Crayfish Neuromuscular Junction Research Articles

An antibody to synaptotagmin I facilitates synaptic transmission

Proper functioning of the nervous system requires precise control of neurotransmitter release. Synaptotagmin, a synaptic vesicle protein, is crucial for the temporal control of neurotransmitter release. The mechanism of synaptotagmin function is still under debate. To investigate the mechanism by which synaptotagmin controls neurotransmitter release, we injected an antibody of rat synaptotagmin I into a crayfish motor axon. We found that the antibody enhanced synaptic transmission at crayfish neuromuscular junctions by increasing the amplitude of the evoked synaptic response. This effect was antibody-dose dependent. The antibody also reduced the rise time of the synaptic potentials. These effects were accompanied by a reduction in the Hill coefficient for Ca(2+)-dependence of synaptic transmission. Our findings support the hypothesis that synaptotagmin inhibits neurotransmitter release in the absence of Ca(2+).

CHARACTERIZATION OF THE EFFECTS OF DL-2-AMINO-4-PHOSPHONOBUTYRIC ACID AT A CRAYFISH NEUROMUSCULAR JUNCTION.

Purpose The crayfish (Procambarus clarkii and simulans) neuromuscular junction (NMJ) is an established model of synaptic transmission. Glutamate is its excitatory neurotransmitter. The purpose of this experiment is to confirm the validity of the crayfish NMJ as a viable model for follow-on experiments addressing the prevention or reduction of glutamate excitotoxicity secondary injury following spinal cord trauma. Specifically, the effects of DL-2-amino-4-phosphonobutyric acid (AP4) on the excitatory junctional potentials (EJP) of NMJs of the dactylopodite opener muscle of the first and second walking limbs of crayfish will be studied. Efficacy of known NMDA agonist/antagonist AP4 will verify the existence of ionotropic NMDA glutamate receptors in the crayfish NMJ and characterize the effects of the agonists at an invertebrate NMJ. Methods The first or second walking limb was removed. The meropodite was dissected to expose the single excitatory axon to the opener muscle of the dactylopodite. The propodite was dissected to expose the opener muscle. The preparation was bathed continually in Van Harrevald9s solution (Van H), pH 7.2 ± 0.1. For each preparation, control EJPs were obtained. AP4 was added and EJPs were recorded immediately and at 1 and 2 minutes post addition. AP4 was then washed out with Van H, using three complete exchanges of the bath. EJPs were recorded following three, six, and nine washouts. Serial dilutions were performed for concentrations of 1 mM to 3.75 μM AP4. Results Based on the serial dilutions, AP4 acts as an antagonist at a concentration of 1,000 μM and an agonist at 3.75 μM. Results at intermediate values were equivocal. Qualitatively similar results were obtained in at least three additional preparations at each concentration. Discussion The crayfish NMJ contains glutamate receptors that respond to NMDA agonist/antagonist AP4 in a concentration-dependent, biphasic manner. This study provides additional evidence of the presence of NMDA glutamate receptors at the crayfish NMJ. Further studies to construct a complete concentration-response curve are warranted. Supported by the Arnold C., Barbara M., and Georgianna Fossa Spinal Cord Injury Research Fund and the Indiana University School of Medicine Brain and Spinal Cord Injury Research Program.

INITIAL CHARACTERIZATION OF EFFECTS OF THE N-METHYL-D-ASPARTATE AGONIST 1-AMINOCYCLOBUTANE-CIS-1,3-DICARBOXYLIC ACID ON GLUTAMATE-EVOKED EXCITATORY JUNCTIONAL POTENTIALS AT A CRAYFISH NEUROMUSCULAR JUNCTION.

