Liwang Liu

Arachidonic acid mediates muscarinic inhibition and enhancement of N-type Ca2+ current in sympathetic neurons

N-type Ca2+ channels participate in acute activity-dependent processes such as regulation of Ca2+-activated K+ channels and in more prolonged events such as gene transcription and long-term depression. A slow postsynaptic M1 muscarinic receptor-mediated modulation of N-type current in superior cervical ganglion neurons may be important in regulating these processes. This slow pathway inhibits N-type current by using a diffusible second messenger that has remained unidentified for more than a decade. Using whole-cell patch–clamp techniques, which isolate the slow pathway, we found that the muscarinic agonist oxotremorine methiodide not only inhibits currents at positive potentials but enhances N-type current at negative potentials. Enhancement was also observed in cell-attached patches. These findings provide evidence for N-type Ca2+-current enhancement by a classical neurotransmitter. Moreover, enhancement and inhibition of current by oxotremorine methiodide mimics modulation observed with direct application of a low concentration of arachidonic acid (AA). Although no transmitter has been reported to use AA as a second messenger to modulate any Ca2+ current in either neuronal or nonneuronal cells, we nevertheless tested whether a fatty acid signaling cascade was involved. Blocking phospholipase C, phospholipase A2, or AA but not AA metabolism minimized muscarinic modulation of N-type current, supporting the participation of these molecules in the slow pathway. A role for the G protein Gq was also confirmed by blocking muscarinic modulation of Ca2+ currents with anti-Gqα antibody. Our finding that AA participates in the slow pathway strongly suggests that it may be the previously unknown diffusible second messenger.

Regulation of voltage-gated Ca2+ channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca(2+) channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acids carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca(V)2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP(2)), a palmitoylated accessory subunit (beta(2a)) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M(1) muscarinic receptors may stimulate multiple lipases to break down the PIP(2) associated with VGCCs and leave PIP(2)s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.

Nicotine persistently activates ventral tegmental area dopaminergic neurons via nicotinic acetylcholine receptors containing α4 and α6 subunits

Nicotine is reinforcing because it activates dopaminergic (DAergic) neurons within the ventral tegmental area (VTA) of the brains mesocorticolimbic reward circuitry. This increase in activity can occur for a period of several minutes up to an hour and is thought to be a critical component of nicotine dependence. However, nicotine concentrations that are routinely self-administered by smokers are predicted to desensitize high-affinity α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) in seconds. Thus, how physiologically relevant nicotine concentrations persistently activate VTA DAergic neurons is unknown. Here we show that nicotine can directly and robustly increase the firing frequency of VTA DAergic neurons for several minutes. In mouse midbrain slices, 300 nM nicotine elicited a persistent inward current in VTA DAergic neurons that was blocked by α-conotoxin MII[H9A;L15A], a selective antagonist of nAChRs containing the α6 subunit. α-conotoxin MII[H9A;L15A] also significantly reduced the long-lasting increase in DAergic neuronal activity produced by low concentrations of nicotine. In addition, nicotine failed to significantly activate VTA DAergic neurons in mice that did not express either α4 or α6 nAChR subunits. Conversely, selective activation of nAChRs containing the α4 subunit in knock-in mice expressing a hypersensitive version of these receptors yielded a biphasic response to nicotine consisting of an acute desensitizing increase in firing frequency followed by a sustained increase that lasted several minutes and was sensitive to α-conotoxin MII[H9A;L15A]. These data indicate that nicotine persistently activates VTA DAergic neurons via nAChRs containing α4 and α6 subunits.

Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety during nicotine withdrawal.

Increased anxiety is a predominant withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here, we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signaling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a meso-interpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knock-down of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signaling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

M1 muscarinic receptors inhibit L-type Ca2+ current and M-current by divergent signal transduction cascades.

