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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Neuropharmacology. 2020 Apr 5;171:108089. doi: 10.1016/j.neuropharm.2020.108089

Examining the role of muscarinic M5 receptors in VTA cholinergic modulation of depressive-like and anxiety-related behaviors in rats.

Eric J Nunes 1, Laura E Rupprecht 1, Daniel J Foster 2,3,4, Craig W Lindsley 2,3,5, P Jeffrey Conn 2,3,4, Nii A Addy 1,6,7,*
PMCID: PMC7313677  NIHMSID: NIHMS1585669  PMID: 32268153

Abstract

Acetylcholine is implicated in mood disorders including depression and anxiety. Increased cholinergic tone in humans and rodents produces pro-depressive and anxiogenic-like effects. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate these responses in male rats, as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and the forced swim test (FST). However, these effects have not been examined in females, and the VTA muscarinic receptor subtype(s) mediating the pro-depressive and anxiogenic-like behavioral effects of increased cholinergic tone are unknown. We first examined the behavioral effects of increased VTA cholinergic tone in male and female rats, and then determined whether VTA muscarinic M5 receptors were mediating these effects. VTA infusion of the acetylcholinesterase inhibitor physostigmine (0.5 μg, 1 μg and 2 μg/side) in males and females produced anhedonic-like, anxiogenic, pro-depressive-like responses on the SPT, EPM, and FST. In females, VTA administration of the muscarinic M5 selective negative allosteric modulator VU6000181 (0.68 ng, 2.3 ng, 6.8 ng/side for a 3 μM, 10 μM, 30 μM/side infusion) did not alter SPT, EPM nor FST behavior. However, in males intra-VTA infusion of VU6000181 alone reduced time spent immobile on the FST. Furthermore, co-infusion of VU6000181 with physostigmine, in male and female rats, attenuated the pro-depressive and anxiogenic-like behavioral responses induced by VTA physostigmine alone, in the SPT, EPM, and FST. Together, these data reveal a critical role of VTA M5 receptors in mediating the anhedonic, anxiogenic, and depressive-like behavioral effects of increased cholinergic tone in the VTA.

1. Introduction

In the United States, about 20% of the adult population has some form of mental illness (Hasin and Grant, 2015). Major depressive disorder (MDD) is a common mental illness that can be chronic, reoccurring, and resistant to current treatments. Symptoms of depression such as low mood, anxiety, and psychomotor slowing are associated with personal, occupational, and social problems, which can lead to suicidal ideation and behavior (Fava et al., 2004; Melhem et al., 2019). Furthermore, depression is highly co-morbid with anxiety-related disorders, which makes remission of depressive symptoms more challenging and presents a more pernicious clinical symptomology (Kessler et al., 2005; Twenge et al., 2019). Current pharmacological treatment options to treat depression and anxiety are of limited efficacy (Bares et al., 2017; Rush et al., 2004). Therefore, new mechanistic insight into the underlying etiology of depression and anxiety are required for the development of new and more effective treatments.

The mesolimbic dopamine system, which comprises the ventral tegmental area (VTA) to nucleus accumbens (NAc) pathway, has been implicated in depression and anxiety in both pre-clinical and clinical studies (Addy et al., 2015; Friedman et al., 2014; Small et al., 2016; Treadway and Zald, 2011). Phasic dopamine (DA) activity, which produces a rapid but transient increase in extracellular DA concentrations in the NAc has been demonstrated to play a casual role in pro-depressive and anti-depressant-like behavioral responses in rodents (Chaudhury et al., 2013; Friedman et al., 2014; Tye et al., 2013). The VTA receives cholinergic input from the laterodorsal tegmentum, which regulates phasic dopamine activity within the VTA to NAc pathway (Chen and Lodge, 2013; Lodge and Grace, 2006; Xiao et al., 2016). Prior work from our laboratory has demonstrated that VTA muscarinic and nicotinic receptors regulate phasic DA release in the NAc (Solecki et al., 2013). Furthermore, cholinergic receptor mechanisms in the VTA have been shown to regulate not only phasic DA release, but also depressive and anxiety-like behaviors in rodents (Addy et al., 2015; Forster and Blaha, 2000; Nunes et al., 2019; Small et al., 2016; Steidl et al., 2011; Yeomans et al., 2001). Specifically, intra-VTA infusion of the anti-cholinesterase inhibitor, physostigmine, or the muscarinic receptor agonist, pilocarpine, induces anhedonic-like, anxiogenic, and pro-depressive-like behavioral responses in the sucrose preference test (SPT), elevated plus maze (EPM) and forced swim test (FST) (Addy et al., 2015; Nunes et al., 2019; Small et al., 2016). However, it is still unknown what muscarinic receptor subtypes are mediating these behavioral responses. There are five known muscarinic receptor subtypes (M1-M5) (Thomsen et al., 2018), and M5 muscarinic receptors have been shown to regulate dopamine activity in the VTA to NAc pathway (Garzon and Pickel 2013; Yeomans et al., 2001). Furthermore, muscarinic M5 receptors are expressed in the midbrain region, including the VTA, and are expressed on dopaminergic neurons (Weiner et al., 1990). Thus, VTA M5 receptors are potential critical mediators of depression and anxiety-related behavior. Here, we sought to determine the role of muscarinic M5 receptors in the VTA in the ability to regulate behavioral responses in the SPT, EPM and FST in male and female rats.

