Central regulation of the hypothalamic-pituitary-adrenocortical axis

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plasma, as well as ACTH in the pituitary, was quantified by Western blot (see 2.4.1). An overview of the experimental schedule is depicted in Figure 10.

Figure 10: Schematic overview of the experimental schedule with time and intervals of the collected blood samples. Stressed animals were subjected to 15 min of restraint stress and returned back to their homecages for 15 min before they were sacrificed. The unstressed control group was killed under basal conditions. t, time.

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2009; Surget et al., 2011). This is of interest since normalisation of HPA axis function was found to be a potent predictor for the successful treatment of MD (Ising et al., 2005;

Ising et al., 2007) and can significantly reduce the risk of relapse (Zobel et al., 2001), emphasising the importance of a balanced HPA system.

Therefore, in two translational experimental approaches, we investigated the impact of chronic Flx treatment on the neuroendocrine and behavioural phenotype of HR, IR and LR mice. In the first experiment, we focused on the effects of chronic Flx treatment on the emotional behaviour as well as HPA axis regulation. In the second experiment, we were interested whether animals of the SR mouse model were affected by chronic Flx treatment regarding their cognitive performance as well as HPA axis reactivity and neuronal activation in response to stress.

2.5.2.1 Experiment 1: Effects of chronic fluoxetine treatment on emotional behaviour and hypothalamic-pituitary-adrenal axis regulation in HR, IR and LR mice

HR, IR and LR mice derived from Gen XVII of the SR mouse model were chronically administered 10 µg/g BW Flx or vehicle by means of a daily intraperitoneal (i.p.) injection over a time period of 34 days. The injection was given once daily at 16:00. Flx was dissolved in Ringer solution. Control animals (Veh) only received Ringer solution. A detailed outline of the experimental schedule is depicted in Figure 11. After 25 days of treatment, a reference blood sample was collected at 9:00 from all animals to assess basal CORT conditions. In order to exclude interfering effects of the blood sampling procedure with the behavioural testing, the animals were allowed to recover for 3 days before the start of the behavioural test battery. However, the daily injection was also maintained on these days.

2.5.2.1.1 Assessment of emotional behaviour and hypothalamic-pituitary-adrenal axis activity in response to stress

On day 28, a behavioural test battery was performed to assess the animals’ locomotor activity and exploratory drive in a 10-min OF test (see 2.3.1). Subsequently, depression-like behavioural parameters were analysed by means of a 6-min FST (see 2.3.2). Right after the FST, a blood sample was collected in order to assess stress-induced plasma CORT concentrations.

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2.5.2.1.2 Assessment of hypothalamic-pituitary-adrenal axis regulation by means of a combined dexamethasone/corticotropin-releasing hormone test

On day 32, a reference blood sample was taken at 15:00, resembling basal/untreated conditions. After allowing the animals to recover for 3 days, a combined Dex/CRH test was performed (see 2.2.2). Subsequently, the animals were sacrificed and the hippocampus and the pituitary were dissected and immediately frozen on dry ice. One hippocampal hemisphere was used for protein expression analysis using Western blot (see 2.4.1). From the other hippocampal hemisphere, as well as from the pituitary, RNA was isolated and gene expression analysis was performed (see 2.4.2). All blood samples were centrifuged for 10 min (4000 x g at 4 °C) and CORT concentrations were determined from the plasma (see 2.4.3.1).

2.5.2.1.3 Gene expression analysis

After the Dex/CRH test, animals were sacrificed and the hippocampus and the pituitary were dissected for qPCR-based gene expression analysis. In the hippocampus, the following candidate genes were investigated known to be involved in HPA axis function as well as negative feedback regulation: the glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1, Nr3c1), the mineralocorticoid receptor (nuclear receptor subfamily 3, group C, member 2, Nr3c2), the glucocorticoid receptor co-chaperones FK506 binding protein 4 (Fkbp4) and FK506 binding protein 5 (Fkbp5), the multidrug resistance transporters ATP-binding cassette, sub-family B, member 1A (Abcb1a) and 1B (Abcb1b), the corticotropin-releasing hormone receptor 1 (Crhr1), the actin modulating protein cofilin-1 (Cfl1) and the glucocorticoid induced leucine zipper (Gilz).

