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General and Comparative Endocrinology
journal homepage:www.elsevier.com/locate/ygcenProfound e
ffects of glucocorticoid resistance on anxiety-related behavior in
zebra
fish adults but not in larvae
Jenni Sireeni, Nina Bakker, Gayathri Jaikumar, Daisy Obdam, Hans Slabbekoorn,
Christian Tudorache, Marcel Schaaf
⁎Institute of Biology, Leiden University, Leiden, The Netherlands
A R T I C L E I N F O Keywords: Glucocorticoids Glucocorticoid receptor Zebrafish Behavior Anxiety Depression A B S T R A C T
Previously, adult zebrafish with a mutation in the gene encoding the glucocorticoid receptor (Gr) were de-monstrated to display anxiety- and depression-like behavior that could be reversed by treatment with anti-depressant drugs, suggesting that this model system could be applied to study novel therapeutic strategies against depression. Subsequent studies with zebrafish larvae from this grs357line and a different gr mutant have
not confirmed these effects. To investigate this discrepancy, we have analyzed the anxiety-like behavior in 5 dpf grs357larvae using a dark/tapping stimulus test and a light/dark preference test. In addition, grs357adultfish were subjected to an openfield test. The results showed that in larvae the mutation mainly affected general locomotor activity (decreased velocity in the dark/tapping stimulus test, increased velocity in the light/dark preference test). However, parameters considered specific readouts for anxiety-like behavior (response to dark/ tapping stimulus, time spent in dark zone) were not altered by the mutation. In adults, the mutants displayed a profound increase in anxiety-like behavior (time spent in outer zone in openfield test), besides changes in locomotor activity (decreased velocity, increased angular velocity and freezing time). We conclude that the neuronal circuitry involved in anxiety- and depression-like behavior is largely affected by deficient Gr signaling in adultfish but not in larvae, indicating that this circuitry only fully develops after the larval stages in zebrafish. This makes the zebrafish an interesting model to study the ontology of anxiety- and depression-related pathology which results from deficient glucocorticoid signaling.
1. Introduction
Glucocorticoids are steroid hormones that are secreted by our adrenal gland. Basal circulating levels of cortisol, the main endogenous glucocorticoid in humans,fluctuate in a diurnal rhythm, and these le-vels strongly increase upon perception of a stressful stimulus (Russell and Lightman, 2019). The secretion of cortisol is controlled by the hypothalamus-pituitaryadrenal (HPA) axis (Smith and Vale, 2006). The hypothalamus produces the neuropeptide CRH which induces the pi-tuitary to secrete the peptide hormone ACTH into the bloodstream which stimulates the adrenal gland to secrete cortisol. Besides mod-ulating a variety of physiological processes throughout our body, cor-tisol also exerts negative feedback on the activity of the HPA axis by interfering with the production of CRH and ACTH, thereby limiting its own secretion.
Steroid hormone function in the body is mediated by intracellular steroid receptors. The effects of cortisol are mediated by two receptors (Reul and de Kloet, 1985; de Kloet, 2014), the mineralocorticoid
receptor (MR) and the glucocorticoid receptor (GR). MR has a ten-fold higher affinity for cortisol than GR (Reul and de Kloet, 1985), and as a result of its high affinity, MR is tonically activated under basal condi-tions. GR gets activated when circulating cortisol levels increase, as a result of stress or at the diurnal peak (Reul and de Kloet, 1985). Cortisol exerts large effects on mood and behavior through activation of MRs and GRs that are expressed in various regions of the brain (Muller et al., 2002).
Like all steroid receptors, GR is a nuclear receptor that acts as transcription factor upon ligand binding (Meijer et al., 2019). It binds to specific glucocorticoid response elements in the genome, and upon in-teraction with transcriptional cofactors, it regulates gene transcription in either a positive or a negative way. Additionally, GR interacts with other transcription factors and can thereby modulate the activity of these factors, again both positively and negatively. In recent years it has become clear that GR also acts in a more rapid, so-called nongenomic way (Groeneweg et al., 2012). The effects of GR deficiency or over-expression has been studied in a variety of transgenic and knockout
https://doi.org/10.1016/j.ygcen.2020.113461
Received 17 September 2019; Received in revised form 26 January 2020; Accepted 10 March 2020
⁎Corresponding author.
