a 25W bulb. Habituation was carried outfor10minfor2daysduring which each mouse was placed individually in the open feld in the absence of objects and allowed to explore the environment. Training and test sessions were performed24h afterhabituation.Inthe frst session(5min) mice explored twoidentical objects(redglass cylinders6.6cm indiameter and4.5cmhigh). Duringthe second session(1h later), the familiar object was replaced by a novel object (a transparent elongated sphere-like object with an orange cap, 5.5cmindiameter,8.5cmhigh). Micewereallowedtoexplore this environmentfor5min.They were administeredhyperforin (30mininterval)followedby MK-801 which wasadministered 30min before the frst training session. Time spent exploring (i.e., sniyng or touching) the familial (T familial) or novel object(T novel) wasmeasuredby atrained observerfollowed by calculationof the recognitionindex[(Tfamilial–T novel)/(T familial+ Tnovel)]. 


WesternBlotting 
Western blotting was conducted as previously described (Szewczyk et al., 2014; Rafalo et al., 2017). Briefy, after 

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TABLE1| Pertinentinformation on the various compounds/drugs. 

Compound Hyperforin Lanicemine MK-2206 MK-801 Larixyl NMDA Fluoxetine sodium salt acetate 
Doses 1,2.5;5;10 2,10 20 0.1;0.3 10 nM/2 µL 75 10 mg/kg i.p. i.p. i.p. i.p. i.c.v i.p. i.p. 
Solvent Aquapro Aquapro 10% Aquapro Phosphate Aquapro-Aquapro injection injection DMSO injection buffer + 1% injection injection DMSO 
Source Wilmar Sigma Selleckchem Sigma Sigma Sigma Selleckchem Schwabe Aldrich Aldrich Aldrich Aldrich 
i.p., intraperitoneal; i.c.v, intracerebroventricular. 
TABLE2| The effect of used compounds andprocedures on thelocomotor activity of mice. 
Treatment mg/kg Locomotor activity (%of control) 
A.  Control  100.0 ± 5.46  
Lanicemine 2  105.1 ± 9.16  
Lanicemine 2 + Hyperforin 1 Lanicemine 10  129.2 ± 13.88 87.77 ± 4.70  
Lanicemine 10 + Hyperforin 5  82.8 ± 12.96  
B.  Control  100.0 ± 4.89  
MK-801 MK-801 + Hyperforin  123.8 ± 15.24 76.44 ± 12.29#  
C.  Control  100.0 ± 7.21  
Hyperforin 2.5 Lanicemine  91.74 ± 8.12 131.1 ± 13.97  
Fluoxetine  104.2 ± 7.87  
Lanicemine + Hyperforin CORT  90.74 ± 12.2 122.9 ±6.45  
CORT + Hyperforin CORT + Lanicemine  90.88 ±9.60 130.2 ± 15.71  
CORT + Fluoxetine  97.94 ± 2.35  
CORT + Lanicemine+Hyperforin  101.6 ± 11.69  
D.  Control  100.0 ± 3.16  
Hyperforin 5 NMDA 75  55.83 ± 5.72** 69.25 ± 8.00*  
NMDA 75 + Hyperforin 5 MK 2206  57.01 ± 11.45** 82.99 ± 6.98  
MK 2206 20 + Hyperforin 5  73.4 ± 6.67*  
E.  Control  100.0 ± 9.60  
Larixyl acetate 10nmol/2ul Hyperforin 2.5 Larixyl acetate 10nmol/2ul + Hyperforin 2.5  84.5 ± 9.51 87.97 ± 21.6 40.31 ± 10.81*  

Locomotor activitywasmeasuredimmediatelyafterTST.*p< 0.05; **p< 0.01vs.Control; #p < 0.05vs. MK-801.DatawasanalyzedbyonewayANOVA (A–D) or twowayANOVA andNewman-Keulsmultiplecomparisons test.Allvaluesareexpressed asmean ± S.E.M. 
(A) 
F(4,20) = 3.719; p = 0.02; (B) F(2,12) = 4.191; p = 0.04; (C) [CORT: F(1,47) = 0.659; p= 0.421; Treatment F(4,47) = 4.476;p = 0.004;InteractionF(4,47) = 0.716;p = 0.585]. 

(D) 
F(5,28) = 5.668;p = 0.001; (E) F(3,13) = 3.766;p = 0.038. n = 4–6. 


