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Heantos-4, a natural plant extract used in the treatment of drug addiction, modulates T-type calcium… Cain, Stuart M; Ahn, Soyon; Garcia, Esperanza; Zhang, Yiming; Waheed, Zeina; Tyson, John R; Yang, Yi; Van Sung, Tran; Phillips, Anthony G; Snutch, Terrance P Dec 5, 2016

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RESEARCH Open AccessHeantos-4, a natural plant extract used inthe treatment of drug addiction, modulatesT-type calcium channels andthalamocortical burst-firingStuart M. Cain1, Soyon Ahn2, Esperanza Garcia1, Yiming Zhang1, Zeina Waheed1, John R. Tyson1, Yi Yang1,Tran Van Sung3, Anthony G. Phillips2 and Terrance P. Snutch1,2*AbstractHeantos-4 is a refined combination of plant extracts currently approved to treat opiate addiction in Vietnam. Inaddition to its beneficial effects on withdrawal and prevention of relapse, reports of sedation during clinical treatmentsuggest that arousal networks in the brain may be recruited during Heantos administration. T-type calcium channelsare implicated in the generation of sleep rhythms and in this study we examined whether a Heantos-4 extractionmodulates T-type calcium channel currents generated by the Cav3.1, Cav3.2 and Ca3.3 subtypes. Utilizing whole-cellvoltage clamp on exogenously expressed T-type calcium channels we find that Heantos inhibits Cav3.1 and Cav3.3currents, while selectively potentiating Cav3.2 currents. We further examined the effects of Heantos-4 extract onlow-threshold burst-firing in thalamic neurons which contribute to sleep oscillations. Using whole-cell current clampin acute thalamic brain slices Heantos-4 suppressed rebound burst-firing in ventrobasal thalamocortical neurons, whichexpress primarily Cav3.1 channels. Conversely, Heantos-4 had no significant effect on the burst-firing properties ofthalamic reticular neurons, which express a mixed population of Cav3.2 and Cav3.3 channels. Examining Heantos-4effects following oral administration in a model of absence epilepsy revealed the potential to exacerbate seizureactivity. Together, the findings indicate that Heantos-4 has selective effects both on specific T-type calcium channelisoforms and distinct populations of thalamic neurons providing a putative mechanism underlying its effects onsedation and on the thalamocortical network.Keywords: Burst firing, Thalamocortical, Thalamus, T-type, Calcium, Epilepsy, Addiction, Heantos-4IntroductionOpiate dependence is estimated to affect 15 million peopleworldwide and opiate overdose is believed to result inapproximately 69,000 mortalities per year [1]. Whilepharmacological therapies can be utilized during rehabili-tation to reduce cravings (methodone, buprenorphine,suboxone), block of the rewarding effects (naltrexone) orlessen the negative symptoms of opioid withdrawal (anti-emetics, sedatives, antidepressants), relapse rates remainunfortunately high [2, 3]. As such, there is a pressing needto discover and develop new therapies for the treatmentof opiate addiction.Heantos-4, which is the Greek term for “Plants” is amixture of organic herbs developed in Vietnam andrecently approved for the clinical alleviation of with-drawal symptoms in individuals dependent upon opiates[4]. Additionally, preliminary observations indicate thatit aids in the reduction in relapse rates. A sedative effectof Heantos has been reported by patients during the firstfew days of treatment. Mechanistically, there is littlecurrent understanding of how Heantos mediates itseffects on opioid withdrawal, relapse or sedation. Recentfindings show that oral administration of Heantos-4reduces drug-seeking behaviors in animal models of* Correspondence: snutch@msl.ubc.ca1Michael Smith Laboratories and Djavad Mowafaghian Centre for BrainHealth, University of British Columbia, 219-2185 East Mall, Vancouver, BC V6T1Z4, Canada2Department of Psychiatry, University of British Columbia, Vancouver, CanadaFull list of author information is available at the end of the article© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Cain et al. Molecular Brain  (2016) 9:94 DOI 10.1186/s13041-016-0274-7morphine addiction [5]. Further, microdialysis experi-ments have revealed that oral administration of Heantosin rats enhances dopamine efflux in the nucleus accum-bens, providing a mechanistic correlate for its effects onaddiction [5]. The current study examines the effects ofHeantos on burst-firing of thalamic neurons due to theinvolvement of this brain region in non-REM sleep andthe control of arousal [6].Voltage-gated calcium channels are a class of mem-brane bound proteins