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Stress adaptation in rats associate with reduced expression of cerebrovascular Kv7.4 channels and biphasic neurovascular responses

Abstract Neurovascular coupling ensures rapid and precise delivery of O2 and nutrients to active brain regions. Chronic stress is known to disturb neurovascular signaling with grave effects on brain integrity. We hypothesized that stress-induced neurovascular disturbances depend on stress susceptibility. Wistar male rats were exposed to 8 weeks of chronic mild stress. Stressed rats with anhedonia-like behavior and with preserved hedonic state were identified from voluntary sucrose consumption. In brain slices from nonstressed, anhedonic, and hedonic rats, neurons and astrocytes showed similar intracellular Ca2+ responses to neuronal excitation. Parenchymal arterioles in brain slices from nonstressed, anhedonic, and hedonic rats showed vasodilation in response to neuronal excitation. This vasodilation was dependent on inward rectifying K+ channel (Kir2) activation. In hedonic rats, this vasodilation was transient and followed by vasoconstriction insensitive to Kir2 channel inhibition with 100 µM BaCl2. Isolated arteries from hedonic rats showed increased contractility. Elevation of bath K+ relaxed isolated middle cerebral arteries in a concentration-dependent and Kir2-dependent manner. The vasorelaxation to 20–24 mM K+ was reduced in arteries from hedonic rats. The expression of voltage-gated K+ channels, Kv7.4, was reduced in the cerebral arteries from hedonic rats, whereas the expression of arterial inward-rectifying K+ channels, Kir2.1 was similar to that of nonstressed and anhedonic rats. We propose that preserved hedonic state is associated with increased arterial contractility caused by reduced hyperpolarizing contribution of Kv7.4 channels leading to biphasic cerebrovascular responses to neuronal excitation. These findings reveal a novel potential coping mechanism associated with altered neurovascular signaling.


Introduction
Chronic stress is among the most important risk factors for a variety of psychiatric disorders including depression (de Kloet et al., 2005). Some subjects exposed to chronic stress are, nevertheless, able to cope with stressful conditions (Bergstrom et al., 2008). However, the mechanism behind the ability to cope with stress is poorly understood. It has been proposed that disturbance in the communication between neuronal tissue and the vasculature, i.e. neurovascular signaling, plays a central role in the pathogenesis of psychiatric manifestations of stress (Longden et al., 2014). Neurovascular signaling ensures rapid and precise delivery of O 2 and nutrients in addition to the removal of waste products from active brain regions (Longden et al., 2016). Neurovascular signaling is believed to be mediated through the release of vasoactive substances, e.g. K þ ions, from active neuronal tissue. The elevation of K þ in the perivascular space activates vascular inward-rectifying K þ channels (K ir 2) leading to smooth muscle cell hyperpolarization, vasodilation, and thus increased blood flow to active brain regions (Longden et al., 2016). On the other hand, the elevation of interstitial K þ has an overall depolarizing action that counterbalances K ir 2-mediated hyperpolarization (Jackson, 2018). Therefore, interstitial K þ elevation has a narrow window where arteries dilate prior to vasoconstriction induced by further K þ elevation (Girouard et al., 2010). This K þ -induced vasoconstriction depends on voltage-gated K þ channels (Jackson, 2018). Under physiological conditions, interstitial K þ does not raise to the concentrations that might constrict vascular wall as this will have devastating consequences for active neuronal tissue. Neurovascular signaling has been shown to be modified upon psychological stress (Han et al., 2019;Longden et al., 2014). However, these previous studies did not question whether neurovascular changes were associated with variable stress susceptibility. Stress susceptibility can be assessed in a rodent model of chronic mild stress (CMS) based on their preference for sucrose consumption . In this CMS model, rodents are exposed to unpredictable stressors and develop symptoms similar to depression (Strekalova et al., 2022;Wiborg, 2013;, such as impaired physical activity and inability to experience pleasure from normally pleasurable stimuli (anhedonia). Hence, rats exhibiting anhedonia-like behavior show a reduction in the preference for consumption of palatable sucrose solution over water. The model is considered to be realistic and with a high validity . In the CMS model, anhedonialike symptoms are detected in approximately 30% of stressed rats, while another 30% of rats show less than 10% reduction in the preference of sucrose intake, i.e., preserve their hedonic state (Wiborg, 2013). Rats with preserved hedonic state are normally characterized with behavior, biochemical parameters, histological, and molecular changes in the brain that are more similar with the non-stressed rats than with rats showing anhedonia-like symptoms (Strekalova et al., 2022;Wiborg, 2013;. In the present study, we hypothesized that chronic mild stress affects neurovascular signaling differently in subjects developing anhedonia-like behavior compared with subjects that preserve their hedonic state upon CMS exposure. We used the conventional CMS protocol with unpredictable stressors and assessed rats' hedonic state using sucrose preference test (Wiborg, 2013). We tested the hypothesis by comparing neurovascular signaling in brain slices from hedonic and anhedonic stressed rats and from nonstressed controls. Cerebrovascular function and expression of K þ channels important for neurovascular signaling were assessed in isolated middle cerebral arteries.