decapitation, the frontal cortex was rapidly dissected from each mouse, frozen on dry ice and stored at -80.C. Next, tissue was homogenized in a 2% solution of sodium dodecyl sulfate (SDS), denatured at 95.C for 10min and centrifuged for 5min at 10,000 rpm. The protein in the supernatant was assayed by the bicinchoninic acid method (Pierce). Proteins were fractionated on a 10 or 12% SDS polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad). Non-specifc binding was blocked by 1% blocking solution (BM ChemiluminescenceWesternBlottingKit Mouse/Rabit, Roche). Following blocking, membranes were incubated overnight with the following antibodies: BDNF (~15 kD, 1:500, Monoclonal, SantaCruz), phosphorylatedCREB(p-CREB);(~43kD,1:1,000, monoclonal, Millipore), GluA1-AMPA (~100 kD, 1:1,000, polyclonal, Abcam), Synapsin I(~74 kD, 1:1,000, monoclonal, Abcam,), total CREB (~43 kD, 1:1,000. monoclonal, Santa Cruz), b-actin (~42 kD, 1:10,000, monoclonal, Sigma). The nextday, membranes were washed(3 times) withTris-buered saline containing Tween(TBST) andincubated with secondary mouse/rabbit antibodies (1:7,000, Roche). Finally, blots were washed 3 times in TBST and incubated in the detection reagent (Roche). The signal from each protein was measured and visualized using the Fuji-Las 1000 system and Image Gauge v 4.0. b-actin was used as a loading control and for normalization(FigureS4). The densities of the bands obtained for phosphorylated and total CREB were frst normalized to appropriate actin bands and then the ratio of normalized p-CREB/CREB was calculated. Data on thegraph are expressed as %of change vs. control. 


WholeCellPatchClampStudies 
Brain Slice Preparation for Patch Clamp Experiments 
Mice were decapitated under isofurane anesthesia (Aerrane, Baxter) between 9 and 10a.m. Brains were quickly removed and placed in ice-cold artifcial cerebrospinal fuid (aCSF) containing(in mM):130NaCl,5KCl,2.5CaCl2,1.3MgSO4, 
1.25 KH2PO4, 26 NaHCO3, and 10 D-glucose. Coronal slices (300µm) containing the mPFC were cut using a vibrating microtome (Leica VT1000) and subsequently incubated in carbonated aCSF at30 ± 0.5.Cfor atleast1hbefore recording. 
Whole-Cell Patch Clamp Recording 
To record NMDA currents, slices were placed in the recording chamber superfused at 3 ml/min with warm (32 ± 0.5.C), modifed Mg2+-free aCSF with the following composition (in mM) 132 NaCl, 2 KCl, 2.5 CaCl2, 1.25 KH2PO4, 26 NaHCO3, 10 D-glucose, continuously bubbled with a mixture of 95% O2 and 5% CO2. NBQX (5µM) was added to block AMPA/kainate receptors. Neurons were visualized using a Zeiss AxoExaminer.A1 upright microscope equipped with IR DIC 

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optics, a 40× water immersion lens and an infrared camera (Sony). Patch pipettes pulled from borosilicate glass capillaries (HarvardInstruments)using aSutterInstrumentP97 pullerhad open tip resistances of approximately 3–5 M when flled with a solution containing (in mM): 130 K-gluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 10 HEPES, 5 Na2-ATP, 0.4 Na-GTP, and 1 EGTA.Osmolarity and pH were adjusted to290 mOsm and7.2, respectively. Signals were recorded with the Multiclamp 700B amplifer(MolecularDevices), flteredat2kHzanddigitized at20kHz using theDigidata1,550interface andpCLAMP10 software(MolecularDevices). 
Stimulation and Recording of NMDA Postsynaptic Currents 
Synaptic NMDA currents were evoked using a concentric platinum/stainless steel electrode (FHC, USA) by stimulating (every30s,duration0.2ms)layerVofthe mPFCwhile recording from mPFC layer II/III pyramidal neurons in a voltage clamp modewith aholdingpotential of -70mV.Stimulationintensity was adjusted so that a stable NMDA current of approximately 100pA could be recorded for at least 10min before drug application. 
Currents were percentage-normalizedwith referenceto5min of the recording right before drug application. Percentage of the response was measured by averaging 5 consecutive currents after10min ofdrug application. Atthistimethe responseto lanicemine (2µM) treatment plateaued while the response to hyperforin(0.5µM) remained stable. 


ExtracellularField PotentialRecordings 
Mice were decapitated, their frontal cortices were dissected and cut into 400 µm-thick slices which were stored in a gassed (95% O2 and5% CO2) ACSF, consisting of (in mM): 127 NaCl, 5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 KH2PO4, 24NaHCO3, and 10glucose. Individual slices were placedin the recording chamber of an interface type which was superfused 
(2.5 ml/min) with a modifed ACSF, (temperature 32.0 ± 0.5.C) containing (in mM) NaCl (132), KCl (2), CaCl2 (2.5), KH2PO4(1.25),NaHCO3(26), andD-glucose(10),bubbledwith 95% O2 and 5% CO2 (temperature 32.0 ± 0.5.C). To study the NMDA receptor-mediated component of feld potentials (FP), the slices were perfused with ACSF devoid of Mg2+ ions and supplemented with 5µm of 2,3-dioxo-6-nitro-1,2,3,4­tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX, Tocris Bioscience), anAMPAreceptor antagonist. 
Aconcentricbipolarstimulating electrode(FHC, USA) was placed in cortical layer V. The recording electrode was placed inlayersII/IIIofthe mPFC(prelimbic cortexregion).Stimuli of 0.033Hz frequency and duration of 0.2ms were applied using a constant-current stimulus isolation unit (WPI). Glass micropipettes flledwithACSF(2–5M)were used to record feld potentials. The responses were amplifed(EXT 10–2F amplifer,NPI), fltered(1Hz-1kHz),A/D converted(10kHz sampling rate),and stored onPC using the Micro1401interface andSignal4 software(CED). 
After the 30min, of incubation and after stabilizing the responses, a stimulus was adjusted to evoke a response of 40% of the maximum amplitude.After15min ofbaseline recording a hyperforin 1 uM was added(20min) and recording was continuedfor70min. 