that regulate the cellular entry ofcalcium ions upon depolarization [7]. T-type calciumchannels (CaV3.1- CaV3.3) are a sub-class of calciumchannels that activate at more hyperpolarized membranepotentials than their High Voltage-Activated (HVA)counterparts (CaV1.1- CaV1.4 and CaV2.1- CaV2.3). As aresult, T-type calcium channels open in response tosmaller depolarizations than HVA calcium and sodiumchannels, endowing them with a unique the ability tomodulate cellular excitability at near-resting membranepotentials [8]. Low-threshold burst-firing is a neuronalfiring mode wherein a short duration of high frequencyaction potentials occur upon the crest of a “Low Thresholdcalcium Spike” (LTS) generated by T-type calcium currents[8, 9]. Thalamic neurons display a well-characterized switchbetween burst- and tonic-firing depending on a combin-ation of resting membrane potential and excitatory/inhibi-tory input [10]. While burst-firing occurs under normalconditions in the brain, in particular during sleep [6, 11] itis also associated with pathophysiological neuronal disor-ders, such as epilepsy [12–14].In this study we examined whether Heantos-4 candirectly modulate exogenously expressed T-type calciumchannels (CaV3.1- CaV3.3). Further, we correlated theeffects observed for cloned channels with Heantos-mediated modulation of ventrobasal (VB) and reticularthalamic nucleus (TRN) neurons in acute rat brain slices.Finally, we examined the effects of Heantos-4 oral admin-istration on seizure activity in the absence epilepsy model,Genetic Absence Epilepsy Rats from Strasbourg (GAERS).ResultsHeantos differentially modulates T-type calcium channelsubtypesTo assess whether Heantos directly alters T-type calciumchannel currents, individual CaV3.1, CaV3.2 and CaV3.3isoforms were exogenously expressed in HEK293 cells.Following a 5 min application of Heantos-4 extracted inaCSF (0.1 mg/ml, see Methods) CaV3.1 and CaV3.3 currentswere significantly inhibited from the baseline (CaV3.1 =−66.2 ± 3.8%, P < 0.001 paired T-test; CaV3.3 = −41.2 ± 3.9%P < 0.05 paired T-test), whereas, CaV3.2 currents were sig-nificantly potentiated (35.0 ± 14.9%, P < 0.05 paired T-test;Fig. 1). Furthermore, a concentration-dependent effect wasobserved with a lower concentration (0.01 mg/ml) acrossall three isoforms (n = 4), and a higher concentration(1 mg/ml) in the CaV3.1 and CaV3.3 channels (Fig. 1c).Voltage-dependence effects of the Heantos-4 were alsoassessed by examining the current-voltage relationshipat the 0.1 mg/ml concentration (Fig. 2). FollowingHeantos-4 application, a significant leftward shift in theactivation curve was observed for both CaV3.1 (V50 control= −46.2 ± 2.4 mV, V50 Heantos-4 = −51.0 ± 2.9 mV; P < 0.05paired T-test) and CaV3.2 (V50 control = 33.3 ± 1.1 mV, V50Heantos-4 = 42.0 ± 1.9 mV; P < 0.05 paired T-test), but notfor CaV3.3. (V50 control = 38.2 ± 1.8 mV, V50 Heantos-4 =37.4 ± .9 mV) (Fig. 2c). In addition, the voltage-dependentkinetics of CaV3.2 currents were also altered by Heantos-4application with the tau of activation increased between−45 mV and -20 mV and the tau of inactivation increasedbetween −40 mV and 0 mV (Fig. 2d and e). The tauof inactivation for CaV3.3 was significantly smaller(faster inactivating) between −35 mV and 10 mV followingHeantos-4 application (Fig. 2e).Heantos-4 inhibits burst-firing in VB neuronsGiven that Heantos-4 inhibited CaV3.1 and CaV3.3currents but potentiated CaV3.2 currents, we sought toevaluate its effects in a native system wherein these T-typeisoforms are differentially expressed. VB neurons are glu-tamatergic, sensory thalamocortical neurons that primarilyexpress the CaV3.1 channel [15–17] and we hypothesizedthat Heantos-4 would suppress burst-firing in this neur-onal class. VB neurons can display tonic-firing or reboundburst-firing upon depolarization or hyperpolarization fromtheir resting membrane potential, respectively [9, 18]. Ifthe resting membrane potential is in range wherein thepopulation of T-type calcium channels are balanced in theinactivated and closed states, a LTS can also occur viadepolarization (as shown in Fig. 3a). For the purposes ofthis study we examined only rebound bursts since not allVB neurons displayed depolarizing bursts.Current clamp recordings were performed on VBneurons using an acute brain slice preparation (Fig. 3). Re-bound burst-firing threshold (≥3 action potentials in 50 ms)was determined by applying incremental current injectionsteps from the resting membrane potential (Fig. 3a).Heantos-4 had little effect on current required to generaterebound burst-firing at concentrations at or below 0.1 mg/ml (not shown). However, at 1 mg/ml Heantos-4 induced asignificant increase the burst threshold (baseline = −34 ± 9.3pA, Heantos-4 = −176 ± 56.6 pA, P < 0.05 paired T-test;Fig. 3a and b) which was not observed in control (aCSF ap-plied) neurons (baseline = −58 ± 17.0, control = −66 ± 19.6;P > 0.05, paired T-test). This equated to a significant % in-crease in burst threshold in Heantos-4 treated neuronscompared to control (control = 17.3 ± 17.6%, Heantos-4 =504.7 ± 136.2%, P < 0.05 T-test). Heantos-4 also induced amodest but significant depolarization of the restingCain et al. Molecular Brain  (2016) 9:94 Page 2 of 12membrane potential (baseline = −72.2 ± 1.0 mV, Heantos =−67.2 ± 1.8 mV; P < 0.05, paired T-test, Fig. 3c), that wasnot observed in control neurons (baseline = −72.3 ± 2.3,control = −71.7 ± 1.8; P > 0.05, paired T-test). Heantos-4had no effect on the number of action potentials per burst(Fig. 3d) or on the input resistance of VB neurons (Fig. 3e).Heantos-4 does not affect burst-firing in TRN neuronsWhile VB neurons primarily express CaV3.1 channels,the GABAergic TRN neurons that project to VB neuronsexpress a combination of CaV3.2 and CaV3.3 [15, 17, 19].Since Heantos-4 potentiates CaV3.2 but inhibits CaV3.3(Figs. 1 and 2) either a modest inhibition/potentiationor no effect could be hypothesized depending uponthe relative co-expression of CaV3.2 and CaV3.3 inTRN neurons. The resting membrane potential of TRNneurons is hyperpolarized in comparison to VB neuronsand as a result these neurons burst-fire in response todepolarization, but not hyperpolarization under normalconditions (Fig. 4a).Current clamp recordings were performed on TRNneurons under the same conditions as described for VBneurons. At a concentration of 1 mg/ml Heantos-4 hadno effect on TRN burst-firing threshold or number ofaction potentials per burst. Similarly, no effect was ob-served on resting membrane potential or input resist-ance. A higher concentration of 5 mg/ml Heantos wasalso tested on TRN neurons. No significant effects wereseen on burst firing or passive membrane properties atthe 5 mg/ml Heantos concentration (Fig. 4b-e).Heantos-4 exacerbates absence seizures in GAERSBurst-firing has been observed in key epileptogenic neuronsin a number of animal epilepsy models [12, 13]. The GAERSabsence model displays spontaneous 5–9 Hz Spike-WaveDischarges (SWDs), the electroencephalographic correlateof absence seizures, from a juvenile age that intensify withdevelopment [20, 21] and burst-firing in GAERS TRN neu-rons occurs in a phase-locked manner with SWDs [22].While a polygenic etiology is believed to underlie seizures inFig. 1 Heantos differentially modulates current density of T-type calcium channel isoforms. a Timecourse of action of Heantos (0.1 mg/ml) on individualT-type calcium channel isoforms (n = 5 per T-type isoform) exogenously expressed in HEK293 cells. b Representative traces of CaV3.1, CaV3.2 andCaV3.3 T-type calcium currents before and after Heantos application. c Histograms summarizing concentration-dependent action ofHeantos on individual T-type calcium channel isoformsCain et al. Molecular Brain  (2016) 9:94 Page 3 of 12this model, approximately 65-75% of the epileptic phenotypecan be attributed to a gain-of-function missense mutation inthe domain III-IV linker of the CaV3.2 channel gene [23].Within the thalamocortical network that drives absence sei-zures CaV3.2 channels are expressed both in layer V of thecortex and in TRN neurons,[15] and CaV3.2 channels areupregulated in the GAERS model [24, 25]. As such, it islikely that seizures in this model are caused by hyperexcit-able cortico-reticular burst-firing.Heantos-4 in its dry powdered form suspended inCMC/saline (see Methods) was orally administered toGAERS rats 30 min prior to wireless EEG recording anddata acquisition for 60 min. Spontaneous seizuresoccurred throughout the recording period (Fig. 5).Heantos-4 had no effect on any of the seizure parametersmeasured at the 250 mg/kg dose compared to control ani-mals. However, at a dose of 500 mg/kg there was a sig-nificant increase in seizure duration (control = 12.3 ± 0.9 s(n = 5), 250 mg/kg = 12.0 ± 1.4 s (n = 3), 500 mg/kg = 18.6± 2.3 s (n = 3); control vs 250 mg/kg P = 0.986, controlvs 500 mg/kg P = 0.004, 250 mg/kg vs 500 mg/kg P =0.011 ANOVA); Fig. 5c). We also observed a variableFig. 