Rest Induces a Distinct Transcriptional Program in the Nervous System of the Exercised L. stagnalis

Abstract

In the freshwater snail L. stagnalis, two hours of shallow water crawling exercise are accompanied by the formation of memory, metabolic, neuronal, and behavioral changes, such as faster orientation in a novel environment. Interestingly, rest following exercise enhances serotonin and dopamine metabolism linked to the formation of memory and adaptation to novel conditions. However, the underlying transcriptional responses are not characterized. In this paper, we show that, while two hours of forced crawling exercise in L. stagnalis produce significant changes in nervous system gene expression, the subsequent rest induces a completely distinct transcriptional program. Chromatin-modifying, vesicle transport, and cell cycle genes were induced, whereas neurodevelopmental, behavioral, synaptic, and hormone response genes were preferentially repressed immediately after two hours of exercise. These changes were normalized after two hours of the subsequent rest. In turn, rest induced the expression of genes functioning in neuron differentiation and synapse structure/activity, while mitotic, translational, and protein degradation genes were repressed. Our findings are likely relevant to the physiology of exercise, rest, and learning in other species. For example, chronic voluntary exercise training in mice affects the expression of many homologous genes in the hippocampus. Moreover, in humans, homologous genes are pivotal for normal development and complex neurological functions, and their mutations are associated with behavioral, learning, and neurodevelopmental abnormalities.

Keywords: L. stagnalis; autism; exercise; nervous system; neurodevelopmental diseases; rest.

Figure 5

Genes from the annotation clusters that are misregulated after exercise or rest. The majority of transcripts that are repressed or induced upon exercise are normalized in the rested animals. Colors represent log₁₀(p-values) for changes in gene expression in the rested relative to the control animals. (A) Changes in gene expression after rest versus exercise for the chromatin regulation and cell cycle related annotation clusters induced after exercise (Figure 4B, cluster 1); (B) Changes in gene expression after rest versus exercise for the nervous system and synaptic transmission related clusters repressed after exercise (Figure 4B, cluster 2); (C) Changes in gene expression after rest versus exercise for the neuron differntiation and synapse structure related clusters induced after rest (Figure 4D, cluster 3); (D) Changes in gene expression after rest versus exercise for the cell cycle related clusters repressed after rest (Figure 4D, cluster 4).

Figure 1

Experimental setup. Two types of experiments were performed: exercise and exercise followed by rest. (A) Exercise trails. The snail is forced to crawl for 2 h using intense muscle contraction to compensate for the lack of a water column supporting the shell. (B) Exercise followed by rest trails. The snail is forced to crawl for 2 h using intense muscle contraction to compensate for the lack of a water column supporting the shell. This was followed by rest in the normal aquarium conditions for 2 h. Mock-handled animals were kept in the normal aquarium conditions for 2 or 4 h.

Figure 2

Genes differentially expressed in L. stagnalis in response to exercise and exercise–rest represent distinct groups. In colors are genes regulated by exercise and exercise–rest (FC > 2 or FC < 0.5, p < 0.01) and in gray are genes whose changes are not significant. (A) Scatterplot of binary logarithm for fold changes of isoform expression levels after rest versus exercise relative to the control values for all transcripts. (B) The same data plotted only for transcripts containing ribosomal internal transcribed spacer (ITS). (C) The same data as in (A), plotted for the putative protein-coding transcripts without ITS. (D) Number of protein-coding transcripts in groups commonly or differentially regulated by exercise or exercise–rest. NC stands for “no changes”. The intersections between groups were random according to the chi-squared test (p = 0.65).

Figure 3

Changes in the normalized expression for selected ribosomal transcripts and transcript homologous to Glut1. The data are normalized to a reference gene EF1a. Colored circles are mean values, bars are standard deviations, and black dots individual animals. Stars highlight significant changes (* p < 0.05, *** p < 0.001, t-test), and numbers are the number of animals in groups.

Figure 4

Clustering of genes misregulated by exercise or rest by the functional properties of the human homologous genes revealed the overrepresentation of annotations related to neuronal, developmental, and disease-related gene clusters. Dots represent annotation groups that are connected if there are 5 or more genes in common [15]. Annotation clusters are labelled by the most significantly overrepresented annotation. (A,B) Annotations of genes up- or downregulated by exercise relative to control revealed clusters of repressed genes related to ion transport, vesicle synaptic transport, neuronal system, and brain development, whereas annotation related to chromatin organization and cell division were induced. Other gene clusters contained both induced and repressed genes, including vesicle-mediated transport and nervous system development. (C,D) Annotations of genes up. or downregulated by rest relative to control revealed clusters of repressed genes related to translation, protein ubiquitination, mitotic cell cycle, Parkinson disease and apoptosis, whereas synapse assembly and neuron differentiation related genes were induced. Numbers on the right panels represent clusters annotated in Figure 5.

Figure 6

Annotation clusters of rest-regulated genes. The majority of transcripts that are repressed or induced upon exercise are normalized in the rested animals, while rest regulates distinct transcripts. Colors represent log₁₀(p-values) for changes in gene expression in the rested relative to the control animals. (A) Changes in gene expression after rest versus exercise for the DNA damage response related annotation cluster repressed after rest (regulation of DNA metabolic process; Figure 4C); (B) Changes in gene expression after rest versus exercise for the apoptosis cluster repressed after rest (Figure 4C); (C) Changes in gene expression after rest versus exercise for the ubiquitin dependent protein catabolism cluster repressed after rest (Figure 4C); (D) Changes in gene expression after rest versus exercise for the translation related genes repressed after rest (Figure 4C).

Figure 7

Annotation of genes regulated by exercise in snails and in mice. (A) Clustering of annotations overrepresented in the lists of misregulated genes. (B) Clustering of genes and corresponding annotations. Colors represent log₂(Fold Change) in the snail after exercise.