CalciumImaging 
Primary cultures of cortical neurons were prepared from embryonic(E13)C57BL6/Jmice as previouslydescribed(Bouron et al., 2005) and approved by the animal care committee of the CEA’s Life Sciences Division (CEtEA). Experiments were conducted in accordance with French legislation and the European Community Council Directive of 24 November 1986 (86/609/EEC). Calcium imaging experiments were performed at room temperature as previously described (Gibon et al., 2013; Chauvet et al., 2015, 2016). Briefy, corticalneurons were incubatedin a saline solution containing(mM)150NaCl,5KCl, 1MgCl2,2CaCl2,5.5glucose,10HEPES(pH7.4) supplemented with5µMFluo4/AM.After20min ofincubation, neurons were rinsedtwiceandincubatedfor10mininaFluo-4/AM-freesaline. The imaging system consisted of an inverted Axio Observer A1 microscope equipped with a Fluar 40× oil immersion objective lens(1.3NA)(CarlZeiss,France) andaCCDCoolSnapHQ2 camera(PrincetonInstruments, RoperScientifc,France).ADG­4wavelength switcher(PrincetonInstruments, RoperScientifc, France) was used with lEX = 470nm and lEM = 525nm. Thesetup wasdrivenby MetaFluor(UniversalImaging, Roper Scientifc,France). 


RadioligandBindingStudies 
Radioligand binding assay was performed according to the method of (Fischer et al., 1997) with slight modifcations. Binding experiments were conducted in 96-well microplates in a total volume of 300 µL. The reaction mix included 30 µL amounts of test compounds, 30 µL of radioligand and 240 µL oftissue suspension. Tissue(rat cortex) washomogenized in50 volumes ofice-cold50mMTris-HClbuer with10mM ethylenediaminetetraacetic acid, pH 7.4 using an Ultra Turrax T25B(IKA) homogenizer.Thehomogenate was centrifuged at 35,000 × gfor15min.Theresulting pelletwasresuspendedin the same quantity of buer and centrifuged two more at the same speed and frozen at -80.Cfor atleast16h and not more than 2 weeks. For binding experiments, the membranes were washedthreetimes(homogenizationin25volumes ofcold5mM Tris-HCl (pH 7.4) with an Ultra-Turrax at maximum speed for 30s). The fnal pellet was resuspended in an appropriate volume ofbuer(10 mg/1ml) for useinthe assay.Incubation was performed in the presence of 10µM added glutamate and glycine.Theligand,[3H]-MK-801(spec. act.40Ci/mmol,Perkin Elmer) wasused ata fnal concentrationof5nM.Non-specifc bindingwasdeterminedinthe presence of10µMMK-801.After a 2 h-incubation at room temperature, the reaction mix was fltered immediately onto GF/B flter mate. Ten rapid washes were performed with chilled 5mM Tris pH 7.4 buer, using an automated harvesting system Harvester-96 MACH III FM (Tomtec, USA).Filter mates weredriedat37.Cin aforcedairfan incubatorCLW32STD(Pol-EkoAparatura,Poland) andthen solid scintillator was melted onto the flter mates at 100.C for 5min. The radioactivity retained on the flter was counted in a 

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MicroBeta2LumiJET scintillationcounter(PerkinElmer, USA). The compounds analyzedrangedin concentrationfrom10-10 to 10-3M.All assays weredoneinduplicates. 


StatisticalAnalysis 
Statistical analysis of behavioral and Western blot studies was performed using GraphPad Prism. We used one or two way ANOVAwhen appropriate,followedbytheNewman-Keuls post-hoctest.Statistical signifcance was set at p< 0.05. 
Analysis of recorded NMDA currents was carried out using the pCLAMP 10 and Graphpad Prism software programs. The obtained NMDA current values were analyzed using a one-sample Wilcoxon Signed Rank Test against 100% separately for hyperforin-andlanicemine. 
Radioligandbindingdata were analyzed usingiterative curve ftting routines GraphPAD/Prism – San Diego, CA, USA). Ki values were calculated from the Cheng and Pruso (1973) equation. 
Calciumimagingdata were analyzedbyStudent’st-test. 
Extracellular feld potential recordings were analyzed by Wilcoxon matched-pairs signedranktest. 
RESULTS 

