Orchestrated ensemble activities constitute a hippocampal memory engram

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

Hippocampal function is not required for the precision of remote place memory

Background: During permanent memory formation, recall of acquired place memories initially depends on the hippocampus and eventually become hippocampus-independent with time. It has been suggested that the quality of original place memories also transforms from a precise form to a less precise form with similar time course. The question arises of whether the quality of original place memories is determined by brain regions on which the memory depends. Results: To directly test this idea, we introduced a new procedure: a non-associative place recognition memory test in mice. Combined with genetic and pharmacological approaches, our analyses revealed that place memory is precisely maintained for 28 days, although the recall of place memory shifts from hippocampus-dependent to hippocampus-independent with time. Moreover, the inactivation of the hippocampal function does not inhibit the precision of remote place memory. Conclusion: These results indicate that the quality of place memories is not determined by brain regions on which the memory depends.Japan. Science and Technology Agency (Core Research for Evolutional Science and Technology (CREST) program)Mitsubishi FoundationSasagawa Scientifi

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

<div><p>During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure <i>in vitro</i>, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.</p></div

Efficiency and specificity of CFL1 KD through miR-CFL1.

<p>(A) CFL1 mRNA KD efficiency of two miRNAs (miR-CFL1_1 and miR-CFL1_2) targeted against different sequences within the CFL1 gene, assessed by using CFL1-overexpressing HEK 293T-cells. CFL1 mRNA levels were normalized to those of the miR-Neg group. <i>n</i> = 3 for all groups; miR-CFL1_1, <i>p</i> = 2.7 × 10^-8; miR-CFL1_2, <i>p</i> = 2.8 × 10^-8 versus miR-Neg, Dunnett’s multiple comparison test. (B) KD efficiency of miR-CFL1_1 and miR-CFL1_2 for endogenous CFL1 protein, assessed by using PC-12 cells. “WT” (wild type) indicates PC-12 cells that were not infected with lentivirus. (C) Two neighboring coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against CFL1 (left) and the other stained with an antibody against NeuN (right). The eYFP fluorescence image (middle) was obtained from the NeuN-stained section. Scale bar, 300 μm. (D) Magnified view of the rectangular region indicated in (C). Scale bar, 150 μm. (E, F) Same as (C and D), but of two neighboring coronal sections derived from a miR-Neg virus-injected rat. (G, H) Confocal images of eYFP+ or eYFP− region in a coronal section obtained from a miR-CFL1_1-expressing rat stained with antibodies against NeuN (blue) and CFL1 (red). Scale bar, 50 μm. (I) Percentage of CFL1+ cells in NeuN+ cells measured in miR-CFL1_1- or miR-Neg-expressing rats. <i>n</i> = 3 for all groups. *<i>p</i> = 0.0003, t-test with Bonferroni’s correction. (J) Effects of miR-CFL1 on mRNA expression of genes related to CFL1, assessed in PC-12 cells. <i>n</i> = 3 for all groups. miR-CFL1_1 of CFL1, <i>p</i> = 2.6 × 10^-7; miR-CFL1_2 of CFL1, <i>p</i> = 5.2 × 10^-7 versus miR-Neg, Dunnett’s multiple comparison test. (K) Effects of miR-CFL1 on expression of ADF protein in PC-12 cells. (L) Three successive coronal sections obtained from a miR-CFL1_1-expressing rat are shown, one stained with an antibody against ADF (middle) and another stained with an antibody against CFL1 (right). Scale bar, 300 μm.</p

Effects of CFL1 KD on spine density during sensory deprivation.

