Authors: Megan Kelly (1Department of Psychology, The University of Texas at Austin, Austin TX, 78712), Merrick Garner (1Department of Psychology, The University of Texas at Austin, Austin TX, 78712), Emily M. Cooper (1Department of Psychology, The University of Texas at Austin, Austin TX, 78712), Caitlin A. Orsini (1Department of Psychology, The University of Texas at Austin, Austin TX, 78712; 2Department of Neurology, The University of Texas at Austin, Austin TX, 78712; 3Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin TX, 78712)
Categories: Article, punishment, decision making, sex differences, acetylcholine, cholinergic receptors
Source: Neurobiology of learning and memory
Authors: Megan Kelly, Merrick Garner, Emily M. Cooper, Caitlin A. Orsini
The ability to choose between options that differ in their risks and rewards depends on brain regions within the mesocorticolimbic circuit and regulation of their activity by neurotransmitter systems. Dopamine neurotransmission in particular plays a critical role in modulating such risk-taking behavior; however, the contribution of other major modulatory neurotransmitters, such as acetylcholine, is not as well-defined, especially for decision making in which the risk associated with more rewarding outcomes involves adverse consequences. Consequently, the goal of the current experiments was to examine how cholinergic signaling influences decision making involving risk of explicit punishment. Male and female rats were trained in a decision-making task in which they chose between a small safe food reward and a larger food reward accompanied by a risk of footshock punishment. After training in this task, the effects of nicotinic and muscarinic agonists and antagonists on risk-taking performance were evaluated. Neither nicotine, a nicotinic receptor agonist, nor mecamylamine, a nicotinic receptor antagonist, affected preference for the risky lever, although mecamylamine did alter latencies to press the risky lever and the percentage of omissions. The muscarinic receptor agonist oxotremorine decreased preference for the large, risky lever; similar effects on behavior were observed with the administration of the muscarinic receptor antagonist scopolamine. Control experiments were therefore conducted in which these same muscarinic receptor ligands were administered prior to testing in a reward discrimination task. These experiments revealed that the effects of oxotremorine and scopolamine on risk taking may be due to altered motivational processes rather than to changes in sensitivity to risk of punishment. Importantly, there were no sex differences in the effects of cholinergic manipulations on preference for the large, risky lever. Collectively, these findings suggest that in both males and females, cholinergic signaling via muscarinic receptors is involved in decision making involving risk of explicit punishment, with a specific role in modulating sensitivity to differences in reward magnitude. Future studies will expand upon this work by exploring whether targeting cholinergic receptors has therapeutic potential for psychiatric conditions in which risk taking is pathologically altered.
People frequently encounter situations in which they must make decisions characterized by varying benefits and the potential risks that may accompany them. Such decisions involve a cost-benefit analysis, wherein individuals must evaluate the available options in terms of their rewards and the relative risks associated with these outcomes. Although multiple factors can influence this evaluation process (e.g., internal states, environmental factors), most individuals are able to adaptively weigh rewards against their potential costs and make a choice that is optimal for them in that current situation. Individuals with certain neurological or psychiatric conditions, however, exhibit impairments in this decision-making process, leading to elevated risk taking (e.g., substance use disorder; Chen et al., 2020; Gowin, Mackey, & Paulus, 2013) or pathological risk aversion (e.g., anorexia nervosa; Bernardoni et al., 2020; Lorian & Grisham, 2011). To understand the mechanisms by which these decision-making processes go awry in psychiatric diseases, it is necessary to first understand biological mechanisms underlying decision making in non-disease states.
