We found clear region-specific differences between fish that had been previously subjected to an early life stress (ELS) regime, compared to control groups at both basal and post-acute stress levels. The ELS fish were characterized by an overall higher brain catecholaminergic signalling (as indicated by expression of adrenergic receptors and overall higher dopaminergic activity in ELS fish), as well as lower bdnf and higher cfos expression. Taken together, these results show how ELS treatment has long-term consequences in the way individuals respond to their environment later in life. Interestingly, these differences appear to be mainly related to catecholaminergic systems, since we found no differences in cortisol or serotonergic activity between treatment groups. This is surprising, since both serotonin and cortisol regulate the hypothalamic pituitary interrenal (HPI) axis28,29,30,31,32, and as expected, both groups react with increased levels/activity to stress. However, we expected that ELS would directly lead to a difference in how treated individuals regulate these systems at both basal and post-stress conditions, similar to what has been reported in mammals13. We believe that a more exhaustive analysis of region-specific serotonergic and cortisol signalling is necessary to shed light on possible effects of ELS, particularly in limbic brain areas. Furthermore, in our experiment we chose a mild chronic ELS in order to avoid mortality in fish groups at these sensitive early stages. Therefore, it is possible that a more severe ELS may lead to more dramatic neurobiological changes, including effects on serotonergic and cortisol regulation. However, further studies are necessary in order to elucidate the possible effects of the magnitude of the ELS on the studied neurobiological parameters.

The brain catecholaminergic systems are fundamental in the control of behavioural flexibility through their role in attention, perception and impulse control20,33,34,35,36. In this context, high levels of dopamine (DA) and noradrenaline (NA) have been associated with increased arousal during stressful situations37,38. These processes are regulated by the interaction of catecholamines with a variety of cellular receptors. Amongst these, the brain adrenergic receptors have been strongly associated with stress coping responses (for a review see39). The adrenergic receptors are classified into 2 main classes, α (inhibiting) and β (stimulating), which are in turn, divided into several subtypes39,40,41,42. Adrenergic receptors are found both pre- and postsynaptic acting as auto- or heteroreceptors, respectively. In mice, it has been found that the majority of the adrenergic autoreceptors are of the α 2Α type, which inhibit the transmission of NA42. In addition, α 2 receptors acting as heteroreceptors modify the release of other neurotransmitters such as serotonin, glutamate and acetylcholine39. Meanwhile, β 2 auto- and heteroreceptors have a stimulating effect on their target neurotransmitters42. Teleostean brain α and β-adrenergic receptors have been shown to have similar characteristics to those of mammals43,44,45,46. In this experiment, we found that there was an overall higher brain DA activity in ELS fish, as well as higher expression of α 2Α in the ventral part of the ventral telencephalon (Vv) and in the dorsomedial pallium subdivision 3 (Dm3) post-acute stress. Unfortunately, we were not able to determine the NA activity, since it was not possible to quantify the NA’s main catabolite MHPG, due to an increase of interacting peaks (i.e. noise) in the area were MHPG is expressed in the HPLC chromatogram. However, the adrenergic receptors have been found to regulate both NA and DA signalling42,47,48 and their expression can therefore be considered to be indicative of catecholaminergic system activity. Specifically, the α 2Α receptor has been shown to regulate both brain NA and DA in a region-specific manner. That is, while α 2Α receptors appear to exclusively regulate NA transmission in the cortex and the nucleus accumbens, they regulate DA transmission in the basal ganglia and the ventral tegmental area42,48. Therefore, it is tempting to speculate that the higher α 2Α expression in the Vv may be part of a regulating mechanism in ELS fish to inhibit catecholaminergic signalling. In other words, the overall higher brain DA activity found in ELS fish may have led to a higher expression of the inhibiting α 2Α adrenergic receptor in the Vv, as a regulating mechanism to reduce