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. Author manuscript; available in PMC: 2025 Oct 1.
Published in final edited form as: Neuropharmacology. 2024 Jun 12;257:110031. doi: 10.1016/j.neuropharm.2024.110031

L-type calcium channel regulation of depression, anxiety and anhedonia-related behavioral phenotypes following chronic stress exposure

Eric J Nunes 1, Nardos Kebede 1, Anjali M Rajadhyaksha 2, Nii A Addy 1,3,4,5
PMCID: PMC11334593  NIHMSID: NIHMS2005988  PMID: 38871116

Abstract

Exposure to chronic and unpredictable stressors can precipitate mood-related disorders in humans, particularly in individuals with pre-existing mental health challenges. L-type calcium channels (LTCCs) have been implicated in numerous neuropsychiatric disorders, as LTCC encoding genes have been identified as candidate risk factors for neuropsychiatric illnesses. In these sets of experiments, we sought to examine the ability of LTCC blockade to alter depression, anxiety, and anhedonic-related behavioral responses to chronic unpredictable stress (CUS) exposure in female and male rats. Rats first underwent either 21 days of CUS or no exposure to chronic stressors, serving as home cage controls (HCC). Then rats were examined for anhedonia-related behavior, anxiety and depression-like behavioral responses as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). CUS exposed females and males showed anhedonic and anxiogenic-like behavioral responses on the SPT and EPM, respectively, when compared to HCCs. In female and male rats, systemic administration of the LTCC blocker isradipine (0.4 mg/kg and 1.2 mg/kg, I.P.) attenuated the CUS-induced decrease in sucrose preference and reversed the CUS-induced decrease in open arm time. In the FST, systemic isradipine decreased immobility time across all groups, consistent with an antidepressant-like response. However, there were no significant differences in forced swim test immobility time between HCC and CUS exposed animals. Taken together, these data point to a role of LTCCs in the regulation of mood disorder-related behavioral phenotype responses to chronic stress exposure.

Introduction

Physical and psychological stressors are ever present in our environment, and these stressors are often unpredictable and can evoke physiological and psychological changes (Selye, 1936, 1956; Gerra et al., 2001; Kudielka et al., 2004; Giles et al., 2014). This stress response can be adaptive when made in response to acute challenges. For example, during a state of hunger, food motivation increases as a response to hunger to promote food seeking behaviors (Salamone et al., 1991; Hanlon et al., 2004; Randall et al., 2012). Moreover, the anxiety of a looming grant submission deadline may motivate us to work longer hours to complete it in time. In this context, stress can serve a beneficial role to reduce hunger and promote attentional resources to finish a grant submission. This form of stress, which serves to promote adaptive behaviors and is considered beneficial for the survival of the organism, has been referred to as ‘eustress’ (Villalba and Manteca, 2019; Lu et al., 2021). Yet, exposure to unresolved and persistent psychological stressors correlates with increased incidence of mood-related disorders, particularly in individuals with genetic vulnerabilities and preexisting mental health conditions (Caspi et al., 2003; Cohen et al., 2007; Lex et al., 2017).

In humans, L-type calcium channels (LTCCs) have been implicated in neuropsychiatric disorders including drug addiction and depression (Pinggera and Striessnig, 2016; Kabir et al., 2017a; Terrillion et al., 2017). Findings from genome-wide association studies (GWAS) have demonstrated that CACNA1C and CACNA1D, which code for the Cav 1.2 and Cav1.3 LTCC subtypes respectively, are top candidate risk genes for neuropsychiatric diseases, including mood disorders (Sklar et al., 2008; Cosgrove et al., 2017; Kabir et al., 2017a; Liu et al., 2022). Furthermore, imaging studies in humans have linked risk variants of LTCC subtypes with alterations in neuronal processing and network connectivity among patients with bipolar disorder, schizophrenia, and substance use disorders (Gurung and Prata, 2015; Kabir et al., 2017a; Romme et al., 2017). Acute and chronic stress has been shown to increase Cav1.2 mRNA in the hippocampus and amygdala (Maigaard et al., 2012). Furthermore, chronic stress in mice also increases Cav1.2 protein levels in the pre-frontal cortex that is correlated with pro-depressive and anxiogenic-like behavioral responses (Bavley et al., 2017). Functionally, chronic infusion of the LTCC activator Bay K8644 into the VTA of Cav1.2 DHP−/− mice, leads to depressive behaviors in both the SPT and FST tests and diminishes social interaction as observed in the social approach paradigm (Martínez-Rivera et al., 2017a). In contrast to LTCC activation, mice deficient in Cav1.3 display behaviors resembling anti-depressant and anti-anxiety-like effects, as assessed through the tail suspension test, FST, and EPM tests (Busquet et al., 2010). Together, data from these preclinical and clinical studies suggests that LTCCs may mediate behavioral responses induced by chronic stressors, and that LTCC mechanisms may mediate symptoms associated with neuropsychiatric illnesses. However, it is unclear whether pharmacological manipulation of LTCCs, with an LTCC inhibitor, alters mood disorder-related phenotypes resulting from chronic stress exposure. Notably, this research question has translational relevance, given the availability of FDA approved LTCC inhibitors that are currently prescribed as antihypertensive medications (Arzilli et al., 1993; Chrysant and Cohen, 1997).

