Facilitated glutamate release at Schaffer collateral to CA1 synapses has access to an exclusive population of NMDA receptors.

In order to explore short-term facilitation of the Schaffer collateral to CA1 synapse in mouse hippocampal brain slices, we measured the time course of the decay of the peak amplitude of successive EPSCs during progressive MK-801-dependent block (PMDB) of NMDAR responses to paired (R1 and R2) stimuli. We made the unexpected observation that the R2 response exhibited a slower PMDB decay constant than that of the R1 response. This indicated that the facilitated R2 response engages release sites with NMDARs that are protected from opening and consequent MK-801 block during the basal R1 response. We then utilized conditions that affect synaptic glutamate distribution to dissect the components of the distinct PMDB decay constants of the first and second of paired pulses. While extra-synaptic NMDARs and glutamate transporters appear to play only minor roles in the differences of the PMDB decay constant, we showed important roles for the R1 response itself and for glutamate diffusion in determining the PMDB decay constant of R2. We used a simple computational model with realistic parameters that allowed us to predict the time course of R2 decay based on the R1 decay time course.

Phylogenomically guided identification of industrially relevant GH1 ß-glucosidases through DNA synthesis and nanostructure-initiator mass spectrometry.

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ß-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different ß-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

Modulation by pregnenolone sulfate of filtering properties in the hippocampal trisynaptic circuit.

Short-term synaptic plasticity alters synaptic efficacy on a timescale that is relevant to encoding information in spike trains. The dynamics of this plasticity, combined with that of the feedback and feedforward contributions of local interneurons, impose frequency-dependent properties on neuronal networks with implications for nervous system function. The trisynaptic network of the hippocampus is especially well suited to selectively filter components of frequency-dependent signals that are transmitted from the entorhinal cortex. We measured presynaptic [Ca(2+)](i) in perforant path, mossy fiber, or Schaffer collateral terminals while simultaneously measuring field potentials of principal cells of the dentate, CA3, or CA1 synaptic fields over a range of stimulus frequencies of 2 to 77 Hz. In all three synaptic fields, the average [Ca(2+)](i) during a 500 ms stimulus train rose monotonically with stimulus frequency. The average population spike amplitude during this stimulus train, however, exhibited a non-linear relationship to frequency that was distinct for each of the three synaptic fields. The dentate synaptic field exhibited the characteristics of a low pass filter, while both CA synaptic fields had bandpass filter characteristics with a gain that was greater than 1 in the passband frequencies. Importantly, alteration of the dynamic properties of this network could significantly impact information processing performed by the hippocampus. Pregnenolone sulfate (PregS), has frequency-dependent effects on paired- and multipulse plasticity in the dentate and CA1 synaptic fields of the hippocampal formation. We investigated the PregS-dependent modulation of the dynamic properties of transmission by the principal cells of the three hippocampal synaptic fields. Importantly, PregS is capable of altering the pattern separation capabilities that may underlie hippocampal information processing.

Presynaptic residual calcium and synaptic facilitation at hippocampal synapses of mice with altered expression of SNAP-25.

Paired pulse facilitation (PPF) is a form of short-term synaptic plasticity that results from an interaction of residual presynaptic Ca(2+) ([Ca(2+)](res)), number of release-competent vesicles, and the sensitivity of the vesicle release mechanisms to Ca(2+). While PPF is predominant at hippocampal Schaffer collateral-CA1 (SC-CA1) synapses, facilitation is greater in adult mice (designated Tkneo) that over express an isoform of the plasma membrane-targeted SNARE protein, SNAP-25a, which is normally predominantly expressed in juvenile animals. SNAP-25 is essential for action potential-dependent neuroexocytosis, yet the significance of the shift between the alternatively spliced variants SNAP-25a and SNAP-25b is not fully understood. This alteration of a key component of the protein machinery required for neurotransmitter release in Tkneo mice, therefore, provides a useful tool to further investigate presynaptic mechanisms that influence short-term plasticity. To explore this link between SNAP-25 and PPF, we simultaneously measured postsynaptic potentials and presynaptic [Ca(2+)](res) during paired-pulses in adult Tkneo, heterozygote null (HET), and wild type (WT) mice. We demonstrate that enhanced PPF is maintained at mature hippocampal synapses of Tkneo mice that predominantly express SNAP-25a, and that [Ca(2+)](res) kinetics are altered at synapses of Tkneo and HET mice, both of which exhibit reduced levels of total SNAP-25 expression. To evaluate the role of SNAP-25 in short-term plasticity and [Ca(2+)](res) regulation, we applied a vesicular release probability model for neurotransmission. Our results suggest that the isoform expression and total level of SNAP-25 affect both [Ca(2+)](res) dynamics and the ability of releasable vesicles to enter into a facilitated state.

