Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons

Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na+ channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (IAChs) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric α3β4 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and IAChs were elicited by fast application of ACh (100-300 μM), either alone or in presence of lidocaine at different concentrations. IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50 = 41 μM; nH close to 1) and the inhibition was, at least partially, voltage-dependent, indicating an open-channel blockade. Besides, lidocaine blocked resting (closed) nAChRs, as evidenced by the increased inhibition caused by a 12 s lidocaine application just before its co-application with the agonist, and also enhanced IAChs desensitisation, at concentrations close to the IC50. These results indicate that lidocaine has diverse inhibitory actions on neuronal heteromeric nAChRs resembling those previously reported for Torpedo (muscle-type) nAChRs ( Alberola-Die et al., 2011). The similarity of lidocaine actions on different subtypes of heteromeric nAChRs differs with the specific effects of other compounds, restricted to particular subtypes of nAChRs.This work was supported by the following MICINN (Spanish government) grants: CONSOLIDER-INGENIO 2010 (CSD2008-00005), BFU2011-25371 and BFU2012-31359

Activation of TREK currents by the neuroprotective agent riluzole in mouse sympathetic neurons.

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (IRIL), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that IRIL flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K+ channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. IRIL was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.Publicado

Activation of TREK currents by riluzole in three subgroups of cultured mouse nodose ganglion neurons

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes

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Involvement of TRPC4 and 5 Channels in Persistent Firing in Hippocampal CA1 Pyramidal Cells

Persistent neural activity has been observed in vivo during working memory tasks, and supports short-term (up to tens of seconds) retention of information. While synaptic and intrinsic cellular mechanisms of persistent firing have been proposed, underlying cellular mechanisms are not yet fully understood. In vitro experiments have shown that individual neurons in the hippocampus and other working memory related areas support persistent firing through intrinsic cellular mechanisms that involve the transient receptor potential canonical (TRPC) channels. Recent behavioral studies demonstrating the involvement of TRPC channels on working memory make the hypothesis that TRPC driven persistent firing supports working memory a very attractive one. However, this view has been challenged by recent findings that persistent firing in vitro is unchanged in TRPC knock out (KO) mice. To assess the involvement of TRPC channels further, we tested novel and highly specific TRPC channel blockers in cholinergically induced persistent firing in mice CA1 pyramidal cells for the first time. The application of the TRPC4 blocker ML204, TRPC5 blocker clemizole hydrochloride, and TRPC4 and 5 blocker Pico145, all significantly inhibited persistent firing. In addition, intracellular application of TRPC4 and TRPC5 antibodies significantly reduced persistent firing. Taken together these results indicate that TRPC4 and 5 channels support persistent firing in CA1 pyramidal neurons. Finally, we discuss possible scenarios causing these controversial observations on the role of TRPC channels in persistent firing

Involvement of TRPC4 and 5 channels in persistent firing in hippocampal CA1 pyramidal cells

Persistent neural activity has been observed in vivo during working memory tasks, and supports short-term (up to tens of seconds) retention of information. While synaptic and intrinsic cellular mechanisms of persistent firing have been proposed, underlying cellular mechanisms are not yet fully understood. In vitro experiments have shown that individual neurons in the hippocampus and other working memory related areas support persistent firing through intrinsic cellular mechanisms that involve the transient receptor potential canonical (TRPC) channels. Recent behavioral studies demonstrating the involvement of TRPC channels on working memory make the hypothesis that TRPC driven persistent firing supports working memory a very attractive one. However, this view has been challenged by recent findings that persistent firing in vitro is unchanged in TRPC knock out (KO) mice. To assess the involvement of TRPC channels further, we tested novel and highly specific TRPC channel blockers in cholinergically induced persistent firing in mice CA1 pyramidal cells for the first time. The application of the TRPC4 blocker ML204, TRPC5 blocker clemizole hydrochloride, and TRPC4 and 5 blocker Pico145, all significantly inhibited persistent firing. In addition, intracellular application of TRPC4 and TRPC5 antibodies significantly reduced persistent firing. Taken together these results indicate that TRPC4 and 5 channels support persistent firing in CA1 pyramidal neurons. Finally, we discuss possible scenarios causing these controversial observations on the role of TRPC channels in persistent firing