In the current study, we first examined the behavioral effects of 21 days of CUS, on pro-depressive and anxiogenic behaviors in male and female rats. Rats exposed to stress displayed pro-depressive and anxiogenic-like behavioral responses. Administration of the LTCC blocker isradipine attenuated the CUS-induced decrease in sucrose preference and time spent in the open arm, indicating an anti-depressant like effect. LTCC inhibition with isradipine also decreased time spent immobile in the FST in both CUS and HCC rats. Taken together, these data suggest that LTCCs mediate pro-depressive and anxiogenic-like behaviors induced by exposure to CUS and that the antihypertensive LTCC blocker, isradipine can alleviate these conditions and phenotypes.

Materials and Methods

2.1. Subjects

Across all experiments, age matched adult male (n = 98) and female (n = 95) Sprague Dawley rats (250–279 g for males, 179–199 g for females; Charles River Laboratories, Wilmington, MA, USA) were used. Throughout the study, cohorts of male and female rats were run separately, alternating between male and female cohorts to prevent sex-specific odor cues and to ensure that males and females were run throughout the year, accounting for potential time of year variations. Upon arrival rats were doubly housed and allowed to acclimate to the housing facility for 7 days. Rats were maintained under a 12h light/dark cycle with climate control between 22 and 24 °C. Food and water was available ad libitum, unless noted otherwise. Rats were weighed every other day and experiments were conducted according to the ethical guidelines and protocols approved by the Institutional Animal Care and Use Committee at Yale University (#2022–11366).

2.2. Pharmacological agents

The non-selective L-type calcium channel blocker, isradipine, (Sigma Aldrich, St. Louis, MO, USA) was dissolved in 1.7 mM EtOH Intraperitoneal doses of isradipine (0.0 mg/kg, 0.4 mg/kg and 1.2 mg/kg, I.P.) were selected based on pilot experiments and previously published behavioral and physiologically effective doses, which demonstrated behaviorally relevant effects on drug and mood-related behavioral responses (Pucilowski et al., 1995; Cohen and Sanger, 1997; Addy et al., 2018). In rats, the half-life of isradipine has been estimated at 3–4 hours (Tse et al., 1989).

2.3. Chronic unpredictable stress

Female and male rats were exposed to 21 days of chronic unpredictable stress (CUS). CUS rats were housed in a separate room from the home cage control (HCC) rats, which were doubly housed and remained in the housing colony. The CUS protocol consisted of the following stressors which were randomly applied twice a day that followed a semi-random order to reduce their predictability: light off during the day, 24 hr social isolation, 12 hr food and water deprivation, cage spray with peppermint, overnight cage tilt, 15 minutes of forced swimming, overnight social crowding, exposure to 4°C for 1 hr, 12 hr exposure to strobe light, and cage rotation at 60 rotations per minute for 1 hr. Multiple experimenters applied the CUS stressors to further reduce predictability of the experimenter and stressor. Different experimenters performed CUS exposure in a counterbalanced order, randomizing the experimenter applying the stressors both within and across cohorts.

2.4. Sucrose preference test

Following CUS exposure, the initial behavioral assessment conducted was the sucrose preference test (SPT). Each experimental cohort consisted of CUS and HCC rats with 24 to 30 rats in each group. These rats first underwent a habituation phase where, for 48 hours in their home cage, they were provided with a 1% sucrose solution (Sigma, St Louis, MO, USA) as an alternative to their regular water bottle. To account for potential side biases, the positioning of the sucrose bottles in the cage was alternated among the animals. Throughout the habituation phase, unrestricted access to their usual chow was provided. For the test, rats first underwent a 4-hour water deprivation period and were subsequently presented with two identical bottles for an hour: one with a 1% sucrose solution and the other with water. Before this hour-long test, 15 minutes prior to the test, rats received a systemic injection of isradipine at varying doses (0.0 mg/kg, 0.4 mg/kg, or 1.2 mg/kg, I.P.). To determine the sucrose and water intake, the bottles' weight difference before and after the test was measured. The preference for sucrose was ascertained as the weight ratio of consumed sucrose versus water during this hour. After the test, rats were immediately given access to their water bottles.

