Crayfish Neuromuscular Junction Research Articles

Ultrasound-Induced Membrane Hyperpolarization in Motor Axons and Muscle Fibers of the Crayfish Neuromuscular Junction.

Focused ultrasound (FUS) can modulate neuronal activity by depolarization or hyperpolarization. Although FUS-evoked depolarization has been studied extensively, the mechanisms underlying FUS-evoked hyperpolarization (FUSH) have received little attention. In the study described here, we developed a procedure using FUS to selectively hyperpolarize motor axons in crayfish. As a previous study had reported that these axons express mechano- and thermosensitive two-pore domain potassium (K2P) channels, we tested the hypothesis that K2P channels underlie FUSH. Intracellular recordings from a motor axon and a muscle fiber were obtained simultaneously from the crayfish opener neuromuscular preparation. FUSH was examined while K2P channel activities were modulated by varying temperature or by K2P channel blockers. FUSH in the axons did not exhibit a coherent temperature dependence, consistent with predicted K2P channel behavior, although changes in the resting membrane potential of the same axons indicated well-behaved K2P channel temperature dependence. The same conclusion was supported by pharmacological data; namely, FUSH was not suppressed by K2P channel blockers. Comparison between the FUS-evoked responses recorded in motor axons and muscle fibers revealed that the latter exhibited very little FUSH, indicating that the FUSH was specific to the axons. It is not likely that K2P channels are the underlying mechanism for FUSH in motor axons. Alternative mechanisms such as sonophore and axon-specific potassium channels were considered. Although the sonophore hypothesis could account for electrophysiological features of axonal recordings, it is not consistent with the lack of FUSH in muscle fibers. An axon-specific and mechanosensitive potassium channel is also a possible explanation.

The effects of levetiracetam on glutamatergic synaptic transmission: Crayfish and Drosophila NMJs

Epilepsy is a neurological disorder characterized by recurring, unpredictable seizures. Its disease burden is high, seeing as it ranks fourth in the world's neurological disorders burden following: tension-type headaches, migraines, and Alzheimer's disease. The commonly used anti-epileptic drug levetiracetam (Keppra) reduces epileptic seizures, but the exact mechanism through which it works is not known. Some studies suggest sodium and/or potassium ionic channels are directly altered reducing membrane excitability, while others suggest the drug interacts with SV2 protein to alter synaptotagmin (a calcium sensor protein) action in the presynaptic nerve terminal (reducing excitability). The glutamatergic synapses at crayfish and larval Drosophila neuromuscular junctions (NMJs) were used to assess the drug's action. The evoked excitatory junction potentials (EJPs) of the crayfish NMJ were enhanced through exposure to 1 mM of the drug, but not lower doses, with 20 minutes of stimulation after 10 minutes of static incubation. However, no significant alterations were noted in the amplitude of the EJPs at the Drosophila NMJ during the 20 minutes of stimulation at 5 Hz. The crayfish model effects of levetiracetam were then used as an authentic curriculum undergraduate research experience (ACURE) in a neurophysiology teaching laboratory with 16 students. It appears levetiracetam acts differently in different animal models, or varied experimental conditions are required to note its effects.

Bacterial endotoxin lipopolysaccharide enhances synaptic transmission at low-output glutamatergic synapses

The endotoxin lipopolysaccharides (LPS), secreted from gram-negative bacteria, has direct effects on synaptic transmission independent of systemic secondary cytokine responses. High concentration of LPS (500 μg/mL) from Serratia marcescens increased synaptic efficacy at glutamatergic low-output synapses more than for high-output synapses. Over an hour of exposure was not toxic to the preparation and continued to enhance synaptic transmission. A small but significant rapid hyperpolarization of the post-synaptic cells occurred, in addition to a slower enhancement of in the amplitude of evoked excitatory junction potentials. LPS may promote reserve pool vesicles to the readily releasable pool for low-output synapses. The action of LPS at the glutamatergic synapses of the crayfish neuromuscular junction is unique in promoting synaptic transmission as compared to other glutamatergic synapses in Drosophila and mammals, where synaptic transmission is depressed.