Methods
All animal experiments conformed to guidelines from the European Communities Council Directive (86/609/EEC) for the Protection of Animals used for Experimental and other Scientific Purposes; and were conducted with permission from the Animal Experiments Inspectorate of the Danish Ministry of Environment and Food.

CMS protocol and sucrose preference test
Male Wistar rats, 6 weeks old, were purchased from Taconic, Denmark, and housed individually in 12-h light/dark cycle. Food and water were available ad libitum. All rats were singly housed. The standard conditions were changed only in the course of the stress regime. Upon arrival to the animal facility, all rats were adapted and trained to consume a palatable sucrose solution prior CMS protocol was initiated (Bouzinova et al., 2014). During this period, the sucrose test was made twice a week during the first three weeks and once a week during the last two weeks. Rats were food and water deprived 14 h prior the test and then exposed to a bottle with sucrose solution (1.5%) for 1 h (Wiborg, 2013). Five weeks later, when rats were 11 weeks old, the CMS protocol was initiated. During the CMS period, the sucrose consumption test was performed once a week.
Rats were randomly sub-divided into a group exposed to CMS and a nonstressed control group. The rats from nonstressed group were housed in animal facility room different from the one, where the CMS protocol was applied. The experimental CMS protocol was applied and continued for 8 weeks, as described previously (Matchkov et al., 2015). The stress protocol consisted of 12-h periods of intermittent illumination, stroboscopic light, grouping, food or water deprivation, 45 � box tilting, soiled cage, and periods of no stress. Grouping was done by housing rats in pairs with different partners alternately being a resident or intruder. All procedures are well established and validated by the research group (Wiborg, 2013).
During the first four weeks of the stress protocol, some rats (approximately 20%) did not change their sucrose consumption more than 10% and were considered hedonic, while the rats with a reduction in sucrose intake exceeding 30% (approximately 30% of rats exposed to CMS) were characterized as the group with anhedonia-like behavior. Rats with 10-30% reduction in sucrose intake were excluded from the study. In total, 14 nonstressed, 11 hedonic rats, and 16 rats with anhedonia-like behavior were distributed between in situ brain slice studies and the experiments for ex vivo assessment of middle cerebral artery function.

Brain slice experiments
At the end of the 8-week CMS paradigm, rats were euthanized by decapitation under pentobarbital anesthesia (0.75 mg/kg, i.p.; Euthasol vet., Virbac, Denmark). The brain was quickly moved into ice-cold artificial cerebrospinal fluid (aCSF) aerated with 95% O 2 and 5% CO 2 . The cerebral cortex was cut into 160 mm slices using vibratome (1200 s, Leica Biosystems, Germany) and incubated for 90 min in 5.33 mg/L Calcium Green AM (ThermoFisher Scientific, MA, USA) diluted into aCSF. The slices were continuously superfused with aerated aCSF at 37 � C and examined by simultaneous contrast and fluorescence microscopy (Zeiss LSM 5 Pascal, Germany) as described previously (Staehr et al., 2020).
Slices were pretreated with thromboxane A2 analog, U46619 (10 nM) for 30 min prior to the experiment in order to achieve sufficient vascular tone. Neurons were stimulated with electric field stimulation (32 Hz, 14 V, 0.3 ms pulses, stimulation period 3 s) using two platinum electrodes. A baseline period before stimulation (15 s