2.5. Elevated plus maze

Following the SPT, the same rats were then tested on the elevated plus maze (EPM). Each experimental cohort contained between 24 and 30 rats consisting of CUS and HCC rats. The EPM is a plus-shaped maze elevated 70 cm above the floor, that contains a center zone, two open arms, and two enclosed arms. Underneath the EPM, a rubber mat was placed to cushion the rat if it falls off the maze. 15 minutes prior to the 5 min test, rats were given a systemic injection of isradipine (0.0 mg/kg, 0.4 mg/kg, or 1.2 mg/kg, I.P). Drug assignments were counterbalanced from the previous SPT experiment to minimize repeated drug effects. Rats were placed in the center of the maze and behavior was monitored by an overhead video camera. Data was quantified by AnyMaze computer software (Stoelting Company, Wooddale, IL, USA) to determine time spent in center zone, open arms, and closed arms. The maze was wiped down with a 70% ethanol solution between test sessions to prevent bias from the scent of the previous rat.

2.6. Forced swim test

Two to three days post-EPM test, the same group of rats previously tested on the SPT and EPM underwent the forced swim test (FST). This test was conducted consistent with earlier protocols from our lab and others (Rada et al., 2006; Duman, 2010; Voleti et al., 2013; Addy et al., 2015).). Each of these experimental groups contained 24 to 30 rats, inclusive of both CUS and HCC rats. For the FST's pre-test phase, the rats were individually introduced to a transparent polypropylene cylindrical water tank (with a diameter of 30 cm, height of 60 cm, water depth surpassing 40 cm, and water temperature maintained between 23 and 26 °C). This 15-minute pre-test was meant to set a consistent baseline for immobility. Notably, during this pre-test, no pharmacological interventions were applied, and behavioral actions were not captured. The main FST was conducted 24 hours post-pre-test and spanned a total of 10 minutes, all of which were video recorded. However, for the purpose of analysis, only minutes 1–6 was considered, in alignment with our lab's and other established FST analytical procedures (Addy et al., 2015; Duman, 2010). A systemic injection of isradipine (at doses of 0.0 mg/kg, 0.4 mg/kg, or 1.2mg/kg, I.P.) was administered to the rats 15 minutes prior to the FST. The drug assignments were counterbalanced to diminish potential effects from repeated drug administration. During the FST, immobility was characterized as a cessation of the swimming activity, evident when the rats displayed no hind or forepaw movement. The rats were considered immobile when they adopted a passive float, making only the slightest movements to keep their snouts above water. A blinded experimenter, unaware of the treatment specifics, quantified each FST session using a stopwatch. After each raťs test, the tank water was refreshed. Once the session concluded, the rats were towel-dried and then placed in a heated cage for another 30 minutes to ensure they were thoroughly dry before returning them to their regular enclosures.

2.7. Statistical analysis

GraphPad Prism 10 (Graph Pad Software, San Diego, CA., USA) was used to conduct all statistical analyses. For initial CUS experiments, a two-way Analysis of Variance (ANOVA) was used to determine if there were any significant main effects of stress exposure or sex, or any significant interactions. For weight analysis, a three-way (ANOVA) was used to determine if there were any significant main effects of weight, day, and sex and if significant interactions were present. CUS and LTCC inhibition experiments, a three-way (ANOVA) was used to determine if there were any significance main effects of stress exposure, drug administration, or sex, and whether there were any significant interactions. If significant main effects were observed, comparison analyses were then performed with Bonferroni t-test correction, to compare between specific groups. All groups were tested for normal distributions and equal variance using the Shapiro-Wilk test.

3. Results

3.1. Chronic unpredictable stress decreases body weight in both female and male rats.

Female and male rats in both the HCC and CUS group were weighed every other day during the experimental timeline. A 3-way factorial ANOVA with stress exposure, sex, and day as factors was performed to examine weight outcome. The analysis revelated significant main effects of stress exposure F (1, 810) = 23876, p < 0.0001), sex (F (1, 810) = 198.1, p < 0.0001), and day (F (9, 810) = 389.2, p < 0.0001). A significant interaction between stress exposure, sex, and day also emerged F (9, 810) = 1.926, p = 1.92, p = 0.453. Post hoc analysis revealed significant differences in the effect of stress exposure on the weights of female and male rats. Female rats began to show significant weight differences in response to stress on Day 12, while male rats began to show these differences on Day 10 (p < 0.01, t-test with a Bonferroni correction, Fig. 1A).

Fig. 1.

