Natural Amyloid-Beta Oligomers Acutely Impair the Formation of a Contextual Fear Memory in Mice

Memory loss is one of the hallmark symptoms of Alzheimer's disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss

Social Stimulus Causes Aberrant Activation of the Medial Prefrontal Cortex in a Mouse Model With Autism-Like Behaviors

Autism spectrum disorder (ASD) is a highly prevalent and genetically heterogeneous brain disorder. Developing effective therapeutic interventions requires knowledge of the brain regions that malfunction and how they malfunction during ASD-relevant behaviors. Our study provides insights into brain regions activated by a novel social stimulus and how the activation pattern differs between mice that display autism-like disabilities and control littermates. Adenomatous polyposis coli (APC) conditional knockout (cKO) mice display reduced social interest, increased repetitive behaviors and dysfunction of the β-catenin pathway, a convergent target of numerous ASD-linked human genes. Here, we exposed the mice to a novel social vs. non-social stimulus and measured neuronal activation by immunostaining for the protein c-Fos. We analyzed three brain regions known to play a role in social behavior. Compared with control littermates, APC cKOs display excessive activation, as evidenced by an increased number of excitatory pyramidal neurons stained for c-Fos in the medial prefrontal cortex (mPFC), selectively in the infralimbic sub-region. In contrast, two other social brain regions, the medial amygdala and piriform cortex show normal levels of neuron activation. Additionally, APC cKOs exhibit increased frequency of miniature excitatory postsynaptic currents (mEPSCs) in layer 5 pyramidal neurons of the infralimbic sub-region. Further, immunostaining is reduced for the inhibitory interneuron markers parvalbumin (PV) and somatostatin (SST) in the APC cKO mPFC. Our findings suggest aberrant excitatory-inhibitory balance and activation patterns. As β-catenin is a core pathway in ASD, we identify the infralimbic sub-region of the mPFC as a critical brain region for autism-relevant social behavior

A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; Translating Ribosome Affinity Purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types

Natural Abeta oligomer solution and injection.

<p>A) Blot image showing the presence of Abeta monomers, dimers, trimers, and tetramers in the Abeta solution. The 6E10 antibody was used for detection of Abeta oligomers that were removed from the Abeta solution using immunoprecipitation with A/G beads and 4G8 antibody (IP1, IP2, IP3: oligomers bound to beads used for first, second, and third immunoprecipitation). No oligomers were detected after three rounds of immunoprecipitation, which confirmed the absence of Abeta oligomers in the control solution (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029940#s4" target="_blank">Materials and Methods</a>” for a detailed description of how Abeta and control solutions were generated). B) Diagram showing the location of the guide cannula (green) and the injector cannula (red) in a Nissl-stained coronal section of the mouse brain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029940#pone.0029940-Lein1" target="_blank">[38]</a>. The tip of the guide cannula stopped just above the corpus callosum. The tip of the injection cannula extended into the lateral ventricle.</p

A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system. Frontiers in molecular neuroscience

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; translating ribosome affinity purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types

Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

<div><p>Abstract</p><p>Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a <i>Drosophila</i> cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes <i>Tdc</i>2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.</p></div

Experiment 2: single Abeta injection 1 hour before fear conditioning.

<p>Top) Diagram showing the design of experiment 2. Separate groups of mice were injected one time with either control or Abeta solution 1 hour before fear conditioning. Bottom) Graphs showing average freezing scores during fear conditioning on day 1 and the two retrieval trials on day 2. Mice injected with the Abeta solution (n = 8) had significantly lower freezing scores during the context fear retrieval trial as compared with mice injected with the control solution (n = 10). Error bars are standard errors of means. * <i>P</i><0.05.</p

Experiment 1: repeated Abeta injection.

<p>Top) Diagram showing the design of experiment 1. Separate groups of mice were injected three times with either control or Abeta solution. Bottom) Graphs showing average freezing scores during fear conditioning on day 1 and the two retrieval trials on day 2 (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029940#s4" target="_blank">Materials and Methods</a>: Analysis of freezing behavior” for explanation of intervals on the X axis). Mice injected with the Abeta solution (n = 5) had significantly lower freezing scores during the context fear retrieval trial as compared with mice injected with the control solution (n = 6). Error bars are standard errors of means. * <i>P</i><0.05.</p

Experiment 3: single Abeta injection 2 hours before fear conditioning.

<p>Top) Diagram showing the design of experiment 3. Separate groups of mice were injected one time with either control or Abeta solution 2 hours before fear conditioning. Bottom) Graphs showing average freezing scores during fear conditioning on day 1 and the two retrieval trials on day 2. There was no difference during any of the intervals analyzed between mice injected with the Abeta solution (n = 10) and mice injected with the control solution (n = 8). Error bars are standard errors of means.</p

Biological processes represented by the rhythmically translated mRNAs.

<p>(A) Pie chart showing different represented processes. The number of mRNAs belonging to each category is shown next to each slice of the pie. (B) Translational profile of thioredoxin system mRNAs.</p