Responses of neurogenesis and neuroplasticity related genes to elevated CO₂ levels in the brain of three teleost species

Abstract

The continuous increase of anthropogenic CO₂ in the atmosphere resulting in ocean acidification has been reported to affect brain function in some fishes. During adulthood, cell proliferation is fundamental for fish brain growth and for it to adapt in response to external stimuli, such as environmental changes. Here we report the first expression study of genes regulating neurogenesis and neuroplasticity in brains of three-spined stickleback (Gasterosteus aculeatus), cinnamon anemonefish (Amphiprion melanopus) and spiny damselfish (Acanthochromis polyacanthus) exposed to elevated CO₂. The mRNA expression levels of the neurogenic differentiation factor (NeuroD) and doublecortin (DCX) were upregulated in three-spined stickleback exposed to high-CO₂ compared with controls, while no changes were detected in the other species. The mRNA expression levels of the proliferating cell nuclear antigen (PCNA) and the brain-derived neurotrophic factor (BDNF) remained unaffected in the high-CO₂ exposed groups compared to the control in all three species. These results indicate a species-specific regulation of genes involved in neurogenesis in response to elevated ambient CO₂ levels. The higher expression of NeuroD and DCX mRNA transcripts in the brain of high-CO₂–exposed three-spined stickleback, together with the lack of effects on mRNA levels in cinnamon anemonefish and spiny damselfish, indicate differences in coping mechanisms among fish in response to the predicted-future CO₂ level.

1. Introduction

Increasing atmospheric CO₂ is causing a concurrent decrease in ocean pH, a process referred to as ocean acidification. The current 400 µatm atmospheric pCO₂ is projected to reach to 800–1150 µatm by the end of this century [1] and lead to a decrease in average ocean pH of up to 0.32 [1].

Altered behaviours have been observed in many (but not all) marine fish exposed to projected future CO₂ levels [2]. A common cause could be a shift in function of the γ-aminobutyric acid receptor A (GABA_A receptor), the main inhibitory neurotransmitter receptor in the vertebrate brain [3]. Altered gradients of [Cl⁻] and [] over neuronal membranes, brought about by pH-regulatory mechanisms responding to elevated pCO₂, have been suggested to shift the action of GABA_A receptors in the direction of being excitatory [3]. While GABA_A receptors obviously play important roles to convey inhibitory neural signals, GABA_A receptors acting excitatory due to high intracellular [Cl⁻] or [] have been shown to be essential for proper neural development, differentiation and plasticity in both immature and mature CNS systems [4,5]. In both cases, depolarizing GABAergic transmission promotes an increase in intracellular Ca²⁺ through the activation of voltage-gated Ca²⁺ channels, followed by a cascade of molecular events involved in neurogenesis [6].

Proliferating cell nuclear antigen (PCNA), neurogenic differentiation factor (NeuroD), doublecortin (DCX) and brain-derived neurotrophic factor (BDNF) are four key factors involved in proliferation, differentiation, migration and survival of newborn neurons [7–10]. NeuroD is a basic helix-loop-helix (bHLH) transcription factor involved in the later stages of neurogenesis and is required for determination, differentiation and survival of neural precursor cells [8]. DCX is a microtubule-associated protein that is highly expressed in progenitor cells and neuroblasts [9]. PCNA is a key processivity factor for DNA synthesis, DNA repair and cell cycle regulation [11]. BDNF belongs to the neurotrophin family of growth factors that play important roles during brain development and in synaptic plasticity, and influences neural transmission via modulation of GABAergic synapses [12].

Consequently, in the light of the widespread behavioural alterations reported in fish exposed to elevated CO₂, we hypothesized that fish brain plasticity and neurogenesis are also altered during such conditions. As a first test of this hypothesis we quantified mRNA expression levels of NeuroD, DCX, PCNA and BDNF in brains of adult three-spined stickleback (Gasterosteus aculeatus), cinnamon anemonefish (Amphiprion melanopus) and spiny damselfish (Acanthochromis polyacanthus) exposed to present day or predicted future CO₂ levels according to species-specific protocols reported to cause behavioural changes in these species [13–15].

2. Material and methods

We exposed adult marine three-spined stickleback, cinnamon anemonefish and spiny damselfish to control or elevated pCO₂ (electronic supplementary materials). After exposure, brains were collected and frozen. Total RNA was extracted using TRIzol^® reagent (Invitrogen), and cDNA synthesized using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT)₁₈.