Fig. 1.

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Chronic unpredictable stress (CUS) alters weight gain in female and male rats. (A) Female rats exposed to CUS had a significantly reduced body weight vs. home cage controls (HCC) (**p < 0.01, days 12–20, post hoc with Bonferroni correction). (B) Male rats exposed to CUS had a significant reduction in body weight compared to CUS controls (**p < 0.01, days 10–20 with Bonferroni correction). Error bars indicate the standard error from the mean (SEM).

3.2. Chronic unpredictable stress induces a pro-depressive and anxiogenic-like behavioral phenotype in female and male rats.

We first sought to determine whether 21 days of CUS would alter behavioral responses in female and male rats, as assessed by the SPT, EPM, and FST. On day 21, CUS rats received their final set of stressors before the start of the SPT. A 2x2 factorial ANOVA with sex and stress exposure as factors was performed on outcome measures in the SPT, EPM and FST. A significant main effect of stress was observed on sucrose preference (F(1,33) = 41.0, p =0.006, Fig 1A) but there was no significant main effect of sex (F(1, 33) = 1.751, p > 0.05, Fig 1A). Post hoc analysis further revealed a significant difference in females and males who received the CUS vs HCC (p < 0.01, t-test with a Bonferroni correction, Fig 1A), showing that exposure to CUS decreased sucrose preference in female and male rats. On the elevated plus maze a significant main effect of stress on time spent in the open arm was observed (F(1, 38) = 40.14, p = 0.002, Fig 1B), with no effect of sex (F (1, 38) = 0.2891, p > 0.05, Fig 1B. Post hoc analysis further revealed a significant difference in females and males who received the CUS vs HCC (p < 0.01, t-test with a Bonferroni correction, Fig 1B), showing that exposure to CUS decreased time spent in the open arms in female and male rats. To determine whether exposure to CUS affects locomotion, we examined total distance traveled on the EPM. No significant main effect of stress was observed (F (1, 36) = 0.1533, p > 0.01, Fig 1C). Finally, on the forced swim test, no significant main effect of stress was observed on immobility time in female or male rats. However, a significant main effect of sex on immobility time emerged (F (1, 36) = 11.52, p = 0.003, Fig 1D). Post hoc analysis revealed that males in both the CUS and HCC showed more immobility time than female CUS and HCC rats (p < 0.01, t-test with a Bonferroni correction, Fig 1D).

3.3. L-type calcium channel blockade with isradipine attenuates CUS-induced anhedonia in female and male rats as measured by the sucrose preference test.

Next, we examined whether the LTCC blocker isradipine could alter the behavioral phenotype induced by CUS exposure in female and male rats. A significant main effect of CUS exposure was observed on sucrose preference (F(1, 116) = 19.68; p = 0.006, Fig 2A, 2B), revealed by a decrease in sucrose preference after stress exposure. There was also a significant effect of isradipine on preference for sucrose (F(2, 116) = 7.975; p = 0.004, Fig 2.). These effects were observed irrespective of sex, as there was no significant main effect of sex (F–(1, 116) = 0.01; p > 0.05, Fig 2.).

Fig. 2.

Fig. 2.

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Chronic unpredictable stress induced a pro-depressive and anxiogeniclike behavioral phenotype in female and male rats. (A) Female and male rats exposed to CUS showed a reduction in sucrose preference compared to HCC and (B) reduced time spent in the open arm in the EPM compared to HCC (*p < 0.05, CUS vs HCC with post-hoc with Bonferroni correction). (C) Stress exposure did not alter total distance traveled. (D) Stress exposure did not alter immobility time in the forced swim test (FST). However, male rats showed greater time immobile in the FST compared to female rats (*p < 0.05, CUS vs HCC with post-hoc with Bonferroni correction). Error bars indicate the standard error from the mean (SEM).

A significant interaction between isradipine and stress also emerged (F(2,116) = 17.84; p = 0.002), indicating that the behavioral effect of isradipine was dependent on CUS exposure. Post hoc analysis revelated a significant decrease in sucrose preference in CUS exposed vs HCC rats in the 0.0mg/kg isradipine group for both females (p < 0.05. t-test with Bonferroni correction, Fig 2A) and males (p < 0.05. t-test with Bonferroni correction, Fig 2B). For female rats exposed to CUS, a significant difference was observed between those receiving 0.0mg/kg isradipine vs. 1.2mg/kg isradipine (p < 0.05. t-test with Bonferroni correction, Fig 2A). Male rats exposed to CUS showed a significant increase in sucrose preference at the 0.4mg/kg and 1.2mg/kg compared to the 0.0mg/kg dose of isradipine (p < 0.05. t-test with Bonferroni correction, Fig 2B).–Thus, CUS female rats only showed a reversal of the sucrose preference deficit at the high dose of isradipine, while male CUS rats showed increases in sucrose preference at both isradipine doses. Isradipine did not alter sucrose preference in female and male HCC rats (p > 0.05. t-test with Bonferroni correction, Fig 2), further highlighting that the behavioral effect of isradipine on sucrose preference only emerged in CUS exposed rats.