In this study, we first sought to examine the effects of increased cholinergic tone in the VTA, using the anti-cholinesterase inhibitor physostigmine, on pro-depressive and anxiogenic behaviors in male and female rats. In both males and females, increasing VTA cholinergic tone produced anhedonic-like, anxiogenic and pro-depressive-like responses on the SPT, EPM, and FST. We also investigated the role of VTA muscarinic M5 receptors in mediating these behaviors. At baseline, VTA infusion of a M5 negative allosteric modulator (NAM) did not alter most behaviors tested in males and females, except for decreasing FST immobility time in males. However, M5 NAM administration was sufficient to reverse the anhedonic, anxiogenic, and pro-depressive-like effects of increased cholinergic tone in the SPT, EPM and FST in male and female rats. Taken together, these findings have important implications regarding cholinergic receptors mechanisms, particularly the muscarinic M5 receptor, which underlie anxiety and depression and could serve as possible therapeutic targets for depression and anxiety.

2. Materials and Methods

2.1. Subjects and Surgery

Across all experiments, adult male (n = 122) and female (n = 120) Sprague Dawley rats (250–279 g for males, 179–199 g for females; Charles River Laboratories, Wilmington, MA, USA) were used and 24 were removed from the final analysis due to cannula misplacements, damage around the injection site, or clogged cannula. Each experimental cohorts was run as a single large group (n= 32–39 rats per cohort). 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 22–24°C. Food and water was available ad libitum, unless noted otherwise. All surgical procedures were performed using aseptic techniques. In preparation for surgery, rats were anesthetized with ketamine HCl (100 mg/kg, i.p., Henry Schein, NY, USA) and xylazine (10 mg/kg, i.p., Henry Schein, NY, USA), and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). Prior to surgical incision, rats received administration of the long-acting nonsteroidal anti-inflammatory drug, carprofen (5 mg/kg s.c., Henry Schein, NY, USA) and were implanted with a bilateral cannula targeting the VTA. All coordinates were obtained from the rat brain atlas (Paxinos and Watson, 2007) with anteroposterior (AP), mediolateral (ML) and dorsoventral (DV) positions referenced from Bregma. A bilateral cannula (Plastics One, Roanoke, VA, USA) spaced 1 mm apart was placed 1 mm above the VTA (AP −5.2 to −5.5 mm, ML ± 0.5 mm, DV −8.1 mm from skull) (Paxinos and Watson 2007). The cannula was secured to the skull using screws (Gexpro, High Point, NC, USA) and dental cement (Dentsply, Milford, DE, USA). After surgery, rats were singly housed and allowed to recover for 5–7 days before behavioral training and/or testing. Experiments were conducted according to the ethical guidelines of the Institutional Animal Care and Use Committee at Yale University.

2.2. Pharmacological Agents

To increase cholinergic tone in the VTA, the anti-cholinesterase inhibitor physostigmine (VMR, Bridgeport, NJ) was dissolved in a 21.1% beta-cyclodextrin, DMSO and saline mix and infused directly into the VTA. Physostigmine was infused at doses that we and others have previously shown to modulate behavior when infused into the VTA (Addy et al., 2015; Small et al., 2016; Zhou et al., 2007). To selectively target the muscarinic M5 receptor, the negative allosteric modulator VU6000181 ((S)-9b-(4-methoxy-3-methylphenyl)-1-(3,4,5-trifluorobenzoyl)-1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-one, first described in (Kurata et al., 2015) was also dissolved in a 21.1% beta-cyclodextrin, DMSO and saline mix. VU6000181 has high selectivity at high nanomolar and low micromolar concentrations (rat IC50 = 516nM), and remains selective for the M5 receptor at concentrations up to 30 μM (Kurata et al., 2015). Therefore, we selected 30 μM as the highest dose used.

2.3. Drug Administration

In preparation for VTA drug infusions, bilateral internal cannula, that extended 1 mm beyond the guide cannula, were inserted into the guide cannula for drug infusion directly into the VTA (~ −9 mm ventral from skull). Drugs were delivered in a 0.3 μL total volume over 2 min via a micro-infusion pump and syringe (25 gauge, Hamilton Syringe, Reno, NE, USA). After the 2 min drug infusion, the internal cannula was left in place for an additional 1 min to allow for complete diffusion of drug into the brain tissue. The four different drug groups were counterbalanced across the three behavioral assays in an incomplete Latin square design. Rats were randomized based on body weight to drug group order. Sample size calculations for these experiments were performed to determine group size with a minimum of 7 per group to have a 95% probability of finding a statistically significant difference at p = 0.05 (Charan and Kantharia, 2013).

