Synaptic Plasticity and NO-cGMP-PKG Signaling Regulate Pre- and Postsynaptic Alterations at Rat Lateral Amygdala Synapses Following Fear Conditioning

In vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. In the present study, we show that auditory Pavlovian fear conditioning is associated with significant and long-lasting increases in the expression of the postsynaptically-localized protein GluR1 and the presynaptically-localized proteins synaptophysin and synapsin in the lateral amygdala (LA) within 24 hrs following training. Further, we show that rats given intra-LA infusion of either the NR2B-selective antagonist Ifenprodil, the NOS inhibitor 7-Ni, or the PKG inhibitor Rp-8-Br-PET-cGMPS exhibit significant decreases in training-induced expression of GluR1, synaptophysin, and synapsin immunoreactivity in the LA, while those rats infused with the PKG activator 8-Br-cGMP exhibit a significant increase in these proteins in the LA. In contrast, rats given intra-LA infusion of the NO scavenger c-PTIO exhibit a significant decrease in synapsin and synaptophysin expression in the LA, but no significant impairment in the expression of GluR1. Finally, we show that intra-LA infusions of the ROCK inhibitor Y-27632 or the CaMKII inhibitor KN-93 impair training-induced expression of GluR1, synapsin, and synaptophysin in the LA. These findings suggest that the NO-cGMP-PKG, Rho/ROCK, and CaMKII signaling pathways regulate fear memory consolidation, in part, by promoting both pre- and post-synaptic alterations at LA synapses. They further suggest that synaptic plasticity in the LA during auditory fear conditioning promotes alterations at presynaptic sites via NO-driven “retrograde signaling”

Rationale and design of The Delphi Trial – I(RCT)(2): international randomized clinical trial of rheumatoid craniocervical treatment, an intervention-prognostic trial comparing 'early' surgery with conservative treatment [ISRCTN65076841]

BACKGROUND: Rheumatoid arthritis is a chronic inflammatory disease, which affects 1% of the population. Hands and feet are most commonly involved followed by the cervical spine. The spinal column consists of vertebrae stabilized by an intricate network of ligaments. Especially in the upper cervical spine, rheumatoid arthritis can cause degeneration of these ligaments, causing laxity, instability and subluxation of the vertebral bodies. Subsequent compression of the spinal cord and medulla oblongata can cause severe neurological deficits and even sudden death. Once neurological deficits occur, progression is inevitable although the rapidity of progression is highly variable. The first signs and symptoms are pain at the back of the head caused by compression of the major occipital nerve, followed by loss of strength of arms and legs. The severity of the subluxation can be observed with radiological investigations (MRI, CT) with a high sensitivity. The authors have sent a Delphi Questionnaire about the current treatment strategies of craniocervical involvement by rheumatoid arthritis to an international forum of expert rheumatologists and surgeons. The timing of surgery in patients with radiographic instability without evidence of neurological deficit is an area of considerable controversy. If signs and symptoms of myelopathy are present there is little chance of recovery to normal levels after surgery. DESIGN: In this international multicenter randomized clinical trial, early surgical atlantoaxial fixation in patients with rheumatoid arthritis and radiological abnormalities without neurological deficits will be compared with prolonged conservative treatment. The main research question is whether early surgery can prevent radiological and neurological progression. A cost-effectivity analysis will be performed. 250 patients are needed to answer the research question. DISCUSSION: Early surgery could prevent serious neurological deficits, but may have peri-operative morbidity and loss of rotation of the head and neck. The objective of this study is to identify the best timing of surgery for patients at risk for the development of neurological signs and symptoms

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide

Carboxyl-Group Footprinting Maps the Dimerization Interface and Phosphorylation-induced Conformational Changes of a Membrane-associated Tyrosine Kinase*

Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5–6.8 × 1012 dm2/mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the full-length Her4 protein

Chronic Corticosterone Exposure Persistently Elevates the Expression of Memory-Related Genes in the Lateral Amygdala and Enhances the Consolidation of a Pavlovian Fear Memory

