Ca(2+)-calmodulin signalling pathway up-regulates GABA synaptic transmission through cytoskeleton-mediated mechanisms

We investigated the role of calcium (Ca(2+))/calmodulin (CaM) signaling pathways in modulating GABA synaptic transmission at CA1 pyramidal neurons in hippocampal slices. Whole-cell pipettes were used to record type A GABA receptor (GABA(A)R)-gated inhibitory postsynaptic currents (IPSCs) and to perfuse intracellularly modulators in the presence of glutamate receptor antagonists. GABA(A)R-gated IPSCs were enhanced by the postsynaptic infusions of adenophostin (1 microM), a potent agonist of inositol-1,4,5-triphosphate receptor (IP(3)R) that induces Ca(2+) release. The enhancement was blocked by co-infusing either 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (10 mM) or CaM-binding peptide (100 microM). Moreover, the postsynaptic infusion of Ca(2+)-CaM (40/10 microM) enhanced both evoked and spontaneous GABA(A)R-gated IPSCs. The enhancement was attenuated by co-infusing 100 microM CaM-KII(281-301), an autoinhibitory peptide of CaM-dependent protein kinases. These results indicate that postsynaptic Ca(2+)-CaM signaling pathways essentially enhance GABAergic synaptic transmission. In the investigation of synaptic targets for the enhancement, we found that IP(3)R agonist-enhanced GABA(A)R-gated IPSCs were attenuated by co-infusing colchicine (30 microM), vincristine (3 microM) or cytochalasin D (1 microM) that inhibits tubulin or actin polymerization, implying that actin filament and microtubules are involved. We conclude that postsynaptic Ca(2+)-CaM signaling pathways strengthen the function of GABAergic synapses via a cytoskeleton-mediated mechanism, probably the recruitment of receptors in the postsynaptic membrane.     

Cholinergic control of firing pattern and neurotransmission in rat neostriatal projection neurons: role of CaV2.1 and CaV2.2 Ca2+ channels

Besides a reduction of L-type Ca2+-currents (Ca(V)1), muscarine and the peptidic M1-selective agonist, MT-1, reduced currents through Ca(V)2.1 (P/Q) and Ca(V)2.2 (N) Ca2+ channel types. This modulation was strongly blocked by the peptide MT-7, a specific muscarinic M1-type receptor antagonist but not significantly reduced by the peptide MT-3, a specific muscarinic M4-type receptor antagonist. Accordingly, MT-7, but not MT-3, blocked a muscarinic reduction of the afterhyperpolarizing potential (AHP) and decreased the GABAergic inhibitory postsynaptic currents (IPSCs) produced by axon collaterals that interconnect spiny neurons. Both these functions are known to be dependent on P/Q and N types Ca2+ channels. The action on the AHP had an important effect in increasing firing frequency. The action on the IPSCs was shown to be caused presynaptically as it coursed with an increase in the paired-pulse ratio. These results show: first, that muscarinic M1-type receptor activation is the main cholinergic mechanism that modulates Ca2+ entry through voltage-dependent Ca2+ channels in spiny neurons. Second, this muscarinic modulation produces a postsynaptic facilitation of discharge together with a presynaptic inhibition of the GABAergic control mediated by axon collaterals. Together, both effects would tend to recruit more spiny neurons for the same task.     

Cannabinoids depress inhibitory synaptic inputs received by layer 2/3 pyramidal neurons of the neocortex

Using whole cell voltage-clamp recordings we investigated the effects of a synthetic cannabinoid (WIN55,212-2) on inhibitory inputs received by layer 2/3 pyramidal neurons in slices of the mouse auditory cortex. Activation of the type 1 cannabinoid receptor (CB1R) with WIN55,212-2 reliably reduced the amplitude of GABAergic inhibitory postsynaptic currents evoked by extracellular stimulation within layer 2/3. The suppression of this inhibition was blocked and reversed by the highly selective CB1R antagonist AM251, confirming a CB1R-mediated inhibition. Pairing evoked inhibitory postsynaptic currents (IPSCs) at short interstimulus intervals while applying WIN55,212-2 resulted in an increase in paired-pulse facilitation suggesting that the probability of GABA release was reduced. A presynaptic site of cannabinoid action was verified by an observed decrease in the frequency with no change in the amplitude or kinetics of action potential-independent postsynaptic currents (mIPSCs). When Cd(2+) was added or Ca(2+) was omitted from the recording solution, the remaining fraction of Ca(2+)-independent mIPSCs did not respond to WIN55,212-2. These data suggest that cannabinoids are capable of suppressing the inhibition of neocortical pyramidal neurons by depressing Ca(2+)-dependent GABA release from local interneurons.     