Hyperforin PotentiatestheEffectsof Lanicemine andMK-801in theTST 
As mentioned earlier, the functional relationship between NMDAR and TRPC6 receptor has been described previously (Qu Z. et al., 2017). Hyperforin and NMDAR antagonists’ antidepressant activity have also been previously reported (Zanoli, 2004; Sanacora and Schatzberg, 2015). Both hyperforin and NMDAR antagonists activate similar Ca2+-dependent intracellular processes(Duman andVoleti,2012;Heiseretal., 2013). Therefore, we decided to investigate whether the combined administration of hyperforin and lanicemine or MK­801 can providesomebenefcial antidepressanteects(stronger eects or long-lasting activity). We choose lanicemine and MK­801 two structurally dierent NMDAR antagonists. In this part of our studies, we examined the antidepressant-like activity of single activedoses ofhyperforin(2.5 and5 mg/kg),lanicemine (10 mg/kg) and a combined administration ofhyperforin(2.5 or 5 mg/kg) and lanicemine (10 mg/kg) at three dierent time points; 1, 24, and 72h after treatment. Additionally, we evaluated the eects of the combined administration of non-active-doses ofhyperforin(1 mg/kg) andlanicemine(2 mg/kg) 1h after treatment. Both at the 1 and 24h time points all drugs evoked antidepressant activity(Figures1A–C). Also, the administration of a combined non-activedose oflanicemine(2 mg/kg) and non-active dose of hyperforin (1 mg/kg) induced antidepressant-like activity after1h(Figure1B).Atthe72htime point, hyperforin and lanicemine administered alone did not evoke any antidepressant eects,but a combined treatment with hyperforin and lanicemine signifcantly reduced the immobility timeintheTST(Figure1D). 
In our preliminary studies combined treatment with hyperforin (2.5 mg/kg) + lanicemine (10 mg/kg) signifcantly reducedtheimmobilitytimeinfemale miceboth atthe1h(TST) and72h(FST)time points(FigureS1). 
The eects observed in the TST were not associated with increasedlocomotor activity(Table2A). Moreover, hyperforin 
(2.5 mg/kg) potentiated short-lasting antidepressant eects inducedby activedoseof MK-801(0.1mg/kg)(Figure1E). A single dose of MK-801 did not evoke any eects in the TST 72hafter administration.In contrast,a combinedadministration of hyperforin and MK-801 evoked an antidepressant response (Figure1F). MK-801 and hyperforin alone had no eect on locomotoractivity.However,acombinedadministrationofMK­801andhyperforindecreasedlocomotoractivitycomparedtothe MK-801group(Table2B). 

CombinedTreatmentof aSingleDose of Hyperforin andLanicemineReversed BehavioralDisturbancesinMiceInduced byChronicCorticosteroneAdministration 
Because the TST is a simple behavioral screening test, in next phase of the studies we wanted to fnd out if the benefcial antidepressant-like activity of hyperforin and lanicemine seen in the TST also occurs under complex conditions such as those evoked by chronic corticosterone administration which mimics stressful conditions where corticosterone is released in response to stressful stimuli(Wilner,2017).Thehypothalamic pituitary adrenal (HPA) axis is the central stress response system. Disturbances in the functioning of the HPA axis have been described in MDD patients and in mice subjected to chronic stress(Keller et al.,2017; Wilner,2017).We examined the eects of combined administration of active doses of hyperforinandlanicemine,lanicemine,hyperforin,or fuoxetine given to control and Cort-treated mice. Doses of hyperforin and lanicemine were selected on the basis of the respective activity of each drug in the TST in naive mice. Single doses of hyperforin(2.5 mg/kg),lanicemine(10 mg/kg), fuoxetine(10 mg/kg) and lanicemine (10 mg/kg) + hyperforin (2.5 mg/kg) were administered 7 days after Cort withdrawal. The TST and splash test were conducted 72 and 144h, respectively, after drug administration (Figure2A). Cort-treated mice displayed increased immobility times compared to controls in the TST (Figure2B). Administration of hyperforin, lanicemine, and fuoxetine did not reverse these eects. However, a combined treatment with hyperforin and lanicemine abolished these eects(Figure2B). In the splashtest, Cort-treated mice showed decreased grooming time compared to controls (Figure2C). This depressive-like behavior was reversed only by a combined treatment of hyperforin and lanicemine (Figure2C). Next, 9 days(216h) after drug administration, FST was performed (Figure2D). We observed increased immobilitytimes in Cort­treated mice and no eects of the antidepressant treatment strategies(Figure2D).Eects observedintheTSTandFST were specifc because changes in locomotor activity were not noticed (Table2C). 


HyperforinReversedMK-801Induced Effectsin theNovelObjectRecognition Test 
NMDAR antagonists, including ketamine, have been shown toinduce some cognitiveimpairment(Rajagopal et al.,2016). 

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FIGURE1| The effect of combined administration of a singledose ofhyperforin(Hyp) andNMDAR antagonists:lanicemine(Lan) andMK-801inTSTin mice. *** 
(A,B)Lanicemine(2 or10 mg/kg; i.p.) was administered90minbefore theTST and30minbeforehyperforin(1,2.5, or5 mg/kg; i.p.) treatment. p < 0.001 vs. control, ** p < 0.01 vs. control, * p < 0.05 vs. control, #p < 0.05 vs.lanicemine10 + Hyp5, n = 7–8;[A: F(3,22) = 10.65; p = 0.0002],[B: F(3,22) = 7.405; p = 0.0013]. (C,D)Hyperforin andlanicemine were administeredin the same way asin (A,B)but theTST was carried out24h (C)or72h (D)afterhyperforin 
** 