Cloning and sequencing were required to design species- and gene-specific primers for quantitative real-time PCR (qPCR) for DCX, PCNA, NeuroD, BDNF, and for two reference genes ubiquitin (ubc) and ribosomal protein L13A (rpl13A) (electronic supplementary material, table s1a–c).

qPCR was carried out in duplicates using 1 : 30 diluted cDNA (3 µl), LightCycler 480 SYBR Green I Master Mix (5 µl; Roche Diagnostic), primers (1 µl; 5 µM) and nuclease-free water (1 µl; Ambion Applied Biosystem). Reaction mix and samples were loaded onto 384 multiwell plates (Roche Diagnostics) using Bravo robot (Agilent Technologies). The following qPCR programme was used: (i) 95°C for 10 min, (ii) 95°C for 10 s, (iii) 60°C for 10 s, (iv) 72°C for 13 s, (v) repeat steps 2–4 42×.

Statistical analyses were performed using GraphPad Prism 6.0d. Normality and variance homogeneity were assessed using D'Agostino & Pearson omnibus normality test and F-test. For each species, a multiple two-tailed t-test was used for comparisons between groups. p-Values were corrected using the Holm–Sidak method. p < 0.05 was considered significant. Data are presented as means ± s.e.m.

3. Results

Exposure of three-spined stickleback to elevated pCO₂ (approx. 990 µatm) significantly increased NeuroD mRNA expression level by 28.4% (p = 0.0094) and DCX mRNA expression level by 51.5% (p = 0.0135; figure 1). By contrast, there were no significant effects of elevated pCO₂ on the mRNA expression levels of BDNF or PCNA (p = 0.4452 and p = 0.7000, respectively).

Figure 1.

mRNA expression levels of NeuroD, DCX, PCNA and BDNF in the three-spined stickleback. Data were normalized to the geometric average of the reference genes rpl13A and ubc. Values are means ± s.e.m. from 11 individuals. *p < 0.05 (see text for p-values).

No significant differences in mRNA expression levels were detected between groups for the examined genes in cinnamon anemonefish (PCNA: p = 0.9284; NeuroD: p = 0.8809; DCX: p = 0.5773; BDNF: p = 0.7809) or in spiny damselfish (PCNA: p = 0.8089, NeuroD: p = 0.8775, DCX: p = 0.7839, BDNF: p = 0.2896; figure 2).

Figure 2.

mRNA expression levels of NeuroD, DCX, PCNA and BDNF in the cinnamon anemonefish (n = 12) and spiny damselfish (n = 8). Data were normalized to the geometric average of the reference genes rpl13A and ubc. Values are means ± s.e.m.

For both three-spined stickleback and spiny damselfish, NeuroD was found to be the highest-expressed gene examined. For cinnamon anemonefish, NeuroD and DCX were expressed at comparable levels. In all three species investigated, BDNF was the least-expressed gene.

4. Discussion

In teleosts, new neurons are formed in the CNS throughout life to allow brain growth and normal brain function [16]. Our results suggest potential species-specific differences in how neurogenesis might be affected by exposure to elevated CO₂ levels. Thus, after exposure to elevated CO₂, three-spined stickleback showed increased mRNA expression levels of both NeuroD and DCX, while no changes in the expression of these factors were detected in cinnamon anemonefish or spiny damselfish.

At the present stage, we can only speculate on the cause of the differential mRNA expression levels among the three species in response to high CO₂ exposure, which could potentially be related to factors such as interspecific variation in coping style, tolerance and brain plasticity. The three-spined stickleback is a temperate species with a remarkable physiological plasticity to various environmental conditions [17], which might be reflected in greater plasticity in gene expression. In particular, the increased mRNA expression levels of NeuroD and DCX in high-CO₂–exposed three-spined stickleback might indicate a larger pool of newborn neurons and different degree of neurogenesis compared to the two tropical species in high CO₂. An increase in NeuroD expression in the brain correlates with neuronal proliferation and differentiation of neural stem/progenitor cells (NCPs) [18]. Interestingly, a study on rainbow trout (Oncorhynchus mykiss) has shown that NeuroD, DCX and PCNA mRNA transcripts are more abundant in brains of individuals from a high-stress response line (confinement stress) than in individuals from a low-stress response line [19]. These differences were suggested to be caused by variations in coping styles that were at the base of divergent behavioural properties among different lines [19].