Cost/benefit decision making is largely mediated by brain regions within the mesocorticolimbic circuit, such as the amygdala, prefrontal cortex and striatum (Orsini, Moorman, Young, Setlow, & Floresco, 2015; Piantadosi, Halladay, Radke, & Holmes, 2021). Critically, neurotransmitters like dopamine and acetylcholine (Ach) interact with these brain regions to influence decision-making processes (Fobbs & Mizumori, 2014). Although the role of dopamine signaling in various forms of risk-based decision making has been well established (Blaes et al., 2018; Di Ciano et al., 2015; Hynes et al., 2021; Simon et al., 2011; St Onge & Floresco, 2009; Zeeb, Robbins, & Winstanley, 2009), the contribution of Ach and cholinergic receptors is surprisingly less clear, despite the fact that brain regions within the mesocorticolimbic circuit receive heavy cholinergic innervation and densely express cholinergic receptors (Feduccia, Chatterjee, & Bartlett, 2012). Further, many psychiatric conditions that are associated with dysfunctional risk taking, such as attention-deficit hyperactivity disorder and substance use disorder (DeVito et al., 2008; Ersche et al., 2005; Gowin et al., 2013; Potter & Newhouse, 2008), have been linked to altered cholinergic transmission and/or receptor expression (Breese et al., 2000; Court et al., 1998; Leonard et al., 2000; Mukhin et al., 2008), and treatments that target the cholinergic system attenuate decision-making impairments (Jensen, DeVito, Yip, Carroll, & Sofuoglu, 2018; Li, Li, & France, 2010; Potter & Newhouse, 2008; Potter, Newhouse, & Bucci, 2006; Wilens & Decker, 2007).
Ach modulates neural activity by acting through two primary cholinergic receptor nicotinic receptors (nAChR) and muscarinic receptors (mAChR). Prior studies using rodent models of decision making have revealed that both nAChRs and mAChRs play a role in certain forms of decision making. For example, Mendez et al. (2012) found that activation of nAChRs increased choice of a large, uncertain reward (i.e., increased risk taking) in a probability discounting task. Further, greater binding at the α4β2* nicotinic receptor subtype in brain regions involved in decision making was associated with decreased choice of the large, uncertain reward (Mendez, Damborsky, Winzer-Serhan, Bizon, & Setlow, 2013). With respect to mAChRs, previous work reported that blocking, but not activating, these receptors decreased choice of the large, uncertain reward (Mendez, Gilbert, Bizon, & Setlow, 2012). Consistent with these findings in the probability discounting task, recent studies have found comparable effects of mAchR antagonism on decision-making performance in a rodent gambling task in which choice outcomes are associated with different probabilities of reward delivery and reward magnitudes (Silveira, Malcolm, Shoaib, & Winstanley, 2015). Specifically, administration of the mAchR antagonist scopolamine impaired decision making, resulting in a decrease in choice of the most advantageous option in this task. Interestingly, scopolamine improved performance in this task when audiovisual cues were integrated into the task (Betts, Hynes, & Winstanley, 2021). In contrast to the effects on choice behavior in the probability discounting task, however, nAchR agonists and antagonists had no effect on behavior in the rodent gambling task. Collectively, this body of work suggests that a decrease in cholinergic tone at mAchRs is sufficient to disrupt decision making involving uncertainty of reward delivery.
The biological mechanisms that mediate risk-based decision making differ based on the nature of the risk associated with the available choices. For example, whereas the administration of the indirect dopamine agonist amphetamine increases choice of the large, risky reward in a decision making task in which the risk is that of reward omission (i.e., probability discounting; St Onge, Chiu, & Floresco, 2010), it decreases choice of the large, risky reward in a decision making task in which the risk is that of explicit punishment (Orsini, Willis, Gilbert, Bizon, & Setlow, 2016; Simon et al., 2011). Consistent with this, acute administration of nicotine increases choice of a large reward associated with risk of punishment but is ineffective in altering risk taking in other risk-based decision-making tasks (Mendez et al., 2012; Mitchell, Vokes, Blankenship, Simon, & Setlow, 2011). Beyond work showing that nAchR activation can alter punishment-based risk-taking behavior (Mitchell et al., 2011), little is known about how the cholinergic system influences decision making involving risk of explicit punishment. Hence, one goal of the current study was to expand upon this initial work and examine the role of both nAChRs and mAChRs in this form of decision making.
In addition to considering the nature of the cost inherent in a decision, it is also necessary to consider the influence of biological sex on cholinergic modulation of decision making. Although there are robust sex differences in risk-based decision making (Islas-Preciado et al., 2020; Orsini et al., 2016; van den Bos, Jolles, van der Knaap, Baars, & de Visser, 2012), the majority of the studies that have examined the role of cholinergic transmission in risk taking have exclusively used male rats (Betts et al., 2021; Hosking, Lam, & Winstanley, 2014; Mendez et al., 2012; Silveira et al., 2015). Further, there are robust sex differences in cognitive processes dependent on central cholinergic function, such as fear learning (Yavas, Trott, & Fanselow, 2021), working memory (Hall et al., 2017) and effortful food-seeking behavior (Nunes et al., 2023). Given that there are sex differences in the effects of dopaminergic manipulation on decision making involving risk of punishment (Orsini et al., 2016), it is conceivable that Ach may also regulate this form of risk taking in a sex-dependent manner. Thus, a second goal of the current study was to determine whether Ach modulates risk taking differently in males and females.
