Antonio Reboreda

TRPC channels underlie cholinergic plateau potentials and persistent activity in entorhinal cortex

Persistent neuronal activity lasting seconds to minutes has been proposed to allow for the transient storage of memory traces in entorhinal cortex and thus could play a major role in working memory. Nonsynaptic plateau potentials induced by acetylcholine account for persistent firing in many cortical and subcortical structures. The expression of these intrinsic properties in cortical neurons involves the recruitment of a non‐selective cation conductance. Despite its functional importance, the identity of the cation channels remains unknown. Here we show that, in layer V of rat medial entorhinal cortex, muscarinic receptor‐evoked plateau potentials and persistent firing induced by carbachol require phospholipase C activation, decrease of PIP2 levels, and permissive intracellular Ca2+ concentrations. Plateau potentials and persistent activity were suppressed by the generic nonselective cation channel blockers FFA (100 μM) and 2‐APB (100 μM), as well as by the TRPC channel blocker SKF‐96365 (50 μM). However, plateau potentials were not affected by the TRPV channel blocker ruthenium red (40 μM). The TRPC3/6/7 activator OAG did not induce or enhance persistent firing evoked by carbachol. Voltage clamp recordings revealed a carbachol‐activated, nonselective cationic current with a heteromeric TRPC‐like phenotype. Moreover, plateau potentials and persistent firing were inhibited by intracellular application of the peptide EQVTTRL that disrupts interactions between the C‐terminal domain of TRPC4/5 subunits and associated PDZ proteins. Altogether, our data suggest that TRPC cation channels mediating persistent muscarinic currents significantly contribute to the firing and mnemonic properties of projection neurons in the entorhinal cortex.

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.

Newly developed blockers of the M-current do not reduce spike frequency adaptation in cultured mouse sympathetic neurons

The M‐current (IK(M)) is believed to modulate neuronal excitability by producing spike frequency adaptation (SFA). Inhibitors of M‐channels, such as linopirdine and 10,10‐bis(4‐pyridinylmethyl)‐9(10H)‐anthracenone (XE991), enhance depolarization‐induced transmitter release and improve learning performance in animal models. As such, they are currently being tested for their therapeutic potential for treating Alzheimers disease. The activity of these blockers has been associated with the reduction of SFA and the depolarization of the membrane observed when IK(M) is inhibited. To test whether this is the case, the perforated patch technique was used to investigate the capacity of IK(M) inhibitors to alter the resting membrane potential and to reduce SFA in mouse superior cervical ganglion neurons in culture. Linopirdine and XE991 both proved to be potent blockers of IK(M) when the membrane potential was held at −30 mV (IC50 2.56 and 0.26 µm, respectively). However, their potency gradually declined upon membrane hyperpolarization and was almost null when the membrane potential was kept at −70 mV, indicating that their blocking activity was voltage dependent. Nevertheless, IK(M) could be inhibited at these hyperpolarized voltages by other inhibitors such as oxotremorine‐methiodide and barium. Under current‐clamp conditions, neither linopirdine (10 µm) nor XE991 (3 µm) was effective in reducing the SFA and both provoked only a small slowly developed depolarization of the membrane (2.27 and 3.0 mV, respectively). In contrast, both barium (1 mm) and oxotremorine‐methiodide (10 µm) depolarized mouse superior cervical ganglion neurons by about 10 mV and reduced the SFA. In contrast to classical IK(M) inhibitors, the activity of linopirdine and XE991 on the IK(M) is voltage dependent and, thus, these newly developed IK(M) blockers do not reduce the SFA. These results may shed light on the mode of action of these putative cognition enhancers in vivo.

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.

TRP channels and neural persistent activity.

One of the integrative properties of the nervous system is its capability to, by transient motor commands or brief sensory stimuli, evoke persistent neuronal changes, mainly as a sustained, tonic action potential firing. This neural activity, named persistent activity, is found in a good number of brain regions and is thought to be a neural substrate for short-term storage and accumulation of sensory or motor information [1]. Examples of this persistent neural activity have been reported in prefrontal [2] and entorhinal [3] cortices, as part of the neural mechanisms involved in short-term working memory [4]. Interestingly, the general organization of the motor systems assumes the presence of bursts of short-lasting motor commands encoding movement characteristics such as velocity, duration, and amplitude, followed by a maintained tonic firing encoding the position at which the moving appendage should be maintained [5, 6]. Generation of qualitatively similar sustained discharges have also been found in spinal and supraspinal regions in relation to pain processing [7, 8]. Thus, persistent neural activity seems to be necessary for both behavioral (positions of fixation) and cognitive (working memory) processes. Persistent firing mechanisms have been proposed to involve the participation of a non-specific cationic current (CAN current) mainly mediated by activation of TRPC channels. Because the function and generation of persistent activity is still poorly understood, here we aimed to review and discuss the putative role of TRP-like channels on its generation and/or maintenance.