2.3. Sucrose Preference Test

The first behavioral examination we performed was the sucrose preference test (SPT). Each experimental group contained between 31–39 rats divided into four different drug groups with 8–10 rats each, which were ran together to avoid potential cohort differences. Before testing in SPT, rats received a habituation period where they were given access to a 1% sucrose solution (Sigma, St Louis, MO, USA) for 48 h in their home cage, instead of their standard water bottle. The bottles containing the 1% sucrose solution were counterbalanced on each side of the home cage to control for side preference. Normal ad-lib access to chow was maintained throughout the habituation period. On test day, rats first underwent 4 h of water deprivation and were then given 1 h access to two identical bottles, one filled with 1% sucrose solution and the other with water. Immediately prior to the 1 h experiment, all rats were infused with either 21.1% beta cyclodextrin vehicle or drug into the VTA. Sucrose and water consumption was determined by measuring the change in bottle weight. The sucrose preference was defined as the ratio of the weight of sucrose vs. water consumed during the 1 h test. The relative consumption of 1% sucrose vs. water was calculated by weighing the bottles before and after 1 h test duration. Access to water bottles and chow was resumed immediately after the test.

2.4. Elevated plus maze

Following 3 to 4 days after the sucrose preference test, the same rats were then tested on the elevated plus maze (EPM). Each experimental group contained between 31–39 rats divided into four different treatment groups with 8–10 rats each, which were ran together to avoid potential cohort differences. 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 is placed to cushion the rat if it falls off the maze. Immediately prior to the 5-minute experiment, all rats were infused with either 21.1% beta cyclodextrin vehicle or drug into the VTA. Drug treatments were counterbalanced from the previous SPT experiment to minimize repeated drug effects. In pilot experiments, when performing VTA physostigmine infusions in a counterbalanced design across 3 behavioral tests (SPT, EPM, FST), we found that physostigmine effects on EPM behavior did not differ when the behavior was performed as the 1st or the 2rd behavioral test (with a 2rd set of VTA infusions) (data not shown). Following either vehicle or drug infusion into the VTA, 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.

2.5. Forced Swim Test

Following 2 to 3 days from EPM test completion, the same cohort of rats that were tested on the SPT and EPM, underwent the forced swim test (FST), in a manner similar to previously published protocols from our lab and others (Addy et al., 2015; Duman, 2010; Rada et al., 2006; Voleti et al., 2013). Each experimental group contained between 31–39 rats divided into four different groups with 8–10 rats, which were ran together to avoid potential cohort differences. During the pre-test, no pharmacological manipulation was given, and behavior was not recorded. In the pre-test, rats were individually placed into a clear polypropylene, cylindrical water tank (diameter 30 cm; height 60 cm; water depth > 40 cm; water temperature between 23–26°C) for 15 min, to establish a stable baseline of immobility for the subsequent test. The FST test session occurred during the second swim session, which took place 24 hr after the pre-test. The length of the FST test session was 10 min, which was filmed and recorded through the session. For quantification purposes, only minutes 1–6 were analyzed, consistent with published FST analysis methodology from our lab and others (Addy et al., 2015; Duman, 2010). Immediately before the test session, rats were infused with drug into the VTA. Drug treatments were counterbalanced to minimize repeated drug effects. In similar experiments, when performing VTA physostigmine infusions in a counterbalanced design across 3 behavioral tests (SPT, EPM, FST), we found that physostigmine effects on FST behavior did not differ when the behavioral was performed as the 1st or the 3rd behavioral test (with a 3rd set of VTA infusions) (data not shown). FST was recorded by video camera and immobility was defined as an interruption of swimming behavior, when rats showed a lack of hind and fore paw paddling. Thus, scoring of immobility time started when rats assumed a passive floating position, using only minimal movements required to keep their heads above water. For FST analysis, each test session was quantified by stopwatch by an experimenter blind to the treatment condition. Tank water was cleaned after each rat. At the end of the test session, rats were dried with a towel and placed in a warmed cage to completely dry off for 30 min before being returned to their home cages.

2.6. Histological verification

At the completion of all behavioral experiments, rats received a systemic, lethal dose of pentobarbital (150 mg/kg, i.p.) and 0.3 μL of Chicago Blue Dye was infused into the VTA over 2 min via a micro-infusion pump and syringe (25 gauge, Hamilton Syringe, Reno, NE, USA). Rats were then sacrificed, brains were removed, transferred to 3.2% paraformaldehyde for 24 hr and then stored in 30% sucrose. Rat brains were placed in a rat brain matrix and sectioned to identify the blue dye placements indicating infusion sites. In order to get a representative photomicrograph of cannula and blue dye placements, rat brains were cut in 40 μM sections using a microtome. These sections were then mounted on a glass microscope slide and images taken on a bright field microscope at 1.25x magnification.

