Zebrafish mecp2 null-mutation increases anxiety and cortisol levels but no change in adult social preference and larval chemically-induced hyperlocomotion

Abstract

Background: Methyl CpG binding protein 2 (MECP2) is an essential global modulator of transcription and mutations in MECP2 are the most common cause of Rett syndrome, an X-linked neurodevelopmental disorder. Patients diagnosed with Rett syndrome have increased risk for epilepsy as well as problems with anxiety and social communication. Using the zebrafish mecp2^Q63X line, this study aimed to increase our understanding of the role of Mecp2 function in regulation of pharmacologically-induced hyperlocomotion, developmental social preference, and adult socialization, anxiety-related behaviour, and baseline cortisol levels. To determine responses of mecp2^-/- zebrafish to a stimulating convulsant, general locomotor activity was measured at 5 days post-fertilization (dpf) in sibling mecp2^+/+, mecp2^+/-, and mecp2^-/- fish after treatment with a GABA_A receptor antagonist pentylenetetrazol (PTZ) at varying concentrations. Responses to social stimulus were investigated in juvenile (21 dpf) and adult mecp2^-/- and mecp2^+/+ fish. Anxiety responses to a novel tank and whole-body cortisol levels were also measured in adult mecp2^-/- and control mecp2^+/+ zebrafish.

Results: The behavioural tests showed that mecp2^-/- zebrafish displayed hypolocomotion at the larval stage, along with increased freezing time and thigmotaxis, and higher whole-body cortisol levels in adulthood. However, the hyper-locomotion response to PTZ at 5 dpf and social preference for visual social stimulus at 21 dpf and in adulthood were not affected by the lack of functional Mecp2.

Conclusions: Functional Mecp2 modulated larval locomotion and behavioural anxiety at different ages and adult cortisol levels, but mecp2 null-mutation did not alter adult locomotion and socialization, and developmental sociability and PTZ-induced hyperlocomotion in zebrafish. Given the variability reported in patients and in rodent Mecp2 knockout models, studies using zebrafish can explore vital elements of MECP2's role across development and improve our understanding of neural mechanisms underlying neurodevelopmental disorders.

Keywords: Anxiety; Cortisol; Locomotion; Pentylenetetrazol; Rett syndrome model; Seizure; Social behaviour.

Fig. 1

Schematic representation of the experimental protocol at 5 days post-fertilization (dpf), 21 dpf, and 6-months old adult fish. Experiments done at the three ages are separated and denoted with purple outline. Blue arrows show timeline of procedures while orange dotted lines represent virtual division of testing arenas into relevant areas. For 5 dpf larval fish (a), general locomotion was observed during a baseline period, followed by PTZ-induced hyperlocomotion. Representative examples of behavioural traces are provided to illustrate typical locomotion (wells A1 and B2), immobility (well B1), and hyperlocomotion (well A2). For 21 dpf fish (b), three traces illustrate preference (top), indifference (middle), and aversion (bottom) to social stimuli. The dotted orange lines mark the social and non-social zone used for calculation of social preference index [SPI]. For adult fish, the dotted orange lines indicate the line between border and center during (c) baseline open field testing, five zones during social preference testing (d) with zone 1 being closest to the shoal [social zone] and zone 5 being the furthest away from the shoal during social behavioural testing, and the division of water column into vertical top, middle and bottom areas (e) during novel tank diving test. Representative traces below show bottom-dwelling vs. exploration in higher water column in the open-field, no preference vs. social preference during the social behaviour test, and exploration of bottom only, middle, and top areas of the novel tank. After the behavioural testing, all fish were (f) fin-clipped under anaesthesia for genotyping. Separate group of experimentally naïve adult fish were used to measure whole-body cortisol levels (g) using enzyme immunoassay

Fig. 2

Larval locomotion behaviour was similarly affected in mecp2^−/− fish bred from homozygous parents (top panel) and heterozygous parents (bottom panel). a Baseline locomotion prior to PTZ-treatment for 5 dpf larvae from homozygous breeding showed that mecp2^−/− (n = 117) larvae moved significantly shorter distances than mecp2^+/+ (n = 203). b Distances moved by mecp2^+/+ (blue; n = 33–35) and mecp2^−/− (red; n = 19–20) fish from homozygous parents were similar during baseline (20 min before; lighter colours) and showed similar increases during PTZ-treatment (20 min, darker colours). c Baseline locomotion prior to PTZ-treatment for 5 dpf larvae from heterozygous mecp2^+/− crosses showed that mecp2^−/− (n = 68) larvae moved significantly shorter distances than mecp2^+/+ (n = 90). d The distances moved were similar during baseline (20 min before; lighter colours) and the hyperlocomotion during PTZ-treatment (20 min, darker colours) was also similar in 5 dpf mecp2^+/+, mecp2^+/−, and mecp2^+/+ fish derived from heterozygous breeding. Sample sizes were mecp2^+/+ (blue; n = 41–65), mecp2^+/− (purple; n = 86–90) and mecp2^−/− (red; n = 35–37). Significant effects of PTZ-treatment within genotypes are indicated in grey. ****p < 0.0001 and *p < 0.05. Mean values shown as dotted lines and brackets mark SEM (a and c) and bars correspond to mean ± SEM (b and d)

Fig. 3

Social preference index, time spent in the social zone, and total distance moved by 21 dpf zebrafish during baseline and social stimulus phases. Social preference (a) was not altered in mecp2 (n = 77) or mecp2^−/− (n = 37) larvae compared to mecp2^+/+ (n = 23) zebrafish. Additionally, sibling zebrafish of all three genotypes spent similar length of time in the social zone (b) and swam similar distances (c) during the baseline and stimulus phases of the experiment. ****p < 0.0001 ***p < 0.001 **p < 0.01 and *p < 0.05. Mean ± SEM

Fig. 4

Anxiety-related behaviours and whole-body cortisol levels were altered in adult mecp2^−/− fish while locomotion and social preference were unaffected. Locomotion in the open-field (a) was not affected but adult mecp2^−/− fish spend significantly less time in the (b) centre zone in the open field compared to the mecp2^+/+ group and (c) male mecp2^−/− fish showed somewhat less time in the periphery, compared to female mecp2^−/− fish. During the presentation of social stimuli, time spent in the social zone (d) was not significantly different in adult mecp2^−/− (n = 43) as compared to mecp2^+/+ (n = 22). While mecp2^−/− fish generally trended towards more time closer to social stimuli than mecp2^+/+ fish across the 15 min (e) the SPI was not different between the two groups of fish (f). In a novel tank diving task, the mecp2^−/− (n = 22) zebrafish made less bottom-to-middle transitions (g) and spent less time in high-mobility state (h) and more time in the immobile state (i) as compared to mecp2^+/+ (n = 10) indicating an increase in bottom-dwelling and freezing behaviours, but overall time spent in bottom, middle, and top areas (j) was not different between the two genotypes. Baseline cortisol levels (k) were higher in experimentally naïve mecp2^−/− (n = 6) fish compared to mecp2^+/+ (n = 6) zebrafish. *p < 0.05 and ****p < 0.0001. Mean ± SEM