Distinct muscarinic receptors enhance spontaneous GABA release and inhibit electrically evoked GABAergic synaptic transmission in the chick lateral spiriform nucleus.

The effects of muscarinic agonists on GABAergic synaptic transmission were examined using whole-cell patch-clamp recording in chick brain slices containing the lateral spiriform nucleus. Bath application of muscarine (10 microM) both increased the frequency of spontaneous GABAergic postsynaptic currents and reduced the amplitude of evoked GABAergic polysynaptic postsynaptic currents elicited by focal afferent fiber electrical stimulation. Both of these muscarinic actions were reversible and dose-dependent. Two M(1) antagonists, telenzepine and pirenzipine, and to a lesser extent the M(2) antagonist methoctramine, protected against muscarine's inhibition of the evoked polysynaptic currents. Other M(2) antagonists (tripitramine and gallamine) as well as the M(3) antagonist 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride) and an M(4) antagonist (tropicamide) provided little or no protection against muscarine in this assay. In contrast, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, tropicamide and telenzepine, but not pirenzepine, methoctramine, tripitramine and gallamine, blocked muscarine's enhancement of spontaneous GABAergic currents. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-m-chlorocarbanilate chloride] and CDD-0097 (5-propargyloxycarbonyl-1,4,5,6-tetrahydropyrimidine hydrochloride), two M(1) agonists, mimicked muscarine's inhibition of the evoked polysynaptic GABAergic currents but did not mimic muscarine's enhancement of spontaneous GABAergic currents. Both actions of muscarine persisted when slices were pretreated with pertussis toxin or N-ethylmaleimide, which inactivate G-proteins coupled to M(2) and M(4) receptors while leaving G-proteins coupled to M(1), M(3) and M(5) receptors intact. Muscarine had no significant effect on the amplitude of the direct postsynaptic current elicited by exogenous GABA in the presence of tetrodotoxin.The results demonstrate that distinct muscarinic receptors oppositely modulate GABAergic transmission in the lateral spiriform nucleus. The receptor mediating the inhibition of evoked GABAergic polysynaptic currents is pharmacologically similar to an M(1) receptor, while the enhancement of spontaneous GABAergic currents appears to be mediated by an M(3) receptor.     

M(1) and M(2) muscarinic acetylcholine receptor subtypes mediate Ca(2+) channel current inhibition in rat sympathetic stellate ganglion neurons

Muscarinic acetylcholine receptors (mAChRs) are known to mediate the acetylcholine inhibition of Ca(2+) channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca(2+) channels in SG neurons. The purpose of this study was to identify the mAChR subtypes that modulate Ca(2+) channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca(2+) channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M), and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca(2+) currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Pre-exposure of SG neurons to the M(1) receptor blocker, M(1)-toxin, resulted in VD inhibition of Ca(2+) currents after Oxo-M application. On the other hand, VI modulation of Ca(2+) currents was observed after pretreatment of cells with methoctramine (M(2) mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M(1) and M(2) mAChR blockers but was unaltered when SG neurons were exposed to the M(4) mAChR toxin, M(4)-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M(1) and M(2) receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons that innervate cardiac muscle express M(1) and M(2) mAChR, and activation of these receptors leads to inhibition of Ca(2+) channel currents through VI and VD pathways, respectively.     

Blocker-resistant Ca2+ currents in rat CA1 hippocampal pyramidal neurons

Ca(2+) currents resistant to organic Ca(2+) channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca(2+) currents were isolated by combined application of N-, P/Q- and L-type Ca(2+) current antagonists (omega-conotoxin GVIA 2 microM; omega-conotoxin MVIIC 3 microM; omega-agatoxin IVA 200 nM; nifedipine 10 microM) and constituted approximately 21% of the total Ba(2+) current.The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of -76 and -17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca(2+) currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca(2+) channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni(2+) (half-maximal block at 28 microM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 microM, respectively).In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca(2+) channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming alpha subunits encoding high-threshold Ca(2+) channels, Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons.In summary, CA1 neurons display blocker-resistant Ca(2+) currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca(2+) channel messenger RNAs that give rise to R-type Ca(2+) currents in expression systems.     

Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.     

Mitral cell presynaptic Ca(2+) influx and synaptic transmission in frog amygdala

Dextran-conjugated Ca(2+) indicators were injected into the accessory olfactory bulb of frogs in vivo to selectively fill presynaptic terminals of mitral cells at their termination in the ipsilateral amygdala. After one to three days of uptake and transport, the forebrain hemisphere anterior to the tectum was removed and maintained in vitro for simultaneous electrophysiological and optical measurements. Ca(2+) influx into these terminals was compared to synaptic transmission between mitral cells and amygdala neurons under conditions of reduced Ca(2+) influx resulting from reduced extracellular [Ca(2+)], blockade of N- and P/Q-type channels, and application of the cholinergic agonist carbachol. Reducing extracellular [Ca(2+)] had a non-linear effect on release; release was proportional to Ca(2+) influx raised to the power of approximately 3.6, as observed at numerous other synapses. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1 microM), blocked 77% of Ca(2+) influx and 88% of the postsynaptic field potential. The P/Q-type Ca(2+) channel blocker, omega-agatoxin-IVA (200 nM), blocked 19% of Ca(2+) influx and 25% of the postsynaptic field, while the two toxins combined to block 92% of Ca(2+) influx and 97% of the postsynaptic field. The relationship between toxin blockade of Ca(2+) influx and synaptic transmission was therefore only slightly non-linear; release was proportional to Ca(2+) influx raised to the power approximately 1.4. Carbachol (100 microM) acting via muscarinic receptors had no effect on the afferent volley, but rapidly and reversibly reduced Ca(2+) influx through both N- and P/Q-type channels by 51% and postsynaptic responses by 78%, i.e. release was proportional to Ca(2+) raised to the power approximately 2.5.The weak dependence of release on changes in Ca(2+) when channel toxins block channels suggests little overlap between Ca(2+) microdomains from channels supporting release or substantial segregation of channel subtypes between terminals. The proportionately greater reduction of transmission by muscarinic receptors compared to Ca(2+) channel toxins suggests that they directly affect the release machinery in addition to reducing Ca(2+) influx.     

Dopamine D1/5 receptor-mediated long-term potentiation of intrinsic excitability in rat prefrontal cortical neurons: Ca2+-dependent intracellular signaling

Prefrontal cortex (PFC) dopamine D1/5 receptors modulate long- and short-term neuronal plasticity that may contribute to cognitive functions. Synergistic to synaptic strength modulation, direct postsynaptic D1/5 receptor activation also modulates voltage-dependent ionic currents that regulate spike firing, thus altering the neuronal input-output relationships in a process called long-term potentiation of intrinsic excitability (LTP-IE). Here, the intracellular signals that mediate this D1/5 receptor-dependent LTP-IE were determined using whole cell current-clamp recordings in layer V/VI rat pyramidal neurons from PFC slices. After blockade of all major amino acid receptors (V(hold) = -65 mV) brief tetanic stimulation (20 Hz) of local afferents or application of the D1 agonist SKF81297 (0.2-50 microM) induced LTP-IE, as shown by a prolonged (>40 min) increase in depolarizing pulse-evoked spike firing. Pretreatment with the D1/5 antagonist SCH23390 (1 microM) blocked both the tetani- and D1/5 agonist-induced LTP-IE, suggesting a D1/5 receptor-mediated mechanism. The SKF81297-induced LTP-IE was significantly attenuated by Cd(2+), [Ca(2+)](i) chelation, by inhibition of phospholipase C, protein kinase-C, and Ca(2+)/calmodulin kinase-II, but not by inhibition of adenylate cyclase, protein kinase-A, MAP kinase, or L-type Ca(2+) channels. Thus this form of D1/5 receptor-mediated LTP-IE relied on Ca(2+) influx via non-L-type Ca(2+) channels, activation of PLC, intracellular Ca(2+) elevation, activation of Ca(2+)-dependent CaMKII, and PKC to mediate modulation of voltage-dependent ion channel(s). This D1/5 receptor-mediated modulation by PKC coexists with the previously described PKA-dependent modulation of K(+) and Ca(2+) currents to dynamically regulate overall excitability of PFC neurons.     