2 Voltage-dependent effects of Heantos of T-type calcium channel isoforms. a Representative current traces to various test potentials from aholding potential of −110 mV before (black) and after (grey) Heantos (0.1 mg/ml). b Current density-voltage relationship, c voltage dependenceof activation, d activation kinetics and e inactivation kinetics for the effect of Heantos (0.1 mg/ml) on CaV3.1, CaV3.2 and CaV3.3 T-type calciumchannel currents (n = 5 per isoform)Cain et al. Molecular Brain  (2016) 9:94 Page 4 of 12albeit non-significant increase in the % time spent inthe seizure state and in the number of seizures follow-ing 500 mg/kg Heantos-4 administration (Fig. 5b, d). Con-versely, the spike frequency associated with seizures wasunaffected by Heantos-4 at either dose (Fig. 5e). Togetherthese findings indicate that at high doses of oral adminis-tration Heantos-4 may exacerbate seizure activity inanimals with a predisposal to cortico-reticular seizures.Mechanistically in GAERS this could occur as a result ofenhancing the gain-of-function alteration in CaV3.2 chan-nels within the cortical seizure initiation focus.DiscussionHeantos-4 is approved by the Vietnamese Food and DrugAdministration and is used clinically to treat opiate ad-dicts in Vietnam [4], although has not yet received formalapproval for use in Western addiction treatment pro-grams. During periods of Heantos-4 treatment, patientsreport not only a reduction in withdrawal symptoms andpossibly craving, but also strong sedative effects. Whilestudies are ongoing in an effort to decipher its mechanismof action with respect to drug-seeking behaviour andneurotransmitter release [5], we sought to examine itseffects on burst-firing in the thalamus due to the role ofthis region in the control of arousal [26, 27]. We firstestablished that Heantos inhibits CaV3.1 and CaV3.3 butpotentiates CaV3.2 channels, and subsequently found thatburst-firing was differentially modulated in two distinctclasses of thalamic neurons. In VB neurons that primarilyexpress CaV3.1 burst firing was inhibited by Heantos, butin TRN neurons that express a combination of CaV3.2 andCaV3.3 burst-firing was not affected.Fig. 3 Heantos inhibits burst firing in VB thalamic neurons. a Representative voltage traces showing input-output response of the same VB thalamicneuron before (left) and after (right) application of Heantos (1 mg/ml). Lower panels show current injection protocol. Black traces show the thresholdburst voltage and corresponding current injection. The same voltage traces are displayed at a higher time resolution of the region delineated by greybox for clarity. b Histograms summarizing mean burst threshold and c resting membrane potential before and after Heantos (1 mg/ml) or control(aCSF). d Histograms summarizing mean number of action potentials per burst and e input resistance for VB thalamic neurons before and after a5 min application of aCSF (n = 5) or Heantos (1 mg/ml (n = 5))Cain et al. Molecular Brain  (2016) 9:94 Page 5 of 12Differential modulation of T-type calcium channelisoformsTo date only a few pharmacological agents have beendiscovered that can selectively distinguish between thethree distinct T-type calcium channel isoforms. CaV3.2channels display a degree of redox sensitivity due to thepresence of extracellular cysteine residues and/or a histi-dine residue found only in CaV3.2, but not CaV3.1 orCaV3.3 channels [19, 28, 29]. As a result CaV3.2 channelsare inhibited by oxidation and potentiated by reductionof at least one of these residues. In addition, CaV3.2 dis-plays a higher efficacy to inhibition by nickel (<100 μM)and other trace metals due to the presence of a highaffinity binding and metal-catalyzed oxidation site pro-vided by the same extracellular histidine residue [30].Heantos-4 is a mixture of 12 organic herbs [4, 5] andthe potentiation of CaV3.2 could occur as a result of oneor more active components. Of note, a shift in the acti-vation curve to hyperpolarized potentials is observed inCaV3.1 and CaV3.2 channels, but not in CaV3.3 channels.Redox modulation of CaV3.2 channels induces onlynominal effects on the voltage-dependence of activation[29]. However, reduction of recombinant CaV3.2 chan-nels with L-cysteine has been reported to cause anapproximate 5 mV shift towards hyperpolarized poten-tials [19]. This is in agreement with Heantos-mediatedpotentiation of CaV3.2 currents via reduction of redox-sensitive channel residues. However, the mechanismunderlying the Heantos-mediated differential modula-tion of voltage-dependence of activation in CaV3.1 andCaV3.3 channels is unknown. It is possible that Heantoscontains certain components that inhibit both CaV3.1Fig. 4 Heantos does not affect burst firing in TRN neurons. a Representative voltage traces showing input-output response of the same TRN thalamicneuron before (left) and after (right) application of