The Effect of Bacterial Endotoxin LPS on Serotonergic Modulation of Glutamatergic Synaptic Transmission.

The release of the endotoxin lipopolysaccharides (LPS) from gram-negative bacteria is key in the induction of the downstream cytokine release from cells targeting cells throughout the body. However, LPS itself has direct effects on cellular activity and can alter synaptic transmission. Animals experiencing septicemia are generally in a critical state and are often treated with various pharmacological agents. Since antidepressants related to the serotonergic system have been shown to have a positive outcome for septicemic conditions impacting the central nervous system, the actions of serotonin (5-HT) on neurons also exposed to LPS were investigated. At the model glutamatergic synapse of the crayfish neuromuscular junction (NMJ), 5-HT primarily acts through a 5-HT2A receptor subtype to enhance transmission to the motor neurons. LPS from Serratia marcescens also enhances transmission at the crayfish NMJ but by a currently unknown mechanism. LPS at 100 µg/mL had no significant effect on transmission or on altering the response to 5-HT. LPS at 500 µg/mL increased the amplitude of the evoked synaptic excitatory junction potential, and 5-HT in combination with 500 µg/mL LPS continued to promote enhanced transmission. The preparations maintained responsiveness to serotonin in the presence of low or high concentrations of LPS.

Effects of levetiracetam on axon excitability and synaptic transmission at the crayfish neuromuscular junction.

Levetiracetam (LEV) is a widely prescribed antiepileptic drug, but its actions on neuronal function are not fully characterized. Since this drug is believed to enter neurons by binding to a vesicular protein during endocytosis, we used motor axons of the crayfish opener neuromuscular junction to examine potential impacts of LEV on axon excitability. Two electrode current clamp from the inhibitory axon of the opener showed that LEV reduced action potential (AP) amplitude (APamp ) and suppressed synaptic transmission, although the latter occurred with a longer delay than the reduction in APamp . Comparison of antidromic and orthodromic conducting APs in LEV suggested that this drug preferentially reduced excitability of the proximal axon, despite the expectation that it entered the axon at the terminals and should affect the distal branches first. Results presented here suggest that LEV modulates axonal excitability, which may in turn contribute to its antiepileptic effects.

Focused ultrasound transiently increases membrane conductance in isolated crayfish axon.

We report a novel phenomenon produced by focused ultrasound (US) that may be important for understanding its effects on cell membranes. When a US burst (2.1 MHz, 1-mm focal diameter, 0.1-1 MPa) was focused on a motor axon of the crayfish neuromuscular junction, it consistently produced a fast hyperpolarization, which was followed or superseded by subthreshold depolarizations or action potentials in a stochastic manner. The depolarization persisted in the presence of voltage-gated channel blockers [1 µM TTX ( INa), 50 µM ZD7288 ( Ih), and 200 µM 4-aminopyridine ( IK)] and typically started shortly after the onset of a 5-ms US burst, with a mean latency of 3.35 ± 0.53 ms (SE). The duration and amplitude of depolarizations averaged 2.13 ± 0.87 s and 10.1 ± 2.09 mV, with a maximum of 200 s and 60 mV, respectively. The US-induced depolarization was always associated with a decrease in membrane resistance. By measuring membrane potential and resistance during the US-induced depolarization, the reversal potential of US-induced conductance ( gus) was estimated to be -8.4 ± 2.3 mV, suggesting a nonselective conductance. The increase in gus was 10-100 times larger than the leak conductance; thus it could significantly influence neuronal activity. This change in conductance may be due to stimulation of mechanoreceptors. Alternatively, US may perturb the lateral motion of phospholipids and produce nanopores, which then increase gus. These results may be important for understanding mechanisms underlying US-mediated modulation of neuronal activity and brain function. NEW & NOTEWORTHY We report a specific increase in membrane conductance produced by ultrasound (US) on neuronal membrane. When a 5-ms US tone burst was focused on a crayfish motor axon, it stochastically triggered either depolarization or a spike train. The depolarization was up to 60 mV in amplitude and 200 s in duration and therefore could significantly influence neuronal activity. Depolarization was still evoked by US burst in the presence of Na+ and Ca2+ channel blockers and had a reversal potential of -8.4 ± 2.3 mV, suggesting a nonselective permeability. US can be applied noninvasively in the form of a focused beam to deep brain areas through the skull and has been shown to modulate brain activity. Understanding the depolarization reported here should be helpful for improving the use of US for noninvasive modulation and stimulation in brain-related disease.