To achieve these goals, the current study used a risk-based decision-making task in which rats choose between a small, safe food reward and a larger food reward accompanied by mild footshock delivery, the probability of which systematically increases over the test session. To identify the roles of cholinergic receptors in this behavior, rats were treated with nAChRs or mAChRs agonists and antagonists prior to being tested in the decision-making task. Because we observed that mAChR-targeting ligands reduced lever pressing for the larger, riskier reward, subsequent experiments were conducted to determine whether these manipulations affected reward motivation and/or reward discrimination in general.
Subjects were divided into two cohorts and consisted of adult male (n = 16) and female (n = 16) Long-Evans rats. Half of the subjects from cohort 1 (male, n = 6; female, n = 6) and all subjects from cohort 2 (male, n = 5; female, n = 5) were obtained from Charles River (Kingston, NY). The remaining subjects from cohort 1 (male, n = 5; female, n = 5) were wild-type Long-Evans rats that were bred in-house as part of a transgenic rodent colony. All rats were individually housed in ventilated cages with Sanichip bedding, and each cage had a Nylabone and wooden block for enrichment. Rats were maintained on a reversed 12h light/dark cycle (lights off at 00am) and had ad libitum access to water. Prior to the start of experimental procedures, rats were handled for a minimum of three days to acclimate them to the experimenters. One week prior to the onset of behavioral testing, rats were food restricted to 85% of their free-feeding weight, which was measured just before they were food restricted and adjusted weekly to account for growth (5g increase per week) until they were fully grown (~250g for females and ~350 g for males). Rats were fed soy-free food (Envigo Teklad Irradiated Global 19% Protein Extruded Rodent Diet, #2919) after behavioral testing each day, with the timing of daily feeding varying across the week. All procedures were conducted in accordance with the University of Texas at Austin Institutional Animal Care and Use Committee and adhered to the guidelines of the National Institutes of Health.
Behavioral testing occurred in ten identical operant chambers (Coulbourn Instruments, Harvard Apparatus) enclosed in sound-attenuating cabinets outfitted with noise-insulating foam. Each cabinet was illuminated with red lights and contained a 1.12 W house light positioned on the back wall. Every operant chamber consisted of metal front and back walls and Plexiglas side walls. Located in the center of the front wall was a food trough into which soy-free 45-mg food pellets (LabSupply, 5TUL, Houston, TX) were delivered from a food hopper mounted on the other side of the front wall. Food troughs were equipped with photosensors to detect nosepokes as well as LED lights that were illuminated to indicate the start of a trial and the delivery of a food pellet. Flanking each side of the food trough were two retractable levers, each of which were positioned 11 cm above the bottom of the operant chamber. The floor of the chamber (on which rats were placed) consisted of stainless steel rods through which scrambled shocks were delivered via a connection to shock generators (Coulbourn Instruments). Positioned directly over the center of the operant chamber was a monitor that recorded locomotor activity. This monitor consisted of an array of infrared detectors that tracked body heat in the test chamber. Movement in x, y, or z planes was defined in a change in infrared energy across the array of different detectors and quantified as arbitrary units. Each operant chamber was interfaced with a computer running Graphic State 4.0 (Coulbourn Instruments) that controlled task events and recorded behavioral data.
There were three experiments that examined the role of acetylcholine receptors in risky decision making. Two separate cohorts of rats were used for these cohort 1 (males n = 11; females n = 11) was used in Experiments 1 and 2, whereas cohort 2 (males n = 5; females n = 5) was used in Experiment 3.
The goal of Experiment 1 was to determine the effects of a nicotinic receptor agonist (nicotine) and a nicotinic receptor antagonist (mecamylamine) on decision-making behavior involving risk of punishment (i.e., risk taking) using The Risky Decision-making Task (RDT). Rats were trained in the RDT until behavioral stability emerged, after which they received systemic injections of nicotine or vehicle prior to testing in the RDT. Following completion of the nicotine injection regimen, rats were re-trained in the RDT until stability re-emerged (Figure 1A). Rats then received systemic injections of the nicotinic receptor antagonist mecamylamine or vehicle prior to testing on the RDT.