Heantos (1 mg/ml, 5 mg/ml). Lower panels show current injection protocol. Blacktraces show the threshold burst voltage and corresponding current injection. The same voltage traces are displayed at a higher timeresolution of the region delineated by grey box for clarity. b Histograms summarizing mean current injection for burst threshold,c resting membrane potential, d number of action potentials and e mean input resistance for TRN thalamic neurons before and aftera 5 min application of Heantos (1 mg/ml (n = 5), 5 mg/ml (n = 3))Cain et al. Molecular Brain  (2016) 9:94 Page 6 of 12and CaV3.3, and other components that modulate thegating dynamics of CaV3.1 only. Alternately, a singlecomponent of Heantos may have a differential effect onthe gating of CaV3.1 and CaV3.3, while simultaneouslyinhibiting both channels. Once analysis of the activecomponents in Heantos has been performed it will bepossible to dissect out how this differential modulationoccurs. Further research is required to determine theexact compound(s) that induce the potentiation ofCaV3.2 and also the inhibition of CaV3.1 and CaV3.3. Tothis end, we are currently conducting mass spectro-graphic analyses of Heantos-4 compounds that pass intothe cerebrospinal fluid compartment in vivo. Preliminaryanalyses confirm the presence in brain CSF of > 98% ofphytochemical classes identified with the components ofHeantos-4 (data not shown). In the meanwhile, giventhat existing T-type channel pharmacological tools arelimited to the redox-mediated inhibition or potentiationof CaV3.2 without affecting CaV3.1 or CaV3.3, Heantos-4provides a novel pharmacological tool to inhibit CaV3.1or CaV3.3 and potentiate CaV3.2.Selective inhibition of VB thalamic neuronsAgents that block all three T-type channel isoformsabolish both depolarizing burst-firing in TRN neuronsand rebound burst-firing in thalamocortical neurons[18, 31–34]. Of note, to date no pharmacologicalagents have been identified that can selectively inhibitburst-firing in thalamocortical neurons without alsoinhibiting TRN neurons.The T-type calcium currents that underlie low-threshold burst-firing in thalamic neurons have beenstudied extensively (for review see [8, 14]). In thalamocor-tical neurons the relatively fast and short burst durationsupports a role of CaV3.1 due to its fast activation/inacti-vation kinetics and hyperpolarized voltage-dependence ofactivation [35]. This is further supported by analysis ofT-type calcium channel mRNA expression using bothFig. 5 Heantos exacerbates seizures in the GAERS model. a Representative EEG traces from GAERS orally administered with control (0.5% CMC;upper traces) or Heantos (500 mg/kg; lower traces) at low (left trace) and high (right traces) time resolution. Histograms displaying mean data forb % time spent in seizure state, c seizure duration, d number of seizures and e spike frequencyCain et al. Molecular Brain  (2016) 9:94 Page 7 of 12in situ hybridization [15] and quantitative PCR [17, 36].In addition, genetic ablation of the Cacna1g gene thatencodes CaV3.1 in mice abolishes burst-firing in thalamo-cortical neurons [16].In TRN neurons the burst duration activates quicklybut is of longer duration than in VB neurons indicatingthat the combined CaV3.2/ CaV3.3 currents underlie theLTS since CaV3.2 channels activate quickly and CaV3.3channels inactivate slowly [8, 37]. This notion issupported by mRNA analyses [15] and by redox pharma-cological evidence. As discussed, CaV3.2 is redox sensi-tive and TRN neurons display enhanced or suppressedburst-firing upon application of reducing and oxidizingagents, respectively [19]. In support, T-type currents inTRN neurons can be potentiated up to 50% by reducingagents and inhibited up to 50% by oxidizing agents. Fur-ther, in mice following genetic ablation of the Cacna1igene, encoding CaV3.3 the T-type current is approxi-mately 40% of the total T-type current in TRN neuronsof wild-type mice [38]. In a separate study investigatinga strain of mice lacking the CaV3.3 channel, T-typecalcium currents were attenuated by approximately 80%in comparison to wild-type mice, although low thresholdbursts could still be elicited in 75% of animals [39]. Ofnote, the remaining T-type calcium current is absentand burst-firing abolished in double CaV3.2/CaV3.3knockout mice [39]. Taken together CaV3.2 currents arepredicted to contribute 20-40% of the total T-typecalcium channel current in TRN neurons, with theremaining current contributed by the CaV3.3 isoform.This assumption is supported by our finding thatHeantos-4 has no effect on burst-firing in TRN neuronsand agrees with our in vitro