Ex vivo studies on isolated middle cerebral arteries
Middle cerebral arteries were cleaned of connective tissue and mounted in a wire myograph (Danish Myo Technology A/S, Denmark) for isometric force measurements (Staehr et al., 2020). Active force (mN) was recorded with a PowerLab 4/25, Chart7 acquisition system (ADInstruments Ltd., New Zealand) and converted to wall tension by dividing the force with two times the arterial segment length. Middle cerebral arteries were constricted with a thromboxane A2 receptor agonist, U46619. The U46619-preconstricted arteries were challenged with an increased bath concentration of K þ ions that substituted Na þ ions in the physiological salt solution (PSS).

Western blot
Lysate of middle cerebral arteries was centrifuged at 10,000g to collect the supernatant that was adjusted 10:3:3 with 1 M/ L DTT and 2� Tris-glycine SDS sample buffer (Thermo Fisher Scientific, Denmark). The expression of K ir 2.1 channel protein was semi-quantified as described previously (Staehr et al., 2020). The membrane above 25 kDa was incubated with primary antibody against K ir 2.1 (1:200; sc-18708; Santa Cruz Biotechnology, TX, USA), and the lower part was incubated with antibody against thioredoxin-2 (1:10,000; ab185544; Abcam, UK) overnight at 5 � C. The next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Dako, Denmark) for 1 h. Bound antibody was detected by an enhanced chemiluminescence (ECL) kit (Sigma Aldrich, Denmark). Detected protein was semi-quantified (ImageJ, NIH, MD, USA) relative to thioredoxin-2 in the same probe. The K ir 2.1 expression in nonstressed controls was taken as 100%.
The voltage-gated potassium channel subunit K v 7.4 was semi-quantified by loading 15 mg of middle cerebral artery lysate to 4-20% precast polyacrylamide stain-free gels (CriterionTM TGX Stain-freeTM precast gel, BioRad, CA, USA). The proteins were electrotransferred to nitrocellulose membranes that were blocked in 5% bovine serum albumin (BSA) with 0.5% v/v Tween 20. The membrane was incubated overnight at 5 � C with K v 7.4 antibody (monoclonal, 1:1000 in 3% BSA; S43-6, Thermo Fisher Scientific Inc., MA, USA). After washing, the membranes were incubated with HRP-conjugated secondary antibody. Bound antibody was detected by an ECL kit and normalized using ImageJ software as a ratio to total protein load measured for the same probe.

Statistical analyses
GraphPad Prism software (v.7.03) was used for graphing and statistical analysis. Data are summarized as the mean value ±standard error of mean (SEM) of the sample group. Contractile concentration-response curves were fitted to experimental data using four-parameter, nonlinear regression curve fitting. From these curves, pD 2 (-log to the concentration required to produce a half-maximal response) and maximal response were derived and compared using an extra sum-of-squares F test. Ex vivo K þ induced relaxation curves were drawn with centered third order polynomial function. Significant differences between means of other values were determined by either one-way or two-way ANOVA, where appropriate, followed by correction for multiple comparison as indicated. A probability (p) level of <.05 was considered significant.