Effect of temperature change on synaptic transmission at crayfish neuromuscular junctions.

ABSTRACTEctothermic animals in areas characterised by seasonal changes are susceptible to extreme fluctuations in temperature. To survive through varied temperatures, ectotherms have developed unique strategies. This study focuses on synaptic transmission function at cold temperatures, as it is a vital component of ectothermic animals' survival. For determining how synaptic transmission is influenced by an acute change in temperature (20°C to 10°C within a minute) and chronic cold (10°C), the crayfish (Procambarus clarkii) neuromuscular junction (NMJ) was used as a model. To simulate chronic cold conditions, crayfish were acclimated to 15°C for 1 week and then to 10°C for 1 week. They were then used to examine the synaptic properties associated with the low output nerve terminals on the opener muscle in the walking legs and high output innervation on the abdominal deep extensor muscle. The excitatory postsynaptic potentials (EPSPs) of the opener NMJs increased in amplitude with acute warming (20°C) after being acclimated to cold; however, the deep extensor muscles showed varied changes in EPSP amplitude. Synaptic transmission at both NMJs was enhanced with exposure to the modulators serotonin or octopamine. The membrane resistance of the muscles decreased 33% and the resting membrane potential hyperpolarised upon warm exposure. Analysis of haemolymph indicated that octopamine increases during cold exposure. These results suggest bioamine modulation as a possible mechanism for ensuring that synaptic transmission remains functional at low temperatures.

New insights into the acute actions from a high dosage of fluoxetine on neuronal and cardiac function: Drosophila, crayfish and rodent models

The commonly used mood altering drug fluoxetine (Prozac) in humans has a low occurrence in reports of harmful effects from overdose; however, individuals with altered metabolism of the drug and accidental overdose have led to critical conditions and even death. We addressed direct actions of high concentrations on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine's action as a selective 5-HT reuptake inhibitor. There appears to be action in blocking action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events in the presynaptic terminals at NMJs of both Drosophila and crayfish. In rodent neurons, cytoplasmic Ca2+ rises by fluoxetine and is thapsigargin dependent. The Drosophila larval heart showed a dose dependent effect in cardiac arrest. Acute paralytic behavior in crayfish occurred at a systemic concentration of 2mM. A high percentage of death as well as slowed development occurred in Drosophila larvae consuming food containing 100μM fluoxetine. The release of Ca2+ from the endoplasmic reticulum in neurons and the cardiac tissue as well as blockage of voltage-gated Na+ channels in neurons could explain the effects on the whole animal as well as the isolated tissues. The use of various animal models in demonstrating the potential mechanisms for the toxic effects with high doses of fluoxetine maybe beneficial for acute treatments in humans. Future studies in determining how fluoxetine is internalized in cells and if there are subtle effects of these mentioned mechanisms presented with chronic therapeutic doses are of general interest.

