Young adult male rats were either left in their home cage (no stress), exposed to a novel context (mild stressor), or subjected to acute restraint stress (a more severe stressor) to examine how stressors alter nuclear immunostaining for the immediate-early genes zif268 and Fos, and the corticosteroid receptors MR and GR. Corticosterone levels tended to be higher in the restrained animals but differences were not statistically significant due to the return to baseline over the post-stress interval (controls: 114±16 ng/ml, context: 82±27 ng/ml, restraint: 186±76 ng/ml).
All gene products were observed in the DG of all groups, but levels were graded and patterns varied significantly across groups. The majority of DG neurons, including immature BrdU+ neurons, displayed graded levels of MR and GR, with the exception of cells bordering the hilus, where precursor cells reside (Fig. 1B, 1C). This is consistent with previous reports that both MR and GR are highly expressed in the DG except in very immature neurons and precursor cells. Generally, a small proportion of DG neurons expressed graded levels of zif268 and Fos. While levels of the IEGs zif268 and Fos likely reflect recent transcriptional history, MR and GR levels could reflect both expression and translocation. We, therefore, use the generic term “signal” when referring to immunostaining levels for each antigen.
To capture graded, experience-dependent changes we measured the immunostaining signals of each immature BrdU+ adult-born neuron and each mature non-BrdU+ cell, located in the superficial granule cell layer. In BrdU+ cells, zif268 signal was greatest in cells from control rats; it was reduced by both behavioral manipulations and was lowest in BrdU+ cells after restraint (44% reduction compared to controls). These data are consistent with previous findings that zif268 expression in immature neurons is reduced following water maze training and exploration of a novel environment. That zif268 reduction was greatest in restrained rats suggests that stress may be responsible. In contrast to the adult-born neuronal population, behavioral manipulations did not alter zif268 signal in mature, non-BrdU+ cells (Fig. 1D). In both BrdU+ and non-BrdU+ cells, Fos signal was not altered by behavioral manipulations (Fig. 1E).
The lack of difference in zif268 and Fos signal in mature non-BrdU+ cells might seem at odds with previous studies showing experience-dependent increases in IEGs. This apparent discrepancy is likely due to methodological differences in quantification. Since there is only sparse activation of DG neurons during behavior, it is possible that only a small proportion of DG neurons show IEG upregulation. Indeed, in the scatterplots it is clear that a handful of non-BrdU+ cells show high levels of Fos but only in the context-exposed and restraint groups. Thus, IEG upregulation in a small proportion of neurons is masked by a lack of upregulation in the majority of DG neurons. A similar effect may be occurring with zif268 in non-BrdU+ cells. In contrast, zif268 appears to be reduced in a large proportion of immature BrdU neurons. The reason for the different patterns is unclear but, since zif268 levels are highest in immature neurons from control rats, this may suggest that immature neurons are active during "offline" processes and actively suppressed during stressful experiences.
In BrdU+ cells, MR signal was reduced following experience in a stepwise fashion, with a significant ~20% reduction following restraint stress relative to controls. In non-BrdU+ cells, neither context exposure nor restraint stress altered MR signal (Fig. 1F). Finally, GR signal was increased by both context exposure and restraint stress, in both BrdU+ and non-BrdU+ cells (Fig. 1G). While the experience-dependent increase in GR signal in adult-born cells was statistically significant, the effect was small in comparison to non-BrdU+ cells (10% vs 35%).
Since MRs and GRs both regulate cellular activity and plasticity, including the expression of IEGs, we examined co-expression of IEGs and corticosteroid receptors at the single cell level. Across all groups and both cell types, there were significant correlations between zif268 and MR, and between Fos and GR (supplementary material). Due to antibody and fluorophore limitations, we did not examine zif268-GR and Fos-MR relationships. Nonetheless, these data provide preliminary support for the possibility that IEGs and corticosteroid receptors cooperate in both immature and mature DG neurons cells to promote plasticity or other physiological processes.