<div><p>Chronic exposure to stress has been widely implicated in the development of anxiety disorders, yet relatively little is known about the long-term effects of chronic stress on amygdala-dependent memory formation. Here, we examined the effects of a history of chronic exposure to the stress-associated adrenal steroid corticosterone (CORT) on the consolidation of a fear memory and the expression of memory-related immediate early genes (IEGs) in the lateral nucleus of the amygdala (LA). Rats received chronic exposure to CORT (50 μg/ml) in their drinking water for 2 weeks and were then titrated off the CORT for an additional 6 days followed by a 2 week ‘wash-out’ period consisting of access to plain water. Rats were then either sacrificed to examine the expression of memory-related IEG expression in the LA or given auditory Pavlovian fear conditioning. We show that chronic exposure to CORT leads to a persistent elevation in the expression of the IEGs Arc/Arg3.1 and Egr-1 in the LA. Further, we show that rats with a history of chronic CORT exposure exhibit enhanced consolidation of a fear memory; short-term memory (STM) is not affected, while long-term memory (LTM) is significantly enhanced. Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine following the chronic CORT exposure period was observed to effectively reverse both the persistent CORT-related increases in memory-related IEG expression in the LA and the CORT-related enhancement in fear memory consolidation. Our findings suggest that chronic exposure to CORT can regulate memory-related IEG expression and fear memory consolidation processes in the LA in a long-lasting manner and that treatment with fluoxetine can reverse these effects.</p></div

Fluoxetine treatment following chronic CORT exposure reverses the CORT-induced enhancement of fear memory consolidation.

<p>(<b>A</b>) Schematic of the behavioral protocol. Rats received either Water (n = 7) or CORT (n = 7; 50 μg/ml) for 2 weeks followed by CORT titration (25 μg/ml, 12.5 μg/ml). Rats then received 3 weeks of either Water (n = 6) or fluoxetine (FLX; n = 7; 333 μg/ml). At the end of the fluoxetine exposure period, rats were tested in the elevated plus maze (EPM). Rats were then fear conditioned with 2 tone-shock pairings and tested for STM (2 hr later) and LTM (24 hr later). (<b>B</b>) Mean (±SEM) time spent exploring the open and closed arms of the EPM in each group. (<b>C</b>) Mean (±SEM) post-shock freezing scores in each group following each conditioning trial. (<b>D</b>) Mean (±SEM) STM scores across each trial in each group. (<b>E</b>) Mean (±SEM) LTM scores across each trial in each group.</p

Chronic exposure to CORT persistently enhances the expression of memory-related IEGs and synaptically-localized proteins in the LA.

<p>(<b>A</b>) Schematic of the behavioral protocol. Rats received either Water or CORT in their drinking water (50 μg/ml) for 2 weeks. Half the rats were sacrificed at the end of CORT exposure period. The other half was titrated off the CORT (25 μg/ml, 12.5 μg/ml) and given a 2 week period of exposure to water alone (‘wash-out’) prior to being sacrificed. (<b>B</b>) Mean (±SEM) immunoreactivity for GluR1, synaptophysin and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of CORT exposure. (<b>C</b>) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 9), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 9), Egr-1 (Water: n = 9; CORT: n = 9), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 7; CORT: n = 9), phospho-ERK 1 and phospho-ERK 2 (Water: n = 8; CORT: n = 9) from the LA in rats sacrificed on the last day of CORT exposure. (<b>D</b>) Representative Western blots for each protein in (C). (<b>E</b>) Mean (±SEM) immunoreactivity for GluR1, synaptophysin, and BDNF (Water: n = 9; CORT: n = 9) from area CA3 in rats sacrificed on the last day of the wash-out period. (<b>F</b>) Mean (±SEM) immunoreactivity for GluR1 (Water: n = 9; CORT: n = 8), synaptophysin (Water: n = 9; CORT: n = 9), Arc/Arg3.1 (Water: n = 9; CORT: n = 8), Egr-1 (Water: n = 9; CORT: n = 8), BDNF (Water: n = 9; CORT: n = 9), acetyl-H3 (Water: n = 9; CORT: n = 8), phospho-ERK 1 and phospho-ERK 2 (Water: n = 9; CORT: n = 8) from the LA in rats sacrificed on the last day of the wash-out period. (<b>G</b>) Representative Western blots for each protein in (F). For each experiment, protein levels have been normalized to GAPDH levels for each sample and expressed as a percentage of the Water group. (*) <i>p</i> < 0.05 relative to Water group.</p