treatment. p < 0.01 vs. control, * p < 0.05 vs. control, n = 7–8;[C: F(5,38) = 4.376; p = 0.003],[D: F(5,39) = 5.063; p = 0.0011]. (E,F)MK-801(0.1 mg/kg; i.p.) 
was administered30minbeforehyperforin(2.5 mg/kg; i.p.),1h (E)or72h (F)afterhyperforintreatmentTST wascarried out. *** p < 0.001 vs. control, ** p < 0.01 vs. control, #p < 0.05 vs.MK-801, n = 7–8;[E: F(2,21) = 17.20; p = 0.0001],[F: F(2,19) = 3.696; p = 0.0441].Alldata was analyzedby one-wayANOVA and Newman-Keuls multiple comparisons test.Values are expressed as mean ± S.E.M. 
Cognitivedefcitshavealsobeenidentifedinpatientsdiagnosed potential antidepressant.Theprocognitiveeectsofhyperforin withMDD(Lametal.,2014). Therefore,theimprovementof havebeenpreviouslyreported(Klusaetal.,2001;Liuetal., cognitive function is a very desirable feature for any novel or 2015). In the present studies, a single dose of hyperforin 
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FIGURE2| Theeffect of theadministrationof singledosesofhyperforin(Hyp),lanicemine(Lan), fuoxetine(Flx),andhyperforin + lanicemine(Hyp + Lan) on behavioraldisturbancesinducedby chronic corticosterone(Cort) treatment n = 9–11.Singledoses of thefollowingdrugs:hyperforin(2.5 mg/kg; i.p.),lanicemine(10 mg/kg; i.p.), fuoxetine(10 mg/kg; i.p.) were used. (A)Experimental schedule ofdrug treatments andbehavioral tests; (B)TST was carried out72h after treatment. ***p < 0.001 vs. control, ###p < 0.001 vs. cort.Two-wayANOVA showed cort effect[F(1,91) = 24.42; p = 0.0001], treatment effect[F(4,91) = 4.55; p = 0.002], 
andinteraction effect[F(4,91) = 3.248; p = 0.015]. (C)Splash test(SPT) wasdone144h aftertreatment. *p < 0.05 vs. control, #p < 0.05 vs. cort.Two-wayANOVA showedCort effect[F(1,91) = 12.725;p = 0.00057], no treatmenteffect[F(4,91) = 1.252; p = 0.294]andinteraction effect[F(4,91) = 3.058;p = 0.0205].(D)FST was carried out216h aftertreatment. *p < 0.05 vs. control, two-wayANOVA showed cort effect[F(1,91) = 4.398; p = 0.03875], no treatment effect[F(4,91) = 1.84; p = 0.24578] andinteraction effect[F(4,91) = 3.412; p = 0.012].Alldata was analyzedby two-wayANOVA andNewman-Keuls multiple comparisons test.All values are expressed as mean ± S.E.M. 
(2.5, 5, 10 mg/kg) reversed cognitive disturbances in mice as measured by the cognitive index evoked by a single dose of MK-801(0.3 mg/kg) intheNORtest(Figure3A). Hyperforin, administered alone did not have any eects on the recognition index(Figure3B). 


Pretreatment WithN-Methyl-D-Aspartic AcidAbolishedAntidepressant-Like Activity ofHyperforin 
A few in vitro studies have shown that hyperforin can block the NMDAR (Chatterjee et al., 1999; Kumar et al., 2006). Thus, we pretreated mice with NMDA and monitored them for changes in the behavioral response to hyperforin in the TST. Figure4 shows that hyperforin (5 mg/kg) induced antidepressant-like activity in the TST which was abolished by the pretreatment with NMDA (75 mg/kg/body weight). The NMDA dose used was selected based on previous studies (Poleszak et al., 2007; Wolak et al., 2013). Also, NMDA/CNS related responses after i.p. administration have beendescribedby Budziszewska et al.(1998).Hyperforingiven alone and in combination with NMDA decreased locomotor activity in comparison to control group. Therefore, decreased locomotor activity is not related to NMDA co-administration (Table2D). 


Administration ofLarixylAcetate and MK-2206AbolishedEffects ofHyperforin Observedin theTST 
Results obtained from previous in vitro studies indicated that hyperforin-induced cellular eects are Ca2+-dependent(Leuner et al., 2007; Tu et al., 2009, 2010), thus we wanted to fnd out whether Ca2+-dependent processes are involved in the behavioral response of hyperforin. In particular, TRPC6 channels and AKT kinase are thought to be involved in these eects. Preinjection with larixyl acetate a potent TRPC6 

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FIGURE3| The effect ofpretreatment withglutamate system modulator onhyperforin activityin theNORin mice. (A,B)Hyperforin(2.5,5,10 mg/kg; i.p.)was administered60minbeforethe frst session.MK-801(0.3 mg/kg; i.p.)wasadministered30minbeforethe frsttraining sessionof theNOR test. ###p < 0.001 vs. control, ***p < 0.001 vs.MK-801, n = 6–8;[A: F(4,32) = 70.04; p = 0.0001],[B: F(4,30) = 36.08; p = 0.0001].Alldata was analyzedby one-wayANOVA and Newman-Keuls multiple comparisons test.All values are expressed as mean ± S.E.M. 

antagonist(Urban et al.,2016)(10nM/2 µL) and pretreatment with Akt 1/2/3 kinase inhibitor -MK-2206 (Cheng et al., 2012) (20 mg/kg) abolished the hyperforin induced eects in the TST (Figures5A–C). However, these results should be interpretedcarefullybecausehyperforin+ larixylacetateinduced decreased locomotor activity when compared to hyperforin group(Table2E). 