Calcium, Calpain, and Calcineurin in Low-Frequency Depression of Transmitter Release

Low-frequency depression (LFD) of transmitter release occurs at phasic synapses with stimulation at 0.2 Hz in both isolated crayfish (Procambarus clarkii) neuromuscular junction (NMJ) preparations and in intact animals. LFD is regulated by presynaptic activity of the Ca(2+)-dependent phosphatase calcineurin (Silverman-Gavrila and Charlton, 2009). Since the fast Ca(2+) chelator BAPTA-AM inhibits LFD but the slow chelator EGTA-AM does not, the Ca(2+) sensor for LFD may be close to a Ca(2+) source at active zones. Calcineurin can be activated by the Ca(2+)-activated protease calpain, and immunostaining showed that both proteins are present at nerve terminals. Three calpain inhibitors, calpain inhibitor I, MDL-28170, and PD150606, but not the control compound PD145305, inhibit LFD both in the intact animal as shown by electromyograms and by intracellular recordings at neuromuscular junctions. Analysis of mini-EPSPs indicated that these inhibitors had minimal postsynaptic effects. Proteolytic activity in CNS extract, detected by a fluorescent calpain substrate, was modulated by Ca(2+) and calpain inhibitors. Western blot analysis of CNS extract showed that proteolysis of calcineurin to a fragment consistent with the constitutively active form required Ca(2+) and was blocked by calpain inhibitors. Inhibition of LFD by calpain inhibition blocks the reduction in phosphoactin and the depolymerization of tubulin that normally occurs in LFD, probably by blocking the dephosphorylation of cytoskeletal proteins by calcineurin. In contrast, high-frequency depression does not involve protein phosphorylation- or calpain-dependent mechanisms. LFD may involve a specific pathway in which local Ca(2+) signaling activates presynaptic calpain and calcineurin at active zones and causes changes of tubulin cytoskeleton.

Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses.

The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14°C but not from those acclimatized to 21°C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.

Spatial variation in membrane excitability modulated by 4-AP-sensitive K+ channels in the axons of the crayfish neuromuscular junction

Current-clamp recordings were made from the primary (1°) and secondary (2°) branching points (BPs) of axons at the crayfish neuromuscular junction. Action potential (AP) firing initiated by current injected at the 2° BP showed strong adaptation or high-frequency firing at threshold current, whereas AP firing frequency at the 1° BP exhibited a gradual rise with increasing current amplitude. The voltage threshold for AP (V(TH)) was higher at the 2° BP than the 1° BP. 4-Aminopyridine (4-AP) at 200 μM increased AP amplitude and duration at both BPs but reduced threshold current at the 2° BP more than at the 1° BP. This blocker lowered V(TH) at both BPs, but the difference between the BPs remained. Firing patterns evoked at the 2° BP became similar to those evoked at the 1° BP in 4-AP. Thus 4-AP-sensitive channels may be more concentrated in the distal axon and control AP initiation and firing patterns there. Orthodromic APs between the two BPs were also compared. There was no difference in AP amplitude between the two BPs, but AP half-width recorded at the 2° BP was longer than that at the 1° BP. AP duration at both BPs increased gradually, by ∼17%, during a 100-Hz, 500-ms train (in-train rise). Normalized AP half-widths revealed a smaller fractional in-train rise at the 2° BP. Thus, although distal APs were broader, AP duration there was under more stringent control than that of the proximal axon. 4-AP increased AP amplitude and duration of the entire orthodromic train and reduced the magnitude of the in-train rise in AP half-width at both BPs. However, this blocker did not uncover a clear difference between the two BPs. Thus 4-AP-sensitive channels concentrated in distal axon may be essential in preventing unintended firing and modulating AP waveform without interfering with orthodromic AP propagation.