2.7. Statistical Analysis

GraphPad Prism 8 (Graph Pad Software, San Diego, CA., USA) and SPSS 26 (IBM, Armonk, NY, USA) were used to conduct statistical analyses. A one-way ANOVA was used to analyze the SPT, EPM, and FST data for both the physostigmine and VU6000181 dose response experiments. If the one-way ANOVA revealed a significant main effect, a Bonferroni post-hoc analysis was performed to compare between specific drug groups. A 2-way ANOVA design was used to assess drug interaction in groups that received two drug exposures (physostigmine vs. vehicle as factor 1, and VU6000181 vs. vehicle as factor 2). If the two-way ANOVA revealed a significant drug interaction, simple main effects were performed to compare between different treatment groups. All groups were tested for normal distributions and equal variance using the Shaprio-Wilk test. No cohorts were found to violate normal distributions.

3. Results

3.1. Increased cholinergic tone in the VTA produces an anhedonic, anxiogenic, and depressive-like behavioral response in male rats.

We have previously demonstrated that increasing VTA cholinergic tone with physostigmine reduces sucrose preference, decreases time spent in the open arm in the EPM, and increases immobility time in the FST in male rats (Addy et al., 2015; Small et al., 2016). Here, we expanded the physostigmine dose response curve in males and also examined whether physostigmine altered SPT, EPM, and FST behavior in female rats. Intra-VTA physostigmine, immediately before the SPT, significantly decreased sucrose preference in male rats, as demonstrated by a significant main effect of drug (F3,30) = 8.720, p = 0.0003, Fig. 1B). Post-hoc analysis further revealed a significant difference in sucrose preference between rats that received 0 μg and 2 μg (p = 0.0001, t-test with a Bonferroni correction, Fig. 1B), 0.5 μg and 2 μg (p = 0.008, t-test with a Bonferroni correction, Fig.1B), and 1.0 μg and 2 μg (p = 0.032, t-test with a Bonferroni correction, Fig 1B) physostigmine doses. Following a period of 3–4 days, rats were then assessed for anxiogenic-like behavioral responses on the EPM. Intra-VTA infusion of physostigmine was administered immediately before the EPM test. Physostigmine significantly decreased time spent in the open arms as demonstrated by a significant main effect of drug (F3, 30) = 7.310; p = 0.0008, Fig. 1C). Post-hoc analysis further revealed a significant difference in time spent in the open arms between rats that received 0 μg and 1 μg (p = 0.0062, t-test with a Bonferroni correction, Fig. 1C), and 0 μg and 2 μg (p = 0.0009, t-test with a Bonferroni correction, Fig. 1C) physostigmine. Following a 2–3 day waiting period after the EPM test, rats were then tested in the FST test. Intra-VTA infusion of physostigmine, immediately before test, led to significantly increased time spent immobile as demonstrated by a significant main effect of drug (F3, 30) = 6.70; p = 0.0014, Fig. 1D). Post-hoc analysis further revealed a significant difference in immobility time between doses of physostigmine at 0 μg and 2 μg (p = 0.0085, t-test with a Bonferroni correction, Fig. 1D), and 0.5 μg and 2 μg (p = 0.0012, t-test with a Bonferroni correction, Fig. 1D).

Fig. 1.

Fig. 1

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Intra-VTA physostigmine produces anhedonic, anxiogenic-like, and pro-depressive behavioral responses on SPT, EPM, and FST in male rats. (A) Experimental timeline. (B) VTA physostigmine decreases sucrose preference. (C) VTA physostigmine decreases time spent in the open arms. (D) VTA physostigmine increases time spent immobile. (E) Histological representation of VTA infusion site. Error bars indicate the standard error from the mean (SEM). *p < 0.05, ***p < 0.001 (1 μg and 2 μg vs. 0 μg, post-hoc with Bonferroni correction).

3.2. Increased cholinergic tone in the VTA produces anhedonic, anxiogenic, and depressive-like behavioral responses in female rats.

To first assess anhedonic-like behavior in female rats, intra-VTA infusion of physostigmine was administered immediately before the SPT session. Intra-VTA physostigmine significantly decreased sucrose preference as demonstrated by a significant main effect of drug (F3,32) = 8.08, p = 0.0004, Fig. 2B). Post-hoc analysis further revealed a significant difference between rats that received 0 μg and 0.5 μg (p = 0.0153, t-test with a Bonferroni correction, Fig. 2B.), 0 μg and 1 μg (p = 0.0159, t-test with a Bonferroni correction, Fig. 2B.), and 0 μg and 2 μg (p = 0.0002, t-test with a Bonferroni correction, Fig. 2B ). Following a period of 3–4 days after the SPT, animals were tested in the EPM. Intra-VTA infusion of physostigmine, immediately before the EPM test, significantly decreased time spent in the open arms as demonstrated by a significant main effect of drug (F3, 28) = 10.04; p = 0.0001, Fig. 2C). Post-hoc analysis further revealed a significant difference in time spent in the open arms between rats that received 0 μg and 1 μg (p = 0.0227, t-test with a Bonferroni correction, Fig. 2C), and 0 μg and 2 μg (p = 0.0206, t-test with a Bonferroni correction, Fig. 2C.) physostigmine. Finally, 2 to 3 days after the EPM, intra-VTA infusion of physostigmine was administered immediately before the FST test. Intra-VTA infusion of physostigmine significantly increased time spent immobile as demonstrated by a significant main effect of drug (F3, 29) = 9.13; p = 0.0002, Fig 2D). Post-hoc analysis further revealed a significant increase in immobility time between rats that received 0 μg and 2 μg physostigmine (p = 0.0069, t-test with a Bonferroni correction, Fig 2D).

