Expression of genes involved in brain GABAergic neurotransmission in three-spined stickleback exposed to near-future CO2

Abstract

Change in the activity of the main inhibitory receptor, GABA_A, has been suggested to be a general mechanism behind the behavioural alterations reported in ocean acidification studies on fish. It has been proposed that regulatory acid-base mechanisms in response to high CO₂ alter the neuronal Cl^- and HCO₃^- gradients that are important for GABA_A receptor function. Here, we report a comprehensive analysis of gene expression of GABA_A receptor subunits and of genes involved in GABAergic transmission in the brain of fish exposed to near-future CO₂. Altogether, 56 mRNA transcripts were quantified in brains of three-spined stickleback (Gasterosteus aculeatus) kept in control pCO₂ (333 ± 30 μatm CO₂) or at high pCO₂ levels (991 ± 57 μatm) for 43 days. The gene expression analysis included GABA_A receptor subunits (α1-6, β1-3, γ1-3, δ, π and ρ1-3), enzymes and transporters involved in GABA metabolism (GAD1-2, GABAT and GAT1-3), GABA_A receptor-associated proteins (GABARAP and GABARAPL), ion cotransporters (KCC1-4, NKCC1, ClC21-3, AE3 and NDAE) and carbonic anhydrase (CAII). Exposure to high CO₂ had only minor effects on the expression of genes involved in GABAergic neurotransmission. There were significant increases in the mRNA levels of α family subunits of the GABA_A receptor, with a more pronounced expression of α1₂, α3, α4 and α6b. No changes were detected in the expression of other GABA_A subunits or in genes related to receptor turnover, GABA metabolism or ion transport. Although the minor changes seen for mRNA levels might reflect compensatory mechanisms in the high-CO₂ conditions, these were apparently insufficient to restore normal neural function, because the behavioural changes persisted within the time frame studied.

Keywords: GABAA receptor; GABAergic system; ion cotranporters; ocean acidification; quantitative polymerase chain reaction; three-spined stickleback.

Figure 1:

Messenger RNA expression levels of GABA_A receptor subunits. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc) and grouped into four families as follows: α subunits (A); β subunits (B); γ subunits (C) and δ, π and ρ subunits (D). Each family was analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water (n = 12) and high-CO₂ water (n = 12) for 43 days. Values are shown as means + SEM.

Figure 2:

Messenger RNA expression levels of GAT, GABAT and GAD genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water (n = 12) and high-CO₂ water (n = 12) for 43 days. Values are shown as means + SEM.

Figure 3:

Messenger RNA expression levels of GABARAP and GABARAPL genes. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water (n = 12) and high-CO₂ water (n = 12) for 43 days. Values are shown as means + SEM.

Figure 4:

Messenger RNA expression levels of KCCs, NKCC1, ClC2, NDAE, AE3 ion cotransporters and CAII enzyme. Data were normalized to the geometric average of the reference genes ribosomal protein L13A (rpl13A) and ubiquitin (ubc). Data were analysed by two-way ANOVA followed by Sidak post hoc test. Open and filled columns represent three-spined sticklebacks exposed to control water (n = 12) and high-CO₂ water (n = 12) for 43 days. Values are shown as means + SEM.