<p>(A, B) Lentiviral vectors employed for co-expressing eGFP and miRNAs (A) and for expressing tdTomato (B). (C) The D1 and D2 barrel columns were identified via intrinsic signal optical imaging. (D) Virus injection was targeted to L2/3 of the D1 and D2 columns. (E) A representative parasagittal section showing expression of tdTomato (D1) and eGFP (D2). Scale bar, 300 μm. (F) Magnified view of an eGFP-expressing region in the parasagittal section shown in (E). Scale bar, 100 μm. (G) Confocal images of a rectangle region shown in (F). Scale bar, 20 μm. (H) A representative dendritic branch in the D2 column is shown that made a putative synaptic connection with a tdTomato+ D1 axonal bouton. Dendritic spines were counted that were localized at a distance less than 15 μm from an identified putative synaptic connection. A magnified view of the putative synaptic connection is shown in the inset. Scale bar, 10 μm. (I) Representative images of the dendritic branches within the distal portion in the D2 column. Scale bar, 10 μm. (J) Spine densities measured in the distal portion. <i>n</i> = 18, 17, 19, and 19 branch segments for miR-Neg non-deprived (ND), miR-Neg deprived (D), miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 1.1 × 10^-4; miR-CFL1_1 ND, <i>p</i> = 1.8 × 10^-5; miR-CFL1_1 D, <i>p</i> = 0.0027 versus WT D group, Tukey-Kramer’s multiple comparison test. (K) Cumulative frequency histogram of spine density. WT ND, <i>p</i> = 0.037; miR-CFL1_1 ND, <i>p</i> = 3.6 × 10^-4; miR-CFL1_1 D, <i>p</i> = 3.6 × 10^-4 versus WT D group, Kolmogorov-Smirnov test with Bonferroni’s correction. (L−N) Same as (I−K) but of dendritic branches measured within the proximal portion. <i>n</i> = 14, 18, 16, and 10 branch segments for miR-Neg ND, miR-Neg D, miR-CFL1_1 ND, and miR-CFL1_1 D groups, respectively. WT ND, <i>p</i> = 0.86; miR-CFL1_1 ND, <i>p</i> = 0.97; miR-CFL1_1 D, <i>p</i> = 0.93 versus WT D group, Tukey-Kramer’s multiple comparison test.</p

Effects of CFL1 expression rescue on impaired experience-dependent plasticity.

<p>(A) Design of three mutant CFL1s that are resistant to miR-CFL1_1 (resCFL1s). Seven or eight nucleic acids within the target sequence of miR-CFL1_1 were mutated such that amino acid sequence did not change. (B) An <i>in vitro</i> test of CFL1 expression rescue by each resCFL1 in rat CFL1 (WT)- and miR-CFL1_1-expressing HEK293T cells. The mock group expressed only WT CFL1. <i>n</i> = 4 for all groups. resCFL1_1, <i>p</i> = 5.8 × 10^-6; resCFL1_2, <i>p</i> = 2.2 × 10^-5; resCFL1_3, <i>p</i> = 1.3 × 10^-4 versus miR-CFL1_1 group, Tukey-Kramer’s multiple comparison test. (C) An <i>in vitro</i> test of the effect of miR-CFL1_1 on resCFL1_1 expression. <i>n</i> = 3 for both groups. <i>p</i> = 0.30, <i>t</i>-test. (D) A schematic diagram of the bicistronic lentiviral vector that co-expresses resCFL1_1 and mCherry via a P2A peptide. (E) Targeted injection of Lenti-CaMKIIα-ChR2-eYFP-miR-CFL1_1 and Lenti-CaMKIIα-mCherry-P2A-resCFL1 to D2 column identified with intrinsic signal imaging induced focal expression of ChR2-eYFP and mCherry. Scale bar, 500 μm. (F) Fluorescent images of a coronal section infected with the two vectors. Scale bar, 300 μm. (G) Confocal images of an infected area showing co-expression of ChR2-eYFP and mCherry in L2/3 neurons. Scale bar, 20 μm. (H) A representative raster plot (100 trials are shown in horizontal row) and peristimulus time histogram (PSTH) of a putative ChR2+ neuron recorded from L2/3 in D2 column of the rat showed in E. (I) A representative raster plot (50 trials) and PSTH of the same neuron with (H), showing responses to D1 whisker deflections. (J) Comparison of average responses recorded from ChR2+ neurons in D2 L2/3 of deprived rats expressing miR-CFL1_1 and resCFL1 with those recorded from ChR2+ neurons in deprived rats expressing only miR-CFL1_1 and those recorded from WT deprived rats. Data of miR-CFL1_1 deprived and WT deprived groups were the same with those shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002070#pbio.1002070.g003" target="_blank">Fig. 3D</a>. <i>n</i> = 17 units (from three rats) for the miR-CFL1_1+resCFL1 deprived group. *<i>p</i> = 0.0069, Tukey-Kramer’s multiple comparison test.</p

Schematic illustration of the circuit-specific cofilin action on barrel map plasticity.

<p>Cross-sectional view of the D1 and D2 barrel columns. L2/3 neurons in the D2 column exhibit a CFL1-dependent neuronal response increase to horizontal transcolumnar inputs from the spared D1 column. By contrast, the response decrease to ascending intracolumnar inputs from L4 is CFL1 independent. In the distal portion of the supragranular layer of the D2 column, spine densities of dendrites receiving transcolumnar inputs from the D1 column increase in a CFL1-dependent manner during sensory deprivation.</p