3.4: L-type calcium channel blockade in female and male rats reverses CUS-induced anxiogenic effects in the elevated plus maze.

On the EPM, 24 hours after the SPT experiment, analysis of open arm time revealed a significant main effect of CUS exposure (F–(1, 124) = 15.83, p = 0.001, Fig 3) and isradipine (F(2, 124) = 7.676, p 0.002, Fig 3)–but no effect of sex −F(1, 124) = 0.6256, p > 0.05, Fig 3). A significant interaction between isradipine and CUS exposure was observed (F(2, 124) = 15.70, p = 0.004 Fig 3) indicating that the behavioral effect of isradipine was dependent on CUS exposure. Post hoc analysis revealed significant a decrease in time spent in the open arm in rats exposed to CUS vs HCC in the 0.0mg/kg isradipine groups in both female and male rats (p < 0.05. t-test with Bonferroni correction, Fig 3), demonstrating the anxiogenic-like effect of CUS exposure. In female CUS exposed rats, a significant difference was observed between those treated with 0.0 mg/kg isradipine and 1.2 mg/kg isradipine (p < 0.05. t-test with Bonferroni correction, Fig 3A). In male rats exposed to CUS, both the 0.4mg/kg and 1.2mg/kg isradipine doses increased open arm time compared to the 0.0mg/kg dose of isradipine (p < 0.05. t-test with Bonferroni correction, Fig 3B). In summary, the anxiogenic effects of CUS exposure were reversed by isradipine administration, as revealed by a significant stress x isradipine interaction, and by post-hoc analyses. In contrast, isradipine did not alter open arm time in HCC female and male rats. (p > 0.05. t-tests with Bonferroni corrections, Fig 3).

Fig. 3.

Fig. 3.

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Isradipine attenuated the CUS-induced decrease in sucrose preference in (A) female and (B) male rats. Isradipine did not alter sucrose preference in HCC subjects. (**p < 0.01, ***p < 0.001, post-hoc with Bonferroni correction). HCC vs. CUS subjects at 0.0 mg/kg isradipine. CUS subjects at 0 mg/kg vs. 0.4 mg/kg or 1.2 mg/kg isradipine. Error bars indicate the standard error from the mean (SEM).

3.5: L-type calcium channel blockade decreases immobility time in female and male rats in the forced swim test.

On the FST, 48 hours after the EPM test, there was a significant main effect of isradipine (F–(2, 111) = 12.66, p = 0.006, Fig 4), and sex (F–(1, 111) = 21.82, p = 0.02, Fig 4), but no effect of stress (F–(1, 111) = 2.971, p > 0.05, Fig 4). There was a significant interaction between isradipine and sex (F (2, 111) = 3.785; p = 0.005., Fig 4), and a significant interaction between isradipine and stress F(2,111) = 3.78; p = 0.003, (Fig 4). Specifically, isradipine administration led to an overall decrease in immobility time, as demonstrated by a main effect of isradipine. Furthermore, male rats showed increased immobility time compared to females, as also reflected by a main effect of sex. Finally, isradipine effects on immobility time varied, based on sex and based on stress exposure.

Fig. 4.

Fig. 4.

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Isradipine reversed CUS-induced decrease in time spent in the open are in (A) female and (B) male rats. No effect of isradipine on open arm time in HCC in (A) female or (B) male rats (**p < 0.01, ***p <0.001, post-hoc with Bonferroni correction). HCC vs. CUS subjects at 0.0 mg/kg isradipine. CUS subjects at 0 mg/kg vs. 0.4 mg/kg or 1.2 mg/kg isradipine. Error bars indicate the standard error from the mean (SEM).

In female and male rats, administration of 1.2mg/kg isradipine decreased immobility time in both HCC and CUS rats, compared to 0.0 mg/kg isradipine rats (p < 0.05. t-test with Bonferroni correction, Fig 4A, 4B). Interestingly, the 0.4 mg/kg isradipine dose did not alter FST behavior of female or male HCC rats. In contrast, administration of 0.4 mg/kg isradipine was sufficient to decrease immobility time in both CUS exposed female (p < 0.05. t-test with Bonferroni correction, Fig 4A) and male rats (p < 0.05. t-test with Bonferroni correction, Fig 4B). L-type calcium channel blockade with isradipine was sufficient to decreases time spent immobile in both CUS and HCC females and males. However, CUS exposure shifted the isradipine dose response, leading to a decrease in immobility time at both the 0.4 mg/kg and 1.2 mg/kg doses.