E-mail address:m.j.m.schaaf@biology.leidenuniv.nl(M. Schaaf).
General and Comparative Endocrinology 292 (2020) 113461
Available online 17 March 2020
0016-6480/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
mouse models with altered GR expression patterns.
The genetic studies into GR function in mice have revealed impact on several behavioral patterns, such as locomotion activity, anxiety-and depression-like behavior, anxiety-and learning anxiety-and memory performance (reviewed in (Kolber et al., 2008)). In addition, several lines of evidence suggest a link between glucocorticoid signaling and the pathogenesis of depression in humans (Holsboer, 2000; Pariante and Miller, 2001; Menke, 2019). First, hypercortisolemia, resulting from resistance to glucocorticoid feedback in the HPA axis, is a hallmark of major de-pression. Second, treatment of patients suffering from psychotic de-pression with a GR antagonist has been shown to reduce the psychotic symptoms (Schatzberg, 2015). Third, in preclinical studies CRH was demonstrated to induce depression-like symptoms in humans (Holsboer, 1999). Fourth, polymorphisms in the human gene encoding GR have been shown to be associated with several psychiatric diseases, the most consistent being an association with increased susceptibility to (psychotic) major depression (Koper et al., 2014; Schatzberg et al., 2014).
In recent years, the zebrafish (Danio rerio) has emerged as a novel animal model to study the role of glucocorticoid signaling in vertebrate behavior. Infish, cortisol is the main endogenous glucocorticoid hor-mone, whereas the mineralocorticoid hormone aldosterone is absent. Similar to the mammalian stress response, the zebrafish cortisol secre-tion has been shown to be controlled by the hypothalamus-pituitary-interrenal (HPI) axis (Alsop and Vijayan, 2009a; Alsop and Vijayan, 2009b), and the effects of cortisol are mediated by the zebrafish Mr and Gr, which have been cloned and characterized (Schaaf et al., 2008; Pippal et al., 2011). A zebrafish line has been identified (grs357) with a
Gr that has a mutation in the DNA-binding domain which make it transcriptionally inactive (Muto et al., 2013; Ziv et al., 2013). Thesefish can be studied during larval stages and they also survive into adult-hood, which is a big advantage of the zebrafish model, because GR knockout mice die within a few hours after birth (Cole et al., 1995).
The possibility to study Gr function in zebrafish larvae and adults has yielded some unexpected results. Adult mutantfish were shown to have increased basal cortisol levels as a result of decreased feedback on the HPI axis, which was associated with depression-like behavior (Ziv et al., 2013). The depression-like phenotype was alleviated upon treatment with the anxiolytic drug diazepam and the antidepressant fluoxetine (Ziv et al., 2013). Subsequently, similar studies were per-formed in zebrafish larvae, since their small size and translucency provide interesting possibilities for high-throughput and imaging stu-dies. In 5 dpf larvae of the grs357 mutant line decreased spontaneous
activity (again alleviated by fluoxetine) and an increased startle re-sponse were observed (Griffiths et al., 2012). In contrast, in a more recent study 4 dpf larvae from a different gr knockout line (CRISPR-Cas9-generated, containing a severely truncated receptor) showed no alterations in behavior (Faught and Vijayan, 2018). This is surprising since in a similar CRISPR-Cas9-generated gr knockout line the tran-scriptional response to Gr activation was more strongly affected than in the grs357 mutant (Facchinello et al., 2017), which suggests that the
grs357mutant has some residual transcriptional activity, which does not
involve DNA-binding but rather interaction with other transcription factors.
In the present study, we have further explored the discrepancies in the literature to gain insight into the behavioral effects of Gr deficiency during different life stages. We have analyzed the behavior of the zebrafish grs357mutant in 5 dpf larvae (using a light/dark preference
test and a dark/tapping stimulus test) and in adults (using a novel en-vironment test). Our results show that the observed behavioral changes due to the mutation in larvae were minor and restricted to general lo-comotion, whereas profound anxiogenic effects of the mutation were found in adult fish. These results indicate that the main anxiogenic behavioral changes resulting from deficient Gr signaling mostly develop after the larval stages.