DA signalling. That is, higher activity of this α 2Α autoreceptor in local telencephalic DA populations would lead to increase reuptake of DA by the presynaptic DA neuron which will eventually lead to reduced DA signalling. Notably, similarly to what has been shown in the mammalian lateral septum49, we have previously proposed that the Vv may be an important area in DA regulation, since we found a 9- and 7-fold higher concentration of DA and DOPAC in the this area, compared to the Dl and Dm19. Furthermore, although we did not include this data in the previously aforementioned study (i.e. Vindas et al.19), we also found that there was a 2.5- and 3-fold higher concentration of NA in the Vv, compared to the Dl and Dm. However, this data was not previously published but is now available in the supplementary Figure 1S). Taken together, with the present results, this suggest that the Vv, just like its mammalian functional homologue (i.e. the lateral septum), is an important brain area for catecholamine regulation9,49. We here propose that the inhibiting α 2Α receptor, which showed a mean overall higher density in the Vv compared to all other interest areas, may have a DA and NA inhibiting role in the Vv, similarly to what Smith and colleagues50 have proposed on the role of this receptor in the inhibition of NA release and anxious-like behaviour in the bed nucleus of the stria terminalis. It is however important to point out that in our experiment we measured whole brain DA activity and not region-specific changes. We believe that a more specific analysis is necessary in order to confirm our current hypothesis on the role of the Vv in catecholaminergic systems regulation. Regarding the β 2 receptor expression in the Dm3, we found the lowest expression in control fish at basal levels. Furthermore, while control fish showed a significant increase in β 2 post-acute stress, the same was not true for ELS groups. As stated above, the β 2 receptor has a stimulating effect on neurotransmitter release and it is therefore expected that this receptor would be upregulated allowing for the increased release of neurotransmitters in response to stress. It is particularly interesting that this effect appears to be exclusive to the Dm3 area, since we also found that the α 2Α receptor was downregulated post-stress in this area, possibly resulting in an increase of NA release regulating Dm3 function. In other words, we found a downregulation of an inhibiting receptor of neurotransmitter release, along with an upregulation of a stimulating one. The Dm has been proposed to be the functional homologous to the mammalian amygdala, which is very important in stress reactivity22,24,51. In agreement with this literature, the Dm has been found to be highly active and regulated in response to different types of stress in several fish species19,20,21,52,53,54. However, it is hard to pinpoint which subdivision of the Dm in seabream may be the functional homologous to the amygdala, since there are few functional neuroanatomy studies focused on this topic. However, we propose that since the Dm3 is the most complex of the 4 subdivisions55 and it showed the highest amount of stress activity in our experiment, it may be the most likely amygdala-like candidate. Notably, in mammals the amygdala has been associated with the direct regulation of the HPI axis, increasing its activity during stressful situations56. In this context, it is interesting to note that ELS fish did not display a post-acute stress downregulation of the α 2Α receptor in the Dm3 as control fish did, which could potentially lead to an increase HPI axis activity mediated via the Dm. Taken all together, it is possible that the lack of adrenergic receptor response to stress in this area in ELS fish may be part of the first signs in the Dm3 of cumulative stress overloading physiological systems and compromising their ability to react further to stressors (i.e. allostatic overload57) or mediating stress resilience mechanisms. Noteworthy, anxiety has been found to be a long-term effect of ELS in mammals, which appears to be related to increased activity in the amygdala-septal hypothalamic circuit12. It would therefore be interesting to study this amygdala-septal hypothalamic circuit in fish subjected to ELS to see if our current results could be indicative of this brain mechanism. Sampling at several time points throughout the ELS fish´s life would also be necessary to better elucidate the specific long-term stress plasticity events.