Developmental changes in presynaptic Ca(2 +) clearance kinetics and synaptic plasticity in mouse Schaffer collateral terminals.

Presynaptic Ca(2+) influx pathways, cytoplasmic Ca(2+) buffering proteins and Ca(2+) extrusion processes undergo considerable change during the first postnatal month in rodent neurons. These changes may be critical in establishing short-term plasticity at maturing presynaptic terminals where neurotransmitter release is directly dependent on the dynamics of cytoplasmic residual Ca(2+) ([Ca(2+)](res)). In particular, the robust paired-pulse facilitation characteristic of adult neurons is almost entirely lacking in newborns. To examine developmental changes in processes controlling [Ca(2+)](res), we measured the timecourse of [Ca(2+)](res) decay in presynaptic terminals of Schaffer collateral to CA1 synapses in acute hippocampal slices following single and paired orthodromic stimuli in the stratum radiatum. Developmental changes were observed in both the rise time and slow exponential decay components of the response to single stimuli such that this decay was larger and faster in the adult. Furthermore, we observed a greater caffeine-sensitive basal Ca(2+) store, which was differentially affected when active uptake into the endoplasmic reticulum was blocked, in the presynaptic fields of the Schaffer collateral to CA1 terminals of P6 and younger mice when compared to adults. These transitions in [Ca(2+)](res) dynamics occurred gradually over the first weeks of postnatal life and correlated with changes in short-term plasticity.

Contributions of SERCA pump and ryanodine-sensitive stores to presynaptic residual Ca2+.

The presynaptic Ca2+ signal, which triggers vesicle release, disperses to a broadly distributed residual [Ca2+] ([Ca2+](res)) that plays an important role in synaptic plasticity. We have previously reported a slowing in the decay timecourse of [Ca2+](res) during the second of paired pulses. In this study, we investigated the contributions of organelle and plasma membrane Ca2+ flux pathways to the reduction of effectiveness of [Ca2+](res) clearance during short-term plasticity in Schaffer collateral terminals in the CA1 field of the hippocampus. We show that the slowed decay timecourse is mainly the result of a transport-dependent Ca2+ clearance process; that presynaptic caffeine-sensitive Ca2+ stores are not functionally loaded in the unstimulated terminal, but that these stores can effectively take up Ca2+ even during high frequency trains of stimuli; and that a rate limiting step of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) kinetics following the first pulse is responsible for a large portion of the observed slowing of [Ca2+](res) clearance during the second pulse. We were able to accurately fit our [Ca2+](res) data with a kinetic model based on these observations and this model predicted a reduction in availability of unbound SERCA during paired pulses, but no saturation of Ca2+ buffer in the endoplasmic reticulum.

Neurosteroid-induced enhancement of short-term facilitation involves a component downstream from presynaptic calcium in hippocampal slices.

We used Magnesium Green AM to measure Ca(2+) transients in Schaffer collateral presynaptic terminals simultaneously with postsynaptic field potentials (fEPSPs) to investigate the mechanism of neurosteroid enhancement of short-term synaptic facilitation. Measurement of [Ca(2+)](i), isolated to presynaptic events, using the fluorescence ratio (DeltaF/F(0)) demonstrated that at a constant stimulus intensity there was no change in the excitability of presynaptic fibres between paired stimuli or between ACSF and 1 mum pregnenolone sulphate (PREGS). Paired-pulse facilitation (PPF) was correlated with residual Ca(2+) ([Ca(2+)](res)), and there was an additional increase in the integralDeltaF/F(0) for the [Ca(2+)](res)-subtracted response to the second of paired stimuli, resulting primarily from a slowing of the decay time constant. In addition to the role of presynaptic [Ca(2+)](res) in PPF, we observed a decrease in EC(50) and a greater maximum for Hill function fits to fEPSP versus DeltaF/F(0) during the second of paired responses. The enhancement of fEPSP PPF by PREGS did not result from an increase of DeltaF/F(0). The data presented here support a PREGS-induced increase in presynaptic glutamate release from the second, but not the first, of a pair of stimuli for a given presynaptic [Ca(2+)] because: (a) there is actually a decrease in the integralDeltaF/F(0) of the [Ca(2+)](res)-subtracted second response over that seen in ACSF; (b) PREGS causes no change in presynaptic Ca(2+) buffering; and (c) there is a decrease in EC(50) and an increase of y(max) in the Hill function fits to DeltaF/F(0) versus fEPSP data. We hypothesize that PREGS enhances short-term facilitation by acting on the Ca(2+)-dependent vesicle release machinery and that this mechanism plays a role in the cognitive effects of this sulphated neurosteroid.