Stressed rats with unchanged hedonic state exhibited biphasic neurovascular responses
After 8 weeks of exposure to CMS, the hedonic state of rats was identified based on sucrose consumption test, and hedonic and anhedonic groups were selected for further experiments (Figure 1(A)). We compared these two groups to matching nonstressed rats. Rats were weighed every week prior and during CMS protocol (Figure 1(B)). There was a significant increase in weight over time for all the three experimental groups (F(3.049, 115.9) ¼ 217.2, p<.001); however, this was not significantly different between the groups (F(2,38) ¼ 1.290, p¼.287).
In the brain slices, we assessed intracellular Ca 2þ in neuronal tissue and astrocytes, and neurovascular responses of parenchymal arterioles in brain slices (Figure 2(A)). The arteriole baseline diameter was similar (F(2, 15)¼0.421, p¼.664) in Figure 1. Based on the sucrose preference test, the stressed hedonic (n ¼ 11) and anhedonic rats (n ¼ 16) were identified and compared to nonstressed rats (n ¼ 14) (A). Anhedonia-like behavior was defined as >30% reduction in sucrose consumption compared with baseline (horizontal dotted black line). A significant differences between groups (F(2, 38) ¼ 41.24, p<.0001) and over time (F(4.88, 183.60) ¼ 3.43, p¼.006) was seen. The hedonic group did not differ in sucrose index from the non-stressed group, whereas the group with anhedonialike behavior had a significantly reduced sucrose index after CMS. There was no significant difference in weight changes over time between the groups (B). � p<.01 and ��� p<.001 for the anhedonia-like group vs. nonstressed controls; †p < .05, † †p<.01, and † † †p<.001 for the anhedonia-like group vs. the hedonic group (ANOVA followed by Tukey correction for multiple comparison).
all the three groups after pre-constriction with 10 nM U46619 to achieve a basal tone of approximately 80% of fully relaxed diameter (Figure 2(B)). The relaxed diameter was also similar among all the three experimental groups (F(2, 15)¼0.751, p¼.489) as it was evident after the exposure of brain slices to a Ca 2þ -free aCSF solution (Figure 2(C)). Electric field stimulation (EFS) was associated with similar vasodilation of parenchymal arterioles in all the three groups (Figure 2(D)). However, the initial vasodilation of arterioles in the brain slices from hedonic rats was followed by vasoconstriction, making their vascular response to EFS different from that of nonstressed rats and rats with anhedonia-like behavior (F(50,375) ¼ 2.499, p<.001). The EFS-induced vasodilation was strongly diminished by 100 mM BaCl 2 in all the three experimental groups (Figure 2(E)). This BaCl 2 -sensitive vasodilation was similar in all the three experimental groups (F(2, 13) ¼ 0.229, p ¼ .798) (Figure 2(F)). Importantly, delayed vasoconstriction in response to EFS in the brain slices from hedonic rats remained in the presence of BaCl 2 (Figure 2(E)) and was not different in the presence and absence of BaCl 2 (F(25, 175) ¼ 1.102, p ¼ .345). Thus, the significant difference in EFS responses of hedonic rats in comparison with nonstressed rats and rats with anhedonia-like behavior remained in the presence of BaCl 2 (F(50, 325) ¼ 5.479, p < .001) (Figure 2(E)).
Neuronal excitation was associated with a similar (F(238, 1663) ¼ 0.926, p ¼ .773) increase in intracellular Ca 2þ in the neuronal tissue in all the three experimental groups ( Figure  2(G)). The neuronal Ca 2þ response was followed by an increase in intracellular Ca 2þ in astrocytic endfeet ( Figure  2(H)), which was also similar in all the three groups (F(238, 1172) ¼ 0.651, p ¼ .999). Importantly, we have previously Figure 2. Assessment of neurovascular signaling in brain slices revealed biphasic neurovascular responses in stressed rats with unchanged hedonic state. Representative image of a Calcium Green AM-loaded brain slice from a nonstressed rat shows the neuronal tissue (black tilted arrow) and astrocytic endfood (white tilted arrow) located in a proximity of the adjacent parenchymal arteriole (horizotal arrow indicates arteriole lumen) (A). Brain slices were incubated with 10 nM U46619 for 30 min to pre-constrict parenchymal arterioles to the diameter of approximately 80% of maximally relaxed diameter. Arterioles of the three experimental groups were similar in diameter after pre-constriction with U46619 (B). The fully relaxed arteriole diameter measured in Ca 2þ -free artificial cerebral spinal fluid was also similar in all the three groups (C). Electric field stimulation (EFS, indicated with vertical dashed lines) of the neuronal tissue in brain slices was associated with vasodilation of parenchymal arterioles in nonstressed (n ¼ 6), hedonic (n ¼ 5) and anhedonic rats (n ¼ 7) (D). In hedonic rats, the initial vasodilation was followed by vasoconstriction. Superfusion of the brain slices with 100 mM BaCl 2 strongly diminished neurovascular responses in all groups, but the subsequent vasoconstriction was still observed in hedonic group (E). There was no difference in BaCl 2 -sensitive vasodilation among the three experimental groups (F). All the three groups showed similar increase in intracellular Ca 2 in the neuronal tissue (G) and in astrocytic endfeet (H) in response to EFS. Two nonstressed brain slices and three slices from the anhedonia-like rats were excluded from Ca 2þ astrocytic signaling analyses as no astrocytes were identified in the field of view. The data in (B), (C), and (F) were compared with one-way ANOVA; the data in (D), (E), (G), and (H) were compared with two-way ANOVA. �� p < .01 and ��� p<.001, respectively.
reported (Staehr et al., 2020) that incubation of brain slices with BaCl 2 does not affect transient elevation of [Ca 2þ ] i in neuronal tissue and astrocytic endfeet in response to EFS. This suggests that the effect of BaCl 2 is of vascular origin.

