Molecular detection and characterization of transient bovine viral diarrhea virus (BVDV) infections in cattle commingled with ten BVDV persistently infected cattle.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

Regulation of long-term plasticity induction by the channel and C-terminal domains of GluN2 subunits.

Conventional long-term potentiation (LTP) and long-term depression (LTD) are induced by different patterns of synaptic stimulation, but both forms of synaptic modification require calcium influx through NMDA receptors (NMDARs). A prevailing model (the "calcium hypothesis") suggests that high postsynaptic calcium elevation results in LTP, whereas moderate elevations give rise to LTD. Recently, additional evidence has come to suggest that differential activation of NMDAR subunits also factors in determining which type of plasticity is induced. While the growing amount of data suggest that activation of NMDARs containing specific GluN2 subunits plays an important role in the induction of plasticity, it remains less clear which subunit is tied to which form of plasticity. Additionally, it remains to be determined which properties of the subunits confer upon them the ability to differentially induce long-term plasticity. This review highlights recent studies suggesting differential roles for the subunits, as well as findings that begin to shed light on how two similar subunits may be linked to the induction of opposing forms of plasticity.

Distinct roles of NR2A and NR2B cytoplasmic tails in long-term potentiation.

NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in long-term potentiation (LTP) in organotypic hippocampal slice cultures using RNA interference (RNAi) and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol]. As slices mature and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP, whereas the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus, expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wild-type NR2A failed to rescue LTP in neurons transfected with the NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together, these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. In contrast, NR2A is not essential for LTP, and its cytoplasmic tail seems to carry inhibitory factors for LTP.

Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132.

MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

The influence of multivesicular release and postsynaptic receptor saturation on transmission at granule cell to Purkinje cell synapses.

The properties of a synapse are crucially dependent on whether an action potential can trigger the release of multiple vesicles at an individual release site [multivesicular release (MVR)] and whether fusion of a single vesicle leads to receptor saturation. MVR and receptor saturation both occur at some high p synapses, but it is not known whether they also occur at low p synapses. Here we examine this issue at the low p synapse between parallel fibers and Purkinje cells using the low-affinity antagonist DGG (gamma-D-glutamylglycine) to relieve AMPA receptor saturation. We find that the presence of MVR and receptor saturation at this synapse alters the calcium dependence of synaptic transmission and reduces the extent of facilitation. These findings establish that MVR and postsynaptic receptor saturation can influence transmission even at synapses with a low initial probability of release and suggest that these properties may be common at synapses in the mammalian brain.

Variance-mean analysis in the presence of a rapid antagonist indicates vesicle depletion underlies depression at the climbing fiber synapse.

Many types of synapses throughout the nervous system are transiently depressed during high-frequency stimulation. Several mechanisms have been proposed to account for this depression, including depletion of release-ready vesicles. However, numerous studies have raised doubts about the importance of depletion in depression of central synapses and have implicated alternative mechanisms, such as decreased release probability. We use variance-mean analysis to determine the mechanism of depression at the climbing fiber to Purkinje cell synapse. We find that postsynaptic receptor saturation makes it difficult to distinguish between a decrease in available vesicles and a reduction in release probability. When AMPA receptor saturation is relieved with a low-affinity antagonist, variance-mean analysis reveals that depression arises from a decrease in the number of release-ready vesicles. Vesicle depletion is prominent, despite numerous docked vesicles at each release site, due to multivesicular release. We conclude that vesicle depletion can contribute significantly to depression of central synapses.

Interaction of postsynaptic receptor saturation with presynaptic mechanisms produces a reliable synapse.

Synapses that reliably activate their postsynaptic targets typically release neurotransmitter with high probability, are not very sensitive to changes in calcium entry, and depress. We have determined the mechanisms that give rise to these characteristic features at the climbing fiber to Purkinje cell synapse. We find that saturation of presynaptic calcium entry, of presynaptic release, and of postsynaptic receptors combine to produce a postsynaptic response that is near maximal. Postsynaptic receptor saturation also accelerates recovery from depression, in part by accentuating a rapid calcium-dependent recovery phase. Thus, postsynaptic receptor saturation interacts with presynaptic mechanisms to produce highly reliable synapses that can effectively drive their targets even during sustained activation.

Assessing the role of calcium-induced calcium release in short-term presynaptic plasticity at excitatory central synapses.

Recent evidence suggests that internal calcium stores and calcium-induced calcium release (CICR) provide an important source of calcium that drives short-term presynaptic plasticity at central synapses. Here we tested for the involvement of CICR in short-term presynaptic plasticity at six excitatory synapses in acute rat hippocampal and cerebellar brain slices. Depletion of internal calcium stores with thapsigargin and prevention of CICR with ryanodine have no effect on paired-pulse facilitation, delayed release of neurotransmitter, or calcium-dependent recovery from depression. Fluorometric calcium measurements also show that these drugs have no effect on the residual calcium signal that underlies these forms of short-term presynaptic plasticity. Finally, although caffeine causes CICR in Purkinje cell bodies and dendrites, it does not elicit CICR in parallel fiber inputs to these cells. Taken together, these results indicate that for the excitatory synapses studied here, internal calcium stores and CICR do not contribute to short-term presynaptic plasticity on the milliseconds-to-seconds time scale. Instead, this plasticity is driven by the residual calcium signal arising from calcium entry through voltage-gated calcium channels.