Fig. 2.

Fig. 2

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Intra-VTA physostigmine produces pro-depressive and anxiogenic behavioral responses on SPT, EPM, and FST in female rats. (A) Experimental timeline. (B) VTA physostigmine decreases sucrose preference. (C) VTA physostigmine decreases time spent in the open arms. (D) VTA physostigmine increases time spent immobile. Error bars indicate the standard error from the mean (SEM). *p < 0.05, ***p < 0.001 (0.5 μg, 1 μg, and 2 μg vs. 0 μg, post-hoc with Bonferroni correction).

3.3. Negative allosteric modulation of VTA muscarinic M5 receptors on anhedonic, anxiogenic-like, and depressive-like behavioral responses.

The role of VTA M5 receptors on behavioral responses in the SPT, EPM, and FST are unknown. Therefore, we sought to determine the behavioral effects of VU6000181, a selective muscarinic M5 receptor negative allosteric modulator, infused into the VTA. In male rats, intra-VTA VU6000181 at all doses tested did not affect sucrose preference (F3,33) = 0.71; p = 0.5519, Fig. 3B) or time spent in the open arms on the EPM (F3,33) = 0.63; p = 0.6008, Fig. 3D). In contrast, intra-VTA VU6000181 in males significantly decreased time spent immobile on the FST as indicated by a significant effect of drug (F3,32) = 2.95; p = 0.0473, Fig. 3F) Post-hoc analysis in the males further revealed a significant differences in immobility time rats that received 0 μM and 30 μM VU6000181 (p = 0.0368, t-test with a Bonferroni correction, Fig. 3F).

Fig. 3.

Fig. 3

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Intra-VTA VU6000181 decreases time spent immobile in male but not female rats. (A) Experimental timeline. (B) VTA VU6000181 does not affect sucrose preference in male rats. (C) VTA VU6000181 does not affect sucrose preference in female rats. (D) VTA VU6000181 does not affect EPM behavior in male rats. (E) VTA VU6000181 does not affect EPM behavior in female rats. (F) VTA VU6000181 decrease time spent immobile in male rats. (G) VTA VU6000181 does not affect swim behavior in female rats. Error bars indicate the standard error from the mean (SEM). *p < 0.05, (30 μM vs. 0 μM, post-hoc with Bonferroni correction).

In female rats, intra-VTA VU6000181 at all doses tested did not affect sucrose preference (F3,29) = 1.31; p = 0.5832, Fig 3C), time spent in the open arms on the EPM (F3,28) = 1.20; p = 0.3268, Fig 3E) or time spent immobile in the FST (F3,27) = 1.62; p = 0.2068, Fig 3G) in female rats.

3.4. VTA M5 receptor negative allosteric modulation attenuates the anhedonic, anxiogenic, and depressive-like behavioral responses of intra-VTA physostigmine in male rats.

To assess the potential ability of VTA M5 receptor negative allosteric modulation to attenuate the behavioral effects of intra-VTA physostigmine on the SPT, a cocktail containing both compounds was administered immediately before the test. A two-way ANOVA design was performed, which examined the effects of both drugs (physostigmine vs. vehicle, and VU6000181 vs. vehicle) on the sucrose preference test in males. There was a statistically significant effect of physostigmine F (1,35) = 15.892, p = 0.003 and VU6000181 F (1,35) = 10.512, p = 0.003, Fig.4B. There was also a statistically significant interaction between drug combination given and sucrose preference, F (1, 35) = 9.965, p = 0.003, Fig 4B. Simple main effect analysis showed a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine + 30 μM VU6000181 (p < 0.001, with Bonferroni correction Fig 4B). This indicates an attenuation of the effect of 2 μg physostigmine to reduce sucrose preference in the presence of VU6000181. Next, a two-way ANOVA design was performed, which examined the effects of drug treatment (physostigmine and VU6000181) on time spent in the open arms in males. There was a statistically significant effect of physostigmine F (1,34) = 6.906, p = 0.013 and VU6000181 F (1,34) = 5.099, p = 0.030, Fig.4C. There was also a statistically significant interaction between drug combination given and open arm time in males, F (1, 34) = 5.680, p = 0.023, Fig 4C). Simple main effect analysis showed a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine +30 μM VU6000181 (p <. 001, with Bonferroni correction Fig 4C). This indicates an attenuation of the effect of 2 μg physostigmine to reduce time spent in the open arm in the presence of VU6000181. Finally, a two-way ANOVA design was performed, which examined the effects of drug treatment (physostigmine and VU6000181) on time spent immobile in males. There was a statistically significant effect of physostigmine F (1,30) = 4.271, p = 0.048 and VU6000181 F (1,30) = 12.597, p = 0.001, Fig.4D. There was a statistically significant interaction between drug combination given and time spent immobile, F (1, 30) = 5.852, p = 0.022, Fig 4D). Simple main effect analysis showed a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine +30 μM VU6000181 (p < 0.001, with Bonferroni correction, Fig 4D). This indicates an attenuation of the effect of 2 μg physostigmine to increase time spent immobile in the presence of VU6000181.