Background Secondary injury following spinal cord trauma can include sequelae of glutamate excitotoxicity (GE). A crayfish walking limb neuromuscular junction (NMJ), having NMDA glutamate receptors, may be a useful model for studying potential GE mechanisms and countermeasures. This project examines the effects of 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA), a potent glutamate agonist in vertebrate nervous systems, on excitatory junctional potentials (EJPs) at crayfish (Procambarus clarkii and simulans) NMJs. Methods Crayfish first and second walking limbs were dissected to expose the dactylopodite opener muscle and the single, excitatory axon innervating it. Standard intracellular electrophysiologic techniques were used to measure EJPs. The opener muscle, located in the proprodite, and its excitatory axon, in the meropodite, were exposed and bathed in van Harreveld9s solution (VanH) pH 7.2 ± 0.1. Van H was removed, and ACDA in vanH at 100 μM was added to exposed opener muscles. EJPs were measured at 1- and 2-minute intervals for each data collection. Short-term facilitated EJPs were evoked by 10-second stimulation at 30 Hz, then EJPs were averaged for 10 seconds. Results EJPs in 100 μM ACDA-treated preparations showed partial suppression, ranging from 17 to 44% of control amplitude, with return to nearly control level (approximately 90%) following vanH wash with at least three complete exchanges of the bath. Qualitatively similar results were obtained using six separate limb preparations. Discussion Suppression of EJPs by the NMDA agonist ACDA was unexpected as the crayfish NMJ has NMDA receptors and glutamate is the excitatory neurotransmitter. A complete concentration:response study is warranted to determine if ACDA acts as an agonist at lesser concentrations. It is also possible that the invertebrate and vertebrate receptors have different responses to ACDA. However, at the 100 μM concentration, ACDA should not be ruled out for studies of means of reducing glutamate excitotoxicity. Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund and the Indiana University Brain and Spinal Cord Injury Research Program.

SUPPRESSION OF AP4-ENHANCED EXCITATORY JUNCTIONAL POTENTIALS BY THE D3 STEREOISOMER OF A CARBOXY DERIVATIVE OF BUCKMINSTERFULLERENE.

Purpose In spinal cord injury (SCI), glutamate excitotoxicity can cause secondary injury. NMDA glutamate receptor blockade is a potential protective strategy following SCI. A crayfish neuromuscular junction (NMJ) model is useful for studying NMDA receptor activity. The D3 stereoisomer of the malonic acid (carboxy) derivative of buckminsterfullerene suppresses excitatory junctional potentials (EJPs) of NMJs of the opener muscle of crayfish (Procambarus clarkii and simulans) walking limbs. DL-2-amino-4-phosphonobutyric acid (AP4, a specific NMDA receptor agonist) potentiates EJPs in this preparation. This study examines whether D3 can suppress AP4-enhanced EJPs. Materials/Methods A first or second walking limb was removed, and the meropodite and propodite were dissected to expose the excitatory axon and the opener muscle, respectively. The preparation was bathed in Van Harreveld9s Solution (VH, pH 7.2 ± 0.1) for collection of pretreatment data. Following a pretreatment control, a solution of 3.75 μm AP4 in VH was applied. EJPs were recorded immediately following application and at 1 and 2 minutes, using standard intracellular electrophysiologic techniques. Short-term facilitation (10-second stimulus at 35 Hz) averaging eight tracings per second for 10 seconds was used to record EJPs. AP4/VH was then replaced with 3.75 μm AP4 and 25 μm D3 in VH. EJPs were recorded immediately and at 1, 2, 3, and 4 minutes. Post-treatment control data collection followed three complete exchanges of the bath with VH. Results Data are reported as percent change from control amplitude; 3.75 μm AP4 enhanced EJP amplitude 30%, with a subsequent depression of 35% in 3.75 μm AP4 and 25 μm D3. The final washout returned to within 0.27% of precontrol. Qualitatively similar results were found in three additional preparations. Discussion 25 μm D3 can suppress 3.75 μm of AP4-induced EJP potentiation. This further suggests that D3 may be a useful agent in suppressing glutamate excitotoxicity induced by NMDA receptor stimulation. Supported by the Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund, the IU School of Medicine Brain and Spinal Cord Injury Research Program, and the Hillblom Foundation (L.D.).

EFFECT OF 1-AMINOCYCLOBUTANE-CIS-1,3-DICARBOXYLIC ACID ON GLUTAMATE EXCITATORY JUNCTIONAL POTENTIALS AT A CRAYFISH WALKING LIMB NEUROMUSCULAR JUNCTION.

