Time Course of Immediate Early Gene Protein Expression in the Spinal Cord following Conditioning Stimulation of the Sciatic Nerve in Rats

Long-term potentiation induced by conditioning electrical stimulation of afferent fibers is a widely studied form of synaptic plasticity in the brain and the spinal cord. In the spinal cord dorsal horn, long-term potentiation is induced by a series of high-frequency trains applied to primary afferent fibers. Conditioning stimulation (CS) of sciatic nerve primary afferent fibers also induces expression of immediate early gene proteins in the lumbar spinal cord. However, the time course of immediate early gene expression and the rostral-caudal distribution of expression in the spinal cord have not been systematically studied. Here, we examined the effects of sciatic nerve conditioning stimulation (10 stimulus trains, 0.5 ms stimuli, 7.2 mA, 100 Hz, train duration 2 s, 8 s intervals between trains) on cellular expression of immediate early genes, Arc, c-Fos and Zif268, in anesthetized rats. Immunohistochemical analysis was performed on sagittal sections obtained from Th13- L5 segments of the spinal cord at 1, 2, 3, 6 and 12 h post-CS. Strikingly, all immediate early genes exhibited a monophasic increase in expression with peak increases detected in dorsal horn neurons at 2 hours post-CS. Regional analysis showed peak increases at the location between the L3 and L4 spinal segments. Both Arc, c-Fos and Zif268 remained significantly elevated at 2 hours, followed by a sharp decrease in immediate early gene expression between 2 and 3 hours post-CS. Colocalization analysis performed at 2 hours post-CS showed that all c-Fos and Zif268 neurons were positive for Arc, while 30% and 43% of Arc positive neurons were positive for c-Fos and Zif268, respectively. The present study identifies the spinal cord level and time course of immediate early gene (IEGP) expression of relevance for analysis of IEGPs function in neuronal plasticity and nociception.     

Precise targeted integration by a chimaeric transposase zinc-finger fusion protein

Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer ‘Z-transposases’ that could deliver transgenic cargoes to chosen genomic locations.     

KCNQ Channels Regulate Age-Related Memory Impairment

In humans KCNQ2/3 heteromeric channels form an M-current that acts as a brake on neuronal excitability, with mutations causing a form of epilepsy. The M-current has been shown to be a key regulator of neuronal plasticity underlying associative memory and ethanol response in mammals. Previous work has shown that many of the molecules and plasticity mechanisms underlying changes in alcohol behaviour and addiction are shared with those of memory. We show that the single KCNQ channel in Drosophila (dKCNQ) when mutated show decrements in associative short- and long-term memory, with KCNQ function in the mushroom body α/βneurons being required for short-term memory. Ethanol disrupts memory in wildtype flies, but not in a KCNQ null mutant background suggesting KCNQ maybe a direct target of ethanol, the blockade of which interferes with the plasticity machinery required for memory formation. We show that as in humans, Drosophila display age-related memory impairment with the KCNQ mutant memory defect mimicking the effect of age on memory. Expression of KCNQ normally decreases in aging brains and KCNQ overexpression in the mushroom body neurons of KCNQ mutants restores age-related memory impairment. Therefore KCNQ is a central plasticity molecule that regulates age dependent memory impairment.     

Differential Involvement of the Dentate Gyrus in Adaptive Forgetting in the Rat

How does the brain discriminate essential information aimed to be stored permanently from information required only temporarily, and that needs to be cleared away for not saturating our precious memory space? Reference Memory (RM) refers to the long-term storage of invariable information whereas Working Memory (WM) depends on the short-term storage of trial-unique information. Previous work has revealed that WM tasks are very sensitive to proactive interference. In order to prevent such interference, irrelevant old memories must be forgotten to give new ones the opportunity to be stabilized. However, unlike memory, physiological processes underlying this adaptive form of forgetting are still poorly understood. Here, we precisely ask what specific brain structure(s) could be responsible for such process to occur. To answer this question, we trained rats in a radial maze using three paradigms, a RM task and two WM tasks involving or not the processing of interference but strictly identical in terms of locomotion or motivation. We showed that an inhibition of the expression of Zif268 and c-Fos, two indirect markers of neuronal activity and synaptic plasticity, was observed in the dentate gyrus of the dorsal hippocampus when processing such interfering previously stored information. Conversely, we showed that inactivating the dentate gyrus impairs both RM and WM, but improves the processing of interference. Altogether, these results strongly suggest for the first time that the dentate gyrus could be a key structure involved in adaptive forgetting.     