Muscarinic receptors depress GABAergic synaptic transmission in rat midbrain dopamine neurons

The effects of muscarine and nicotine on evoked and spontaneous release of GABA were studied using intracellular and whole-cell patch-clamp recordings from rat midbrain dopamine neurons in an in vitro slice preparation. Muscarine (30 microM) reversibly depressed the pharmacologically isolated inhibitory postsynaptic potential evoked by local electrical stimulation. The maximal inhibition of the inhibitory postsynaptic potential amplitude was 39.6+/-5%. This depressant effect of muscarine was blocked by the M3/M1 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (100 nM), but was slightly affected by the M1/M3 receptor antagonist pirenzepine (1 microM). In addition, muscarine decreased the frequency of the miniature synaptic currents without any effect on their amplitude. Moreover, muscarine did not change the GABA-induced hyperpolarization, indicating that its effect on the inhibitory postsynaptic potential is mediated by presynaptic receptors. On the contrary, the cholinergic agonist nicotine did not change the frequency or the amplitude of the spontaneous glutamatergic and GABAergic synaptic currents.Our data indicate that a prevalent activation of presynaptic M3 muscarinic receptors inhibits the GABA-mediated synaptic events, while the activation of nicotinic receptors does not affect the release of glutamate and GABA on midbrain dopamine neurons.     

Possible roles of kainate receptors on GABAergic nerve terminals projecting to rat substantia nigra dopaminergic neurons

GABAergic afferent inputs are thought to play an important role in the control of the firing pattern of substantia nigra pars compacta (SNc) dopaminergic neurons. We report here the actions of presynaptic kainite (KA) receptors in GABAergic transmission of rat SNc dopaminergic neurons. In mechanically dissociated rat SNc dopaminergic neurons attached with native presynaptic nerve terminals, GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded by use of conventional whole cell patch recording mode. In the voltage-clamp condition, KA (3 microM) significantly increased GABAergic mIPSC frequency without affecting the current amplitude. This facilitatory effect of KA was not affected in the presence of 20 microM GYKI52466, a selective AMPA receptor antagonist, but was completely inhibited in the presence of 20 microM CNQX, an AMPA/KA receptor antagonist. Presynaptic KA receptors on GABAergic terminals were mainly permeable to Na+ but impermeable to Ca2+ because KA-induced facilitation of mIPSC frequency was completely suppressed in either Na+-free or Ca2+-free external solutions, and in the presence of 200 microM Cd2+, a general voltage-dependent Ca2+ channel blocker. In the slice preparation, KA increased GABAergic spontaneous mIPSC frequency, but significantly suppressed evoked IPSC (eIPSC) amplitude. However, this inhibitory action on eIPSCs was reversed by 10 microM CGP55845, a selective GABAB receptor antagonist, implicating the possible involvement of GABAB autoreceptors in KA-induced modulation of GABAergic transmission. Thus presynaptic KA receptors on GABAergic nerve terminals synapsing onto SNc neurons may play functional roles contributing the fine control of neuronal excitability and firing pattern of SNc.     

Adenosine inhibits GABAergic and glycinergic transmission in adult rat substantia gelatinosa neurons

The effect of adenosine on inhibitory postsynaptic currents (IPSCs) was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole cell patch-clamp technique. Adenosine reversibly reduced the amplitude of GABAergic and glycinergic electrically evoked IPSCs (eIPSCs) in a dose-dependent manner (EC50 = 14.5 and 19.1 microM, respectively). The A1 adenosine-receptor agonist N6-cyclopentyladenosine also reduced the eIPSCs, whereas the A1 antagonist 8-cyclopentyl-1,3-dimethylxanthine reversed the inhibition produced by adenosine. In paired-pulse experiments, the ratio of the second to first GABAergic or glycinergic eIPSC amplitude was increased by adenosine, whereas the response of SG neurons to exogenous GABA or glycine was unaffected. Adenosine reduced the frequency of GABAergic and glycinergic spontaneous IPSCs without changing their amplitude. This reduction in frequency disappeared in the presence of a K+ -channel blocker (4-aminopyridine) but not in the absence of Ca2+. The inhibition by adenosine disappeared in the presence of cyclic-AMP analog (8-Br-cyclic AMP) and adenylate-cyclase activator (forskolin) but not protein-kinase C (PKC) activator (phorbol-12,13-dibutyrate). We conclude that adenosine suppresses inhibitory transmission in SG neurons by activating presynaptic A1 receptors and that this action is mediated by K+ channels and cyclic AMP but not by Ca2+ channels and PKC. This inhibitory action of adenosine probably contributes to the modulation of pain transmission in the SG.     