Purpose To determine the utility of the crayfish walking limb opener muscle (OM) as a model for studying glutamate excitotoxicity, it is necessary to confirm NMDA receptor activity and identify agonist agents that are effective in this preparation. This experiment studies the effects of 1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA), a potent NMDA glutamate receptor agonist, on excitatory junctional potentials (EJPs) of the crayfish walking limb OM. Methods A first or second walking limb from a medium-sized crayfish was dissected to reveal the OM of the walking limb dactylopodite as well as the single excitatory axon that innervates it. Standard intracellular electrophysiologic recording methods were used to examine EJPs of OM fibers. The preparation was bathed in Van Harreveld9s solution (VanH) for control data and washing. The resting membrane potential (RMP) was monitored. The preparation was then bathed in 100, 50, and 25 μM solutions of ACDA in VanH. The axon was stimulated at 30 Hz to evoke short-term facilitated EJPs. EJPs averaged over 8 seconds were recorded, at time immediately upon addition of ACDA and at 1 and 2 minutes postaddition. ACDA was then removed, the preparation was washed with at least three complete exchanges of the bath, and postwash data were collected. Results The RMP in both VanH and ACDA was unchanged during the studies. One-hundred micrometer ACDA showed depression with EJPs ranging from 0 to 70% of control amplitude (CA), with partial recovery upon wash (75-94% of CA). Fifty-micrometer ACDA showed enhancement of EJPs (116-214% of CA), with partial to full recovery upon wash (70-115% of CA). Twenty-five-micrometer ACDA showed a transient enhancement of EJPs initially (110-118% of CA) followed by depression with nearly complete recovery or enhancement upon wash (86-136% of CA). Conclusion ACDA has a concentration-dependent, biphasic effect on EJPs at the concentrations studied. Qualitatively similar results were observed with at least three different limb preparations at each concentration. ACDA enhances EJPs at relatively lower concentrations and depresses at higher concentrations. The effects confirm NMDA receptor activity at the crayfish neuromuscular junction and suggest that ACDA may be useful for future studies of NMDA glutamate receptor activity. Acknowledgment: Arnold C., Barbara M., and Georgianna Fossa Spinal Cord Injury Research Fund and the Indiana University Brain and Spinal Cord Injury Research Program.

Role of NSF in Neurotransmitter Release: A Peptide Microinjection Study at the Crayfish Neuromuscular Junction

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.

Glutamatergic autoinhibition of quantal release augments the early phase of releases after a depolarization pulse

At the crayfish neuromuscular junction, glutamatergic autoinhibition of quantal excitatory postsynaptic current (qEPSC) release is mediated by a presynaptic DL-glutamate transporter and its associated Cl- conductance. I investigated whether it also affects the time course of release. qEPSCs were recorded with a perfused macroelectrode through which depolarization pulses and D- or L-glutamate could be applied to a terminal. In order to represent the time course of release, cumulative delays of qEPSCs were determined and scaled to a common final value. At 10 degrees C, on the application of D- or L-glutamate, release increased relative to the controls especially during its first millisecond, taking the mean of 20 experiments (P < 0.01). Also, in many single experiments the respective shifts in the time courses of release were highly significant. The relative surplus of early releases decreased with time constants tau1 of 86 micros and tau2 of 0.75 ms. At 0 degrees C, in the presence of glutamate, the surplus of early delays was increased relative to the controls to a significantly greater extent and for a longer time than at 10 degrees C. The tau1 of 240 micros was almost three times larger than at 10 degrees C. Autoinhibition was inactivated in Cl(-)-free solution. In such solutions the surplus of early releases also disappeared and the shortening of early delays reverted to a lengthening. Interaction of the inhibitory autoreceptor and its associated Cl- flow with the release machinery is discussed.

