Effects of plus-maze experience and chlordiazepoxide on anxiety-like behavior and serotonin neural activity in the dorsal raphe nucleus in rats.

The extent to which rats express anxiety-like behavior on the elevated plus-maze (EPM) depends on their previous maze experience. Open-arm avoidance develops in maze-experienced rats, and is often accompanied by a diminished anxiolytic response to benzodiazepines. Regions of the dorsal raphe nucleus (DRN) were examined in male Sprague-Dawley rats using c-Fos and serotonin immunohistochemistry following a single exposure, a second exposure or no exposure to the EPM. We then examined the effect of the benzodiazepine anxiolytic chlordiazepoxide (CDP, 5 mg/kg) on EPM behavior and DRN neural activity. Enhanced open-arm avoidance was evident on the second EPM trial in both experiments. The observed pattern of c-Fos expression suggests that the first exposure to the maze activates serotonin cells in the rostral and dorsal regions of the DRN and that only the dorsal subregion is activated by a second exposure. CDP increased open-arm exploration during the first trial, which corresponded to decreased 5-hydroxytryptamine (5-HT) activity in the rostral and ventral subregions of the DRN. However, 5-HT activity in the DRN was reduced in rats on the second maze trial compared with the first trial, when CDP had no effect on open-arm exploration. These results suggest that open-arm avoidance in maze-experienced rats can be characterized as a coping response that is mediated by specific populations of 5-HT neurons in the DRN.

Measuring salivary cortisol in the behavioral neuroscience laboratory.

It is often difficult for instructors teaching laboratory courses in behavioral neuroscience to find appropriate experiments that can ethically examine biological parameters in human participants. In most instances, the default experiments that allow students to act as both experimenter and subject tend to be electrophysiological in nature (e.g., EEG, GSR, etc.). We report here the use of an experiment module that utilizes an easily-obtained enzyme immunoassay (EIA) kit to measure human salivary cortisol. Cortisol is a hormone of the adrenal cortex that can be used as a peripheral indicator of hypothalamic neural activity. Plasma (and salivary) cortisol levels rise due to circadian influences as well as perturbations in the organism's environment (i.e., stressors). The involvement of the hypothalamic-pituitary-adrenal (HPA) axis in the pathophysiology of depression makes this an appealing module to students in behavioral neuroscience laboratories. Measurement of salivary cortisol takes advantage of a simple, painless, non-invasive sampling procedure. The assay can be performed successfully by anyone with access to a plate reader, a shaker or rotary mixer, and a few commonly used pipettors. A single plate assay can be completed in two to three hours. Students in our behavioral neuroscience laboratory class have utilized this kit successfully to examine the circadian cortisol rhythm as well as the effect of stress/relaxation on cortisol levels.

Rapid corticosteroid-dependent regulation of mineralocorticoid receptor protein expression in rat brain.

Corticosteroid hormones regulate many aspects of neural function via mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Although GR expression is negatively regulated by endogenous corticosteroids, the autologous regulation of MR expression has been less well studied, partly due to limitations of receptor binding assays that cannot measure the ligand-activated form of MR. Using MR-reactive antibodies and Western blot, we examined relative MR protein expression in rat brain and its potential autoregulation by corticosteroids. We found that MR protein expression is autoregulated in a negative fashion by adrenal steroids. Compared with GR, we see a more rapid regulation of MR, such that there is a substantial increase in MR protein within 12 h after adrenalectomy, whereas GR levels show very little increase until more than 24 h after adrenalectomy. Also, in contrast to GR, which has been found to be regulated by both MR and GR, adrenalectomy-induced increase in MR was prevented by treatment with the MR selective agonist, aldosterone, but not the GR selective agonist, RU28362. Interestingly, acute treatment of adrenalectomized rats with corticosterone produced a significant decrease in whole-cell MR protein within 45 min, suggesting ligand-induced rapid degradation of MR. Chronic high levels of corticosterone also produced a significant decrease in MR protein levels below adrenal-intact rat levels. These results have important implications for previous studies that estimated the proportion of MR that are occupied in vivo by various circulating levels of corticosterone. Those studies compared available MR binding levels in adrenal-intact rats with 24-h adrenalectomized rats, with the assumption that there were no differences between the various conditions in total receptor expression. Those studies concluded that MR is nearly fully occupied by even the lowest circulating corticosterone levels. Given the 2- to 3-fold increase in MR protein that we have observed within 24 h after adrenalectomy, it is likely that those studies significantly overestimated the proportion of MR that were occupied by low basal corticosterone levels. These results support the prospect that MR as well as GR can participate in the transduction of phasic corticosteroid signals.