Endocannabinoids mediate rapid retrograde signaling at interneuron right-arrow pyramidal neuron synapses of the neocortex

In the neocortex, inhibitory interneurons tightly regulate the firing patterns and integrative properties of pyramidal neurons (PNs). The endocannabinoid system of the neocortex may play an important role in the activity-dependent regulation of inhibitory (i.e., GABAergic) inputs received by PNs. In the present study, using whole cell recordings from layer 2/3 PNs in slices of mouse sensory cortex, we have identified a role for PN-derived endocannabinoids in the control of afferent inhibitory strength. Pairing evoked inhibitory currents with repeated epochs of postsynaptic depolarization led to a transient suppression of inhibition that was induced by a rise in postsynaptic Ca(2+) and was expressed as a reduction in presynaptic GABA release. An antagonist (AM251) of the type-1 cannabinoid receptor blocked the depolarization-induced suppression of evoked inhibitory postsynaptic currents (eIPSCs), and the cannabinoid WIN55,212-2 reduced eIPSC amplitude and occluded suppression. The degree of WIN55,212-2-mediated inhibition of eIPSCs was strongly correlated with the magnitude of depolarization-induced suppression of the eIPSCs, suggesting that the WIN-sensitive afferents are suppressed by PN depolarization. Moreover, blocking endocannabinoid uptake with AM404 strongly modulated the kinetics and magnitude of eIPSC suppression. We conclude that the release of endocannabinoids from PNs allows for the postsynaptic control of presynaptic inhibition and could have profound consequences for the integrative properties of neocortical PNs.     

Control of synaptic strength and timing by the release-site Ca2+ signal

Transmitter release is triggered by highly localized, transient increases in the presynaptic Ca(2+) concentration ([Ca(2+)]). Rapidly decaying [Ca(2+)] elevations were generated using Ca(2+) uncaging techniques, and [Ca(2+)] was measured with a low-affinity Ca(2+) indicator in a giant presynaptic terminal, the calyx of Held, in rat brain slices. The rise time and amplitude of evoked excitatory postsynaptic currents (EPSCs) depended on the half-width of the fluorescence transient, which was predicted by a five-binding site model of a Ca(2+) sensor having relatively high affinity (K(d) approximately 13 microM). Very fast [Ca(2+)] transients (half-width <0.5 ms) evoked EPSCs similar to those elicited by a single action potential (AP) in the same synapse. Triggering release with dual [Ca(2+)] transients of variable amplitudes demonstrated the supralinear transfer function of the sensor. The sensitivity of release to the time course of the [Ca(2+)] transient may contribute to mechanisms by which the presynaptic AP waveform controls synaptic strength.     

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.     

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.     

Inhibition of Ca2+ influx by surfactant in NR8383 alveolar macrophages

OBJECTIVE: Pulmonary surfactants reduce alveolar surface tension and alter inflammatory cell function. We studied the effects of surfactant preparations on Ca2+ influx regulated by protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) and cytokine secretion in the alveolar macrophage (AM) cell line NR8383.METHODS: Fura-2-loaded AMs were stimulated with zymosan (200 microg/ml), 1,2-dioctanoyl-sn-glycerol (DOG, 20 microM) or C6-ceramide (C6C, 10 microM) in the presence of exogenous surfactants (beractant, calfactant or colfosceril) or surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), at 250 microg/ml phospholipid and changes in cytosolic free Ca2+ (Delta[Ca2+]i) and cytokines were measured. RESULTS: Zymosan-induced Delta[Ca2+]i (117 +/- 5 nM) at 3 min was reduced (p <0.001) by beractant (50 +/- 6 nM), colfosceril (61 +/- 2 nM), calfactant (46 +/- 5 nM), and DPPC (52 +/- 5 nM). Beractant inhibited the Delta[Ca2+]i by PKC stimulation with DOG and all preparations reduced the MAPK-induced Ca2+ influx by C6C. Beractant and Ca2+ channel blocker SKF 96365 (10 microM) together abolished the zymosan-stimulated Delta[Ca2+]i. Zymosan-stimulated TNF-alpha and IL-1beta secretion was also inhibited by surfactant pretreatment. CONCLUSIONS: These results indicate that exogenous surfactant inhibits Ca2+ influx and cytokine secretion in zymosan-stimulated AMs. This anti-inflammatory activity may be through an interaction with downstream signaling elements or Ca2+ channels.