Cholesterol and synaptic transmitter release at crayfish neuromuscular junctions

During exocytosis of synaptic transmitters, the fusion of highly curved synaptic vesicle membranes with the relatively planar cell membrane requires the coordinated action of several proteins. The role of membrane lipids in the regulation of transmitter release is less well understood. Since it helps to control membrane fluidity, alteration of cholesterol content may alter the fusibility of membranes as well as the function of membrane proteins. We assayed the importance of cholesterol in transmitter release at crayfish neuromuscular junctions where action potentials can be measured in the preterminal axon. Methyl-beta-cyclodextrin (MbetaCD) depleted axons of cholesterol, as shown by reduced filipin labelling, and cholesterol was replenished by cholesterol-MbetaCD complex (Ch-MbetaCD). MbetaCD blocked evoked synaptic transmission. The lack of postsynaptic effects of MbetaCD on the time course and amplitude of spontaneous postsynaptic potentials or on muscle resting potential allowed us to focus on presynaptic mechanisms. Intracellular presynaptic axon recordings and focal extracellular recordings at individual boutons showed that failure of transmitter release was correlated with presynaptic hyperpolarization and failure of action potential propagation. All of these effects were reversed when cholesterol was replenished with Ch-MbetaCD. However, focal depolarization of presynaptic boutons and administration of a Ca2+ ionophore both triggered transmitter release after cholesterol depletion. Therefore, both presynaptic Ca2+ channels and Ca2+-dependent exocytosis functioned after cholesterol depletion. The frequency of spontaneous quantal transmitter release was increased by MbetaCD but recovered when cholesterol was reintroduced. The increase in spontaneous release was not through a calcium-dependent mechanism because it persisted with intense intracellular calcium chelation. In conclusion, cholesterol levels in the presynaptic membrane modulate several key properties of synaptic transmitter release.

Low Concentration Effects of a Carboxy Derivative of a Buckminsterfullerene on Excitatory Junction Potentials at a Crayfish Neuromuscular Junction

Low Concentration Effects of a Carboxy Derivative of a Buckminsterfullerene on Excitatory Junction Potentials at a Crayfish Neuromuscular Junction

Structural Relationships, Distribution and Biological Activities ofStemonaAlkaloids

Stemona alkaloids represent a unique class of natural products exclusively isolated from the monocotyledonous family Stemonaceae comprising three genera mainly distributed in southeast Asia. Structurally the alkaloids are characterised by a pyrrolo[1,2- a]azepine nucleus usually linked with two carbon chains mostly forming terminal lactone rings. Based on biosynthetic considerations and their various distribution the present review describes 82 Stemona alkaloids grouped into three skeletal types. Due to different carbon chains attached to C-9 of the pyrroloazepine nucleus they were classified into stichoneurine-, protostemonine- and croomine-type alkaloids. The genera Croomia and Stichoneuron only accumulate croomine or stichoneurine derivatives, respectively, whereas the genus Stemona produces all three types of alkaloids. However, species-specific accumulation trends towards certain structural types represent valuable chemosystematic criteria. Bioassays with larvae of Spodoptera littoralis exhibited very high insect toxicity for the roots of Stemona species containing certain protostemonine derivatives, especially didehydrostemofoline, whereas those with dominating stichoneurine or croomine derivatives showed low toxicity but sometimes remarkable repellence due to an accumulation of tuberostemonine. Tuberostemonine also showed effects on the motility of helminth worms and reduced the excitatory transmission at the crayfish neuromuscular junction. Significant antitussive activity was shown for the stereoisomeric neotuberostemonine in guinea-pig after cough induction by citric acid aerosol stimulation. Studies on structure-activity relationship with seven related compounds revealed that the saturated tricyclic pyrrolobenzazepine nucleus of tuberostemonines is the prerequisite for antitussive activity.