Discussion

In these experiments, we sought first to determine and validate that CUS exposure induced pro-depressive and anxiogenic-like behavioral responses in rats in our laboratory. Female and male rats exposed to 21 days of CUS exhibited depressive and anxiogenic-like behaviors compared to non-stressed controls, including a significant reduction in body weight Specifically, female rats had reduced sucrose preference and spent less time in the open arm of the EPM. Administration of the L-type calcium channel blocker (LTCC), isradipine, in female or male rats attenuated the behavioral effects of CUS exposure in the SPT and EPM. In male rats, this reversal of CUS-induced anhedonic and anxiogenic responses was observed with both doses of isradipine. In female rats, in contrast, the isradipine-induced reversal was only observed at the high isradipine dose (1.2 mg/kg). While CUS exposure did not alter immobility time in the FST, isradipine administration led to an antidepressant-like effect, as reflected in a decrease in immobility time of both CUS and HCC. Furthermore, CUS exposure shifted the isradipine dose response in the FST. Specifically, a low dose isradipine administration was sufficient to decrease immobility time in male and female CUS rats, whereas it had no effect in non-exposed HCC rats. Together, these data reveal the ability of systemic LTCC inhibitor administration to alter anhedonia, anxiety and stress-related behaviors in male and female rats, and to specifically reverse the anhedonic and anxiogenic effects of chronic unpredictable stress.

The FST model examines immobility time and struggling behavior, which is correlated with antidepressant efficacy. Specifically, extended immobility reflects a pro-depressive-like phenotype while reduced immobility suggests an antidepressant-like effect (Slattery and Cryan, 2012). In humans, agents eliciting pro-depressive effects typically extend immobility time in the FST, whereas those with antidepressant effects tend to reduce immobility time (Carratalá-Ros et al., 2023). Moreover, immobility time can also reflect other aspects of such as learning and stress coping behaviors (West, 1990; De Kloet and Molendijk, 2016)). Our data demonstrates that female and male rats exposed to 21 days of CUS did not exhibit a significant alteration in immobility time during the FST compared to those in the HCC group. It is possible that the duration of CUS used in our experiments was not sufficient to induce changes in immobility time in our paradigm. Thus, a more extended stress exposure could be employed in future experiments. However, a critical observation in our study was the significant reduction in immobility time in both CUS and HCC groups following the administration of the LTCC blocker, isradipine. This finding is consistent with prior findings showing the ability of LTCC blockers to diminish immobility durations in the FST (Mogilnicka et al., 1987; Czyrak et al., 1990; Aburawi et al., 2007). Given that the FST itself is considered an acute stressor, the FST results, in combination with our SPT and EPM results, demonstrate that LTCC blockade can attenuate adverse behavioral responses to both acute and chronic stress conditions. Furthermore, our FST experiments revealed a significant interaction between isradipine and stress, with CUS-exposed animals exhibiting heightened sensitivity to low-dose isradipine (0.4 mg/kg) in the FST. This is likely due to CUS-induced neural plasticity, with LTCC blockade potentially reversing or compensating for some aspect of this CUS-induced plasticity. Indeed, evidence has demonstrated that CUS affects mesolimbic DA release depending on the intensity, duration, and availability of the stressor ()(Holly and Miczek 2016). Studies have revealed that 3 to 7 weeks of exposure to CUS increased DA levels in the NAc, while other studies have shown a decrease in DA levels (Stamford et al., 1991; Willner et al., 1991; Di Chiara et al., 1999; Mangiavacchi et al., 2001). The effects of chronic stress have also been shown to decrease normal bursting activity of VTA DA neurons (Tye et al., 2013; Chang and Grace, 2014). Other brain regions, like the prefrontal cortex, also experience stress-induced changes. For instance, D1-containing pyramidal neurons in CUS mice showed a reduced firing threshold (Anderson et al., 2019). , Our previous findings showed that systemic isradipine administration altered cue-induced reward seeking and increased dopamine signaling after cocaine exposure and abstinence, but not after sucrose exposure and abstinence, and not during cocaine taking behavior (Addy et al., 2018). These prior behavioral and neurochemical findings suggested LTCC inhibition effects that emerged due to cocaine exposure and abstinence-induced plasticity in the mesolimbic dopamine system. Together, the findings may indicate a specific interaction between LTCC blockade and cocaine and stress-induced plasticity on drug and mood-related behavioral responses. However, additional experiments are needed to determine if similar neuroplastic mechanisms play a role in these isradipine-induced effects after cocaine or chronic stress exposure. The findings in our current study highlights the potential of LTCC blockers to serve as therapeutic agents for stress-related disorders. Indeed, these compounds, some of which are already FDA-approved for hypertension, may offer a promising pathway for the treatment of stress-related illnesses. Future clinical research can further investigate the efficacy of LTCC blockade in attenuating adverse responses to both acute and chronic stressors.

