Effect of size on expression of bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of choline acetyltransferase (ChAT)-green fluorescent protein (GFP) and Hb9-GFP mice during the first 4 wk of life. We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, whereas larger fast α-motoneurons [matrix metalloproteinase-9 (MMP-9)+/Hb9+] with a soma area ≥ 400 µm2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, the thermosensitive Trpm5 Ca2+-activated Na+ current, and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.NEW & NOTEWORTHY Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

Knockdown of calpain1 in lumbar motoneurons reduces spasticity after spinal cord injury in adult rats.

Spasticity, affecting ∼75% of patients with spinal cord injury (SCI), leads to hyperreflexia, muscle spasms, and cocontractions of antagonist muscles, greatly affecting their quality of life. Spasticity primarily stems from the hyperexcitability of motoneurons below the lesion, driven by an upregulation of the persistent sodium current and a downregulation of chloride extrusion. This imbalance results from the post-SCI activation of calpain1, which cleaves Nav1.6 channels and KCC2 cotransporters. Our study was focused on mitigating spasticity by specifically targeting calpain1 in spinal motoneurons. We successfully transduced lumbar motoneurons in adult rats with SCI using intrathecal administration of adeno-associated virus vector serotype 6, carrying a shRNA sequence against calpain1. This approach significantly reduced calpain1 expression in transduced motoneurons, leading to a noticeable decrease in spasticity symptoms, including hyperreflexia, muscle spasms, and cocontractions in hindlimb muscles, which are particularly evident in the second month post-SCI. In addition, this decrease, which prevented the escalation of spasticity to a severe grade, paralleled the restoration of KCC2 levels in transduced motoneurons, suggesting a reduced proteolytic activity of calpain1. These findings demonstrate that inhibiting calpain1 in motoneurons is a promising strategy for alleviating spasticity in SCI patients.

A size principle for bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability related to postural tone is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar ventrolateral α-motoneurons of ChAT-GFP and Hb9-GFP mice across different developmental stages: neonatal (P2-P7), young (P7-P14) and mature (P21-P25). We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast α-motoneurons (MMP-9 + /Hb9 + ) with a soma area ≥ 400µm 2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, thermosensitive Trpm5 Ca 2+ -activated Na + current and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties, but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size dictates this process. New and Noteworthy: Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

Persistent Nav1.1 and Nav1.6 currents drive spinal locomotor functions through nonlinear dynamics.

Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identify the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The inhibition of Nav1.6, but not of Nav1.1, in motoneurons impairs INaP, bistability, postural tone, and locomotor performance. In interneurons of the rhythmogenic CPG region, both Nav1.6 and Nav1.1 equally mediate INaP. Inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG.

Riluzole for treating spasticity in patients with chronic traumatic spinal cord injury: Study protocol in the phase ib/iib adaptive multicenter randomized controlled RILUSCI trial.

BACKGROUND: Satisfactory treatment is often lacking for spasticity, a highly prevalent motor disorder in patients with spinal cord injury (SCI). Low concentrations of riluzole potently reduce the persistent sodium current, the post-SCI increase in which contributes to spasticity. The repurposing of this drug may therefore constitute a useful potential therapeutic option for relieving SCI patients suffering from chronic traumatic spasticity. OBJECTIVE: RILUSCI is a phase 1b-2b trial designed to assess whether riluzole is a safe and biologically effective means of managing spasticity in adult patients with traumatic chronic SCI. METHODS: In this multicenter double-blind trial, adults (aged 18-65 years) suffering from spasticity after SCI (target enrollment: 90 participants) will be randomly assigned to be given either a placebo or a recommended daily oral dose of riluzole for two weeks. The latter dose will be previously determined in phase 1b of the study by performing double-blind dose-finding tests using a Bayesian continuous reassessment method. The primary endpoint of the trial will be an improvement in the Modified Ashworth Score (MAS) or the Numerical Rating Score (NRS) quantifying spasticity. The secondary outcomes will be based on the safety and pharmacokinetics of riluzole as well as its impact on muscle spasms, pain, bladder dysfunction and quality of life. Analyses will be performed before, during and after the treatment and the placebo-controlled period. CONCLUSION: To the best of our knowledge, this clinical trial will be the first to document the safety and efficacy of riluzole as a means of reducing spasticity in patients with chronic SCI. TRIAL REGISTRATION: The clinical trial, which is already in progress, was registered on the ClinicalTrials.gov website on August 9, 2016 under the registration number NCT02859792. TRIAL SPONSOR: Assistance Publique-Hôpitaux de Marseille.

