Asymmetrically Positioned Flagellar Control Units Regulate Human Sperm Rotation.

Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm.

Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.

Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. In the present study, we used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy between fluorescently labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly before endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only present in docked vesicles; unexpected residual "orphan SNARE complexes" also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis-conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatiotemporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid intermolecular interactions between small proportions of protein partners in cellular subcompartments.

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.

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.

Calcium sensitivity of neurotransmitter release differs at phasic and tonic synapses.

The efficacy of synaptic transmission varies greatly among synaptic contacts. We have explored the origins of differences between phasic and tonic crustacean neuromuscular junctions. Synaptic boutons of a phasic motor neuron release three orders of magnitude more quanta to a single action potential and show strong depression to a train, whereas tonic synapses are nearly unresponsive to single action potentials and display an immense facilitation. Phasic and tonic synapses display a similar nonlinear dependence on extracellular [Ca2+]. We imposed similar spatially uniform intracellular [Ca2+] ([Ca2+]i) steps in phasic and tonic synapses by photolysis of presynaptic caged calcium. [Ca2+]i was measured fluorometrically while transmitter release was monitored electrophysiologically from single boutons in which the [Ca2+]i was elevated. Phasic synapses released the readily releasable pool (RRP) of vesicles at a much higher rate and with a shorter delay than did tonic synapses. Comparison of several kinetic models of molecular events showed that a difference in Ca2+-sensitive priming of vesicles in the RRP combined with a revision of the kinetic Ca2+-binding sequence to the secretory trigger produced the best fit to the markedly different responses to Ca2+ steps and action potentials and of the characteristic features of synaptic plasticity in phasic and tonic synapses. The results reveal processes underlying one aspect of synaptic diversity that may also regulate changes in synaptic strength during development and learning and memory formation.

Minis: whence and wherefore?

In this issue of Neuron, Sara et al. find that spontaneously released miniature synaptic potentials arise from a pool of vesicles distinct from those released by neural activity. This modification of a basic tenet of the quantal hypothesis has important implications for the analysis of changes in synaptic transmission.

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.

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.

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.

Calcium influx through HCN channels does not contribute to cAMP-enhanced transmission.

Serotonin is a native neuromodulator of synaptic transmission at glutamatergic neuromuscular junctions of crayfish limb muscles. During times of stress, serotonin binds to presynaptic receptors, which activate adenylyl cyclase to elevate presynaptic levels of cAMP. cAMP binds to two presynaptic target proteins, hyperpolarization and cyclic nucleotide-activated (HCN) ion channels and an exchange protein activated by cAMP (Epac), and activation of these effectors results in enhancement of transmitter release to action potentials. cAMP elevation also results in a small preterminal rise in [Ca(2+)](i), which we show here to result from Ca(2+) influx through the presynaptic HCN channels opened by cAMP. Little or no Ca(2+) influx occurs through voltage-dependent Ca(2+) channels, despite the small presynaptic depolarization caused by current through the HCN channels. Loading terminals with BAPTA delays the rise in preterminal [Ca(2+)](i) without affecting the enhancement of transmission to cAMP elevation. This dissociation of the dynamics of the [Ca(2+)](i) rise and synaptic enhancement, plus the small magnitude and location of [Ca(2+)](i) elevation distant from release sites, seems to preclude any direct role for this [Ca(2+)](i) elevation in cAMP-dependent enhancement of transmission.

Photolysis of postsynaptic caged Ca2+ can potentiate and depress mossy fiber synaptic responses in rat hippocampal CA3 pyramidal neurons.

