Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Cannabinoid-induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212-2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration-dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB₁ receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C-fibre EPSPs than on Aδ-fibre EPSPs. Ba²⁺ (100 μ m ) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CgTX) occluded the WIN-mediated presynaptic inhibition, whereas the P/Q-type Ca²⁺ channel blocker ω-agatoxin TK (ω-Aga) had no effect. These results suggest that cannabinoids preferentially activate CB₁ receptors at the nerve terminal of small-diameter primary afferent fibres. Upon activation, CB₁ receptors may selectively inhibit presynaptic N-type Ca²⁺ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.     

Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.     

Hypoxia-induced secretion of serotonin from intact pulmonary neuroepithelial bodies in neonatal rabbit

We examined the effects of hypoxia on the release of serotonin (5-HT) from intact neuroepithelial body cells (NEB), presumed airway chemoreceptors, in rabbit lung slices, using amperometry with carbon fibre microelectrodes. Under normoxia ( P _O2∼155 mmHg; 1 mmHg ≈133 Pa), most NEB cells did not exhibit detectable secretory activity; however, hypoxia elicited a dose-dependent ( P _O2 range 95–18 mmHg), tetrodotoxin (TTX)-sensitive stimulation of spike-like exocytotic events, indicative of vesicular amine release. High extracellular K⁺ (50 m m ) induced a secretory response similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 m m ) or 4-aminopyridine (2 m m ). Hypoxia-induced secretion was abolished by the non-specific Ca²⁺ channel blocker Cd²⁺ (100 μ m ). Secretion was also largely inhibited by the L-type Ca²⁺ channel blocker nifedipine (2 μ m ), but not by the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 μ m ). The 5-HT₃ receptor blocker ICS 205 930 also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5-HT secretion from intact NEBs via inhibition of K⁺ channels, augmentation of Na⁺-dependent action potentials and calcium entry through L-type Ca²⁺ channels, as well as by positive feedback activation of 5-HT₃ autoreceptors.     

Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

Stimulation-induced changes in NADH fluorescence and mitochondrial membrane potential in lizard motor nerve terminals

To investigate mitochondrial responses to repetitive stimulation, we measured changes in NADH fluorescence and mitochondrial membrane potential (Ψ_m) produced by trains of action potentials (50 Hz for 10–50 s) delivered to motor nerve terminals innervating external intercostal muscles. Stimulation produced a rapid decrease in NADH fluorescence and partial depolarization of Ψ_m. These changes were blocked when Ca²⁺ was removed from the bath or when N-type Ca²⁺ channels were inhibited with ω-conotoxin GVIA, but were not blocked when bath Ca²⁺ was replaced by Sr²⁺, or when vesicular release was inhibited with botulinum toxin A. When stimulation stopped, NADH fluorescence and Ψ_m returned to baseline values much faster than mitochondrial [Ca²⁺]. In contrast to findings in other tissues, there was usually little or no poststimulation overshoot of NADH fluorescence. These findings suggest that the major change in motor terminal mitochondrial function brought about by repetitive stimulation is a rapid acceleration of electron transport chain (ETC) activity due to the Ψ_m depolarization produced by mitochondrial Ca²⁺ (or Sr²⁺) influx. After partial inhibition of complex I of the ETC with amytal, stimulation produced greater Ψ_m depolarization and a greater elevation of cytosolic [Ca²⁺]. These results suggest that the ability to accelerate ETC activity is important for normal mitochondrial sequestration of stimulation-induced Ca²⁺ loads.     

Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

The modulation of synaptic transmission by presynaptic ionotropic and metabotropic receptors is an important means to control and dynamically adjust synaptic strength. Even though synaptic transmission and plasticity at the hippocampal mossy fibre synapse are tightly controlled by presynaptic receptors, little is known about the downstream signalling mechanisms and targets of the different receptor systems. In the present study, we identified the cellular signalling cascade by which adenosine modulates mossy fibre synaptic transmission. By means of electrophysiological and optical recording techniques, we found that adenosine activates presynaptic A₁ receptors and reduces Ca²⁺ influx into mossy fibre terminals. Ca²⁺ currents are directly modulated via a membrane-delimited pathway and the reduction of presynaptic Ca²⁺ influx can explain the inhibition of synaptic transmission. Specifically, we found that adenosine modulates both P/Q- and N-type presynaptic voltage-dependent Ca²⁺ channels and thereby controls transmitter release at the mossy fibre synapse.     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized 'slow wave' current. Reversal of tail current analysis showed this current was due to a Cl⁻ selective conductance. ICC express ANO1, a Ca²⁺-activated Cl⁻ channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca²⁺, replacement of Ca²⁺ with Ba²⁺, or extracellular Ni²⁺ (30 μ m ) blocked the slow wave current. Single Ca²⁺-activated Cl⁻ channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC₅₀= 4.8 μ m ). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca²⁺-activated Cl⁻ conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.     

Parallel decrease in ω-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats

Whole-cell patch-clamp recordings of GABAergic IPSCs were made from cholinergic interneurones in slices of striatum from developing rats aged 21-60 days postnatal. In addition, the Ca²⁺ channel subtypes involved in synaptic transmission, as well as dopamine (DA)-induced presynaptic inhibition, were investigated pharmacologically with development by bath application of Ca²⁺ channel blockers and DA receptor agonists. The IPSC amplitude was reduced by ω-conotoxin GVIA (ω-CgTX) or ω-agatoxin TK (ω-Aga-TK) across the whole age range, suggesting that multiple types of Ca²⁺ channels mediate transmission of the synapse. The IPSC fraction reduced by ω-CgTX significantly decreased, whereas that reduced by ω-Aga-TK remained unchanged with development. DA or quinpirole, a D₂-like receptor agonist, presynaptically reduced the IPSC amplitude throughout development. The DA-induced inhibition decreased with age in parallel with the decrease in N-type Ca²⁺ channels. DA showed no further inhibition of IPSCs after the inhibitory effect of ω-CgTX had reached steady state throughout development. These results demonstrate that there is a functional link between presynaptic N-type Ca²⁺ channels and D₂-like DA receptors at inhibitory synapses in the striatum. They also demonstrate that the suppression of GABAergic transmission by D₂-like receptors is mediated by modulation of N-type Ca²⁺ channels and decreases in parallel with the developmental decline in the contribution of N-type Ca²⁺ channels to exocytosis.     

Ca2+-dependent generation of mitochondrial reactive oxygen species serves as a signal for poly(ADP-ribose) polymerase-1 activation during glutamate excitotoxicity

Mitochondrial Ca²⁺ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca²⁺ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A₂ or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca²⁺ from extracellular medium and blockage of mitochondrial Ca²⁺ uptake by either a mitochondrial uncoupler or a Ca²⁺ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca²⁺ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca²⁺-dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca²⁺ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.     

Molecular aspects of acute inhibition of Na+-H+ exchanger NHE3 by A2-adenosine receptor agonists

Adenosine regulates Na⁺ homeostasis by its acute effects on renal Na⁺ transport. We have shown in heterologously transfected A6/C1 cells (renal cell line from Xenopus laevis ) that adenosine-induced natriuresis may be effected partly via A₂ adenosine receptor-mediated inactivation of the renal brush border membrane Na⁺-H⁺ exchanger NHE3. In this study we utilized A6/C1 cells stably expressing wild-type as well as mutated forms of NHE3 to assess the molecular mechanism underlying A₂-dependent control of NHE3 function. Cell surface biotinylation combined with immunoprecipitation revealed that NHE3 is targeted exclusively to the apical domain and that the endogenous Xenopus NHE is located entirely on the basolateral side of A6/C1 transfectants. Stimulation of A₂-adenosine receptors located on the basolateral side for 15 min with CPA ( N 6-cyclopentyladenosine) acutely decreased NHE3 activity (microspectrofluorimety). This effect was mimicked by 8-bromo-cAMP and entirely blocked by pharmacological inhibition of PKA (with H89) or singular substitution of two PKA target sites (serine 552 and serine 605) on NHE3. Downregulation of NHE3 activity by CPA was attributable to a reduction of NHE3 intrinsic transport activity without change in surface NHE3 protein at 15 min. At 30 min, the decrease in transport activity was associated with a decrease in apical membrane NHE3 antigen. In conclusion, two highly conserved target serine sites on NHE3 determine NHE3 modulation upon A₂-receptor activation and NHE3 inactivation by adenosine proceeds via two phases with distinct mechanisms.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Modulation of Ca channels by activation of adenosine A1 receptors in rat striatal glutamatergic nerve terminals