A novel, extremely fast, feedback inhibition of glutamate release in the crayfish neuromuscular junction

Feedback inhibition serves to modulate release when neurotransmitter levels in the synaptic cleft are elevated. The “classical” feedback auto-inhibition of neurotransmitter release is predominantly mediated by activation of presynaptic G-protein-coupled receptors (GPCRs) and exhibits slow kinetics. In cholinergic and glutamatergic synapses and for focal graded depolarization of the axon terminal, feedback inhibition was found to be voltage-dependent. At high depolarizations, such as the one produced by an action potential, low concentrations of neurotransmitter were insufficient to inhibit release. On the other hand, at higher neurotransmitter concentrations, feedback inhibition was observed also for action potential-evoked release. This finding suggests the presence of an additional mechanism of feedback inhibition that operates also at large presynaptic depolarizations. Using the glutamatergic crayfish neuromuscular junction we discovered a novel, extremely fast, form of feedback inhibition which hampers action potential-evoked release. This novel mechanism is pertussis toxin-insensitive, and is activated already 1 ms after flash photolysis producing glutamate concentrations higher than the ones required to activate the classical feedback inhibition. This finding implies that this mechanism is recruited only when glutamate levels in the synaptic cleft are relatively high (after high-frequency activation or in pathological conditions). We show that both the classical and this novel mechanism operate under physiological conditions.

Different mechanisms of Ca2+ regulation that influence synaptic transmission: Comparison between crayfish and Drosophila neuromuscular junctions

A brief historical background on synaptic transmission in relation to Ca(2+) dynamics and short-term facilitation is described. This study focuses on the mechanisms responsible for the regulation of intracellular calcium concentration ([Ca(2+)](i)) in high output terminals of larval Drosophila compared to a low-output terminal of the crayfish neuromuscular junction (NMJ). Three processes; plasmalemmal Na(+)/Ca(2+) exchanger [NCX], Ca(2+)-ATPase (PMCA), and sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) are important in regulating the [Ca(2+)](i) are examined. When the NCX is compromised by reduced [Na(+)](o), no consistent effect occurred; but a NCX blocker KB-R7943 decreased the excitatory postsynaptic potential (EPSP) amplitudes. Compromising the PMCA with pH 8.8 resulted in an increase in EPSP amplitude but treatment with a PMCA specific inhibitor carboxyeosin produced opposite results. Thapsigargin exposure to block the SERCA generally decreases EPSP amplitude. Compromising the activity of the above Ca(2+) regulating proteins had no substantial effects on short-term depression. The Kum(170TS) strain (with dysfunctional SERCA), showed a decrease in EPSP amplitudes including the first EPSP within the train. Synaptic transmission is altered by reducing the function of the above three [Ca(2+)](i) regulators; but they are not consistent among different species as expected. Results in crayfish NMJ were more consistent with expected results as compared to the Drosophila NMJ. It is predicated that different mechanisms are used for regulating the [Ca(2+)](i) in high and low output synaptic terminals.

A General Model of Synaptic Transmission and Short-Term Plasticity

Some synapses transmit strongly to action potentials (APs), but weaken with repeated activation; others transmit feebly at first, but strengthen with sustained activity. We measured synchronous and asynchronous transmitter release at "phasic" crayfish neuromuscular junctions (NMJs) showing depression and at facilitating "tonic" junctions, and define the kinetics of depression and facilitation. We offer a comprehensive model of presynaptic processes, encompassing mobilization of reserve vesicles, priming of docked vesicles, their association with Ca(2+) channels, and refractoriness of release sites, while accounting for data on presynaptic buffers governing Ca(2+) diffusion. Model simulations reproduce many experimentally defined aspects of transmission and plasticity at these synapses. Their similarity to vertebrate central synapses suggests that the model might be of general relevance to synaptic transmission.

