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Temporal Profile of Brain Gene Expression After Prey Catching Conditioning in an Anuran Amphibian

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Temporal Profile of Brain Gene Expression After Prey Catching Conditioning in an Anuran Amphibian

Vern Lewis et al. Front Neurosci.

Abstract

A key goal in modern neurobiology is to understand the mechanisms underlying learning and memory. To that end, it is essential to identify the patterns of gene expression and the temporal sequence of molecular events associated with learning and memory processes. It is also important to ascertain if and how these molecular events vary between organisms. In vertebrates, learning and memory processes are characterized by distinct phases of molecular activity involving gene transcription, structural change, and long-term maintenance of such structural change in the nervous system. Utilizing next generation sequencing techniques, we profiled the temporal expression patterns of genes in the brain of the fire-bellied toad Bombina orientalis after prey catching conditioning. The fire-bellied toad is a basal tetrapod whose neural architecture and molecular pathways may help us understand the ancestral state of learning and memory mechanisms in tetrapods. Differential gene expression following conditioning revealed activity in molecular pathways related to immediate early genes (IEG), cytoskeletal modification, axon guidance activity, and apoptotic processes. Conditioning induced early IEG activity coinciding with transcriptional activity and neuron structural modification, followed by axon guidance and cell adhesion activity, and late neuronal pruning. While some of these gene expression patterns are similar to those found in mammals submitted to conditioning, some interesting divergent expression profiles were seen, and differential expression of some well-known learning-related mammalian genes is missing altogether. These results highlight the importance of using a comparative approach in the study of the mechanisms of leaning and memory and provide molecular resources for a novel vertebrate model in the relatively poorly studied Amphibia.

Keywords: Bombina orientalis; RNA sequencing; immediate-early genes; learning and memory; neuronal plasticity; transcriptome.

Figures

FIGURE 1
FIGURE 1
Training area for prey-catching conditioning. Toads were required to touch the prey stimulus five times, at which point the video was turned off and the toad fed a cricket (Photo by V. Lewis).
FIGURE 2
FIGURE 2
Prey catching conditioning results. (A) Percent decrease in latency to five snaps from session one to session two for each toad. The dashed line represents the rejection threshold, which was set to 1 standard deviation below the mean of two previous 2-session acquisition experiments (n = 20; 37% ± 15%; Lewis, unpublished). Dark bars are two rejected toads that were not used for transcriptomic analysis. Only toads showing a ≥20% decrease in latency between sessions 1 and 2 were used for transcriptomic analysis. (B) Mean prey-catching performance over 2 sessions of 6 trials for trained toads included in the study. Error bars represent ± Standard Deviation. The decrease in latency between sessions is statistically significant (Paired t-test: t = 9.07, df = 8, p < 0.01).
FIGURE 3
FIGURE 3
Differential gene expression associated with conditioning. (A) Number of differentially expressed genes (DEG) in each treatment (2, 4, 24 h) vs. control (0 h; total of 4692 DEG). The first Venn diagram includes the full data set, while the bottom two diagrams show up/down regulated gene numbers separately. (B) The top three co-expression patterns of all 122981 unsorted DEG based on hierarchical clustering. More than 90% of DEG fall into these three co-expression patterns: 2, 4, and 24 h peak. Line color represents direction and magnitude of gene expression; red is up regulated while green is down regulated. Inset figures are idealized up-regulated co-expression patterns.
FIGURE 4
FIGURE 4
Distribution of Gene Ontology assignments for all annotated DEG transcripts. (A) Biological process functional group. (B) Cellular component group. (C) Molecular function group. Horizontal bars represent the number of DEG found in the associated level 1 Gene Ontology functional classification. (D) The largest Biological Process group, Cellular Process, has been expanded to show relative distribution of DEG at GO levels 2–4. Isolated segments represent expanded subgroup in subsequent charts on the right. The majority of DEG in the Biological Process group belong to cytoskeletal organization and gene expression.
FIGURE 5
FIGURE 5
Differentially expressed gene counts associated with similar functional categories. Gene ontology enriched functional groups for each time-point (2, 4, and 24 h) were grouped by functional similarity and plotted as a function of gene number. Positive bars represent upregulated genes vs. the control, while negative bars are downregulated genes. All genes part of the enrichment analysis as well as manually selected genes (Supplementary Tables S4A–C) are included.

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