HyperforinDoesNotAffectNMDAR SynapticCurrents andNMDAComponent of theField Potential 
Some studies have shown that hyperforin is able to infuence NMDARfunction(Chatterjee et al.,1999; Kumar et al.,2006). Thus, our next set of studies was focused on evaluating the eects of hyperforin and lanicemine on NMDAR-mediated synaptic currents resulting from electrical stimulation. As we described earlier, the behavioral eects of hyperforin were abolished by pretreatment with larixyl acetate and MK-2206 (TRPC6 and Akt 1/2/3 kinase inhibitors, respectively). Thus, for patch clamp studies, hyperforin’s concentration (0.5µM) was selected based on its potential to activate TRPC6 receptors 
(0.3 to 10µM) (Tu et al., 2010; Heiser et al., 2013; Leuner et al.,2013).Electrophysiologicalstudies showedthatlanicemine (2µM) signifcantly attenuated the NMDAR current while hyperforin had no eect (Figure6). Moreover, there was no eect of acute hyperforin (1 µM) administration on NMDA receptor component amplitude (101% vs. 96%, p > 0.2) (FigureS2). 


TheAffnityofLanicemine andHyperforin forNMDAReceptorChannel 
The radioligand receptorbinding studies(3H-MK801 asligand) demonstrated no aynity for NMDA receptor channel of hyperforin In contrast lanicemine shows aynity for NMDAR receptors(Ki = 1.067× 10-5)(FigureS3). 

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TheEffects ofHyperforin,Lanicemine, and CombinedAdministration ofHyperforin andLanicemine on theExpression of SelectedProteinsintheFrontalCortex 
The antidepressant-like activityofNMDAR antagonistsis related tothe enhancedsynthesis ofsynaptic proteins and neurotrophins (Duman etal.,2016).Hyperforin can activate signalingpathways involvedinthe processes ofneuroplasticity in vitro(Heiser etal., 2013).Therefore, wedeterminedthelevels of theGluA1 subunit ofAMPA receptors, synapsinI,BDNF, andCREB aftertreatment of mice with eitherhyperforin orlanicemine or a combination of both drugs(Figures7A,B,G,H).This treatmentstrategydid not signifcantly alter p-CREB/CREBlevels(Figure7C)butelevated 

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BDNF (Figure7D)inthefrontal cortex(1h afterhyperforin and 1.5h after lanicemine administration). A single dose of either hyperforin or lanicemine did not alter the expression of synapsinI andGluA1(Figures7E,F).In contrast, the combined administration ofhyperforin andlanicemineincreasedthelevels of both proteins (Figures7E,F). After 72h, a combination of hyperforin and lanicemine did not alter p-CREB/CREB levels (Figure7I); however, lanicemine and hyperforin+ lanicemine signifcantly increased the levels of BDNF in the test animals compared to controls(Figure7J). The levels of other proteins (synapsinI,GluA1) remained unchanged72h aftertreatment (Figures7K,L). 


Lanicemine PotentiatesIntracellularCa2+ ResponsesTriggeredbyHyperforin 
To better delineate the eects of lanicemine, Ca2+ imaging experiments were carried out on primary cultures of cortical neurons loaded with the fuorescent Ca2+ probe Fluo4. The external application of hyperforin (1µM) generated specifc Ca2+ responses as illustratedin(Figure8)(horizontalgraybar) (Chauvet et al., 2015, 2016). In some instances, lanicemine (5µM) was added frst(Figure8, horizontal hatched bar). This had no eect on the basal Fluo-4 fuorescence, suggesting that basal levels of Ca2+ were not perturbed. However, hyperforin­induced Ca2+ responses were increased by a 4min pre­incubation withlanicemine. 