data showing potentiationof CaV3.2 and inhibition of CaV3.3 currents and pre-dicted to result in opposing modulation that cancels outany global effect on the total T-type current.Heantos and T-type calcium channel blockade in addic-tion and neurological disordersTo date, Heantos-4 has been used clinically solely forthe purpose of treating aspects of drug addiction. Theparaventricular thalamic nucleus (PVN) has been impli-cated in drug-seeking behaviour [40] and T-type calciumchannel activity has been identified in midline PVN neu-rons [41, 42]. While CaV3.1 mRNA is expressed athigher levels in burst-firing PVN neurons than the otherT-type channel isoforms, the PVN LTS is abolished by50 μM nickel implicating CaV3.2 since this concentrationwould not block CaV3.1 currents [43]. Further, CaV3.2,not CaV3.1 appears to be involved in pain transductionin the PVN [44]. With further relevance to addiction,TTA-A2, a pan T-type calcium channel blocker attenu-ates food- versus nicotine-induced cue-potentiated re-instatement for a response previously reinforced by foodadministration in rats [45]. This suggests that T-type an-tagonists have the potential to alleviate nicotine addic-tion. Although limited, these studies provide support fora direct role of T-type calcium channels in the acquisi-tion and/or maintenance of addiction.In addition to addiction, T-type calcium channelsare implicated in a number of other neurological dis-orders including in epileptic seizures [12–14, 46–48],anxiety [49–51], cognitive and memory impairments[52, 53], and in the transmission of pain [54–57]. PanT-type blockers that block burst-firing in both VB[18] and TRN [31] neurons have been shown tosuppress seizures in animal models of absenceepilepsy [31, 58], prevent seizure kindling in a modelof complex-partial seizures [59], and suppress tonic-clonic seizures [60]. In terms of pain signalling, panT-type antagonists have shown efficacy in reducingpain sensation in both animal models [54, 61–63] andhumans [64, 65]. T-type calcium currents underlyingburst-firing and slow oscillations in the thalamocorti-cal system have been extensively linked to non-REMsleep [6]. Somewhat counterintuitively, pan-T-typecalcium channel antagonists have been shown to in-duce sedation [66, 67] rather than the awake state,perhaps indicating that the T-type calcium channelsubtypes individually mediate distinct roles within thethalamocortical system and that block of all threesubtypes shifts equilibrium to the sleep state. Takentogether, Heantos-4 may have potential as a subtype-specific T-type calcium channel antagonist for use asboth a pharmacological tool and potential thera-peutic agent.In summary, Heantos-4 is a clinically utilized treat-ment for drug addiction and is currently the subject ofstudies aimed at determining its mechanism(s) of action[5]. Here we show that Heantos-4 selectively inhibitsCaV3.1 and CaV3.3 but potentiates CaV3.2 T-typecalcium currents. In addition, Heantos-4 selectively in-hibits burst-firing in VB thalamic neurons while havingno significant effect on TRN neurons. This supports thedata from exogenously expressed T-type calcium chan-nel isoforms and their corresponding differential expres-sion in distinct VB and TRN thalamic nuclei. ThatHeantos-4 appears to exacerbate absence seizure activityin GAERS suggest that caution may be warranted in theclinical usage of Heantos-4 in seizure-prone individuals.Determining whether the effect of Heantos-4 on T-typecalcium channels is related to its role in the treatment ofdrug-craving and withdrawal will require additionalstudies. Regardless, our findings that Heantos-4 differen-tially modulates distinct neuronal populations expressingT-type calcium channel isoforms may be relevant withrespect to a number of neurological disorders in whichT-type calcium channels are implicated [13, 68].Cain et al. Molecular Brain  (2016) 9:94 Page 8 of 12MethodsElectrophysiology on exogenously expressed T-typecalcium channel isoformsFlp-In 293 cells (Invitrogen), stably expressing pcDNA5/FRT plasmids containing hCaV3.1, hCaV3.2 or hCaV3.3were grown at 37 °C in DMEM supplemented with 10%heat inactivated fetal bovine serum. Cells expressing T-type calcium channel isoforms were selected by incuba-tion with hygromycin. Cells were seeded on poly-D-Lysine(0.1 mg/ml) coated glass coverslips and hygromycin re-moved from media 48 h before voltage clamp recordings.Calcium currents were recorded at 22–24 °C usingwhole-cell voltage clamp with the following solutions con-taining in mM: Internal: 120 Cs-Methanesulphonate, 11EGTA, 10 HEPES, 2 MgCl2, 5 MgATP and 0.3 