We found that the transcript abundance of the neuronal plasticity marker brain derived neurotrophic marker (bdnf) was overall lower in ELS fish compared to control in the Dm3 and the Dld. The downregulation of bdnf has been previously reported to decrease and increase in response to chronic and acute stress, respectively58. Furthermore, chronic downregulation of BDNF has been strongly associated with several neurobiological diseases, such as depression-like states59. In this context, it appears that the ELS treatment had a long-term downregulating effect on 2 brain areas associated with memory, learning, navigation and emotional reactivity (i.e. the Dl and the Dm)23,24. Since bdnf expression has been strongly associated with promoting neurogenesis, cell survival, and the strengthening of learning and memory60, this region-specific downregulation implies that ELS fish may have a diminished capacity regarding the aforementioned processes. Notably, unpredictable chronic stress during adolescence has been found to have a context dependent long-lasting effect on memory systems. Specifically, it appears that while rats subjected to unpredictable chronic stress showed an enhancement in working memory to the location of a reward at basal levels, this was completely hindered after exposure to a novel acute stressor7. Interestingly, all fish groups responded with an increase in bdnf expression to the acute stressor in the Dlv. As mentioned above, an increase in this factor is expected when animals experience acute stress and the fact that this is strongly expressed in this area for both groups, implies that a higher neural plasticity in this area may help cope with this type of stressor. Notably, in a previous experiment we also found a higher expression of this marker in the Dl (as a whole since we did not differentiate between the dorsal and ventral subregions), after exposing fish to crowding stress19. The difference we found between the Dld and the Dlv in our current results suggests that these 2 areas may indeed be involved in the regulation of different processes, in agreement with what has been proposed by Broglio et al. based on the topography, connections and histochemistry in these 2 subregions61. Further studies are needed including the link between BDNF expression in region-specific areas, memory tasks and ELS in order to understand region-specific neuronal function and the positive and negative effects of ELS in vertebrates.

Neuronal activity can often be characterized by quantifying the expression of immediate early genes62,63,64, such as cfos. We found that cfos mRNA transcript abundance post-acute stress was higher in ELS fish in both the Dlv and the Dm3. It is important to point out that an increase in cfos abundance may reflect either inhibition or excitation of specific neuronal circuitry within each nucleus. In other words, neuronal activity, as inferred by cfos transcript abundance, may be indicative of a stimulating or an inhibiting neuron62. In this context, the increase cfos abundance in ELS fish found in the Dm3 may be directly associated with the higher activity of inhibiting neurons associated with catecholamine regulation, particularly considering the overall elevated expression of both adrenergic receptors in this area. Unfortunately, we were unable to obtain cfos levels at basal conditions. Therefore, we cannot confirm that this activation pattern was also present in ELS fish at basal conditions, as it is suggested from our results on the expression of adrenergic receptors. Further studies should be focused on quantifying the expression of early activity genes at both basal and post-acute stress conditions in order to elucidate the overall activity pattern in ELS fish as compared to control individuals.

In conclusion, we found that seabream that experienced an ELS regime display long-term neurobiological brain-region specific effects as juveniles (approximately 5 months after ELS). These results illustrate how neuronal populations within the telencephalon show a distinct regulation to the same stimuli, which may be associated with the control of stress coping responses, in agreement to results obtained in mammalian studies regarding effects of ELS in limbic areas9,12,13. In this context, physiological and behavioural responses represent trade-offs from life history strategies and should be viewed/interpreted in a context dependent manner7,65, as exemplified by the enhancing and inhibiting effect of unpredictable chronic stress on working memory on rats reported by Chaby et al.7. We here found that ELS fish showed signs suggesting allostatic overload, which is in contrast to what we found for ELS salmon in our previous studies32,66. This may be due to species-specific differences, context-dependent effects to different stressful stimuli or to the exact period in which the ELS regime was given, since it was done at a later age in salmon. We hope that future studies will be focused towards better understanding of allostatic processes and both the possible negative and positive consequences of early life stress in a context- and species-specific dependent manner. Importantly, our data presented here corroborates that the Vv is functionally homologous to the lateral septum, particularly regarding catecholamine regulation.