DISCUSSION 
A rapid onset of action and long-lasting activity after a single dose of a drug are the most desired features sought for in novel antidepressants. These eects have been observed in rodents after treatment withketamine andits metabolite,2R,6R­hydroxynorketamine (Li et al., 2010, 2011; Autry et al., 2011; Zanos et al., 2016; Yang et al., 2017). A similar profle of antidepressant-like activity has also been described for a few NMDARmodulators(Ro25-6981 andGLYX-13)(Liet al.,2010, 2011;Liu etal.,2017). 
In the present study, we showed that a combination of a single dose of hyperforin and the NMDAR antagonist lanicemine evoked long-lasting antidepressant eects 72h after administration in both naive and chronic Cort-treated male mice. Additionally, in our preliminary study, we also observed the long-lasting antidepressant-like eects of hyperforin+lanicemine in naive female mice. It seems that this interaction is not exclusive to lanicemine and hyperforin because the co-administration of hyperforin and MK-801, another NMDAR antagonist also induced the same long-lasting antidepressant-like responses in the TST in naive mice. Lanicemine, MK-801, and hyperforin by themselves did not elicitlong-lasting antidepressant eects after72h.It shouldbe mentionedthat we selected only a singledose each oflanicemine and MK-801; it is thus possible that the induction of the long-lasting antidepressant eects may be dose-dependent. As reported previously, the exceptional profles of antidepressant­like activity of ketamine and NMDAR modulators are related to the immediate activation of molecular processes that lead to enhanced neuroplasticity mainlyin theHp andPFC(Li et al., 2010, 2011; Ardalan et al., 2017). These two brain structures are functionally impaired in MDD (Duman and Voleti, 2012; Duman et al., 2016). Fast and long-acting antidepressants enhanced the expression of synaptic markers like synapsin I or theGluA1 subunit ofglutamateAMPA receptors(Liet al.,2010; Liu etal.,2017).In our study,hyperforin orlaniceminetreatment did not alter the levels of synapsin I and GluA1 subunit in the frontal cortex of mice. However, a combined administration of the two compounds elevated the levels of synapsin I and GluA1 subunit after 1h. Enhanced expression of GluA1 and synapsin I was not sustained72h afterhyperforin + lanicemine treatment. These results maysuggestthatelevatedsynaptic protein synthesis is not involved in the long-lasting eects induced by hyperforin 
+ 
lanicemine. 

Another well-documented explanatory hypothesis of the mechanism of action of long-acting drugs is related to the enhanced activation of the BDNF signaling pathway. Results obtained from several studies have indicated that BDNF plays an important role in the mechanism of action of long-acting antidepressants. Autry et al. (2011) and Zanos et al. (2016) showed that a single dose of ketamine or its metabolite 2R,6R-hydroxynorketamine increased the synthesis of BDNF in the mouse Hp. Both ketamine and GLYX-13 have been shown to induce the release of BDNF in cortical neurons(Lepack et al.,2014,2016).Wefoundthathyperforin, lanicemine, and hyperforin + lanicemine enhanced BDNF expression 1h after administration. Surprisingly, while only combined doses of lanicemine and hyperforin induced long-lasting antidepressant eects, lanicemine alone and lanicemine 

+ 
hyperforin enhancedBDNFlevels72h after administration. Because lanicemine similar to hyperforin + lanicemine also enhancedBDNF synthesis72h aftertreatment,these results may suggest that the enhanced synthesis of BDNF is not suycient to explain the long-lasting antidepressant eects observed only after hyperforin+lanicemine treatment. As indicated in a few other studies, the relationship between BDNF synthesis and antidepressant-like activity may be more complicated than originally thought (Song et al., 2017). There are indications that BDNF may be synthesized in dierent parts of neural cells (glia vs. neurons or presynaptic vs. synaptic elements). These distinctions may underlie the biological role of BDNF in the neural system (Song et al., 2017). Therefore, it is possible that the signifcance of BDNF in the antidepressant­like activity of these compounds is not only related to its levels but also related to the site of synthesis. We should also mention that hyperforin is an agonist of TRPC6 receptors. Zhou et al. (2008) described the relationship between BDNF signaling pathway and TRPC6 receptors and showed that TRPC6 receptors mediated the infuence of BDNF on spine formation in rat hippocampal cultures. The involvement of TRPC6receptorsinthe antidepressant-like activity ofhyperforin wasimplicitlyrevealedin ourbehavioral studies.We showedthat pretreatment withlarixyl acetate(TRPC6 antagonist) abolished the antidepressant-like eectsinducedbyhyperforinin theTST. Also, pretreatment with MK-2206 (Akt1/2/3 kinase inhibitor) 



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FIGURE7| lanicemine(10 mg/kg) was administered90min andhyperforin(2.5 mg/kg)60minbeforeTST. (G)Experimental schedule ofdrug treatments. (H)Representativeblots. (I–L)lanicemine was administered72.5h andhyperforin72hbeforeTST.Frontal cortex wasdissectedimmediately afterTST. (C)p-CREB/CREB[F(3,28) = 0.4795; p=0.6992]. (D)BDNF,[F(3,28) = 4.886; p = 0.0074]. (E)Synapsin I,[F(3,24) = 3.573; p = 0.028]. (F)GluA1[F(3,28) = 3.656, 
$

