Discussion Our studies reveal a complex pattern of changes in kainate receptor subunit expression induced by adrenalectomy and cortico- steroid replacement and a significant and somewhat paradoxical effect of CRS and chronic corticosterone on KA1 mRNA levels, but no effect of CRS upon either KA2 mRNA levels or levels of GluR5-7 mRNA. This pattern, which was found in the dentate gyrus (DG) and CA3 region of the hippocampal formation, demonstrates that CRS involves more than adrenal steroid mediation and that increased KA1 mRNA levels may help explain morphological changes caused by CRS in the DG and CA3. Moreover, the results for KA1 mRNA levels highlight the potential of adrenal steroids to oppose certain actions of stress, which is analogous to their ability to inhibit inflammatory cytokine production. Effects of adrenalectomy and steroid replacement The use of chronic adrenalectomy might have potentially confounded the interpretation of our results as adrenalectomy can produce apoptosis in dentate granule cells [16–18]. In our experiment, adrenalectomy did increase the number of pyknotic cells observed in the dentate gyrus, though the total percentage of pyknotic cells was never higher than 5%. While we cannot exclude dentate apoptosis as the reason for the change in KAR mRNA levels we observe in that region, it seems an improbable explanation for a number of reasons. First, the changes in mRNA levels we observed after adrenalectomy were generally increases. Further, the changes we saw in the dentate were mirrored in the CA3 (GluR6 and KA1) and CA1 (KA1), suggesting that in these cases at least, the change is more likely due to a direct effect of our manipulations of steroid levels, rather than an indirect one due to cell death in the dentate gyrus. GluR6 mRNA levels increased after adrenalectomy in all regions of the hippocampus examined. This effect was reversed by aldosterone treatment, but not by the specific glucocorticoid receptor agonist, RU28362. This implicates the MR in the control of GluR6 mRNA levels in the hippocampal formation. Joels [13], also observed a non-significant increase in GluR6 in the DG after 3 days of adrenalectomy. Collectively, these observations suggest that MR activation inhibits GluR6 expression within the hippocampal formation. KA1, but not KA2, mRNA expression also increased after 7 days of adrenalectomy, but the effect was reversed by high dose corticosterone rather than either the MR or GR selective agonists. GluR7 mRNA expression showed a similar pattern to KA1. We observed no changes in KA2 or GluR5 though expression of the latter was very low, which may have limited our ability to detect subtle changes. That KA1 and GluR7 were regulated by corticosterone but not by selective GR or MR agonists suggests they may be regulated by MR/GR heterodimers, a permutation of classical steroid receptor signaling recently described in cell culture [19], but as yet undescribed in vivo. Effects of chronic restraint stress KA1 expression also increased after CRS; in fact, it was the only KAR subunit to do so. This is interesting because KA1, in contrast to KA2, appears to have a largely pre-synaptic localization at the mossy fiber synapse [20]. Presynaptic KARs have been shown to act as facilitating autoreceptors at the mossy fiber synapse [2,21–23]. These findings, therefore, suggest a potential mechanism for the increase in hippocampal glutamate levels observed after stress [24,25], namely, that they mediate a feed-forward enhancement of glutamate release from mossy fiber terminals. Mossy fiber activation by glutamate has been identified as a key factor in the damaging effects of kainic acid on CA3 neurons [26–28]. KAR, Stress & Steroids Effects of chronic corticosterone treatment Similarly to the effects of CRS, chronic treatment with a moderate dose of corticosterone produced an elevation of KA1 mRNA in the dentate, similar to that produced by chronic restraint stress. In the CA3, which has comparatively little GR [29,30], this effect was not present, suggesting that the changes observed in the CA3 with CRS are the result of other mediators of the response to chronic stress, such as increased activity of the glutamate system in the hippocampus [25] . Interestingly, the response to chronic corticosterone showed an inverted-U shaped dose response, an effect often seen with regard to the effects of glucocorticoids on brain [31]. Chronic restraint, which produces a moderate elevation of corticosterone levels similar to that produced by our low dose treatment, but not as high as those produced by the 400 mg/ml dose [32,33] fits with this interpre- tation, as do the findings of Joels[13], who also found that KA1 mRNA expression was enhanced more by a lower dose of cort than by a high dose. our results suggest that KA1 is also subject to regulation by corticosteroids in an inverted U shaped fashion. Adrenal steroids oppose effects of CRS in CA3 and dentate gyrus The role of adrenal steroids, at least based on the effects of adrenalectomy and hormone replacement reported in this study, is somewhat paradoxical and not unlike their anti-inflammatory effects [34]. Moreover, the observation of increased KA1 expression in both adrenalectomy and CRS, however, is similar to what has been observed for the glutamate transporter, GLT-1, namely, an increased expression of GLT-1 after CRS but also an increase after ADX that is reversed by adrenal steroid replacement [35,36]. It is possible, for both GLT-1 and KA1, that two different processes are operating in the two different treatment schemes. One may speculate that, under basal conditions, adrenal steroids may help to maintain the basal level of kainate receptors, as well as GLT-1, so as to homeostatically regulate the level of glutamate release and glutamatergic activity. According to the present study, this type of regulation also applies to GluR6 and GluR7, but not to GluR5 or KA2 mRNA expression. Yet, at the same time, acute restraint stress elevates extracellular glutamate levels, measured by microdialysis, and these elevations are blocked by adrenalectomy [24]. Moreover, we show in the present study that CRS produces a feed forward, allostatic up-regulation of the KA1 subunit that may contribute to the dendritic retraction caused by CRS, which is mediated in part by excitatory amino acids [37]. Finally, our finding that moderate doses of CORT in the drinking water mimic the CRS induced increase of KA1 mRNA levels whereas high oral doses of CORT fail to elevate KA1mRNA indicates that a hormetic inverted U shaped dose response is operating [38]. Moreover, this hormetic dose response relationship may help explain the paradoxical finding that, while both CRS and chronic CORT each separately cause shrinkage of dendrites of CA3 neurons via a process dependent on glutamate release, the combination of CRS plus chronic CORT treatment, which presumptively elevates CORT levels beyond those pro- duced by either treatment alone, prevented the dendritic remodeling [39]. Future work, when specific antibodies become available, needs to determine whether this up-regulation at the mRNA level is reflected in increased KA1 protein expression (as subunit specific radioligands are as yet unavailable), as well as determine the extent to which stress-induced glucocorticoid secretion may be involved in these changes. Examination of the behavior of KARs after chronic stress using electrophysiology might also provide us with a window on the functional role of these receptors in the adaptation PLoS ONE | www.plosone.org 5 January 2009 | Volume 4 | Issue 1 | e4328