2. Material and methods 2.1. Animals
Zebrafish were maintained and handled according to the guidelines from the Zebrafish Model Organism Database (http://zfin.org) and all experimental procedures were approved by the animal welfare com-mittee of Leiden University (DEC-approval # 11023). Fish homozygous for the grs357mutation and wild type controls (referred to as gr+) were
provided by Dr. H. Baier (Max Planck Institute of Neurobiology, Martinsried, Germany (Muto et al., 2013; Ziv et al., 2013)), and reared in ourfish facility at 28 °C in a 14–10 h light–dark cycle. Fish were fed twice a day, with dry food (Dupla Rin M, Gelsdorf, Germany) and frozen artemias (Dutch Select Food, Aquadistri BV, Klundert, The Netherlands). Fertilization was performed by natural spawning at the beginning of the light period. Eggs were collected and transferred to Petri dishesfilled with egg water (60 μg/ml ‘Instant Ocean’ sea salts (Blacksburg, VA, USA) and 0.0005% methylene blue). Dead and unvi-able eggs were removed from the plates and water was refreshed every day. Larvae were used for experiments at 5 dpf.
2.2. Dark/tapping stimulus test for larvae
The dark/tapping stimulus test was adapted from a test previously described bySchnorr et al. (2012). Larvae (5 dpf) were put into 24 well plates, one larva per well. The plate was placed into the DanioVision Observation Chamber (Noldus Information Technology, Wageningen, The Netherlands),fitted with infrared backlight, adjustable white light, and an infrared-sensitive digital camera. Recording began after 10 min of acclimatization. The wells (Ø 15.4 mm) were defined into inner zone (central part of the well, Ø 7.4 mm) and outer zone (remaining area around the inner zone). The dark/tapping stimulus test consisted of four phases. Thefirst phase was named the Basal phase, during which the light in the apparatus was on. The Basal phase lasted for 10 min, and it was followed by the Dark phase of 4 min, during which the light was turned off. After the Dark phase, a Recovery phase followed, which lasted for 10 min and during which the light was on. The Recovery phase was followed by a Tapping stimulus for 5 s, during which a built-in hammer tapped the setup built-in a consistent way, providbuilt-ing a vibration stimulus. The final phase was a Recovery phase, which lasted for 10 min with the light on. The video recordings were analysed with Ethovision XT 12 (Noldus Information Technology, Wageningen, The Netherlands). The swimming kinematic parameters Mean velocity (in mm/s) and Maximal velocity (mm/s) were measured (both over 1 min periods), and these parameters are considered measures for anxiety-like behavior, especially during the challenge phases (Schnorr et al., 2012). In addition, we measured the Time spent in the outer zone (as % of total time), as a measure for thigmotaxis or‘wall-hugging’, considered to represent anxiety-related behavior (Schnorr et al., 2012). A total of seven plates were tested, spread over three experimental days (so three different batches of eggs were used). Each of the plates had an equal number of the gr+and the grs357larvae, thus yielding a total N = 168
(n = 84 per genotype).
In an additional experiment the effect of cortisol was tested. Two hours before the assay, a vehicle or cortisol solution (25μM end con-centration (Sigma-Aldrich, Zwijndrecht, The Netherlands)) was ad-ministered to gr+larvae. The assay was performed as described above, but consisted of only a Basal, Dark and Dark recovery phase. A total of eight plates were tested, spread over two experimental days. Each of the plates had an equal number of vehicle- and the cortisol-treated larvae, thus yielding a total N = 192 (n = 96 per genotype).
2.3. Light/dark preference test for larvae
consisting of four small separate containers (each L42 × W26 mm), of which the walls of one half were clear (light zone) and of the other half black (dark zone). Each small container wasfilled with 5 ml egg water. A removable divider was placed between the light and the dark zone and larvae were transferred to the light zones, one per container. Each plate had 2 gr+and 2 grs357 larvae, spread randomly over the four
containers. The plate was placed in the Daniovision Observation Chamber. After around 2 min of acclimatization, the divider was re-moved and the chamber door was closed and recording was started. Fish were recorded for 15 min, and the video recordings were analysed with Ethovision XT 11 (Noldus Information Technology, Wageningen, The Netherlands). Parameters measured were Mean velocity (in mm/s, for light and dark zone, as a measure for locomotor activity), Distance moved (in cm, for light and dark zone, as a measure for locomotor activity, not corrected for time spent in different zones), and Time spent per zone (as % of the total time, as a measure for zone avoidance/ preference (Steenbergen et al., 2011a)). A total of 18 assays was per-formed, spread over two experimental days (N = 72, per genotype n = 36).