Calcineurin and cytoskeleton in low‐frequency depression

Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 min when stimulated at 0.2 Hz. Inhibition of the protein phosphatase calcineurin by intracellular pre-synaptic injection of autoinhibitory peptide inhibited low-frequency depression (LFD) and resulted in facilitation of transmitter release. Since this inhibitor had no major effects when injected into the post-synaptic cell, only pre-synaptic calcineurin activity is necessary for LFD. To examine changes in phosphoproteins during LFD we performed a phosphoproteomic screen on proteins extracted from motor axons and nerve terminals after LFD induction or treatment with various drugs that affect kinase and phosphatase activity. Proteins separated by PAGE were stained with phospho-specific/total protein ratio stains (Pro-Q Diamond/SYPRO Ruby) to identify protein bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin decreased during LFD, but increased when calcineurin was blocked. Tubulin and phosphoactin immunoreactivity in pre-synaptic terminals were also reduced after LFD. The actin depolymerizing drugs cytochalasin and latrunculin and the microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of pre-synaptic actin and tubulin and consequent changes in the cytoskeleton may regulate LFD. LFD is unlike long-term depression found in mammalian synapses because the latter requires in most instances post-synaptic calcineurin activity.Thus, this simpler invertebrate synapse discloses a novel pre-synaptic depression mechanism.

Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics

Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics Olga Kochan 1 * and Robert S. Zucker** *Molecular & Cell Biology and Psychology Major, College of Letters and Science, **Department of Molecular & Cell Biology, University of California, Berkeley Key words: Synaptic plasticity, synaptic transmission, cAMP dependent enhancement, actin, HCN channels ABSTRACT: Glutamatergic synapses are highly modifiable, making them key targets in processes such as learning and memory. In crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) channels and actin cytoskeleton dynamics are critical intermediate factors in hormonal modulation of glutamatergic synapses which lead to cAMP (3’-5’-cyclic adenosine monophosphate)-dependent enhancement of synaptic transmission. Although models have been proposed, there has been a lack of experimental evidence on the relationship between HCN channels and the integrity of the actin cytoskeleton during cAMP-dependent enhancement. The specific goal of this study is to test the sequence of activation of the aforementioned mediators in synaptic enhancement via precisely controlled pharmacological experiments. At glutamatergic neuromuscular junctions of crayfish limb muscles, HCN channel activator, lamotrigine (50 µM), enhanced synaptic transmission about 20%. This enhancement was completely blocked with actin depolymerizer, latrunculin B (3 µM). These results support previous models of the temporal arrangement of events leading to synaptic enhancement, specifically that changes in actin cytoskeleton follow HCN channel activation. Concurrently, we are also using a spatio-temporal marker called phalloidin, a toxin which binds actin filaments, to further test the hypothesis that activation of HCNCs precedes actin cytoskeleton polymerization. This allows for manipulation of HCN channels and visualization of actin that could propose the associated molecular mechanisms. Preliminary evidence suggests actin reorganization. INTRODUCTION Synaptic plasticity underlies adaptability of the nervous system to changing circumstances and is involved in learning and memory. One such modifiability includes cAMP-dependent enhancement of synaptic transmission. At crayfish neuromuscular junctions (NMJ), the number of glutamate-containing synaptic vesicles available for release by action potentials at the exciter is increased by the circulating neurohormone serotonin (1) acting in part by production of the presynaptic second messenger cAMP (2). This cAMP-dependent enhancement of transmission, which can also be produced directly by stimulating adenylyl cyclase with forskolin, involves the activation of presynaptic hyperpolarization and cyclic nucleotide-activated channels (HCNCs) by cAMP as well as the integrity of the actin cytoskeleton (3). In past studies, it was noted that any of three HCNC blockers (ZD 7288, DK-AH 269, or Cs+) or depolymerization of actin by cytochalasin D, latrunculin B, or swinholide A, greatly reduce the forskolin-induced enhancement of transmission (4). A schematic model of the steps involved in induction of serotonergic (cAMP- dependent) enhancement of transmission has been proposed (4). From the model, it is evident that both HCN channels and actin cytoskeleton dynamics are required to mediate synaptic enhancement; however, the relationship between HCNCs and actin still remains elusive and merely hypothesized (Figure 1). Therefore, in this study, we set out to determine the sequential relationship between the actin cytoskeleton and HCN channels in supporting synaptic plasticity and transmission. We used the crayfish NMJ to study the implicated mediators of synaptic enhancement. This model offers many advantages such as availability, suitability for electrophysiological recording and molecular manipulations, as well as physiological and biochemical properties nearly identical to those of mammalian brain synapse. We investigated the mechanism by which changes in HCN channel activation and actin cytoskeleton dynamics result in synaptic enhancement via two main experimental protocols. The first experimental technique manipulates HCN channels 8-CPT Jasplakinolide Lat B Figure 1: This previously published figure (4) depicts a schematic of the steps involved in induction of serotonergic (cAMP-dependent) enhancement of transmission, tetanic activation of LTF (long term facilitation), and synaptic tagging, which are all mechanisms of synaptic plasticity. cAMP enhances transmission by activating Epac and HCN channels, the latter acting via an actin dependent step. The temporal relationship between HCN channels and actin was still indefinite and needed to be further investigated, leading to the experiments in the present study. Corresponding author: University of California, Department of Molecular and Cell Biology, 111 LSA, Berkeley, Ca 94720. Phone number: (925) 286-8491. E-mail: olga54@berkeley.edu.