Reduced expression of Kv7.4 in cerebral arteries from hedonic rats
We analyzed the expression of K ir 2.1 and K þ voltage-gated channel protein K v 7.4 channels in the middle cerebral artery. No difference in the expression of K ir 2.1 channels was found between the groups (F(1.715, 10.29) ¼ 0.984, p ¼ .394) (Figure 4(A,B)). However, the expression of K v 7.4 was significantly decreased in the arteries from hedonic rats in comparison with arteries from nonstressed rats (q ¼ 2.554, 95% CI [1.62, 56.450], p ¼ .0375) (Figure 4(C,D)). No significant difference between hedonic and anhedonic groups was achieved (q ¼ 1.433, 95% CI [-48.37, 12.32], sp ¼ .2911).

Discussion
In this study, we suggest that unchanged hedonic state of rats after exposure to the CMS protocol is associated with biphasic cerebrovascular responses to neuronal excitation Figure 3. Middle cerebral arteries from stressed rats with unchanged hedonic state exhibited increased arterial contractility. Middle cerebral arteries from hedonic (n ¼ 6) and anhedonic (n ¼ 9) rats showed increased constriction to U46619 compared with nonstressed control rats (n ¼ 8) (A). Middle cerebral arteries pre-constricted with 10 nM U46619 from nonstressed (n ¼ 6), hedonic (n ¼ 9), and anhedonic groups (n ¼ 6) relaxed in response to the elevation of bath K þ concentration up to 18 mM (B). At 20 mM and 24 mM of bath K þ , the arteries from hedonic rats showed a decline in vasorelaxation, whereas arteries from anhedonic and nonstressed rats remained relaxed. The K þ -induced relaxation was inhibited by 100 mM BaCl 2 ; but at 24 mM bath K þ , arteries from hedonic rats constricted (C). The concentration-response curves in (A) were compared with an extra sum-of-squares F test; the responses in (B) and (C) were analyzed with two-way ANOVA followed by Tukey correction for multiple comparison. � p < .05 and �� p< .01 for nonstressed vs. hedonic rats; †p < .05 for anhedonia-like vs. nonstressed rats; þp < .05 for anhedonia-like vs. hedonic rats.
caused by reduced expression of cerebrovascular K v 7.4 channels. We observed different individual responses to stress measured as a sucrose consumption of outbred rats, which is presumably caused by genetic variations. Similar to results of other reports (Bergstrom et al., 2008), approximately 20% of rats did not change their hedonic state. We found that neurovascular signaling was reduced in hedonic rats. While the hyperemic response was sustained one minute after neuronal excitation in brains from rats exhibiting anhedonia-like behavior and nonstressed rats, parenchymal arterioles of hedonic rats constricted after initial transient vasorelaxation. Since neuronal and astrocytic Ca 2þ dynamics in response to neuronal excitation were similar, we suggest that the changes in neurovascular signaling are caused by different vascular properties. Our data suggest that reduced expression of K v 7.4 channels in cerebral arteries from hedonic rats may be responsible for altered neurovascular signaling.
Neurovascular coupling is essential for brain integrity (Longden et al., 2016). It has been shown to be modified by psychological stress in humans (Elbau et al., 2018) and suggested to be diminished upon stress in rodents (Han et al., 2019;Lee et al., 2015;Longden et al., 2014). The present study questions whether changes in neurovascular signaling are associated with variable stress susceptibility. In accordance with other reports, we found that vasodilation in response to neuronal activity was BaCl 2 -sensitive, suggesting the involvement of K ir 2.1 channels (Longden et al., 2016). However, the amplitude of BaCl 2 -sensitive vasodilation and the expression of K ir 2.1 channels were similar in all the three experimental groups, suggesting that this channel is not responsible for the observed difference in neurovascular signaling. This is in contrast to a previous study by Longden et al. (2014) where chronic stress led to reduced cerebrovascular K ir 2-dependent signaling. This previous study suggested that stress causes a glucocorticoid-mediated decrease in functional K ir 2 channels and thereby suppresses neurovascular signaling (Longden et al., 2014). We have previously shown a similar corticosterone level after 8 weeks of CMS in nonstressed and stress-exposed groups, suggesting a functional habituation of the hypothalamic-pituitary-adrenal axis to incessant stress regardless of hedonic status (Christiansen et al., 2012). This argues against glucocorticoid-mediated changes in K ir 2 channels in the present study. A different duration of stress exposure and different stress paradigms may therefore explain the discrepancy between our results and those of the previous study (Longden et al., 2014).