The induction of mossy fiber-CA3 long-term potentiation (LTP) and depression (LTD) has been variously described as being dependent on either pre- or postsynaptic factors. Some of the postsynaptic factors for LTP induction include ephrin-B receptor tyrosine kinases and a rise in postsynaptic Ca2+ ([Ca2+]i). Ca2+ is also believed to be involved in the induction of the various forms of LTD at this synapse. We used photolysis of caged Ca2+ compounds to test whether a postsynaptic rise in [Ca2+]i is sufficient to induce changes in synaptic transmission at mossy fiber synapses onto rat hippocampal CA3 pyramidal neurons. We were able to elevate postsynaptic [Ca2+]i to approximately 1 microm for a few seconds in pyramidal cell somata and dendrites. We estimate that CA3 pyramidal neurons have approximately fivefold greater endogenous Ca2+ buffer capacity than CA1 neurons, limiting the rise in [Ca2+]i achievable by photolysis. This [Ca2+]i rise induced either a potentiation or a depression at mossy fiber synapses in different preparations. Neither the potentiation nor the depression was accompanied by consistent changes in paired-pulse facilitation, suggesting that these forms of plasticity may be distinct from synaptically induced LTP and LTD at this synapse. Our results are consistent with a postsynaptic locus for the induction of at least some forms of synaptic plasticity at mossy fiber synapses.

Can a synaptic signal arise from noise?

The spontaneous fusion of vesicles at nerve terminals produces random miniature postsynaptic potentials (quantal responses) that are thought to have little functional significance. In this issue of Neuron, Sharma and Vijayaraghavan provide evidence that exogenous signals can accelerate and synchronize the occurrence of quanta strongly enough to activate postsynaptic neurons in what may be a new way to transfer information across synapses.

Temporal synaptic tagging by I(h) activation and actin: involvement in long-term facilitation and cAMP-induced synaptic enhancement.

Presynaptic I(h) channels become activated during a tetanus through membrane hyperpolarization resulting from Na(+) accumulation and electrogenic Na(+)/K(+) exchange. I(h) activation is obligatory for inducing long-term facilitation (LTF), a long-lasting synaptic strengthening. cAMP-induced synaptic enhancement also requires I(h) activation, and both processes are sensitive to actin depolymerization. Other mechanisms are responsible for expression of the responses. Once initiated, continued response to cAMP is I(h) and actin independent. Moreover, LTF-induced activation of I(h) renders subsequent cAMP enhancement insensitive to both I(h) blockers and actin depolymerization. This actin-stabilized "temporal synaptic tagging" set by I(h) activation is prolonged when I(h) is activated concurrent with an elevation in presynaptic calcium concentration ([Ca(2+)]i), permitting the further strengthening of synapses given appropriate additional stimuli.

Short-term synaptic plasticity.

Synaptic transmission is a dynamic process. Postsynaptic responses wax and wane as presynaptic activity evolves. This prominent characteristic of chemical synaptic transmission is a crucial determinant of the response properties of synapses and, in turn, of the stimulus properties selected by neural networks and of the patterns of activity generated by those networks. This review focuses on synaptic changes that result from prior activity in the synapse under study, and is restricted to short-term effects that last for at most a few minutes. Forms of synaptic enhancement, such as facilitation, augmentation, and post-tetanic potentiation, are usually attributed to effects of a residual elevation in presynaptic [Ca(2+)]i, acting on one or more molecular targets that appear to be distinct from the secretory trigger responsible for fast exocytosis and phasic release of transmitter to single action potentials. We discuss the evidence for this hypothesis, and the origins of the different kinetic phases of synaptic enhancement, as well as the interpretation of statistical changes in transmitter release and roles played by other factors such as alterations in presynaptic Ca(2+) influx or postsynaptic levels of [Ca(2+)]i. Synaptic depression dominates enhancement at many synapses. Depression is usually attributed to depletion of some pool of readily releasable vesicles, and various forms of the depletion model are discussed. Depression can also arise from feedback activation of presynaptic receptors and from postsynaptic processes such as receptor desensitization. In addition, glial-neuronal interactions can contribute to short-term synaptic plasticity. Finally, we summarize the recent literature on putative molecular players in synaptic plasticity and the effects of genetic manipulations and other modulatory influences.