The behavioral effects of CUS are often evaluated by using the SPT and EPM which have been used extensively in behavioral neuroscience in both mice and rats (He et al., 2020; Nunes et al., 2020). The SPT is traditionally used as a task to assess anhedonia, which is considered a core symptom of major depressive disorder (MDD), characterized by a diminished capacity to experience pleasure (Fawcett et al., 1983; Liu et al., 2018). Our data shows that female and male rats exposed to a 21-day CUS protocol demonstrated a decrease in sucrose preference. The decrease in preference for sucrose was significantly attenuated following the administration of the LTCC blocker isradipine. Interestingly, in female and male controls (HCC), isradipine did not affect behavior on the SPT. Within this context, a ceiling effect on the SPT may have occluded a behavioral effect of isradipine, as HCC rats were already exhibiting sucrose preference rates ranging between 80 to 90 precent. Additionally, LTCC blockade efficacy on behavior may also be linked to the presence of stress-related or drug-induced synaptic plasticity.

To assess anxiogenic-like behavior, we used the EPM, a well-established anxiety model in rats and mice that primarily gauges the avoidance of open spaces, considered a proxy for anxiety-like behavior (Walf and Frye, 2007). Our findings indicate that both female and male rats exposed to a 21-day CUS exhibit an anxiogenic-like phenotype, evidenced by a reduction in open arm time in the EPM. Our data revealed that this increase in anxiety-like behavior was significantly attenuated by isradipine. This outcome suggests a potential anxiolytic effect of isradipine, underscoring its prospective therapeutic value in treating the anxiety component often comorbid with depression and anhedonia. Taken together, this suggests that LTCC blockade's ability to produce some of these behaviorally relevant effects depends on chronic stress exposure. Moreover, these findings suggest a potential therapeutic role for isradipine in alleviating anhedonia-like and anxiogenic-like behavioral phenotypes. While these results are promising, future research should examine the efficacy of isradipine across a broader spectrum of tasks, especially those tailored to discerning motivational aspects of anhedonia and effort-related choice, which are also impaired in MDD (Salamone and Correa, 2012; Treadway et al., 2012) Such studies would be pivotal in elucidating the specific neurobehavioral domains affected by stress and isradipine.

The examination of sex-based differences in mood-related disorders remains a critical focus in neuropsychiatric research. A notable sex difference was observed in the FST data, with male rats exhibiting increased immobility time compared to females, aligning with multiple studies that have reported similar findings in Sprague Dawley male rats (Alonso et al., 1991; Walker et al., 1995; Verma et al., 2010; Simpson et al., 2012). Despite this trend, the literature presents a mixed picture, with some research indicating higher immobility times in female rats and others finding no significant sex differences (Pitychoutis et al., 2011; Rayen et al., 2011; Allen et al., 2012; Brummelte et al., 2012; Hong et al., 2012; Warner et al., 2013) The variability in these outcomes across different laboratories remains unexplained but could potentially stem from variations in experimental methodologies or even the sex of the experimenter, suggesting that subtle environmental or procedural nuances may influence the behavioral responses of rats in a sex-specific manner. However, in our study, the LTCC blocker isradipine, showed efficacy in mitigating pro-depressive and anxiogenic-like behaviors in both female and male rats subjected to a 21-day CUS protocol. Yet, while the behavioral outcomes following CUS exposure were consistent across sexes, our data also revealed sex-specific differences in the dose responsiveness to isradipine on the SPT and EPM. Male rats exhibited attenuation of reduced sucrose preference and an increased time spent in the open arm on the EPM across all administered doses. In contrast in female rats, this isradipine-induced reversal was only observed at the high dose of isradipine that was used in our study. This differential dose efficacy suggests the possibility of sex-dependent differences in LTCC subtype expression or functionality. Nevertheless, isradipine administration in both female and male rats attenuated the pro-depressive and anxiogenic-like phenotype induced by CUS exposure. Interestingly, isradipine did not affect behavior on the SPT or EPM in rats not exposed to CUS. This may suggest changes to LTCCs expression and activity after stress exposure. Future investigations should examine LTCC expression between sexes, particularly in brain regions identified as vulnerable to chronic stress pathology.