Fig. 4.

Fig. 4

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Intra-VTA VU6000181 attenuates physostigmine induced behavioral effects in male rats. (A) Experimental timeline. (B) VTA VU6000181 attenuates physostigmine induced decrease in sucrose preference. (C) VTA VU6000181 attenuates physostigmine effects on the EPM. (D) VTA VU6000181 attenuates physostigmine induced increase in immobility time. Error bars indicate the standard error from the mean (SEM). *p < 0.05, (0 μg physo + 0 μM NAM vs 2 μg physo + 0 μM NAM vs 2 μg physo + 30 μM NAM, post-hoc with Bonferroni correction).

3.5. VTA M5 receptor negative allosteric modulation attenuates the anhedonic, anxiogenic, and depressive-like behavioral responses of intra-VTA physostigmine in female rats.

A two-way ANOVA was performed, which examined the effects of drug treatment (physostigmine vs. vehicle, and VU6000181 vs. vehicle) on sucrose preference test in females. There was a statistically significant effect of physostigmine F (1,36) = 15.274, p < 0.001 and VU6000181 F (1,36) = 5.616, p = 0.023, Fig.5B. Furthermore, there was a statistically significant interaction between drug combination given and sucrose preference, F (1, 36) = 4.704, p = 0.037, Fig 5B.) Simple main effects analysis demonstrated a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine +30 μM VU6000181 (p < 0.001, with Bonferroni correction, Fig 5B). This indicates an attenuation of the effect of 2 μg physostigmine to reduce sucrose preference in the presence of VU6000181. Next, a two-way ANOVA was performed, which examined the effects of drug treatment (physostigmine and VU6000181) on time spent in the open arms in females. There was a statistically significant effect of physostigmine F (1,30) = 12.376, p = 0.001, Fig 5C) and VU6000181 F (1, 30) = 4.211 p = 0.049, Fig 5C). There was a statistically significant interaction between drug combination given and open arm time, F (1, 30) = 8.065, p = 0.008, Fig 5C.) Simple main effects analysis demonstrated a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine +30 μM VU6000181 (p < 0.001, with Bonferroni correction, Fig 5C). This indicates an attenuation of the effect of 2 μg physostigmine to reduce open arm time in the presence of VU6000181. Finally, a two-way ANOVA design was preformed that examined the effects of drug treatment (physostigmine and VU6000181) on time spent immobile in females. There was a statistically significant effect of physostigmine F (1,30) = 12.736, p = 0.001, Fig 5C) and VU6000181 F (1, 30) = 7.330 p = 0.11, Fig 5C). There was a statistically significant interaction between drug combination given and time spent immobile, F (1, 30) = 4.431, p = 0.047, Fig 5C) Simple main effect analysis showed a significant difference between rats that received 2 μg physostigmine + 0 μM VU6000181 versus rats that received 2 μg physostigmine + 30 μM VU6000181 (p < 0.001, with Bonferroni correction Fig 5C). This indicates an attenuation of the effect of 2ug physostigmine to increase time spent immobile in the presence of VU6000181.

Fig. 5.

Fig. 5

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Intra-VTA VU6000181 attenuates physostigmine induced behavioral effects in female rats. (A) Experimental timeline. (B) VTA VU6000181 attenuates physostigmine induced decrease in sucrose preference. (C) VTA VU6000181 attenuates physostigmine effects on the EPM. (D) VTA VU6000181 attenuates physostigmine induced increase in immobility time. Error bars indicate the standard error from the mean (SEM). *p < 0.05, (0 μg physo + 0 μM NAM vs. 2 μg physo + 0 μM NAM vs. 2 μg physo + 30 μM NAM, post-hoc with Bonferroni correction).