Electrophysiological events recorded at presynaptic terminals of the crayfish neuromuscular junction with a voltage indicator

The water-soluble voltage indicator JPW1114 was used to stain thin axons and terminal varicosities of the crayfish neuromuscular junction. A slow, overnight injection protocol was developed to brightly stain fine structures without cytotoxicity. Fluorescence transients filtered at 2 kHz showed that the duration of terminal action potentials was shorter than that of those recorded in the main trunk of the axons. In addition, the repolarization phases of the terminal and axonal action potentials overlapped in time, suggesting that the entire axonal arborization repolarizes simultaneously. Manipulating resting membrane potential, +/-15-20 mV, did not alter the peak level or duration of action potentials if they fired in isolation. A prolongation of action potential, by 23%, could be induced if a 10-spike burst at 100 Hz was fired from depolarized membrane potential. No such change was observed when the high frequency train was fired from resting or hyperpolarized levels. Microelectrodes in the main trunk of axons typically recorded a depolarizing after-potential (DAP) following an action potential initiated from resting membrane potential. The DAP could be inverted and enlarged by depolarization and hyperpolarization, respectively. Fluorescence transients recorded from terminals exhibited similar DAP characteristics. The ratio of DAP to action potential amplitude recorded from terminals was similar to that recorded from the main axon. Thus, the entire axonal arborization returned to resting level in a spatially uniform manner during the DAP. The functional significance of DAP is discussed in the light of these observations.

Facilitating exchange: activity‐dependent regulation of presynaptic Na+/Ca2+ exchange

Alterations in the amount of released neurotransmitter are responsible for multiple forms of short- and long-term synaptic plasticity. At many synapses it is unclear if this presynaptic plasticity is mediated by direct regulation of Ca2+ current through presynaptic channels or through other targets that act more indirectly on the release apparatus. The Zucker laboratory has revisited this question in its investigations of long-term facilitation (LTF) of neurotransmitter release at the crayfish neuromuscular junction. Their surprising results, recently published in The Journal of Physiology (Minami et al. 2007), are likely to have wider implications for studies addressing regulation of presynaptic Ca2+ signalling during synaptic plasticity.

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.

Involvement of L-type calcium channels and mitochondria in post-tetanic potentiation: Is it a general rule for different types of synapses?

Our experiments and studies of a few other authors demonstrated that L-type calcium channels and mitochondria are involved in the induction of post-tetanic potentiation (PTP) in a number of preparations (Aplysia central nervous system, hippocampal cell cultures, crayfish neuromuscular junctions, etc.). We extend this conclusion on cortical synapses by the demonstration that inhibitors of mitochondrial Ca2+ uptake and release suppress PTP in rat neocortical cell cultures.