Contribution of KCNQ and TREK channels to the resting membrane potential in sympathetic neurons at physiological temperature

The ionic mechanisms controlling the resting membrane potential (RMP) in superior cervical ganglion (SCG) neurons have been widely studied and the M-current (IM, KCNQ) is one of the key players. Recently, with the discovery of the presence of functional TREK-2 (TWIK-related K+ channel 2) channels in SCG neurons, another potential main contributor for setting the value of the resting membrane potential has appeared. In the present work, we quantified the contribution of TREK-2 channels to the resting membrane potential at physiological temperature and studied its role in excitability using patch-clamp techniques. In the process we have discovered that TREK-2 channels are sensitive to the classic M-current blockers linopirdine and XE991 (IC50 = 0.310 ± 0.06 µM and 0.044 ± 0.013 µM, respectively). An increase from room temperature (23 °C) to physiological temperature (37 °C) enhanced both IM and TREK-2 currents. Likewise, inhibition of IM by tetraethylammonium (TEA) and TREK-2 current by XE991 depolarized the RMP at room and physiological temperatures. Temperature rise also enhanced adaptation in SCG neurons which was reduced due to TREK-2 and IM inhibition by XE991 application. In summary, TREK-2 and M currents contribute to the resting membrane potential and excitability at room and physiological temperature in the primary culture of mouse SCG neurons.Ministerio de Ciencia e Innovación | Ref. CONSOLIDER CSD2008-00005MINECO | Ref. BFU2014-58999-PXunta de Galicia | Ref. INBIOMED CN2012 / 273Xunta de Galicia | Ref. INB1-131H-

PIP2 mediated inhibition of TREK potassium currents by bradykinin in mouse sympathetic neurons

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.Gobierno español | Ref. CONSOLIDER CSD2008-00005, BFU2014-58999-PXunta de Galicia | Ref. INBIOMED CN2012/273, INB1-131H-

Intrinsic spontaneous activity and subthreshold oscillations in neurones of the rat dorsal column nuclei in culture

The basis of rhythmic activity observed at the dorsal column nuclei (DCN) is still open to debate. This study has investigated the electrophysiological properties of isolated DCN neurones deprived of any synaptic influence, using the perforated-patch technique. About half of the DCN neurones (64/130) were spontaneously active. More than half of the spontaneous neurones (36/64) showed a low threshold membrane oscillation (LTO) with a mean frequency of 11.4 Hz (range: 4.3–22.1 Hz, n = 20; I = 0). Cells showing LTOs also invariably showed a rhythmic 1.2 Hz clustering activity (groups of 2–5 action potentials separated by silent LTO periods). Also, more than one-third of the silent neurones presented clustering activity, always accompanied by LTOs, when slightly depolarised. The frequency of LTOs was voltage dependent and could be abolished by TTX (0.5 μM) and riluzole (30 μM), suggesting the participation of a sodium current. LTOs were also abolished by TEA (15 mM), which transformed clustering into tonic activity. In voltage clamp, most DCN neurones (85 %) showed a TTX-/riluzole-sensitive persistent sodium current (INa,p), which activated at about -60 mV and had a half-maximum activation at −49.8 mV. An M-like, non-inactivating outward current was present in 95 % of DCN neurones, and TEA (15 mM) inhibited this current by 73.7 %. The non-inactivating outward current was also inhibited by barium (1 mM) and linopirdine (10 μM), which suggests its M-like nature; both drugs failed to block the LTOs, but induced a reduction in their frequency by 56 and 20 %, respectively. These results demonstrate for the first time that DCN neurones have a complex and intrinsically driven clustering discharge pattern, accompanied by subthreshold membrane oscillations. Subthreshold oscillations rely on the interplay of a persistent sodium current and a non-inactivating TEA-sensitive outward current