In the tropical species, exposure to high CO₂ levels did not affect the mRNA levels of the four neurogenesis-related factors. While spiny damselfish was exposed to high-CO₂ conditions for 4 days, cinnamon anemonefish was exposed for at least 10 months. For the three-spined stickleback the high-CO₂ treatment lasted 43 days. Obviously, we cannot rule out that the differences observed are related to the treatment regimes. However, we did choose the treatment regimes to assure that the fish displayed behavioural abnormalities at the time of sampling. In the case of the anemonefish and stickleback, the samples were taken from treatment groups that were part of behavioural studies that have now been published [20,21]. Thus, we find it likely that these are indeed species-specific differences, but further experiments would be needed to test this hypothesis, and we suggest that neurogenesis-related factors should receive attention in future transcriptomics or proteomics studies related to ocean acidification. Interestingly, the absence of change in these factors in spiny damselfish is in agreement with a study on juveniles of this species where analysis of the transcriptome and proteome of offspring from CO₂-tolerant and CO₂-sensitive parents reared under control or high-CO₂ conditions revealed no significant changes in mRNA expression levels for neurogenesis genes [22]. On the contrary, genes related to nervous system function were upregulated in the Mediterranean pteropod Heliconides inflatus exposed to acidified seawater for 3 days, where six transcripts were involved in neural differentiation and survival, such as BDNF and neurotrophin NT-3 receptors [23]. This observation is particularly interesting because it suggests that elevated CO₂ may affect neural differentiation in a wide range of marine animals.

In previous studies, the behavioural disturbances seen in fish exposed to elevated CO₂ have been linked to a shift in GABAergic action, from inhibitory (hyperpolarizing) to excitatory (depolarizing) [2]. We have also recently found that the mRNA levels of some GABA_A receptor subunits are increased in stickleback exposed to the present treatment regime [14]. GABAergic depolarization can promote maturation and integration of new neurons in mammalian brains through changes in gene expression [6], for instance the NeuroD gene [18]. It is possible that a shift in the GABA_A receptor activity from hyperpolarizing to depolarizing in three-spined stickleback exposed to high CO₂ is responsible for triggering the increased expression of NeuroD and DCX.

In conclusion, our results indicate a possible species-specific regulation of genes involved in neurogenesis in response to ocean acidification. The apparent absence of change in neurogenesis-related factors in both cinnamon anemonefish and spiny damselfish could indicate that these tropical species are less capable of adaptive neural responses to the predicted future CO₂ level than the temperate three-spined stickleback living under more variable environmental conditions.

Ethics

Ethical permits: Gothenburg University, Sweden (nos. 100-2010 and 151-2011) to F.J. and James Cook University, Australia (A1427) to G.M.M.

Data accessibility

Data were available from the Dryad Digital Repository: (http://dx.doi.org/10.5061/dryad.pn397) [24]. GeneBank accessions supporting this article have been uploaded as part of the electronic supplementary material.

Authors' contributions

The study was conceived and designed by G.E.N. and F.L. Experiments and data analysis were executed by F.L. Advice and assistance on gene expression analyses by C.E.F. Fish collection, rearing and water chemistry by F.J., G.M.M. and P.L.M. RNA extraction and cDNA synthesis by F.L. and N.J.B. Written by F.L., C.E.F. and G.E.N. F.L., C.E.F. and G.E.N drafted the manuscript. F.L., C.E.F., G.E.N., F.J., P.L.M., G.M.M. and N.J.B. contributed on the revising and editing of the manuscript. All authors approved the final version of this manuscript and agreed to be held accountable for all aspects of the work performed.

Competing interests

We have no competing interests.

Funding

This study was funded by the University of Oslo (G.E.N. and F.L.), the Swedish Research Council (G.E.N. and F.J.), the Swedish Research Council FORMAS (F.J.), the ARC Centre of Excellence for Coral Reef Studies (G.M.M. and P.L.M.), GBRMPA Science for Management Awards (G.M.M.), Sea World Research and Rescue Foundation (G.M.M.) and Australian Coral Reef Society (G.M.M.).