Ionic basis of the resting membrane potential in cultured rat sympathetic neurons

&NA; The conductances which determine the resting membrane potential of rat superior cervical ganglia (SCG) neurons were investigated using perforated voltage‐ and current‐clamp whole‐cell techniques. The resting potential of SCG cells varied from ‐47 to ‐80 mV (‐58.3 ± 0.8 mV, n = 55). Blockade of M and h currents induced a depolarisation (7.4 ± 0.7 mV, n = 22) and a hyperpolarisation (7.2 ± 0.7 mV, n = 20) respectively; however, no correlation between the amplitude of these currents and the resting potential was found. The inhibition of the Na/K pump also induced membrane depolarisation (3.2 ± 0.2 mV, n = 8). Inhibition of voltage‐gated currents unmasked a voltage‐independent resting conductance reversing at ‐50 mV. The reversal potential of the voltage‐independent conductance, which included the electrogenic contribution of the Na/K pump, was strongly correlated with the resting potential (R = 0.87, p < 0.0001, n = 30). Ionic substitution experiments confirmed the existence of a voltage‐independent conductance (leakage) with four components, a main potassium conductance, two minor sodium and chloride conductances and a small contribution of the Na/K pump. It is concluded that the resting potential of SCG cells strongly depends on the reversal potential of the voltage‐independent conductance, with voltage‐activated M and h currents playing a prominent stabilising role.

Expression of K2P Channels in Sensory and Motor Neurons of the Autonomic Nervous System

Several types of neurons within the central and peripheral somatic nervous system express two-pore-domain potassium (K2P) channels, providing them with resting potassium conductances. We demonstrate that these channels are also expressed in the autonomic nervous system where they might be important modulators of neuronal excitability. We observed strong mRNA expression of members of the TRESK and TREK subfamilies in both the mouse superior cervical ganglion (mSCG) and the mouse nodose ganglion (mNG). Motor mSCG neurons strongly expressed mRNA transcripts for TRESK and TREK-2 subunits, whereas TASK-1 and TASK-2 subunits were only moderately expressed, with only few or very few transcripts for TREK-1 and TRAAK (TRESK ≈ TREK-2 > TASK-2 ≈ TASK-1 > TREK-1 > TRAAK). Similarly, the TRESK and TREK-1 subunits were the most strongly expressed in sensorial mNG neurons, while TASK-1 and TASK-2 mRNAs were moderately expressed, and fewer TREK-2 and TRAAK transcripts were detected (TRESK ≈ TREK-1 > TASK-1 ≈ TASK-2 > TREK-2 > TRAAK). Moreover, cell-attached single-channel recordings showed a major contribution of TRESK and TREK-1 channels in mNG. As the level of TRESK mRNA expression was not statistically different between the ganglia analysed, the distinct expression of TREK-1 and TREK-2 subunits was the main difference observed between these structures. Our results strongly suggest that TRESK and TREK channels are important modulators of the sensorial and motor information flowing through the autonomic nervous system, probably exerting a strong influence on vagal reflexes.

Cholinergic receptor activation supports persistent firing in layer III neurons in the medial entorhinal cortex.

Medial temporal lobe (MTL) areas are crucial for memory tasks such as spatial working memory and temporal association memory, which require an active maintenance of memory for a short period of time (a few hundred milliseconds to tens of seconds). Recent work has shown that the projection from layer III neurons in the medial entorhinal cortex (MEC) to hippocampal region CA1, the temporoammonic (TA) pathway, might be specially important for these memory tasks. In addition, lesions to the entorhinal cortex disrupt persistent firing in CA1 which is believed to support active maintenance of memory. Injection of cholinergic antagonists and group I mGlu receptor antagonists to the MEC impairs spatial working memory and temporal association memory. Consistent with this, we have shown that group I mGlu receptor activation supports persistent firing in principal cells of the MEC layer III in vitro (Yoshida et al. [39]). However, it still remains unknown whether cholinergic receptor activation also supports persistent firing in MEC layer III neurons. In this paper, we tested this in MEC layer III cells using both ruptured and perforated whole-cell recordings in vitro. We report that the majority of cells we recorded from in MEC layer III show persistent firing during perfusion of the cholinergic agonist carbachol (2-10μM). In addition, repeated stimulation gradually suppressed persistent firing. We further discuss the possible role of persistent firing in memory function in general.

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 (100-300μM), either alone or in presence of lidocaine at different concentrations. IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50=41μM; 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 12s 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.

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M‐currents and voltage‐dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M‐current inhibition. Here, we report the presence of a riluzole‐activated current (IRIL) that flows through the TREK‐2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine‐M (Oxo‐M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single‐channel TREK‐2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2‐aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET‐18‐OCH3. Finally, the phosphatidylinositol‐3‐kinase/phosphatidylinositol‐4‐kinase inhibitor wortmannin strongly attenuated the IRIL, whereas blocking phosphatidylinositol 4,5‐bisphosphate (PIP2) depletion consistently prevented IRIL inhibition by Oxo‐M. These results demonstrate that TREK‐2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC‐dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M‐current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.