A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide

DF 2 (DRNFLRFamide), a FMRFamide-like peptide, has been shown to increase the amount of transmitter released at crayfish neuromuscular junctions. Here, we examined a possible role for the cyclic nucleotide monophosphates, cAMP and cGMP, in DF 2's effects on synaptic transmission. The effects of DF 2 on synaptic transmission were monitored by recording excitatory postsynaptic potentials (EPSPs) in the deep abdominal extensor muscles of the crayfish, Procambarus clarkii. A number of activators and inhibitors were used to determine whether or not cAMP, cGMP, protein kinase A (PKA) and protein kinase G (PKG) mediate the effect of this neuropeptide. Phosphodiesterase inhibitors, known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of DF 2 on synaptic transmission. Activators of PKA (Sp-cAMPS) and PKG (8-pCPT-cGMP) increase EPSP amplitude, mimicking the effects of DF 2. Inhibitors of PKA (Rp-cAMPS) and PKG (Rp-8-pCPT-cGMPS) each block a portion of the response to the peptide, and when applied together these two inhibitors completely block the response. Taken together, these results indicate that cyclic nucleotides and cyclic nucleotide-dependent protein kinases are necessary components of the pathway underlying modulation by this neuropeptide.

Presynaptic mechanism of action induced by 5-HT in nerve terminals: Possible involvement of ryanodine and IP 3 sensitive Ca 2+ stores

Although modulation of transmitter release by serotonin (5-HT) at crayfish neuromuscular junctions has been known since 1965, the mechanisms of action have not been established in this classical synaptic preparation. We show that injections of adenophostin-A (an IP 3 analog) in the nerve terminals greatly enhances synaptic transmission. Exposure to ryanodine (Ry) produces a biphasic response: at low concentration it is excitatory and high concentration it is inhibitory. Likewise, a low concentration (1 μM) of caffeine enhances synaptic transmission, whereas a high concentration (10 mM) has little effect on transmission. The varied responses and sensitivity to Ry and caffeine suggest a Ca 2+-induced Ca 2+-release mechanism and/or the presence of an IP 3-receptor within the terminal. Thus, it is likely 5-HT's response is due to activation of intracellular pathways, which subsequently release Ca 2+ from internal stores.

Probing the Endogenous Ca2+Buffers at the Presynaptic Terminals of the Crayfish Neuromuscular Junction

Ca2+ indicators of varying affinity and mobility were pressure injected into the presynaptic axon of the inhibitor of the crayfish neuromuscular junction (NMJ). Fluorescence transients recorded at a 2-kHz resolution were used to probe physiological parameters governing the decay of fluorescence transients within 100 ms after an action potential (early decay). Blocking Ca2+ extrusion or Ca2+ sequestration processes did not significantly alter early decay, arguing against a role for either mechanism. Fluorescence transients recorded with low mobility or fixed indicators exhibited early decay similar to that recorded with indicators of comparable affinity but high mobility, suggesting that early decay was not due to the rate of Ca2+-indicator diffusion. The extent of early decay correlated closely with the affinity, but not mobility, of the Ca2+ sensitive dyes tested. These results implicate intrinsic buffers with slow Ca2+ binding kinetics as the most likely determinants of early decay. However, computer simulations showed that intrinsic buffers with a slow binding rate are unlikely to be the only ones present in the system because the slow kinetics would be unable to buffer incoming Ca2+ during an action potential and would result in momentary indicator saturation. In fact, experimental data show that the peak amplitude of an action potential activated Ca+ transient is about 20% of the maximal fluorescence intensity activated by prolonged Ca2+ influx. We conclude that endogenous buffering at the crayfish NMJ includes both fast and slow components, the former being fast enough to compete with fast Ca2+ indicators, and the latter dictating the early decay.

Interaction between facilitation and presynaptic inhibition at the crayfish neuromuscular junction