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M1-muscarinic receptors (M1Rs) may inhibit both Ca2+ (L- and N-) and K+ (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate (PIP2). However, we found previously that M1R inhibition of N-current in superior cervical ganglion (SCG) neurons requires loss of PIP2 and generation of a free fatty acid, probably arachidonic acid (AA) by phospholipase A2 (PLA2). It is not known whether PLA2 activity and AA also participate in L- and M-current modulation in SCG neurons. To test whether PLA2 plays a similar role in M1R inhibition of L- and M-currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP2 minimized M-current recovery from inhibition, whereas L-current recovered normally. Second, L-current inhibition required group IVa PLA2 [cytoplasmic PLA2 (cPLA2)], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA2 by muscarinic stimulation. Third, in type IIa PLA2 [secreted (sPLA2)]−/−/cPLA2−/− double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M1R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

Effects of arachidonic acid on unitary calcium currents in rat sympathetic neurons

1 We have characterized the actions of arachidonic acid (AA) on whole cell and unitary calcium (Ca2+) currents in rat neonatal superior cervical ganglion (SCG) neurons using barium (Ba2+) as the charge carrier. 2 Whole cell currents were elicited by stepping the membrane potential from −90 mV to +10 mV. Arachidonic acid (5 μm) was introduced into the bath in the continued presence of 1 μm (+)‐202‐791, an L‐type Ca2+ channel agonist. Under these conditions, the peak current, comprised mainly of N‐type current, and a slow, (+)‐202‐791‐induced component of the tail current were inhibited by 67 ± 6 and 60 ± 10%, respectively, indicating that AA inhibits both N‐ and L‐type currents. 3 At a test potential of +30 mV, AA (5 μm) decreased unitary L‐ and N‐type Ca2+ channel open probability (Po) in cell‐attached patches that contained a single channel. For both channels, the underlying causes of the decrease in Po were similar. Arachidonic acid caused an increase in the percentage of null sweeps and in the number of null sweeps that clustered together. In sweeps with activity, the average number of openings per sweep decreased, while first latency and mean closed time increased. Arachidonic acid had no significant effect on unitary current amplitude or mean open time. 4 Our findings are the first description of the inhibition of unitary L‐ and N‐type Ca2+ channel activity by AA and are consistent with both channels spending more time in their null mode and with increased dwell time in one or more closed states.

The Ca2+ channel β subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AAs concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated CaVβ2a.

Phospholipid metabolism is required for M1 muscarinic inhibition of N-type calcium current in sympathetic neurons

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca2+ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein Gq and identified phospholipase C (PLC), phospholipase A2 (PLA2), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M2/M4 muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M1 muscarinic receptors (M1Rs), Gqα, PLC, PLA2, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA2 cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M1Rs, Gqα, PLC, PIP2, PLA2, and AA for N-current inhibition.

Nicotinic acetylcholine receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100 mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse.

Pharmacological discrimination between muscarinic receptor signal transduction cascades with bethanechol chloride

Muscarinic agonist specificity is limited, making it difficult to match receptor subtypes with signal transduction cascades that mediate ion channel modulation. We have characterized the inhibitory effects of two muscarinic agonists, oxotremorine‐M (Oxo‐M) and bethanechol chloride (BeCh), on Ca2+ currents in neonatal rat superior cervical ganglion neurons. Oxo‐M‐mediated (10 μM) inhibition occurred via two signaling pathways. The first pathway inhibited whole cell peak currents, consisting primarily of N‐type current, but not FPL 64176‐induced, long‐lasting tail currents, comprised entirely of L‐type current. Inhibited currents displayed slowed activation kinetics and voltage dependence, characteristics of membrane‐delimited inhibition. Current inhibition was blocked by the selective M2 receptor antagonist, methoctramine (METH; 100 nM), or following pertussis toxin (PTX) pretreatment. Activation of the second pathway inhibited both peak and long‐lasting tail currents. This pathway was voltage‐independent, PTX‐insensitive, but sensitive to internal Ca2+ chelator concentration. Muscarinic toxin 7 (MT‐7, 100 nM), an irreversible M1 receptor antagonist, eliminated this inhibition. Oxo‐M (100 μM) decreased L‐ and N‐type channel activities in cell‐attached patches, indicating that a diffusible second messenger is involved. BeCh (100 μM) also inhibited whole cell currents via the membrane‐delimited pathway. Blocking M4 receptors with 100 nM pirenzepine (in the presence of MT‐7) had no effect, while antagonizing M2 receptors with METH abolished inhibition. Concentrations of BeCh as high as 3 mM failed to inhibit either peak or long‐lasting tail currents following PTX pretreatment. These results indicate that BeCh may be an effective tool for selectively activating M2 receptor stimulation of the membrane‐delimited pathway.