p = 0.0243]. *p < 0.05 vs. control, **p < 0.01 vs. control, #p < 0.05 vs.Hyp + Lan, p < 0.05 vs.Hyp + Lan.Effects observed72h after treatment: (I) p-CREB/CREB,[F(2,20) = 2.734; p = 0.0892]. (J)BDNF[F(2,21) = 3.764; p = 0.0401]. (K)Synapsin I,[F(2,21) = 0.2062; p = 0.8153]. (L)GluA1,[F(2,21) = 0.3509; p = 0.7081]. *p < 0.05 vs. control.Alldata was analyzedby one-wayANOVA andNewman-Keuls multiple comparisons test.All values are expressed as mean ± S.E.M. 
completely abolishedhyperforin’s eects observedin theTST.In other words, pharmacologicalblockade ofTRPC6 and one ofits mainintracellular eectorsAktkinaseinhibitsthe antidepressant eects of hyperforin induced in mice. These results are in agreement with in vitro studies which have shown that the biologicaleects ofhyperforin are relatedtothe activation ofthe TRPC6 channel(Leuner et al.,2007;Tu et al.,2010)followedby a subsequent activation of theAktkinase(Heiser et al.,2013). Therefore, it is possible that the antidepressant-like activity of combined doses of hyperforin + lanicemine is dependent on crosstalk between the blockade of NMDAR, activation of TRPC6 receptors, and BDNF pathways. Additionally, (Zhou et al., 2008) showed that TRPC6 regulates synaptic plasticity through the CREB signaling pathway. It is also well-established thatthe synthesis ofBDNFisdependent on the phosphorylation ofCREB(Nibuya etal.,1996).Sointhe nextstep of our study, we evaluatedthe eects oftreatments on p-CREB/CREB ratiolevels. We only observed a trend toward an increase in the levels of p-CREB/CREB ratio1h afterhyperforin + lanicemine treatment. Thus,theinvolvementofCREBinthebiosynthesisofBDNFafter hyperforin + lanicemine requires further detailed and precise studies. 
Second possible hyperforin’s antidepressant mechanism of action reported previously is related to blockade of NMDAR (Kumar et al., 2006). Whole cell patch clamp studies showed thathyperforin at 0.5µM concentration did not change cortical NMDAR currents. Moreover, there was no eect of hyperforin (1µM) administration on the NMDA component of the feld potential. Furthermore, radioligand binding studies indicated that hyperforin had no aynity for the 3H-MK-801 labeled site of the NMDAR. Thus, in these particular conditions hyperforin does notinterfere withNMDAR.Concomitantly, wehave shown that pretreatment with NMDA abolished antidepressant-like activityofhyperforininTST.ToanswerthequestionwhyNMDA administrationblocksthe antidepressant-like eect ofhyperforin inTSTin mice requiresfurtherinvestigation. 
Because the biosynthesis and release of synaptic proteins like GluA1 and synapsin I, and BDNF and their release is strictly coupled to the activation of cellular processes that require elevated levels of intracellular Ca2+ (Duman et al., 2016; Finkbeiner, 2016)we sought to fnd out if the combined administration of hyperforin and lanicemine evoked dierent intracellular Ca2+ responses than the administration of either lanicemine or hyperforin alone. Calcium imaging studies revealed that lanicemine potentiated hyperforin-induced Ca2+ signals.However, the mechanismby whichlanicemine regulates hyperforin-dependentCa2+ responsesis currently unknown and requiresfurther studies. 

To summarize, the results obtained in this study showed that treatment with a single active dose of hyperforin and lanicemine induced a long-lasting antidepressant-like activity in mice. These eects were observed in both naive and corticosterone-treated male mice. Hyperforin + lanicemine eects were sustained for up to 6 days after treatment. Interestingly,our preliminary studies also showedthat combined single doses of hyperforin + lanicemine also induced long-lasting eects in naive female mice in the TST. The induction of antidepressant-like eects in both male and female mice is a very desirable feature of any potential, novel antidepressant because sex-related responses to antidepressant therapy have been postulated frequently (Khan et al., 2005). The potential beneft of administration hyperforin + lanicemine is associated with hyperforin’s potential to improve cognitive activity. Because hyperforin attenuates cognitive disturbances induced by MK-801 it can be administered with other NMDAR antagonists which induce cognitive impairments. Furthermore, MDD with cognitive dysfunction has been described (Lam et al., 2014). Therefore, combined doses of hyperforin + laniceminecanbeauseful potential strategyinthetreatment of depressionincluding cognitivedisturbances.Anotherimportant question is what kinds of biological mechanisms are involved in hyperforin + lanicemine induced mechanism of action. 

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Calcium imaging studies have shown that the biological interaction between hyperforin + lanicemine is a Ca2+­dependent process. Our biochemical and behavioral studies indicated that the main mechanism of action can be associated with crosstalk between the TRPC6 receptor and the BDNF signaling pathway. However, further studies are required to better understand the biological foundations of these interactions. 


AUTHORCONTRIBUTIONS 
BP and GN designed the studies. BP, BS, AR-U, and KK performed behavioral studies. MS, JS, and KT conducted electrophysiological studies. AB performed calcium imaging studies. BP and BS performed surgical procedures. BP determined protein expression by Western Blotting. AS conducted radio ligand binding studies. BP, KF, and GN analyzed the results. BP, BS, and GN wrote the manuscript. 

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FUNDING 
This study was supportedby thegrantHYPZITRP(ERA-NET­Neuron/11/2014) and the Statutory activity of the Institute of Pharmacology Polish Academy of Sciences and Jagiellonian UniversityMedicalCollegeinKrakow. 

ACKNOWLEDGMENTS 
The authors thank Dr. Willmar Schwabe GmbH & Co. K, Karlsruhe, Germany for the generous gift of hyperforin. We would also like to thank Dr. Bartosz Bobula for help in the electrophysiological studies. 


SUPPLEMENTARYMATERIAL 
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol. 2018.00456/full#supplementary-material 
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Confict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or fnancial relationships that could be construedas a potential confict ofinterest. 
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