The goal of Experiment 2 was to determine the effects of a muscarinic receptor agonist (oxotremorine) and a muscarinic receptor antagonist (scopolamine) on risk taking in the RDT. The rats used in Experiment 1 were also used in Experiment 2. Following injections of mecamylamine, rats were re-trained in the RDT until stability was achieved and then received systemic injections of oxotremorine or vehicle prior to testing in the RDT (Figure 1A). As in Experiment 1, rats were then re-trained in the RDT to establish a new stable behavioral baseline, after which they received systemic injections of scopolamine prior to being tested in the RDT.
The goal of Experiment 3 was to identify whether the effects of oxotremorine and scopolamine on risk taking were due to motivational deficits and/or impairments in the ability to discriminate between small and large rewards (rather than affecting evaluation of rewards vs. risks). Rats were trained on a Reward Discrimination (RD) task and then received injections of scopolamine prior to behavioral testing using the same doses as those in Experiment 2 (Figure 1B). Following scopolamine injections, rats were re-trained in the RD task to re-establish stable performance, after which they received injections of oxotremorine (Figure 1B) prior to behavioral testing using the same doses as those used in Experiment 2.
Prior to training on the RDT (Experiments 1 and 2) and RD task (Experiment 3), subjects learned to perform the basic elements of these tasks through various shaping procedures. The first phase of shaping consisted of rats learning to nosepoke into the food trough to receive a food reward. During this 64-min session, a single food pellet was delivered every 60–140 seconds, and subjects were required to nosepoke into the trough at least 100 times within the session to progress to the next shaping procedure. After rats met this criterion, they learned to press a lever for the delivery of one food pellet. One retractable lever was extended into the chamber for the entire 30-min session and rats could freely press on this lever for reward delivery. Lever identity was counterbalanced across rats such that half of the rats learned to press the left lever while the other half learned to press the right lever during this procedure. Rats were required to press the lever at least 50 times in one session to move on to lever shaping on the opposite lever. Once rats met the same criterion on the opposite lever, they began the final shaping phase, requiring them to nosepoke into the food trough to initiate the extension of one of the two levers (left or right; randomized across trials), a press on which resulted in the retraction of the lever and the delivery of a food pellet. To move on to RD, rats were required to press each lever at least 30 times within the 60-min session.
Following shaping procedures, rats were trained on the RD task. In this task, rats learned to discriminate between levers associated with a small food reward (1 pellet) and a large food reward (2 pellets). These sessions were 60 minutes in duration and divided into 5 blocks with 18 trials per block. Each 40-sec trial began with illumination of the house light and food trough light. A nosepoke into the trough extinguished the food trough light and triggered the extension of one (forced choice) or both (free choice) levers into the chamber. If the rat did not nosepoke within 10 seconds, both lights were extinguished, and the trial was scored as an omission. A press on one lever yielded 1 food pellet whereas a press on the other lever yielded 2 pellets. The identity of the lever was counterbalanced across rats and remained consistent throughout RD and testing in the Risky Decision-making Task. If a rat failed to press the lever within 15 seconds, the house light was extinguished, and the trial was scored as an omission. Once a rat pressed a lever, levers were retracted, and the food trough was illuminated. Once the reward was retrieved or 10 seconds had elapsed (whichever occurred first), the food trough light and house light were extinguished, and the rat entered the intertrial interval. The first 8 trials of each block were forced choice trials in which only one lever was extended (four trials per lever, randomized across the eight trials). After the forced choice trials, there were 10 free choice trials wherein both levers were extended into the chamber and the rat could choose freely between them. Unlike the RDT, each block of trials in RD were identical to one another; training in RD therefore served to inform subjects of the task structure before shocks were introduced in the RDT. Rats were required to press the large lever on at least 80% of trials for 3 consecutive days to progress to the RDT.
