Effect of serotonin transporter genotype on carbon dioxide-induced fear-related behavior in mice

Abstract

Background: Inhaling 35% carbon dioxide induces an emotional and symptomatic state in humans closely resembling naturally occurring panic attacks, the core symptom of panic disorder. Previous research has suggested a role of the serotonin system in the individual sensitivity to carbon dioxide. In line with this, we previously showed that a variant in the SLC6A4 gene, encoding the serotonin transporter, moderates the fear response to carbon dioxide in humans. To study the etiological basis of carbon dioxide-reactivity and panic attacks in more detail, we recently established a translational mouse model.

Aim: The purpose of this study was to investigate whether decreased expression of the serotonin transporter affects the sensitivity to carbon dioxide.

Methods: Based on our previous work, wildtype and serotonin transporter deficient (+/-, -/-) mice were monitored while being exposed to carbon dioxide-enriched air. In wildtype and serotonin transporter +/- mice, also cardio-respiration was assessed.

Results: For most behavioral measures under air exposure, wildtype and serotonin transporter +/- mice did not differ, while serotonin transporter -/- mice showed more fear-related behavior. Carbon dioxide exposure evoked a marked increase in fear-related behaviors, independent of genotype, with the exception of time serotonin transporter -/- mice spent in the center zone of the modified open field test and freezing in the two-chamber test. On the physiological level, when inhaling carbon dioxide, the respiratory system was strongly activated and heart rate decreased independent of genotype.

Conclusion: Carbon dioxide is a robust fear-inducing stimulus. It evokes inhibitory behavioral responses such as decreased exploration and is associated with a clear respiratory profile independent of serotonin transporter genotype.

Keywords: Panic attacks; carbon dioxide; panic disorder; serotonin transporter.

Figure 1.

Overview of experimental design. (a) Male wildtype (WT), serotonin transporter (5-HTT) heterozygous (+/–) and 5-HTT homozygous (–/–) knockout mice were first behaviorally tested in the modified open field test (OFT), while being exposed to either carbon dioxide (CO₂) (n = 14/genotype) or air (n = 14/genotype), followed by a two-chamber test (TCT) in which a chamber was filled with CO₂ and the other one with air or both chambers were filled with air. Then CO₂-induced plasma corticosterone levels were determined. (b) Based on the results of these experiments, cardio-respiration was assessed in a new cohort of male WT and 5-HTT +/– mice. HPA: hypothalamo-pituitary-adrenal.

Figure 2.

Assessment of the behavioral performance in the modified open field test. (a) Under air exposure serotonin transporter (5-HTT) –/– mice covered less distance than mice of other genotypes. Exposure to carbon dioxide (CO₂) significantly reduced the total distance moved in all genotypes. (b) During air exposure 5-HTT –/– mice spent less time in the center zone in comparison with wildtype (WT) mice. When exposed to CO₂, only WT mice spent less time in the center compared to air exposure, no difference was found in 5-HTT +/– and 5-HTT –/– mice. (c) In all genotypes, CO₂ exposure resulted in a robust freezing response compared to air exposure. Bars represent mean + standard error of the mean (SEM). +/–: heterozygous 5-HTT knockout mice; –/–: homozygous 5-HTT knockout mice. *p < 0.05, ***p < 0.001; same letters indicate a group difference of p < 0.05.

Figure 3.

Assessment of the behavioral performance in the two-chamber test. (a) The total distance moved was strongly reduced under carbon dioxide (CO₂) exposure in all genotypes. Serotonin transporter (5-HTT) –/– mice covered less distance than wildtype (WT) and 5-HTT +/– mice during air exposure. (b) Under CO₂ exposure the number of crossings was significantly lower than during air exposure. (c) When exposed to air, no genotype differences were found. When exposed to CO₂, a marked freezing response was observed only in WT and 5-HTT +/– mice compared to air-exposure. 5-HTT –/– mice froze less to CO₂ than WT mice. (d) The ratio of time spent and freezing per chamber indicated that WT and 5-HTT +/– mice froze longer in the chamber filled with CO₂ than 5-HTT –/– mice. No effect was found within the chamber filled with air. Bars indicate mean + standard error of the mean (SEM). +/–: heterozygous 5-HTT knockout mice; –/–: homozygous 5-HTT knockout mice. ***p < 0.001; a, b, d same letters indicate a group difference of p < 0.05; c, same letters indicate a group difference of 0.05.

Figure 4.

Plasma corticosterone secretion before and after exposure to 10% carbon dioxide (CO₂) for 20 min. CO₂ exposure strongly increased corticosterone levels in mice of all genotypes (exposure effect p = 0.002). Bars represent mean + standard error of the mean (SEM). +/–: heterozygous 5-HTT knockout mice; –/–: homozygous 5-HTT knockout mice; 5-HTT: serotonin transporter; WT: wildtype mice.

Figure 5.

Respiratory and cardiovascular monitoring during exposure to air and carbon dioxide (CO₂). (a) Schematic representation of a 2 s epoch of pressure-induced changes to assess breathing frequency and tidal volume (amplitude) during inhaling air (top) and CO₂ (bottom). CO₂ strongly increased breathing frequency in comparison with air exposure. (b) Heart rate decreased during inhaling CO₂. No genotype effect was present in any outcome measurement. Bars represent mean + standard error of the mean (SEM). +/–: heterozygous 5-HTT knockout mice; 5-HTT: serotonin transporter; WT: wildtype mice. ***p < 0.001.