NaGTP(pH 7.2) External: 2 CaCl2, 1 MgCl2, 10 HEPES, 40TEACl, 92 CsCl and 10 Glucose (pH 7.4). Fire polishedpatch pipettes (borosilicate glass) had typical resistancesof 3 to 5 MΩ when containing internal solution. The re-cording chamber was grounded with a Ag/AgCl pellet.Whole-cell currents were recorded at room temperatureusing an Axopatch 200B amplifier (Axon instrumentsInc., Union City, CA). Data was acquired with pClampsoftware package version 9 (Axon Instruments Inc.).Series resistance (Rs) was compensated by 65–75% andseals with Rs values higher than 20MΩ or cells with peakcurrent lower than 100pA were discarded. Currents sam-pled at 10 kHz and filtered at 2 kHz. Data analysis wascarried out using Clampfit 9 (Axon Instruments Inc.)and software Origin version 7.5 (OriginLab Corp.,Northampton, MA).Timecourse of drug action was obtained by depolariz-ing the membrane with a 200 msec pulse to −30 mVfrom a holding potential of −100 mV every 5 s. The peakcalcium current was taken from each trace for analysis.A two minute stable baseline was acquired before initiat-ing a five minute Heantos application via the perfusate.The current-voltage (I-V) relationship was obtained be-fore and after the timecourse protocol by depolarizing themembrane with pulses (CaV3.1 and CaV3.2 = 150 msec,CaV3.3 = 450 msec) from a holding potential of −110 mV.Test pulses from −90 to +10 mV were applied at 5 mVsteps. Peak amplitude of calcium currents was plottedagainst test pulse potential and I-V curves were fittedusing a modified Boltzmann equation: I = (Gmax*(Vm-Er))/(1 + exp((Vm-V50)/k)), where Gmax is the maximumvalue of membrane conductance, Vm is the test potential,Er is the extrapolated reversal potential, V50 is the half-ac-tivation potential, and k (Slope constant: k = RT/zδF;where R = gas constant, T = absolute temperature, z =valence of conducting ion, δ = electrical distanceacross the membrane, F = Faraday’s constant) reflects thevoltage sensitivity. Activation curves were obtained by cal-culating conductance from the I-V curves and plotting thenormalized conductance as a function of the membranepotential. The data was fitted with the Boltzmannequation: G/Gmax = A2 + (A1-A2)/(1 + exp((Vm-V50)/k)),where A1 is minimum normalized conductance, A2 ismaximum normalized conductance, Vm is the test poten-tial, V50 is the half-activation potential, and k value theslope of the activation curve (Slope constant).Acute brain slice electrophysiologyMale and female Wistar rats (P15–P20) were used in acutebrain slice experiments in accordance with CanadianCouncil for Animal Care guidelines.Animals were anesthetized using isoflurane (5% in O2),sacrificed by decapitation, the brains rapidly removedand transferred to ice cold sucrose-aCSF containing inmM: 214 sucrose, 26 NaHCO3, 1.25 NaH2PO4, 11glucose, 2.5 KCl , 0.5 CaCl2, 6 MgCl2, bubbled with 95%O2:5% CO2. Brain tissue was glued to a cutting chamberin a vibrating microtome (VT 1200, Leica, USA), whichwas then filled with ice cold sucrose-aCSF. Horizontalbrain slices containing the whole thalamus (350 μmthick) were cut from the level of the ventral TRN/VBand incubated for a minimum of 30 min at 34 °C in acurrent clamp recording solution containing in mM: 126NaCl, 2.5 KCl, 26 NaHCO3, 1.5 NaH2PO4, 2 CaCl2, 2MgCl2, 10 glucose; bubbled with 95% O2:5% CO2. Sliceswere transferred to a recording chamber superfusedwith current clamp recording solution and maintainedat 33–35 °C. VB and TRN neurons were visualizedusing a DIC microscope (Axioskop 2-FS Plus, CarlZeiss) and infrared camera (IR-1000, DAGE MTI) andvisually identified by their location, morphology andorientation. All recordings were undertaken using aMulticlamp 700B amplifier and pClamp softwareversion 9 (Molecular devices). The recording chamberwas grounded with a Ag/AgCl pellet.Whole cell current clamp recordings were undertakenusing fire polished borosilicate glass pipettes (4–6 MΩ)filled with the following solution containing in mM:120 K-gluconate, 10 HEPES, 1 MgCl2, 1 CaCl2, 11 KCl,11 EGTA, 4 MgATP, 0.5 NaGTP , pH adjusted to 7.2using KOH, osmolarity adjusted to 290 mOsm/kg usingD-mannitol. The liquid junction potential for currentclamp solutions was calculated as +13.3 mV andcorrected off-line. To evaluate input–output neuronalresponses to hyperpolarization and depolarization, DCcurrent was injected from −100 pA to +200 pA in 10 pAincrements for a duration of 1200 ms at the neuron’s in-trinsic resting membrane potential. VB neurons that didnot rebound burst fire with ≤ −100 pA current injectionwere then evaluated with a current injection protocolwhere DC current was injected