2.4. Openfield test for adult fish
For the openfield test (adapted from a test previously described by
Champagne et al. (2010)), 3 rows of 8 plexiglass tanks
(33 × 15 × 13 cm), visually separated by white walls, were placed on a table surface (1.0 × 1.0 m). A circular plastic ring (Ø 12 cm, height 15 cm) was placed inside each tank. All rings were connected by a system of bars from above. Underneath, a retro-reflecting tape surface (1.2 × 1.2 m, Scotchlite 3 M, St Paul, MN, USA) reflected infrared light from three illuminators (U48R Univivi, Shenzhen, China), placed around an infraredfirewire camera (Dragonfly, Point Grey Research Inc., Richmond, Canada), which was situated 1.8 m above the table surface. Adult gr+and grs357fish (N = 24, per genotype n = 12) were randomly placed inside the rings inside the tanks (containing 3 l water (oxygen concentration 8 mg/l)). They were left overnight in an accli-mated room (28 °C). The next morning, the rings were removed, by lifting the bar system, exposing allfish simultaneously to a novel area, i.e. the actual plexiglass tank. Behavior was recorded at a speed of 15 frames per second (fps), starting 10 s after removal of the ring, and for 70 min. Video recordings were analysed by EthoVision XT 11, by tracking the centre of mass of the individualfish over time. The fol-lowing parameters were measured: Mean velocity (in mm/s, as a measure for locomotor activity); Angular velocity (in °/s, i.e. the angle of locomotion directionality over consecutive frames, divided by the time lag between subsequent frames), as a measure for erratic move-ment, considered to represent anxiety-like behavior (Steenbergen et al., 2011a)); Freezing time (in s, duration of freezing bouts, i.e. time per-iods with swimming velocities below 0.1 mm/s), also considered a measure for anxiety-like behavior (Steenbergen et al., 2011a); Time spent in the outer zone (as % of the total active swimming period (with swimming speeds > 0.1 mm/s) spent in the zone less than 40 mm (~1 body length) from the edge of the tank), as a measure for thigmotaxis.
2.5. Statistical analysis
All data shown are means ± standard error of the mean (s.e.m.). Data shown inFig. 1C, E, G,2B, C, and D were analyzed by two-way ANOVA, and Tukey’s test was used for post-hoc comparisons. Data shown in Fig. 3B, C, D and E were analyzed using unpaired t-tests. Statistical significance was accepted at p < 0.05. Statistical analysis was performed using GraphPad prism software (version 7.00).
3. Results
3.1. Dark/tapping stimulus test for larvae
To study differences in the behavior between wild type (gr+
) and gr mutant (grs357) zebrafish, we first subjected the gr+and grs357larvae to
the dark/tapping stimulus test (Fig. 1A). In this test, larvae were placed in 24 well plates and their behavior was recorded. After 10 min they were exposed to a 4 min dark stimulus followed by a recovery period of 10 min after which the larvae were subjected to a tapping stimulus. During all test periods (Basal, Dark, Dark recovery, Tapping, Tapping recovery), the velocities of the larvae were measured, averaged per minute, and the average values of all larvae are shown inFig. 1B. Ad-ditionally, over each time period the average values were determined, and these values are shown in Fig. 1C. Although two-way ANOVA showed that there was a significant effect of the mutation on these values, post hoc comparisons did not reveal any differences between the gr+and grs357larvae during any phase of the assay. We also determined the maximum velocities of the larvae per minute (Fig. 1D) and for each time period (Fig. 1E). Two-way ANOVA of the maximum values per time period showed a significant effect of the mutation, and post hoc comparisons revealed that the grs357 had a lower maximal velocity
during the Basal period (81.0 ± 11.2 versus 131.6 ± 12.2 mm/s). Furthermore, the relative time spent in the outer zone of the wells was determined per minute (Fig. 1F) and for each time period (Fig. 1G). No effect of the mutation was observed upon two-way ANOVA of the data per time period. However, post hoc comparisons showed a significantly lower percentage of time spent in the outer zone for the grs357larvae
during the Basal period (86.0 ± 3.0 versus 92.2 ± 1.6%). In sum-mary, no large differences were found between the gr+and the grs357
larvae in the dark/tapping stimulus test. The small differences that were found were all observed during the Basal period.