4. Discussion

Our data showed that increased cholinergic tone in the ventral tegmental area (VTA) produces pro-depressive and anxiogenic behavioral responses on the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). Intra-VTA infusion of the anti-cholinesterase inhibitor physostigmine decreased sucrose preference in the SPT, decreased time spent in the open arm in the EPM, and increased time spent immobile in the FST in both male and female rats. We also examined the potential role of VTA muscarinic M5 receptors in these behaviors. VTA infusion of the M5 negative allosteric modulator (NAM) VU6000181 did not affect behavioral responses in female rats, but led to reduced immobility time in male rats in the FST. Furthermore, we found a critical role of VTA M5 muscarinic receptors in mediating VTA physostigmine’s behavioral effects. VTA infusion of physostigmine in combination with the selective M5 NAM VU6000181, attenuated the physostigmine-induced anhedonic, anxiogenic-like, and pro-depressive-like behavioral responses on the SPT, EPM, and FST in both male and female rats. Taken together, our data reveals a novel role of VTA M5 muscarinic receptors in mediating pro-depressive and anxiogenic behaviors in male and female rats when cholinergic tone in VTA is elevated. This study supports future investigations to examine the role of VTA M5 muscarinic receptors in other behavioral responses to stress and anxiety and in other models of mood disorder-related behavior.

In humans, it has been demonstrated that the anti-cholinesterase inhibitor physostigmine is capable of producing symptoms of depression and anxiety (Dulawa and Janowsky, 2018; Janowsky et al., 1980; Risch et al., 1980). In rodents, increasing cholinergic tone produces pro-depressive and anxiogenic behavioral responses on the tail suspension test, FST, and EPM (Addy et al., 2015; Mineur et al., 2018; Small et al., 2016). Yet, all the pre-clinical work on the role of VTA AChRs in mood disorder-related behavioral models had only been performed in male rats. Here in female rats, we demonstrate that increased cholinergic tone in the VTA with physostigmine also induced anhedonic, pro-depressive and anxiogenic-like behavioral responses on the SPT, EPM, and FST. Despite the same dose range of physostigmine used in both male and female rats, there were differences in effective doses between males and females. Females were more sensitive to the effects of physostigmine on the SPT as demonstrated by a significant decrease in sucrose preference across all doses tested. In contrast, only the highest dose of physostigmine tested in males produced a decrease in sucrose preference. The effects of physostigmine on the EPM and FST between females and males were more consistent across doses. It is unclear what the basis is for these differences in VTA physostigmine sensitivity between males and females on the SPT. Sex differences between female and male rats may underlie differential sensitivity to increased cholinergic tone in the VTA. For example, sex hormones such as estrogen and progesterone modulate levels of dopamine, norepinephrine, serotonin and acetylcholine that influence depressive and anxiety-like symptoms (Di Paolo et al., 1985; Haywood et al., 1999; Lapchak et al., 1990; McQueen et al., 1997). Therefore, differences in sex hormone expression could be mediating increased sensitivity to VTA physostigmine in females, particularly in the SPT. Indeed, estradiol has also been shown to mediate cholinergic tone via multiple mechanisms such as regulation of acetylcholinesterase activity, by interactions with nicotinic receptors and via modulation of cholinergic neurons by estradiol receptors (Gibbs, 1996; Hammond and Gibbs, 2011; Kandi and Hayslett, 2011; Luine, 1985).

We previously found that muscarinic receptor mechanisms in the VTA regulate behavioral responses on the SPT, EPM and FST (Addy et al., 2015; Nunes et al., 2019; Small et al., 2016). However, it is unknown what specific VTA muscarinic receptor subtype or subtypes play a critical role in mediating pro-depressive and/or anxiogenic behavioral responses. There are five different muscarinic subtypes M1-M5 that have been identified (Bonner et al., 1987; Bonner et al., 1988; Kubo et al., 1986; Thomsen et al., 2018). The M5 muscarinic receptors are found and expressed in both the VTA/substantia nigra and in the striatum (Levey, 1993; Reever et al., 1997; Vilaro et al., 1990; Weiner et al., 1990). In the VTA, M5 receptors are localized to dopamine (DA) cell bodies and facilitates the release of DA (Forster et al., 2002; Foster et al., 2014; Garzon and Pickel, 2013; Shin et al., 2015; Steidl et al., 2011; Yeomans et al., 2001). In contrast, activation of M5 receptors in the striatum has been shown to decrease DA release (Foster et al., 2014). Furthermore, muscarinic M5 receptors have been implicated in DA-related disorders and as potential drug targets for the development of treatments for schizophrenia and addiction (Anney et al., 2007; Basile et al., 2002; Bender et al., 2019; Fink-Jensen et al., 2003; Gould et al., 2019; Gunter et al., 2017; McGowan et al., 2017; Teal et al., 2019). Moreover, we recently demonstrated an overlapping role of VTA cholinergic receptors in mediating both cocaine-seeking and anxiety-like behavior during periods of abstinence (Nunes et al., 2019). Therefore, VTA M5 receptors are an attractive target to examine their role and ability to regulate behavioral responses on the SPT, EPM and FST. First, we found that the selective muscarinic M5 receptor NAM, VU6000181, when infused into the VTA did not affect female rat behavior on the SPT, EPM or FST. In contrast, when VU6000181 was infused into the VTA of male rats, it decreased time spent immobile on the FST; however, it did not affect behaviors on the SPT or EPM. Thus, one outstanding question is why VU6000181 only produced a behavioral effect in the FST in male rats. A potential explanation may be related to the mechanism of action of VU6000181. Since VU6000181 is a negative allosteric modulator of the muscarinic M5 receptor, its functionality as a negative allosteric modulator is regulated by the presence of acetylcholine (Geanes et al., 2016; Kurata et al., 2015). It is plausible then that the FST procedure may differentially enhance cholinergic tone in VTA in males versus females, leading to different efficacy of VU6000181 in modulating depressive vs. anxiogenic-like behaviors in males versus females. Furthermore, VTA muscarinic M5 receptors may be playing differential roles in depressive vs anxiogenic-like behaviors in male and female rats. An alternative possibility is that males and female rats may have differential levels or density of M5 mAChR expression in the VTA, leading to different sensitivity to VU600181. Thus, future investigations using immunohistochemistry and qPCR will help to determine if there are differences in M5 receptor and mRNA expression between male and female rats.