The voltage-gated K þ channel, K v 7.4 has previously been shown to be one of the most abundant K þ channels in rat . Cerebral arteries from stressed rats with unchanged hedonic state had reduced expression of K v 7.4 channels but unchanged K ir 2.1 expression. Lysate of middle cerebral arteries was blotted for the K ir 2.1 channel expression with thioredoxin as a loading control. Representative blots of K ir 2.1 and thioredoxin from the same lanes (A); the vertical white space between the bands indicates that irrelevant lanes from the same film are not shown. No difference in relative K ir 2.1 expression was seen between middle cerebral arteries from nonstressed (n ¼ 6), hedonic (n ¼ 4), and anhedonic rats (n ¼ 5) (B). Representative blots for K v 7.4 protein detection and total protein load as identified with stain-free gel loading control. The expression of K v 7.4 protein was reduced in arteries from hedonic rats (n ¼ 7) in comparison with nonstressed rats (n ¼ 9). � p < .05 for nonstressed vs. resilient rats (one-way ANOVA followed by Dunnett correction for multiple comparison).
Cerebral arteries from stressed rats with unchanged hedonic state had reduced expression of K v 7.4 channels but unchanged K ir 2.1 expression. middle cerebral artery (Zhong et al., 2010). The K v 7.4 channels were proposed to play an essential role in the control of cerebrovascular resistance and cerebral blood flow (Chadha et al., 2014). Activation of these channels hyperpolarizes cerebrovascular smooth cells, while inhibition of the channels depolarizes and constricts middle cerebral arteries (Zhong et al., 2010). We found a significantly reduced cerebrovascular expression of K v 7.4 channels and increased contractile properties of middle cerebral arteries from hedonic rats. Interestingly, this is not the first time an association between chronic stress and reduced expression of Kv7 channels has been observed. A recent study based on RNA sequencing and pharmacological intervention suggested that K v 7 membrane conductance and expression in the brain was reduced following chronic stress-induced brain injury (Ren et al., 2021).
It remains to be studied whether the association of reduced K v 7.4 channel expression and stress susceptibility is caused by an original intersubjective variability or is a result of stress-mediated expression changes. Circulating angiotensin II level that increases during stress (Yang et al., 1996) has recently been shown to increase the degradation of vascular K v 7.4 channels (Barrese et al., 2018). Whether angiotensin II modifies stress response through its regulation of vascular K v 7.4 channel expression remains, however, to be tested.
Suppression of neurovascular signaling, as we observed in hedonic rats, will diminish hyperemic responses in the brain (Longden et al., 2016). Reduced ability to match blood supply to neuronal metabolic demand might have detrimental impact on neuronal survival and brain integrity (Iadecola, 2017). Hence, hedonic rats exposed to CMS may suffer from neurological deterioration over time (Carrier et al., 2021). The acceleratory effects of stress, including cerebrovascular changes, have previously been shown to be associated with the progression of major depression, anxiety, posttraumatic stress disorder, stroke, dementia, and Alzheimer's disease (Johansson et al., 2010;Rissman et al., 2012). This suggests that stress-induced neurovascular changes may be an important modulatory factor in these pathologies. The finding of an association between decreased expression of K v 7.4 channels and reduced neurovascular signaling in stressed subjects with unchanged hedonic state provides novel knowledge on the mechanisms underlying the ability to cope with stress and how stress susceptibility may be modified.