To investigate if cortisol has any effect on the behavior of the larvae in this assay (and check if the high cortisol levels of the grs357larvae (Griffiths) affected their behavior), we administered a high dose of cortisol (25μM) to gr+larvae and subjected them to the dark stimulus
test (Suppl. Fig. 1). The results showed no differences in behavior be-tween vehicle- and cortisol-treated larvae, indicating that increased cortisol levels do not affect the behavior of the larvae in this assay.
3.2. Light/dark preference test for larvae
Second, 5 dpf larvae were tested in the light/dark preference test (Fig. 2A). In this test, larvae were placed in a small container of which the walls of one half were clear (light zone) and of the other half black (dark zone), and their behavior was recorded for 15 min. The swim-ming velocities of the larvae were measured, both in the light and the dark zone (Fig. 2B). Velocities were similar in the light and the dark zone, but the grs357larvae showed higher velocities that the gr+larvae, both in the light zone (5.5 ± 0.2 mm/s versus 4.4 ± 0.2 mm/s) and in the dark zone (5.9 ± 0.3 mm/s versus 4.8 ± 0.2 mm/s). As a result of the higher velocities of the grs357 larvae, the total distances they moved were larger (Fig. 2C), both in the light zone (263.3 ± 11.6 cm versus 220.6 ± 9.0 cm) and in the dark zone (234.4 ± 14.2 cm versus 179.3 ± 9.0 cm). Finally, the times each larva spent in the light and dark zones were measured and the average times (n = 36) are shown in Fig. 2D. The results show that both the gr+and the grs357larvae had a
preference for the light zone and that the percentages of time spent in this zone were similar (57.4 ± 1.6% and 55.2 ± 1.7% for gr+and grs357respectively). In summary, in the light/dark preference test no
3.3. Openfield test for adult fish
Third, the behavior of adult gr+and grs357fish was investigated in an openfield test (Fig. 3A). In this test, thefish were placed in a ring inside a tank. The following day, the ring was removed, exposing the
fish to a novel area, and their behavior was recorded for 70 min. Three parameters were measured that described the swimming pattern of the fish: the mean velocity (Fig. 3B), angular velocity (i.e. the angle of lo-comotion directionality,Fig. 3C), and the freezing time (i.e. time per-iods with velocities below 0.1 mm/s, Fig. 3D). All three parameters
Fig. 1. Behavior of 5 dpf gr+and grs357larvae in
showed a difference between the gr+
and grs357 fish. The swimming velocity of the grs357 fish was significantly lower (2.2 ± 0.2 mm/s
versus 2.8 ± 0.1 mm/s), their angular velocity was significantly higher (143.0 ± 9.7°/s versus 87.3 ± 7.6°/s), and their freezing time was higher (1585.0 ± 272.3 s versus 235.0 ± 141.0 s). Finally, the times spent in the outer zone of the tank was determined (Fig. 3E), which showed that the grs357fish spent significantly more time in the outer zone (54.0 ± 3.2% versus 34.9 ± 2.3%). Taken together, these data demonstrate large behavioral differences between the adult gr+
and grs357 fish. The grs357 fish displayed a slower, more irregular swimming pattern and spent more time in the outer zone of the tank than the gr+fish.
4. Discussion
In the present study we have performed a behavioral analysis of the grs357mutant zebrafish line. We have used a light/dark preference test, a dark/tapping challenge test for larvae and a novel environment test for adultfish. The results showed profound differences between wild type and mutantfish in the adults, but differences in the larvae ap-peared to be smaller and limited to general locomotion.
4.1. Minor role for Gr in anxiety-like behavior in larvae
In the dark/tapping challenge test in larvae, we found that the mutation decreased the locomotor activity under basal conditions and had no effect on the response to either the dark or the tapping chal-lenge. The observed decrease in mobility under basal conditions is in line with the results ofGriffiths et al. (2012), who found a lower mo-bility of the grs357larvae (also at 5 dpf) in a comparable setup under
basal conditions over a 24 h period, and could abolish this effect by treating the larvae with the antidepressant fluoxetine. In contrast, Faught and Vijayan (2018)did not observe any change during the basal or recovery phases using larvae of a CRISPR-Cas9 generated gr knockout line. The discrepancy with their results may result from the different age of the larvae (4 versus 5 dpf), or the different genetic background and/or mutation of their gr mutant line. Griffiths et al. (2012)demonstrated an increased response of 5 dpf grs357larvae after a tapping stimulus, which in their study was a repeated stimulus. This makes the studies hard to compare, since the increased response in their study was mainly an effect of reduced habituation to the challenge in the mutant larvae. The absence of any difference during the challenge or recovery phases observed in our study suggests that the mutation does not affect anxiety-related behavior in the larvae.
