Effect of Repeated Predator Scent Exposure on Excitability of Serotonin Neurons and Stress Markers in Rats

Exposure to predator scent (PS) has been used aa a model of stress associated with danger to life and body integrity. We tested the hypothesis that repeated PS exposure alters the excitability of 5-HT neurons of the dorsal raphe nucleus. To study the mechanisms involved, we approached serum and adrenal corticosterone and aldosterone concentrations, as well as cortical brain-derived neurotrophic factor (BDNF) expression. Adult male Sprague-Dawleys rats were exposed to PS for ten minutes daily for ten consecutive days. Two weeks after the last exposure, the selected electrophysiological and biochemical assessments were performed. Measurements by in vivo electrophysiology showed increased spontaneous firing activity of 5-HT neurons in rats exposed to repeated PS. Repeated PS exposure resulted in reduced serum corticosterone and aldosterone concentrations. Concentrations of both corticosteroids in the adrenal glands, as well as the relative weight of the adrenals, were unaffected. The gene expression of hippocampal BDNF of rats exposed to PS remained unaltered. In conclusion, repeated exposure of rats to PS leads to enhanced firing activity of 5HT neurons accompanied by reduced serum, but not adrenal aldosterone and corticosterone concentrations. Reduced corticosteroid concentrations in the blood appear to be the result of increased metabolism and/or tissue uptake. The decrease in circulating corticosterone in rats experienced repeated PS may represent part of the mechanisms leading to increased excitability of 5-HT neurons. The increase in 5-HT neuronal firing activity might be an important compensatory mechanism designated to diminish the harmful effects of the repeated PS exposure on the brain.

The excitability of 5-HT neurons of the DRN was affected by used stress stimulus ( Figure 1). The mean spontaneous firing activity of 5-HT neurons in the DRN of rats experienced repeated PS was significantly higher than that in the controls (p<0.01, two-tailed Student's post-hoc test): Interestingly, concentrations of corticosterone in the right adrenal did not differ between the groups ( Figure 2C). The concentration of aldosterone in the adrenal gland was unaffected by the repeated PS exposure ( Figure 2D). The repeated PS exposure failed to modify the relative weight of the adrenal glands. The relative weight of both left and right adrenals was comparable in PS exposed and control rats ( Figure 2E).

Repeated PS exposure failed to modify BDNF gene expression in the prefrontal cortex
The gene expression of BDNF in the prefrontal cortex was unaffected by PS exposure. In animals exposed to repeated PS, the concentrations of mRNA coding for BDNF (0.82±0.05 a.u.) were comparable to those in the control animals (1.06±0.22 a.u.). the adrenal gland corticosterone (C) and aldosterone (D) contents, and on the relative weight of the adrenal glands (E); *p<0.05 and **p<0.01, two-tailed Student's t-test.

Discussion
The results of the present study demonstrate that rats exposed to the repeated PS exert increased spontaneous firing activity of 5-HT neurons of the DRN compared to that in unexposed controls.
Repeated PS exposure resulted in decreased serum concentrations of corticosterone and aldosterone.
The concentrations of both steroid hormones within the adrenal glands, as well as the relative weight of the adrenals, were unaffected by the PS. The expression of BDNF in the hippocampus of rats exposed to the repeated PS was not statistically different from the controls.
In this study we detected, for the first time, the enhancing effect of repeated PS exposure on the excitability of 5-HT neurons of the DRN. This observation is startling, since other stressors, such as maternal immune activation [6] and chronic unpredictable stress [7], led to decreased 5-HT neuronal firing activity. It is possible that increased excitability of 5-HT neurons is lined with decreased brain serotonin concentrations in the midbrain and neocortex, observed in our previous study, though using a rats from a different source [21]. A decrease in midbrain 5-HT can diminish the tonic activation of 5-HT1A autoreceptors of the DRN [24], resulting in an increased firing activity of 5-HT neurons. Cortical 5-HT can attenuate the excitability of 5-HT neurons of the DRN as well, via a mechanism involving 5-HT1A receptor-mediated inhibition of cortical neurons projecting to the DRN and positively regulating 5-HT neuronal firing activity [25]. Alternatively, increased firing activity of BDNF was observed also in studies investigating the consequences of exposure to PS, particularly those using protocols including situational trauma reminder to simulate post-traumatic stress symptoms [26,27]. In a recent study, stress of post-weaning isolation did not alter hippocampal BDNF, but it led to a reduced gene expression of VGF (non-acronymic) and tyrosine receptor kinase B (TrkB) receptor [28]. Thus, the modulation of VGF/BDNF/TrkB signaling by repeated PS exposure cannot be excluded.
Although increased release of glucocorticoids in response to repeated stressors is a well-known phenomenon [29], The present study shows that repeated exposure to PS leads to a decrease in serum concentrations of the mineralocorticoid hormone aldosterone. This finding is surprising, since other stressors, such as insulin-induced hypoglycaemia, and immune challenge were shown to increase aldosterone concentrations in the rat blood [33,34]. Aldosterone is a somewhat neglected stress hormone and its involvement in stress-related mental disorders is being explored only recently [29].
To our best knowledge, concentrations of aldosterone have not been investigated in animal models using PS exposure.
Interestingly, exposure to PS led to decreased corticosterone and aldosterone concentrations blood serum, but not their content in the adrenal gland. The relative weight of the adrenal glands remained unchanged as well. Corticosteroids are not being stored in the adrenal cortex and changes in their circulating levels in response to stress stimuli usually occur with parallel changes in their concentration in the adrenals [35]. The present results thus provide indirect evidence that reduced serum concentrations of these steroids in rats experienced repeated PS resulted from their excessive metabolism and/or tissue uptake, rather than reduced release or synthesis within the adrenal cortex.
It is in agreement with our previous suggestion that increased metabolism of glucocorticoids contributes to decreased glucocorticoid signaling in stress-related psychopathologies [23,36]. Further studies aimed to reveal changes in the expression of hepatic and tissue corticosteroid-metabolizing enzymes are needed to confirm this hypothesis.
In conclusion, repeated exposure of rats to PS leads to enhanced firing activity of 5-HT neurons activity might be an important compensatory mechanism designated to diminish the harmful effects of the repeated PS exposure on the brain.