Action potential-mediated calcium (Ca) entry into excitor nerve terminal boutons during presynaptic inhibition and the effects of co-activation of the inhibitor on the kinetics of muscle contraction were studied at crayfish claw opener muscle. Inhibition reduced postsynaptic excitatory junction potentials (EJPs) below the threshold to initiate contraction. Upon cessation of inhibition, EJP amplitudes immediately increased several-fold due to the build-up of presynaptic facilitation during inhibition. Consequently, muscle contraction was initiated more rapidly after a period of inhibitor-excitor coactivation. Presynaptic inhibition reduced Ca entry into presynaptic excitor terminal boutons (range 0-50%, mean +/-s.e.m.=20+/-1%, N=122 terminals; 12 preparations) and reduced the EJP amplitude (range 30-70%, mean +/- s.e.m.=51+/-2%, N=27 cells). The decline in the EJP was proportional to the reduction of Ca influx raised to the power of 2.8. Since presynaptic inhibition reduces the number of Ca channels opened by an action potential, our data suggest cooperativity between Ca channel microdomains to initiate vesicle fusion at this synapse. The amount of inhibition of Ca influx into an excitor bouton was not correlated with either the distance to the closest inhibitor bouton or the main excitor branch, although slightly more inhibition was seen for excitor boutons on tertiary versus secondary branches. Unlike inhibitor axon stimulation, bath application of GABA caused inhibition of Ca influx that steadily increased from proximal to distal terminal boutons on a branch. We propose a model where presynaptic inhibition causes localized shunting of an actively propagated action potential in the vicinity of release sites, which can recover its amplitude outside the shunted region.

CAMP Acts on Exchange Protein Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Protein to Regulate Transmitter Release at the Crayfish Neuromuscular Junction

Glutamatergic synapses are highly modifiable, suiting them for key roles in processes such as learning and memory. At crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) ion channels mediate hormonal modulation of glutamatergic synapses and a form activity-dependent long-term facilitation (LTF) of synaptic transmission. Here, we show that a new target for cAMP, exchange protein activated by cAMP (Epac) or cAMP-regulated guanine nucleotide exchange protein, is involved in the hormonal enhancement of synaptic transmission by serotonin. Induction of LTF "tags" synapses, rendering them responsive to cAMP in an HCN-independent manner. Epac also mediates the enhancement of tagged synapses. Thus, the cAMP-dependent enhancement of transmission is mediated by two separate pathways, neither of which involves protein kinase A.

Modulation of available vesicles and release kinetics at the inhibitor of the crayfish neuromuscular junction

We have investigated the effect of serotonin (5-HT) and okadaic acid (OA) on presynaptic processes at the crayfish inhibitory neuromuscular junction. Two different physiological parameters of transmitter release were examined: release kinetics and the size of the readily releasable pool of vesicles (RRP). Using a paired pulse stimulus and high frequency trains, we established that a single broad action potential, recorded in 20 mM tetraethylammonium and 1 mM 4-amino-pyridine, released the RRP in its entirety. Thus, by measuring the amplitude of inhibitory postsynaptic potential (IPSC) we were able to directly assess the effects of 5-HT and OA on the RRP. Serotonin at 200 nM and OA at 2.5 μM each significantly increased IPSC above control levels and the effects of these two modulators were comparable. Both modulators also induced a leftward shift in the rising phase of IPSC, i.e. an apparent acceleration in release kinetics. The shift caused by OA was significantly more pronounced than that induced by 5-HT. This apparent acceleration in release was not associated with a corresponding change in the presynaptic Ca 2+ transient measured at a 2 kHz resolution, suggesting that modulation was not due to an acceleration in Ca 2+ channel kinetics. In view of the comparable increase in the size of the RRP by the modulators, the differential modulation of release kinetics suggests that these two parameters may be modulated by separate biochemical processes.

Facilitation through Buffer Saturation: Constraints on Endogenous Buffering Properties