A potential limitation of these sets of experiments includes the set of stressors may affect behavioral outcomes on our measures. For example, food and water deprivation have been linked to physiological changes that may affect sucrose consumption and preference, including a reduction in body weight (Gilbert and Sherman, 1970; Gillette-Bellingham et al., 1986). However other reports indicate that 12 hr food and water deprivation does not affect sucrose consumption or reduce weight gain (He et al., 2020; Fonseca-Rodrigues et al., 2022). Despite these conflicting reports food and water deprivation remain commonly used stressors in chronic unpredictable stress (CUS) protocols. To mitigate the potential impact of food and water deprivation on sucrose preference and body weight, we refrained from subjecting the animals to this stressor for 72 hours preceding any behavioral test. However, it is worth noting that other stressors, cold stress, can also influence energy balance and metabolism, with implications for weight gain and loss (Wang et al., 2015; El Marzouki et al., 2021). Furthermore, disrupting the natural circadian rhythms of rodents has been shown to impact several metabolic parameters including changes in weight, insulin sensitivity, lipid metabolism and hormonal changes (Wideman and Murphy, 2009; Kim et al., 2015; Dauchy et al., 2019). We also acknowledge the limitation of using forced swimming as both a stressor and behavioral measure. Repeated exposure to the swimming may lead to familiarization with the behavioral task and introduce aspects of learning and memory. Moving forward, swimming stress would be replaced by another stressor that is separate from the behavioral assay. Nevertheless, we demonstrate the ability of LTCC blockade with isradipine to attenuate affect immobility time in both stressed and non-stressed rats.

In conclusion, our findings reveal a significant role of LTCC in modulating chronic stress-induced anhedonic and anxiogenic responses in both female and male rats. The behavioral manifestations induced by CUS, notably diminished sucrose preference and altered EPM behavior, were effectively attenuated by LTCC blockade with isradipine. Furthermore, the ability of isradipine to decrease immobility time in both stressed and control cohorts suggests a broader role for LTCCs in response to acute stressors. This is consistent with other findings demonstrating the role of LTCCs in regulation of mood-related behaviors and the ability of LTCC blockers to produce anti-depressant like behavioral responses on models like the FST (Cryan et al., 2005; Kabir et al., 2017b; Martínez-Rivera et al., 2017b). Interestingly, chronic stress exposure also increases the efficacy of isradipine administration in producing antidepressant-like effects, further highlighting the ability of LTCC blockade in mediating mood related behavioral phenotypes, following chronic stress. The consistency of our findings across females and males also supports the potential utility of LTCC blockade for reversing adverse stress related responses across both sexes. However, the potential for cardiac effects is possible given that isradipine has an established use as an antihypertensive. In our behavioral experiments, we used isradipine doses that are lower than those typically utilized in rodent hypertension studies (Levy et al., 1994; Tomassoni et al., 2003). Clinically, in healthy individuals, daily oral administration of 10mg of isradipine was well tolerated, with no participant drop out due to adverse effects (Wang et al., 2013). In non-treatment seeking cocaine dependent individuals 15mg and 30mg of isradipine was shown not to produce significant adverse effects on cardiovascular parameters (Johnson et al., 2005). Taken together, our data provides further evidence that LTCC blockers, already approved by the FDA, could emerge as promising medications for treating acute and chronic stress-related disorders.

Fig. 5.

Fig. 5.

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lsradipine decreased time spent immobile in the FST in HCC and CUS exposed (A) female and male rats (*p < 0.05, **p < 0.01, post-hoc with Bonferroni correction). HCC subjects at 0 mg/kg vs. 0.4 mg/kg or 1.2 mg/kg isradipine. CUS subjects at 0 mg/kg vs. 0.4 mg/kg or 1.2 mg/kg isradipine. Error bars indicate the standard error from the mean (SEM).

Highlights.

  • Chronic stress induced anhedonic and anxiogenic responses in female and male rats.

  • Isradipine inhibition reversed chronic stress-induced behavioral deficits.

  • Isradipine decreased FST immobility time in chronic stress and control rats.

ACKNOWLEDGMENTS

This work was supported by grant R01 DA050454 (NAA) and R01 DA053261 (AMR) from the National Institute of Drug Abuse (NIDA). This work was also funded in part by the State of Connecticut, Department of Mental Health, and Addiction Services, but this publication does not express the views of the Department of Mental Health and Addiction Services or the State of Connecticut. The views and opinions expressed are those of the authors.

Footnotes

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DISCLOSURES

Royalties - Tyndale House Publishers (NAA). Speakers Bureau Consultation Fees - American Program Bureau (NAA).

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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