To further study the behavior in the grs357larvae, we used the light/ dark preference test. The wild type and mutant larvae displayed a si-milar preference for the light zone, indicating no difference in anxiety-related behavior. Similar to the results from the dark/tapping challenge test, in this assay the mutation affected just basal locomotion. Independent of the zone in which they were present, the swimming velocity of the mutant larvae was higher than that of the wild types.
Fig. 2. Behavior of 5 dpf gr+and grs357larvae in the light/dark preference test.
This effect of the mutation was opposite from the effect shown in the dark/tapping test. However, the observed swimming velocities in the light/dark preference test were more than two-fold higher than those seen in the dark/tapping challenge test, which demonstrates that the conditions of these two tests are very different. Apparently, the differ-ence in locomotor activity between gr+ larvae and grs357 larvae is context-dependent.
Taken together, our results from the behavioral assays performed using 5 dpf larvae show that the mutation does not affect anxiety-re-lated behavior, and that observed differences in locomotion between wild type and mutant larvae are dependent on the context of the be-havioral assay. It could be argued that the small effects of Gr mutation observed in this study are due to an inactivity of Gr signaling at this stage of development. Indeed, in early development zebrafish show no or relatively small increases in cortisol levels upon mild stressors like swirling (Alsop and Vijayan, 2008; Steenbergen et al., 2011b), and it has been suggested that zebrafish experience a stress hyporesponsive period during thefirst days of development (Steenbergen et al., 2011b). However, gr expression levels have been shown to be relatively stable after hatching (Alsop and Vijayan, 2008; Schaaf et al., 2008), and en-hanced transcriptional activity has been observed upon exposure to exogenous glucocorticoids like dexamethasone and cortisol at 5 dpf, e.g. in reporterfish lines (Weger et al., 2012; Benato et al., 2014). In the present study we have exposed larvae to a high dose of cortisol to
activate Gr, and found no effect on larval behavior. These results sup-port the conclusion that Gr signaling in larval zebrafish does not affect anxiety-like behavior.
4.2. Major role for gr in anxiety-like behavior in adults
In contrast, when adultfish were investigated in an open field test, a mildly anxiogenic stimulus, a profound effect on thigmotaxis was found, with the mutants spending considerably more time in the outer zone of their tank (~54%) than the wild types (~35%). In line with this more anxiety-like behavior in the mutants, an approximately six-fold higher freezing time was found for the mutants compared to the wild types. In addition, the mutation altered the locomotion pattern of the fish: the grs357fish showed decreased mean velocities and higher
an-gular velocities. Together, these results demonstrate more anxiety-like behavior in the mutants. The different behavioral responses of the mutantfish are not necessarily an effect of the lack of Gr signaling, but may also be due to an increased Mr signaling as a result of the higher circulating cortisol concentrations in the mutantfish (Ziv et al., 2013). The increased freezing behavior of the grs357 adult fish has
pre-viously been observed in a comparable test (exposure to a novel tank) byZiv et al. (2013). It should be noted however that in the latter study the differences in freezing behavior (and thigmotaxis) were mainly observed after repeated exposure to the stressful stimulus, similar to the
Fig. 3. Behavior of adult gr+and grs357fish
larval study ofGriffiths et al. (2012). The authors therefore interpreted these experience-dependent effects as related to learned helplessness or a depression-like phenotype, rather than anxiety (Ziv et al., 2013). The effects on freezing behavior could be reversed by social interactions, acute treatment with both an anxiolytic drug (the GABA antagonist diazepam), and long-term treatment with an antidepressant (the se-lective serotonin reuptake inhibitor fluoxetine). Since the zebrafish grs357mutant mimics the association between glucocorticoid resistance and symptoms of depression observed in humans, it could serve as an interesting model for neuropsychiatric research, e.g. in screening for novel antidepressant drugs.