Astrocytic Kir4.1 channels regulate locomotion by orchestrating neuronal rhythmicity in the spinal network.

Neuronal rhythmogenesis in the spinal cord is correlated with variations in extracellular K+ levels ([K+ ]e ). Astrocytes play important role in [K+ ]e homeostasis and compute neuronal information. Yet it is unclear how neuronal oscillations are regulated by astrocytic K+ homeostasis. Here we identify the astrocytic inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) as a key molecular player for neuronal rhythmicity in the spinal central pattern generator (CPG). By combining two-photon calcium imaging with electrophysiology, immunohistochemistry and genetic tools, we report that astrocytes display Ca2+ transients before and during oscillations of neighboring neurons. Inhibition of astrocytic Ca2+ transients with BAPTA decreases the barium-sensitive Kir4.1 current responsible of K+ clearance. Finally, we show in mice that Kir4.1 knockdown in astrocytes progressively prevents neuronal oscillations and alters the locomotor pattern resulting in lower motor performances in challenging tasks. These data identify astroglial Kir4.1 channels as key regulators of neuronal rhythmogenesis in the CPG driving locomotion.

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1.

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.

Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice.

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.

The M-current works in tandem with the persistent sodium current to set the speed of locomotion.

The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

Alteration of glycinergic receptor expression in lumbar spinal motoneurons is involved in the mechanisms underlying spasticity after spinal cord injury.

Spasticity is a disabling motor disorder affecting 70% of people with brain and spinal cord injury. The rate-dependent depression (RDD) of the H reflex is the only electrophysiological measurement correlated with the degree of spasticity assessed clinically in spastic patients. Several lines of evidence suggest that the mechanism underlying the H reflex RDD depends on the strength of synaptic inhibition through GABAA (GABAAR) and glycine receptors (GlyR). In adult rats with spinal cord transection (SCT), we studied the time course of the expression of GABAAR and GlyR at the membrane of retrogradely identified Gastrocnemius and Tibialis anterior motoneurons (MNs) 3, 8 and 16 weeks after injury, and measured the RDD of the H reflex at similar post lesion times. Three weeks after SCT, a significant decrease in the expression of GABAA and GlyR was observed compared to intact rats, and the H-reflex RDD was much less pronounced than in controls. Eight weeks after SCT, GlyR values returned to normal. Simultaneously, we observed a tendency to recover normal RDD of the H reflex at higher frequencies. We tested whether an anti-inflammatory treatment using methylprednisolone performed immediately after SCT could prevent alterations in GABAA/glycine receptors and/or the development of spasticity observed 3 weeks after injury. This treatment restored control levels of GlyR but not the expression of GABAAR, and it completely prevented the attenuation of RDD. These data strongly suggest that alteration of glycinergic inhibition of lumbar MNs is involved in the mechanisms underlying spasticity after SCI.

Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1G93A mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1G93A mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1G93A mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS.

Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity.

Up-regulation of the persistent sodium current (INaP) and down-regulation of the potassium/chloride extruder KCC2 lead to spasticity after spinal cord injury (SCI). We here identified calpain as the driver of the up- and down-regulation of INaP and KCC2, respectively, in neonatal rat lumbar motoneurons. Few days after SCI, neonatal rats developed behavioral signs of spasticity with the emergence of both hyperreflexia and abnormal involuntary muscle contractions on hindlimbs. At the same time, in vitro isolated lumbar spinal cords became hyperreflexive and displayed numerous spontaneous motor outputs. Calpain-I expression paralleled with a proteolysis of voltage-gated sodium (Nav) channels and KCC2. Acute inhibition of calpains reduced this proteolysis, restored the motoneuronal expression of Nav and KCC2, normalized INaP and KCC2 function, and curtailed spasticity. In sum, by up- and down-regulating INaP and KCC2, the calpain-mediated proteolysis of Nav and KCC2 drives the hyperexcitability of motoneurons which leads to spasticity after SCI.

Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomotion.

Spinal motoneurons are endowed with nonlinear spiking behaviors manifested by a spike acceleration whose functional significance remains uncertain. Here, we show in rodent lumbar motoneurons that these nonlinear spiking properties do not rely only on activation of dendritic nifedipine-sensitive L-type Ca2+ channels, as assumed for decades, but also on the slow inactivation of a nifedipine-sensitive K+ current mediated by Kv1.2 channels that are highly expressed in axon initial segments. Specifically, the pharmacological and computational inhibition of Kv1.2 channels occluded the spike acceleration of rhythmically active motoneurons and the correlated slow buildup of rhythmic motor output recorded at the onset of locomotor-like activity. This study demonstrates that slow inactivation of Kv1.2 channels provides a potent gain control mechanism in mammalian spinal motoneurons and has a behavioral role in enhancing locomotor drive during the transition from immobility to steady-state locomotion.

Activation of 5-HT2A Receptors Restores KCC2 Function and Reduces Neuropathic Pain after Spinal Cord Injury.

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI.

[Calpain as a new therapeutic target for treating spasticity after a spinal cord injury]. / Une nouvelle cible thérapeutique dans le traitement de la spasticité après une lésion de la moelle épinière : la calpaïne.

After a spinal cord injury (SCI), patients develop spasticity, a motor disorder characterized by hyperreflexia and stiffness of muscles. Spasticity results from alterations in motoneurons with an upregulation of their persistent sodium current (I NaP), simultaneously with a disinhibition caused by a reduction of expression of chloride (Cl-) co-transporters KCC2. Until recently the origin of alterations was unknown. After reviewing pathophysiology of spasticity, the manuscript relates our recent work showing a tight relationship between the calpain-dependent proteolysis of voltage-gated sodium channels, the upregulation of I NaP and spasticity following SCI. We also discuss KCC2 as a substrate of calpains which may contribute to the disinhibition of motoneurons below the lesion. This led us to consider the proteolytic cleavage of both sodium channels and KCC2 as the upstream mechanism contributing to the development of spasticity after SCI.

Prochlorperazine Increases KCC2 Function and Reduces Spasticity after Spinal Cord Injury.

In mature neurons, low intracellular chloride level required for inhibition is maintained by the potassium-chloride cotransporter, KCC2. Impairment of Cl- extrusion after KCC2 dysfunction has been involved in many central nervous system disorders, such as seizures, neuropathic pain, or spasticity, after a spinal cord injury (SCI). This makes KCC2 an appealing drug target for restoring Cl- homeostasis and inhibition in pathological conditions. In the present study, we screen the Prestwick Chemical Library® and identify conventional antipsychotics phenothiazine derivatives as enhancers of KCC2 activity. Among them, prochlorperazine hyperpolarizes the Cl- equilibrium potential in motoneurons of neonatal rats and restores the reciprocal inhibition post-SCI. The compound alleviates spasticity in chronic adult SCI rats with an efficacy equivalent to the antispastic agent, baclofen, and rescues the SCI-induced downregulation of KCC2 in motoneurons below the lesion. These pre-clinical data support prochlorperazine for a new therapeutic indication in the treatment of spasticity post-SCI and neurological disorders involving a KCC2 dysfunction.

Cleavage of Na(+) channels by calpain increases persistent Na(+) current and promotes spasticity after spinal cord injury.