Our laboratory has recently identified VTA muscarinic acetylcholine receptors (mAChRs) as having bidirectional control of depression and anxiogenic-like behavioral responses on the SPT, EPM, and FST. VTA administration of mAChR non-selective agonist, pilocarpine, is sufficient to decrease time spent in the open arm on the EPM and to increase time spent immobile on the FST (Small et al., 2016). In contrast, VTA administration of the non-selective mAChR antagonist scopolamine increases time spent in the open arm on the EPM and decreases time spent immobile on the FST (Addy et al., 2015; Nunes et al., 2019). The ability of non-selective mAChR agents, but not the M5 mAChR selective VU6000181 alone, to modulate EPM behavior could suggest that multiple midbrain mAChR subtypes may be involved in regulating anxiogenic-like behaviors. M2 and M4 receptors are expressed on laterodorsal tegmentum neurons that innervate the VTA (Kohlmeier et al., 2012), and it is possible that these receptors may contribute to the effects seen following scopolamine or pilocarpine administration. Given the ability of VU6000181 to reverse physostigmine effects, the results reported here suggest that M5 plays an especially important role in regulating VTA function during hypercholinergic states, while the modulation of these behaviors in the absence of acetylcholinesterase inhibitors may be more complex and regulated by M5 receptors as well as other mAChR subtypes.

We observed robust reversals of the pro-depressive and anxiogenic behavioral responses on the SPT, EPM, and FST by physostigmine with the negative allosteric modulator of the M5 receptor VU6000181. But there are some potential limitations of our study. While we used behavioral models of anhedonia, anxiety and depression, we acknowledge that clinical presentations of depression and anxiety are heterogeneous and show complex behavioral symptoms in humans such as sleep disturbances, cognitive ruminations, and mental and physical psychomotor retardation (Goldberg, 2013; Goldberg, 2014; Goldberg et al., 2014; Treadway et al., 2012; Treadway and Zald, 2013). For example, there is individual variability in sleeping patterns in depressed individuals where some are suffering from insomnia and others from hypersomnia (Nutt et al., 2008). Furthermore, sex differences can influence the amount of time spent ruminating in patients with depression, which tends to be more prevalent in women (Shors et al., 2017). We did not assess sleep behavior in this study, nor can we assess ruminations in rats. However, future studies can investigate the role of VTA muscarinic receptors in motivated and social behaviors in rodents, which is also impaired in depression. Given the potential limitation of repeated VTA drug infusions and potential damage to brain tissue, we administered drugs in a counterbalanced manner to minimize repeated drug effects. Importantly, we saw no differences in drug effects whether rats received drug as the first infusion or the third infusion. Furthermore, rats with significant damage around the injection site were excluded from the analysis.

5. Conclusions

In conclusion, our study showed that infusion of the anti-cholinesterase inhibitor physostigmine into the VTA in male and female rats produced pro-depressive and anxiogenic behavioral responses as measured by the SPT, EPM, and FST. This is the first demonstration for a role of VTA M5 receptors on behavioral responses on the SPT, EPM, and FST. Importantly, we show that muscarinic M5 receptors in the VTA are critical in mediating the pro-depressive and anxiogenic behavioral effects of increased cholinergic tone in the VTA. Given the evidence for restricted localization of M5 mAChR on dopaminergic neurons, these data support future investigations of dopaminergic mechanisms underlying these effects and also supports future clinical investigations of the ability of M5 receptor as targets to treat depression and anxiety.

  • VTA physostigmine induces pro-depressive and anxiogenic-like responses in male and female rats

  • M5 NAM infusion to VTA decreases FST immobility time in male, but not female, rats

  • M5 NAM does not alter behavior in EPM nor sucrose preference test

  • VTA physostigmine and M5 NAM co-infusion attenuates the effects of physostigmine

6. Funding

This research was supported by grant R01 MH108663 from the National Institute of Mental Health (NIMH).

Footnotes

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