Animals
Adult

Repeated exposure to PS
After an acclimatization period of one week, the rats were randomly divided to control (n=20) and PS exposed (n=20) groups. Sand containing fresh cat urine was collected daily from the litterbox used by two domestic cats belonging to one of the authors (ED). This sand was stored at the room temperature in a closed plastic container for 3-5 h before the exposure. This sand was laid in plastic dish and placed in a 20×30×50 cm exposition cage. Rats were transferred to the exposition cage for 10 min daily, between 13:00 and 14:00, for 10 consecutive days. Control unstressed rats were exposed to the sand containing clean water, using the same schedule. Control and PS exposed rats were kept and exposed to the stressor in different rooms with separate air conditioning systems. After the last exposure, the rats were kept under non-stress conditions for 2 weeks.

In vivo electrophysiology
The first half of the animals (10 control and 10 PS exposed rats) was used for electrophysiological measurements. Fourteen days after the last exposure to the sand containing clean water or cat urine, those rats were anesthetized by chloral hydrate (400 mg/kg, i.p.) and mounted in the stereotaxic frame The other half of the animals (10 control and 10 PS exposed rats) was decapitated fourteen days after the last exposure and their blood and tissues were collected. The 2 rats from the same cage were killed within 30 s. Trunk blood was collected into polyethylene tubes without anticoagulant. The clotted blood was spun at 3000 rpm for 15 min at 4 °C and the serum was separated. The separated adrenal glands were quickly weighted, and frozen in liquid nitrogen. The brain was quickly removed from the skull, the prefrontal cortex was dissected and frozen in liquid nitrogen. Serum aliquots and collected tissues were stored at -80 °C until analyzed.
The intra-and inter-assay CVs were 3.8% and 6.2%, respectively and the detection limit was 1.4 pg/ml. For the measurement of adrenal corticosterone and aldosterone concentrations, the right adrenal gland was homogenized (D1000 Handheld Homogenizer, Benchmark Scientific, USA) in necessary amount of saline and processed as described previously [35]. The concentrations of corticosterone and aldosterone were analyzed using the same commercially available kits mentioned above.

BDNF gene expression in the prefrontal cortex
The total mRNA was isolated by TRIzol® Reagent (Life technologies, California, USA) from prefrontal cortex homogenates according to manufacturer protocol as described previously [42].
Concentration and purity of mRNA preparations was measured by absorption spectroscopy described previously [42,43]. Primers (Table 1) were used at a concentration of 0.25 pmol/μl. 5 ng of BDNF gene cDNA was added to the final reaction volume of 10 μl. [44]. Quantitative PCR was performed by QuantStudio 5 Fast Real-Time PCR System (ThermoFisher, USA). All data obtained by quantitative PCR analysis were evaluated as ng of mRNA (cDNA) according to a standard curve and was normalized to gene expression of peptidyl prolyl isomerase A (PPIA) and Ribosomal Protein S29 (RPS29) as reference genes. Gene expressions were evaluated by ∆∆Ct calculation and normalized to PPIA and RPS29 housekeeping genes as arbitrary units.

Statistical analysis
Data was expressed as mean ± SEM. Two-tailed Student's t-test was used to determine the effects of PS exposure on excitability of 5-HT neurons in the DRN and on stress related parameters. The probability of p ≤ 0.05 was considered significant.