We determined that activation of adenosine A₁ receptors in striatal synaptosomes with 100 nM N⁶-cyclopentyladenosine (CPA) inhibited both the release of endogenous glutamate and the increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i), due to 4-aminopyridine (4-AP) stimulation, by 28 and 19%, respectively. Furthermore, CPA enhanced the inhibition of endogenous glutamate release due to ω-conotoxin GVIA (ω-Cgtx GVIA), ω-Cgtx MVIIC or ω-Cgtx GVIA plus ω-Cgtx MVIIC. Similar effects were observed in the [Ca²⁺]_i signal. The inhibitory effects of CPA and ω-Cgtx GVIA were additive, but the effects of CPA and ω-Cgtx MVIIC were only partially additive. These results suggest that P/Q-type Ca²⁺ channels and other type(s) of Ca²⁺ channel(s), coupled to glutamate release, are inhibited subsequently to activation of adenosine A₁ receptors.     

Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

IP3 receptors: some lessons from DT40 cells

Summary:  Inositol-1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels that are regulated by IP₃ and Ca²⁺ and are modulated by many additional signals. These properties allow them to initiate and, via Ca²⁺-induced Ca²⁺ release, regeneratively propagate Ca²⁺ signals evoked by receptors that stimulate formation of IP₃. The ubiquitous expression of IP₃R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP₃R. DT40 cells are a pre-B-lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40-knockout cells with both alleles of each of the three IP₃R genes disrupted provide the only null-background for analysis of homogenous recombinant IP₃R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP₃R behavior. Patch-clamp recording from the nuclear envelope and Ca²⁺ release from intracellular stores loaded with a low-affinity Ca²⁺ indicator address the mechanisms leading to activation of IP₃R. We show that IP₃ causes intracellular IP₃R to cluster and re-tune their responses to IP₃ and Ca²⁺, better equipping them to mediate regenerative Ca²⁺ signals. Finally, we show that DT40 cells reliably count very few IP₃R into the plasma membrane, where they mediate about half the Ca²⁺ entry evoked by the B-cell antigen receptor.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

Mechanisms underlying cannabinoid inhibition of presynaptic Ca2+ influx at parallel fibre synapses of the rat cerebellum

Activation of CB1 cannabinoid receptors in the cerebellum acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses by decreasing the probability of glutamate release. This depression involves the activation of presynaptic 4-aminopyridine-sensitive K⁺ channels by CB1 receptors, which in turn inhibits presynaptic Ca²⁺ influx controlling glutamate release at these synapses. Using rat cerebellar frontal slices and fluorometric measures of presynaptic Ca²⁺ influx evoked by stimulation of parallel fibres with the fluorescent dye fluo-4FF, we tested whether the CB1 receptor-mediated inhibition of this influx also involves a direct inhibition of presynaptic voltage-gated calcium channels. Since various physiological effects of CB1 receptors appear to be mediated through the activation of PTX-sensitive proteins, including inhibition of adenylate cyclases, activation of mitogen-activated protein kinases (MAPK) and activation of G protein-gated inwardly rectifying K⁺ channels, we also studied the potential involvement of these intracellular signal transduction pathways in the cannabinoid-mediated depression of presynaptic Ca²⁺ influx. The present study demonstrates that the molecular mechanisms underlying the CB1 inhibitory effect involve the activation of the PTX-sensitive G_i/G_o subclass of G proteins, independently of any direct effect on presynaptic Ca²⁺ channels (N, P/Q and R (SNX-482-sensitive) types) or on adenylate cyclase or MAPK activity, but do require the activation of G protein-gated inwardly rectifying (Ba²⁺- and tertiapin Q-sensitive) K⁺ channels, in addition to 4-aminopyridine-sensitive K⁺ channels.