Studies in mammals showed that the early social environment persistently affects behaviour, and that this is associated with a changed expression of genes implicated in the HPA axis. We hypothesized that similar joint effects on behaviour and gene expression may exist in fish. More specifically, we predicted that expression of the neuropeptide CRF and of the steroid hormone receptor GR1 would be differentially regulated in N. pulcher
, a cichlid fish showing lasting alterations of its social behaviour depending on its social rearing environment. We found that early-life social experience indeed significantly altered the expression of these two key gene products of the stress response of teleost fish [17
]. Our results suggest that the differential expression of these genes results from irreversible reprogramming of the brain that occurred during the social treatment the fish were exposed to in the first two months of their life. Irreversible programming of the brain is suggested because (i) the gene expression differences induced during early life were still detected at ages of 1.5 years and older (that is, about half a year after sexual maturity), and (ii) previous behavioural experiments revealed that social behaviour was persistently affected by the early social treatments, suggesting underlying irreversible differences in the mechanisms controlling behaviour: +F fish performed better than –F fish in social challenge tests across three different life stages (early juvenile [8
], late juvenile and early adult stages [9
]). Also, (iii) differences in gene expression induced during early life were remarkably robust, as they were detected in adults despite sex differences and differential trajectories of social status occurring among fish belonging to the same treatment group. Alternatively, cascading effects caused by the persistently differing social behaviour of the two treatment groups might have influenced gene expression. This possibility is unlikely, however, as the different social experiences individuals faced after the early social treatment (differences in rank, sex and breeding status) and the ensuing altered frequencies of expressed behaviours are by far stronger than the behavioural differences detected in our behavioural experiments comparing the two treatments groups [8
]. If social experience gained after the early-life treatments had strongly influenced gene expression of the HPI axis in our study, then this later-life experience should have entirely masked all early-life treatment effects.
CRF is the primary hypothalamic neurohormone regulating the activity of the HPA/HPI axis in vertebrates [39
]. Mammalian studies showed that the regulation of CRF expression can be modulated by experiences early in life, and is therefore thought to be involved in setting the ‘tone’ of individual stress responsiveness [40
]. Rodent pups exposed to low stress levels and/or to high levels of maternal care typically express low basal CRF levels and an attenuated stress responsiveness [11
]. In line with these findings, CRF gene expression was lower in the fish reared together with older family members (+F fish). The presence of guarding adults may have signalled to the developing young that they lived in a secure environment relative to a −F environment, with little exposure to stressful experiences (see [8
] for discussion). Juveniles may thus have responded to this environment by developing a lower HPI reactivity.
In mammals, an attenuated HPA reactivity induced by early experience typically involves a higher expression of GR in the hippocampus and several other brain regions [41
]. These receptors mediate an earlier termination of a stress response by a negative feedback on CRF [42
]. Therefore, we had expected that GR1 would be expressed more in +F fish than in −F fish. The opposite was found to be true. There are several possible reasons for this deviation from expectation. First, as we analysed total brain samples, we could not identify the specific brain regions where GR1 was differentially expressed between treatments. Second, the role of GR signalling in teleost fish is likely to differ from mammals as even between fish species the specificity and sensitivity of the different corticoid receptors differ [17
]. Pharmacologically, knocking down both GR receptors in rainbow trout reduces basal CRF transcript abundance, suggesting a role for GR signalling in maintaining basal gene expression of this peptide [19
]. The positive correlation between basal CRF and basal GR expression found in our study is in line with this result. Based on their findings, Alderman et al
] hypothesized that genomic signalling mediated by GRs in fish may be important for maintaining the transcription of basal CRF, while CRF suppression by cortisol through negative feedback control may involve other unidentified pathways. By contrast to GR1, the expression of GR2 did not differ between early social conditions. GR2 sensitivity to corticoids in N. pulcher
is high, such that activation of this receptor by corticosteroids at basal levels can be expected [18
]. Therefore, the absence of expression differences in this receptor would indicate that epigenetic reprogramming as a consequence of early-life social experience occurs specifically in genes involved in the stress response rather than in general corticosteroid signalling.
MRs are known to contribute to the negative feedback on CRF [11
] and are actively involved in stress responsiveness in mammals. While in our study MR transcript levels did not differ between treatments, +F fish had a markedly higher MR/GR1 ratio. The physiological relevance of this ratio in fish has not been investigated so far, but mammalian models suggest its possible implication for human health [37
]. Recent findings suggest that the mammalian MR takes a much more active role in the control of stress responses than previously thought: MRs bound to membranes in the hippocampus amplify the onset of a stress response, and thereby control the initial non-genomic stress reaction, which is important for appraisal and coping with a stressor, whereas GRs are essential for management of the later adaptive phase [37
]. Thus the balance of MR versus GR may be more relevant for the understanding of HPA/HPI reactivity than the basal levels of each receptor in isolation [44
]. We present this ratio analysis while keeping in mind that the functional significance of such a ratio may differ in fish, as (i) aldosterone is not present as a ligand for MR; (ii) two different GRs are found; (iii) non-genomic action of steroid receptors (i.e. membrane-bound steroid receptors not acting as transcription factors) is known to exist as in mammals [45
], but has not yet been thoroughly studied in link with the stress response; and (iv) these ratios may be specific to certain brain regions [37
Epigenetic reprogramming of the stress axis after experimental variation of early-life social experience has so far only been shown in a number of mammalian species, particularly in laboratory rodents, and more recently also in a bird species (zebra finch, T. guttata
]). Here, we show for the first time that the early social environment can alter the programming of the HPI axis in poikilothermic vertebrates. This suggests that the mechanism for reprogramming the stress axis via early social experience is deeply conserved within vertebrates. In order to fully understand possible homologies, convergences and differences between signalling pathways between homeothermic and poikilothermic vertebrates, future research should (i) localize where in the fish brain corticoid receptors are differentially expressed in response to early-life experience, (ii) identify other possible candidate genes involved in the regulation of the HPI axis, and (iii) explore the mechanisms (e.g. epigenetic marks such as DNA methylation) that allow the long-term maintenance of these differences.
Tactile stimulation has been proposed as a key proximate mechanism inducing a persistent reprogramming of the stress axis in rats and mice (reviewed in earlier studies [11
]). Unlike the brood care of mammals and birds, which involves direct tactile contact during grooming and feeding of young, in N. pulcher
, brood care does not involve body contact during the social experience phase [8
]. Thus, our results demonstrate that at least in some vertebrates (fish), a reprogramming of the stress axis is possible without
direct tactile contact between parents and offspring. Rather than parental tactile stimulation, epigenetic changes in N. pulcher
might possibly be induced by the higher frequency of social interactions between siblings that was observed in those fish reared together with adults [8
]. Whether epigenetic changes in DNA methylation as detected in rodents are also involved in the reprogramming of the N. pulcher
HPI axis is currently under investigation.