Zn finger-based direct detection system for PCR products of <Emphasis Type="Italic">Salmonella</Emphasis> spp. and the Influenza A virus

We have detected PCR products from Salmonella spp. and Influenza A virus using Zn finger protein Zif268 and Sp1, respectively. Previously, we demonstrated a novel method of rapid and specific detection of PCR products from Legionella pneumophila genome using Zn finger protein Sp1. In principle, this methodology might be applied to the detection of most bacteria and viruses using various Zn finger proteins. Here, to demonstrate the wider applicability of our method, we detected PCR products from Salmonella spp. and the Influenza A virus. BLAST data indicated the Zif268 and Sp1 recognition sequence were located on the gyrB gene of Salmonella spp. and the nucleoprotein gene of Influenza A virus, respectively. The PCR products from the oligonucleotide corresponding to the gyrB gene of Salmonella spp. or the nucleoprotein gene of the Influenza A virus could be specifically detected by ELISA or fluorescence depolarization measurement using Zif268 or Sp1. These results indicate the wide applicability of our novel methodology.     

New neurons generated from running are broadly recruited into neuronal activation associated with three different hippocampus-involved tasks

Running increases the formation of new neurons in the adult rodent hippocampus. However, the function of new neurons generated from running is currently unknown. One hypothesis is that new neurons from running contribute to enhanced cognitive function by increasing plasticity in the adult hippocampus. An alternative hypothesis is that new neurons generated from running incorporate into experience-specific hippocampal networks that only become active during running. The purpose of this experiment was to determine if new neurons generated from running are selectively activated by running, or can become recruited into granule cell activity occurring during performance on other behavioral tasks that engage the hippocampus. Therefore, the activation of new 5-6 week neurons was detected using BrdU, NeuN, and Zif268 triple-label immunohistochemistry in cohorts of female running and sedentary adult C57BL/6J mice following participation in one of three different tasks: the Morris water maze, novel environment exploration, or wheel running. Results showed that running and sedentary mice displayed a nearly equivalent proportion of new neurons that expressed Zif268 following each task. Since running approximately doubled the number of new neurons, the results demonstrated that running mice had a greater number of new neurons recruited into the Zif268 induction in the granule cell layer following each task than sedentary mice. The results suggest that new neurons incorporated into hippocampal circuitry from running are not just activated by wheel running itself, but rather become broadly recruited into granule cell layer activity during distinct behavioral experiences. ? 2012 Wiley Periodicals, Inc.     

Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.     

The role of group I metabotropic glutamate receptors in memory consolidation and reconsolidation in the passive avoidance task in 1-day-old chicks

Although reconsolidation of memory after reminder does not seem to be the simple reiteration of the sequential stages occurring during memory consolidation, both phenomena probably employ similar mechanisms including activation of glutamate receptors and protein synthesis. It is known that group I metabotropic glutamate receptors (mGluRs) are involved in memory consolidation and modulation of protein synthesis. The aim of present study was to investigate the role of mGluR5 in memory consolidation and reconsolidation and to determine whether inhibition of these receptors may affect protein synthesis in these processes. The one-trial passive avoidance task on chicks was used as the experimental model of learning. Injection of the mGluR5 antagonist MPEP into a specific chick brain region IMM resulted in amnesia, provided the injection was made either shortly before or after training, or approximately 4 h after training. This amnesia was permanent, resembling the effects of protein synthesis inhibitors. MPEP injection immediately after reminder resulted in only a transient amnesia revealed 1h later. Increased expression of Zif/268 and c-Fos proteins 2 h after initial training was abolished bilaterally in chicks injected with MPEP. Injection of MPEP immediately after reminder did not inhibit c-Fos and Zif/268 expression, on the contrary, their expression was increased, specifically in left IMM and was similar to that observed after initial training. These results show that at least in the chick model mGluR5 play an important role in both consolidation and reconsolidation of memory but the mechanisms triggered by their activation in these processes differ. It is suggested that Ca(2+) signal derived from mGluR5 stimulation is necessary for complete memory consolidation, whereas during reconsolidation other mGluR5 triggered mechanisms of protein synthesis activation and regulation may be involved.