Synaptic facilitation (SF) is a ubiquitous form of short-term plasticity, regulating synaptic dynamics on fast timescales. Although SF is known to depend on the presynaptic accumulation of Ca 2+, its precise mechanism is still under debate. Recently it has been shown that at certain central synapses SF results at least in part from the progressive saturation of an endogenous Ca 2+ buffer (Blatow et al., 2003), as proposed by Klingauf and Neher (1997). Using computer simulations, we study the magnitude of SF that can be achieved by a buffer saturation mechanism (BSM), and explore its dependence on the endogenous buffering properties. We find that a high SF magnitude can be obtained either by a global saturation of a highly mobile buffer in the entire presynaptic terminal, or a local saturation of a completely immobilized buffer. A characteristic feature of BSM in both cases is that SF magnitude depends nonmonotonically on the buffer concentration. In agreement with results of Blatow et al. (2003), we find that SF grows with increasing distance from the Ca 2+ channel cluster, and increases with increasing external Ca 2+, [Ca 2+] ext, for small levels of [Ca 2+] ext. We compare our modeling results with the experimental properties of SF at the crayfish neuromuscular junction, and find that the saturation of an endogenous mobile buffer can explain the observed SF magnitude and its supralinear accumulation time course. However, we show that the BSM predicts slowing of the SF decay rate in the presence of exogenous Ca 2+ buffers, contrary to experimental observations at the crayfish neuromuscular junction. Further modeling and data are required to resolve this aspect of the BSM.

Roles of Ca2+, hyperpolarization and cyclic nucleotide-activated channel activation, and actin in temporal synaptic tagging.

At crayfish neuromuscular junctions, cAMP increases transmitter released by action potentials by activating two effectors, hyperpolarization and cyclic nucleotide-activated channels (HCNCs) and a separate target that has been tentatively identified as exchange protein activated by cAMP (Epac). Intense electrical activity in the motor neuron induces a long-term facilitation (LTF) of transmitter release in which hyperpolarization from an electrogenic Na+-K+ exchanger activates HCNCs. The coupling of HCNCs to transmission involves actin. After LTF induction, cAMP further increases transmission in an HCNC-independent manner, activating the second target. This relaxation of the requirement for HCNC activation to enhance release is called temporal synaptic tagging. Tagging lasts at least 1 d but develops only in the 10 min period after electrical activity. The HCNCs are activated by the post-tetanic hyperpolarization occurring during this time. Both synaptic tagging and LTF induction depend on presynaptic Ca2+ accumulation during activity; both are blocked by EGTA-AM, and LTF is also prevented by stimulation in a low-[Ca2+] medium. Actin depolymerizers prevent induction of LTF and tagging, with little effect on HCNCs, whose sensitivity to cAMP and HCNC blockers is unaffected by tagging. Enhancement of actin polymerization can rescue tagging from HCNC block, suggesting that actin acts at a step after HCNC activation. These and other recent results suggest a model in which HCNC activation, followed by a process involving actin polymerization, acts cooperatively with [Ca2+] to induce tagging, after which only Epac activation is required for cAMP to further enhance transmission.

5-HT offsets homeostasis of synaptic transmission during short-term facilitation

In this study, we approach the topic of vesicle recruitment and recycling by perturbing neurotransmission at the crayfish neuromuscular junction with altered electrical activity and the presence of the neuromodulator serotonin (5-HT). After induction of short-term facilitation (STF) with stimulus pulse trains (40 Hz, 20 pulses), the amount of synaptic transmission can be maintained at a relatively constant level, producing a plateau in the amplitude of the excitatory postsynaptic potentials (EPSPs) throughout the remaining stimuli within a train of a few hundred milliseconds. With an increase in the frequency of the stimuli within a train (60 Hz, 20 pulses), an altered plateau of larger EPSP amplitudes occurs. This suggests that differential rates of vesicle recruitment can be rapidly reached and maintained. Exposure of nerve terminals to 5-HT further enhances the EPSP amplitudes to yet a higher plateau level. The effect of 5-HT is more pronounced for 40-Hz pulse trains than for 60-Hz trains. This suggests that 5-HT can recruit vesicles into the readily releasable pool (RRP) and that the recruitment is limited at higher stimulation frequencies. The attainment of a larger amplitude in the plateaus of the EPSPs at 60 Hz compared with 40 Hz also suggests that the rapid induction of STF enhances the entry of vesicles into the RRP. By direct quantal counts, mean quantal content increases linearly during STF, and 5-HT offsets the linear release. We propose that 5-HT and electrically induced recruitment of vesicles from a reserve pool to the RRP may share similar recruitment mechanisms.

Actions of MDMA at glutamatergic neuromuscular junctions.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.