from −100 pA to −500pA in 50 pA increments. Membrane potential responsesunder current clamp conditions were sampled at 50 kHzCain et al. Molecular Brain  (2016) 9:94 Page 9 of 12and filtered at 10 kHz. Bridge balance was monitored dur-ing recordings and any neurons displaying bridge balancevalues greater than 25 MΩ were discarded. Capacitanceneutralization was performed between 3.8 and 4.2 pF.EEG recordingAdult GAERS (4–6 month old) were anesthetized withisoflurane and implanted with skull screw electrodes onthe somatosensory cortex surface (bregma = +1.2 mm,lateral = ± 5.0 mm, depth below skull = 0.1 mm) and areference electrode in the cerebellum (lambda = -0.5mm, lateral = -0.5 mm, depth below skull = 1 mm).Electrodes were connected to a custom EEG interfaceimplanted on the skull. Following a 2 week recoveryperiod the interface was connected to a wireless head-stage (W2100, Multichannel Systems, Germany) andEEG signals acquired in the animals’ home cage for 1 h.DrugsHeantos-4, a brown, tea-like powder was provided byDr. Sung at the Institute of Chemistry, Vietnam Academyof Science and Technology (Hanoi, Vietnam). For in vitroelectrophysiology experiments Heantos-4 was prepared at1 mg/ml by immersing in aCSF, vortexing for severalseconds and incubating at 50 °C for 10 min followed bysonication at 22–24 °C for 5 min. Insoluble componentsof Heantos were then filtered and the flow-through usedin all experiments at this concentration (1 mg/ml) or as aserial dilution.For in vivo experiments Heantos-4 was mixed into 0.5%carboxymethylcellulose (CMC) in 0.9% saline and admin-istered via oral gavage 30 min prior to EEG recording.StatisticsData followed a normal distribution and statistical sig-nificance was calculated using Student’s T-test (pairedwhere appropriate) or ANOVA with Tukey's Post-hoctest taking P value < 0.05 as significant. Data was plottedas mean values ± standard error.AbbreviationsCaV: Voltage-gated calcium channel; GAERS: Genetic Absence Epilepsy Ratsfrom Strasbourg; HVA: High Voltage-Activated; LTS: Low Threshold calciumSpike; SWDs: Spike-Wave Discharges; TRN: Reticular thalamic nucleus;VB: Ventrobasal thalamic nucleusAcknowledgementsNot Applicable.FundingT.P. Snutch is supported by an operating grant from the Canadian Institutesof Health Research (#10677) and the Canada Research Chair inBiotechnology and Genomics-Neurobiology. S.M. Cain is supported by a re-search grant from the B.C. Epilepsy Society. A.G. Phillips is supported by anoperating grant from the Canadian Institutes of Health Research (#101025).Availability of data and materialsAll data generated or analysed during this study are included in thispublished article.Authors’ contributionsSMC undertook in vitro and in vivo electrophysiological experiments,participated in study design and coordination and drafted the manuscript.SA performed in vivo EEG experiments and edited the manuscript. EG, YZand ZW performed in vitro exogenous electrophysiology experiments. JYgenerated CaV clones. YY performed EEG implantation. TVS providedHeantos-4. AGP participated in study design and coordination and editedthe manuscript. TPS participated in study design and coordination anddrafted the manuscript. All authors read and approved the final manuscript.Competing interestsDr. T.V. Sung is a director of Heantos.jsc and a beneficial owner of shares inthis company.Consent for publicationNot applicable.Ethics approvalAll animal procedures were undertaken in accordance with Canadian Councilon Animal Care guidelines.Author details1Michael Smith Laboratories and Djavad Mowafaghian Centre for BrainHealth, University of British Columbia, 219-2185 East Mall, Vancouver, BC V6T1Z4, Canada. 2Department of Psychiatry, University of British Columbia,Vancouver, Canada. 3Institute of Chemistry, Vietnam Academy of Science andTechnology, Hanoi, Vietnam.Received: 29 September 2016 Accepted: 21 November 2016References1. WHO | Information sheet on opioid overdose. WHO. http://www.who.int/substance_abuse/information-sheet/en/.2. Bell J. Pharmacological maintenance treatments of opiate addiction. 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Kraus RL, Li Y, Gregan Y, Gotter AL, Uebele VN, Fox SV, et al. In vitrocharacterization of T-type calcium channel antagonist TTA-A2 and in vivoeffects on arousal in mice. J Pharmacol Exp Ther. 2010;335:409–17.68. Cain SM, Snutch TP. Voltage-gated calcium channels and disease. BioFactorsOxf Engl. 2011;37:197–205.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Cain et al. Molecular Brain  (2016) 9:94 Page 12 of 12


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