4.3. Discrepancies between zebrafish and mouse studies
Our data on increased anxiety in Gr deficient zebrafish are in sharp contrast withfindings from comparable studies in mice. In a variety of transgenic and knockout mouse models the GR gene has been targeted and the behavior of these mice has been studied extensively (Kolber et al., 2008). Conventional heterozygote knockouts have been used (Oitzl et al., 1997; Ridder et al., 2005), as well as mice with a point mutation in the DNA binding domain (Oitzl et al., 2001), specific de-letion of GR in all neuronal and glial cells of the CNS (Tronche et al., 1999), forebrain-specific GR disruption (Boyle et al., 2005; Boyle et al., 2006), antisense knockdown preferentially in the nervous system (Steckler and Holsboer, 1999), and general and brain-specific over-expression of GR (Wei et al., 2004; Ridder et al., 2005). Several of these mouse models have been subjected to an openfield test, and in none of these studies an effect of the altered GR expression on anxiety-like behavior was observed (Oitzl et al., 1997; Tronche et al., 1999; Oitzl et al., 2001; Wei et al., 2004; Boyle et al., 2005; Ridder et al., 2005; Boyle et al., 2006). In addition, decreased GR expression resulted in increased locomotor activity in the openfield test in two studies (Oitzl et al., 1997; Steckler and Holsboer, 1999). In other tests for anxiety-like behavior (elevated plus maze, elevated zero maze and light/dark pre-ference test) mostly decreased anxiety was observed in response to disruption of the GR expression (Steckler and Holsboer, 1999; Tronche et al., 1999; Boyle et al., 2005; Boyle et al., 2006) and increased anxiety upon overexpression (Wei et al., 2004). Apparently, it is difficult to translate results from behavioral studies between zebrafish and mice, either due to the differences in behavioral testing (Champagne et al., 2010), or due to context-specific and species-specific effects of the al-teration in GR expression.
In mice, tests for depression- or despair-like behavior (e.g. forced swim test), performed to study the effect of GR deficiency or over-expression, show inconsistent results. Reduced GR activity in mice was shown to induce no effect (Ridder et al., 2005), decreased despair (Montkowski et al., 1995; Tronche et al., 1999; Ridder et al., 2005), or increased despair (Boyle et al., 2005; Ridder et al., 2005; Boyle et al., 2006), whereas GR overexpression in the forebrain resulted in in-creased despair (Wei et al., 2004). These inconsistencies may be due to the fact that increased and decreased GR activity have similar effects, to artefacts of ectopic overexpression, or to the fact that this type of be-havior is also affected by altered locomotion, appraisal of stressful si-tuations, and memory formation. These inconsistencies make it difficult to compare the increase in depression-like behavior in grs357zebrafish,
in which the Gr is knocked out ubiquitously (Ziv et al., 2013), to the effects observed in mice. Future studies will indicate whether the link between GR deficiency and depression-like behavior is more consistent in zebrafish.
5. Conclusions
The results of the present study show profound effects of gr mutation on anxiety-like behavior in adult zebrafish, whereas in 5 dpf larvae only minor effects on locomotor activity were found, which were dependent on the context of the different tests. Apparently, behavioral changes
resulting from resistance to glucocorticoid signaling develop after the larval stages in zebrafish. This may be due to the relatively late ma-turation of HPI axis reactivity to stress (Alsop and Vijayan, 2009b) and/ or the development of the neuronal circuitry involved in anxiety- and depression-like behavior that is sensitive to deficient Gr signaling. We therefore conclude that studies on the link between Gr signaling and anxiety- or depression-like behavior in zebrafish should be performed in adultfish, but that zebrafish larvae could serve as an interesting model to study the ontology of anxiety- and depression-like behavior resulting from deficiencies in glucocorticoid signaling.
CRediT authorship contribution statement
Jenni Sireeni: Formal analysis, Investigation. Nina Bakker: Formal analysis, Investigation. Gayathri Jaikumar: Formal analysis, Investigation. Daisy Obdam: Investigation. Hans Slabbekoorn: Conceptualization, Writing - review & editing.Christian Tudorache: Conceptualization, Methodology, Formal analysis, Writing - review & editing.Marcel Schaaf: Conceptualization, Methodology, Formal ana-lysis, Writing - original draft, Visualization.
Acknowledgement
The authors would like to thank Sebastian Rock for assistance with the analysis of the behavioral data.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps:// doi.org/10.1016/j.ygcen.2020.113461.
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