Upregulation of the persistent sodium current (I(NaP)) in motoneurons contributes to the development of spasticity after spinal cord injury (SCI). We investigated the mechanisms that regulate I(NaP) and observed elevated expression of voltage-gated sodium (Nav) 1.6 channels in spinal lumbar motoneurons of adult rats with SCI. Furthermore, immunoblots revealed a proteolysis of Nav channels, and biochemical assays identified calpain as the main proteolytic factor. Calpain-dependent cleavage of Nav channels after neonatal SCI was associated with an upregulation of I(NaP) in motoneurons. Similarly, the calpain-dependent cleavage of Nav1.6 channels expressed in human embryonic kidney (HEK) 293 cells caused the upregulation of I(NaP). The pharmacological inhibition of calpain activity by MDL28170 reduced the cleavage of Nav channels, I(NaP) in motoneurons and spasticity in rats with SCI. Similarly, the blockade of I(NaP) by riluzole alleviated spasticity. This study demonstrates that Nav channel expression in lumbar motoneurons is altered after SCI, and it shows a tight relationship between the calpain-dependent proteolysis of Nav1.6 channels, the upregulation of I(NaP) and spasticity.

KCNK5 channels mostly expressed in cochlear outer sulcus cells are indispensable for hearing.

In the cochlea, K(+) is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K(+) channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5(-/-) mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5(-/-) mice have a normal endolymphatic [K(+)] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K(+) secretion in stria vascularis have normal expression in Kcnk5(-/-) mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K(+) recycling along the outer sulcus lateral route.

Sensitization of neonatal rat lumbar motoneuron by the inflammatory pain mediator bradykinin.

Bradykinin (Bk) is a potent inflammatory mediator that causes hyperalgesia. The action of Bk on the sensory system is well documented but its effects on motoneurons, the final pathway of the motor system, are unknown. By a combination of patch-clamp recordings and two-photon calcium imaging, we found that Bk strongly sensitizes spinal motoneurons. Sensitization was characterized by an increased ability to generate self-sustained spiking in response to excitatory inputs. Our pharmacological study described a dual ionic mechanism to sensitize motoneurons, including inhibition of a barium-sensitive resting K(+) conductance and activation of a nonselective cationic conductance primarily mediated by Na(+). Examination of the upstream signaling pathways provided evidence for postsynaptic activation of B2 receptors, G protein activation of phospholipase C, InsP3 synthesis, and calmodulin activation. This study questions the influence of motoneurons in the assessment of hyperalgesia since the withdrawal motor reflex is commonly used as a surrogate pain model.

Sodium-mediated plateau potentials in lumbar motoneurons of neonatal rats.

The development and the ionic nature of bistable behavior in lumbar motoneurons were investigated in rats. One week after birth, almost all (∼80%) ankle extensor motoneurons recorded in whole-cell configuration displayed self-sustained spiking in response to a brief depolarization that emerged when the temperature was raised >30°C. The effect of L-type Ca(2+) channel blockers on self-sustained spiking was variable, whereas blockade of the persistent sodium current (I(NaP)) abolished them. When hyperpolarized, bistable motoneurons displayed a characteristic slow afterdepolarization (sADP). The sADPs generated by repeated depolarizing pulses summed to promote a plateau potential. The sADP was tightly associated with the emergence of Ca(2+) spikes. Substitution of extracellular Na(+) or chelation of intracellular Ca(2+) abolished both sADP and the plateau potential without affecting Ca(2+) spikes. These data suggest a key role of a Ca(2+)-activated nonselective cation conductance ((CaN)) in generating the plateau potential. In line with this, the blockade of (CaN) by flufenamate abolished both sADP and plateau potentials. Furthermore, 2-aminoethoxydiphenyl borate (2-APB), a common activator of thermo-sensitive vanilloid transient receptor potential (TRPV) cation channels, promoted the sADP. Among TRPV channels, only the selective activation of TRPV2 channels by probenecid promoted the sADP to generate a plateau potential. To conclude, bistable behaviors are, to a large extent, determined by the interplay between three currents: L-type I(Ca), I(NaP), and a Na(+)-mediated I(CaN) flowing through putative TRPV2 channels.