Additionally, the following candidate genes were analysed in the pituitary of the animals:

Nr3c1, proopiomelanocortin (Pomc), Fkbp5, Abcb1a, Abcb1b, Crhr1 and Gilz (for abbreviations see above).

2.5.2.1.4 Protein expression in the hippocampus

In addition to the hippocampal gene expression analysis, GR and FKBP51 protein abundance was assessed in Veh- and Flx-treated HR, IR and LR animals in the hippocampal hemisphere which was not used for qPCR analysis.

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Figure 11: Schematic overview of the experimental schedule showing the day of behavioural testing (day 28) and the Dex/CRH test (day 35). The detailed experimental schedules on the respective days are given in the timelines on top or below. OF, open field test; FST, forced swim test; Dex, dexamethasone; CRH, corticotropin-releasing hormone; i.p., intraperitoneal; t, time; d, day; BW, body weight.

2.5.2.2 Experiment 2: Effects of chronic fluoxetine treatment on spatial reference memory, hypothalamic-pituitary-adrenal axis function and neuronal activation in HR, IR and LR mice

In the second Flx experiment, HR, IR and LR mice derived from Gen XIX of the SR mouse model were used. Similarly to the first experiment, animals were chronically administered 10 µg/g BW of Flx by means of a daily intraperitoneal (i.p.) injection over a time period of 34 days. The injection was given once daily at 16:00. Flx was dissolved in Ringer solution. Control animals (Veh) only received Ringer solution. In this experiment, two control groups consisting of animals of the HR, IR and LR line, were used (control I and control II, see Figure 12). Similar to the experimental animals, mice of control I received a daily injection of Flx or Veh over a period of 34 days and were subjected to the Y-maze test. This group served to control for effects of the daily injection procedure on (i) CORT levels and (ii) C-fos expression, while control II consisted of entirely untreated, naïve HR, IR and LR mice reflecting basal plasma CORT levels and brain C-fos expression.

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2.5.2.2.1 Assessment of spatial reference memory by means of the Y-maze test

After 28 days of chronic Flx treatment, mice of the experimental and control I group were subjected to the Y-maze test to assess their hippocampus-dependent, spatial reference memory (see 2.3.3). The Y-maze test has been validated previously as a hippocampus-dependent spatial task (Conrad et al., 1996; Conrad et al., 1997) and previous experiments in HR, IR and LR mice showed that animals of the HR line display cognitive deficits regarding their hippocampus-dependent memory performance (Knapman et al., 2010a; Knapman et al., 2012). Thus, we were interested whether the cognitive deficits in HR mice could be rescued by Flx treatment.

2.5.2.2.2 Assessment of stress-induced hypothalamic-pituitary-adrenal axis function and neuronal activation in the central nervous system

After allowing the animals to recover for seven days to avoid confounding influences of the Y-maze test on plasma CORT concentrations and brain C-fos expression, they were subjected to a SRT starting at 9:00. A blood sample was collected immediately before and after the 15-min restraint period in order to assess unstressed and stress-induced CORT levels, respectively. Subsequently to the SRT, animals were transferred back to their homecages for 45 min. Sixty minutes after the onset of the stressor, the mice were sacrificed, trunk blood was collected and the brain was dissected. The brain was immediately shock-frozen in dry-ice cooled methylbutane (Carl Roth GmbH, Karlsruhe, Germany), wrapped in aluminium foil, and stored at -80 °C until analysis of C-fos expression by means of in-situ hybridisation (see 2.4.4). All blood samples were centrifuged for 10 min (4000 x g at 4 °C) and CORT concentrations were determined from the plasma (see 2.4.3.1). An overview of the experimental schedule is depicted in Figure 12.

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Figure 12: Schematic overview of the experimental schedule of the experimental group, control I and control II, respectively. The day of Y-maze testing (day 28) and the SRT (day 35) are given. The detailed experimental schedule of the SRT is outlined in the timeline on top. i.p., intraperitoneal; t, time; d, day; BW, body weight.