The task structure of the RDT was identical to that of RD, with the exception that the delivery of the large reward was associated with variable probabilities of footshock delivery. The probability of footshock systematically increased across the 5 trial blocks in 25% increments (i.e., 0, 25, 50, 75, 100%). Rats were informed of the shock probability during the 8 forced choice trials preceding the free choice trials. In the forced choice trials, the probability of footshock delivery depended on the outcome of previous outcomes of pressing the large, risky lever. For example, in the 25% trial block, one and only one of the four forced choice trials in which the large lever was extended would result in footshock upon a lever press. In contrast, in the 75% trial block, three out of the four forced choice trials in which the large lever was extended would result in footshock upon a lever press. Unlike the forced choice trials, the probability of footshock delivery during the free choice trials was independent of the outcomes of other trials in which the large, risky lever was selected. Hence, the risk of punishment was equivalent across all 10 free choice trials. Irrespective of whether footshock was delivered, two food pellets were always delivered after selection of the large, risky lever. Shock intensities were initially set at 150 µA for females and 250 µA for males but were subsequently adjusted for each rat individually during training to ensure that there would be sufficient parametric space to observe drug-induced increases or decreases in lever pressing. Rats were trained on the RDT until behavioral stability emerged (see Data Analysis section for a definition of stability), after which systemic injections began.
All drugs were administered using a randomized within-subjects design such that each rat received each dose of the drug and vehicle, with a minimum of 24 hours between successive injections (i.e., washout). All drugs were dissolved in 0.9% saline and administered subcutaneously at a volume of 1 mL/kg. The selection of drugs and their doses were based on previous work that showed that these doses were effective in altering other forms of decision making (Mendez et al., 2012).
The nicotinic receptor agonist nicotine (Sigma-Aldrich; 0.1, 0.3, 1.0 mg/kg) or vehicle (0.9% saline) was administered to rats in Experiment 1. Nicotine was injected 10 minutes prior to testing in the RDT, and each successive injection day was separated by one washout day in which rats were tested on the RDT drug-free. These doses and injection parameters were based on previous work showing that nicotine increased choice of a large uncertain reward in a probability discounting task (Mendez et al., 2012).
The nicotinic receptor antagonist mecamylamine (Tocris Biosciences; 0.5, 1.0, 2.0 mg/kg) or vehicle (0.9% saline) was administered to rats in Experiment 1. Mecamylamine was injected 10 minutes before testing in the RDT, and each successive injection day was separated by one washout day in which rats were tested drug-free on the RDT. These doses and injection parameters were based on previous work showing that the highest dose mecamylamine administered with these same parameters altered choice behavior in a probability discounting task and delay discounting task (Mendez et al., 2012). Others have also shown that similar doses of mecamylamine administered using identical parameters decreases impulsive action (Betts et al., 2021).
The muscarinic receptor agonist oxotremorine (Sigma-Aldrich; 0.01, 0.03, 0.1 mg/kg) or vehicle (0.9% saline) was administered to rats in both Experiments 2 and 3. Oxotremorine was injected 15 minutes prior to behavioral testing, and each successive injection day was separated by one washout day in which rats were tested drug-free on the RDT or RD. The doses and timing of injections were chosen based on previous work examining the role of cholinergic manipulations on other forms of decision making (Betts et al., 2021; Mendez et al., 2012). Although these studies did not find an effect of any dose of oxotremorine on overt choice behavior, there were effects of oxotremorine on locomotor activity (Mendez et al., 2012) and latencies to make a choice (Betts et al., 2021), indicating these doses are physiologically effective.
The muscarinic receptor antagonist scopolamine (Sigma-Aldrich; 0.03, 0.1, 0.3 mg/kg) or vehicle (0.9% saline) was administered to rats in Experiments 2 and 3. Scopolamine was injected immediately before rats were tested in the RDT or RD and each successive injection day was separated by three washout days in which rats were tested drug-free on the RDT or RD. These doses and injection parameters were chosen based on previous work showing that they have robust effects on choice behavior in a probability discounting task and a delay discounting task (Mendez et al., 2012).
Data were extracted using customized Graphic State and Excel templates and then analyzed using SPSS 29.0. All figures were created with GraphPad Prism 10.0. The primary dependent variable for the RDT and RD was the number of lever presses on the large, risky lever (RDT) or large lever (RD) in each trial block. Although percent choice of the large, risky reward in free choice trials is a more standard dependent variable in similar pharmacology experiments (Blaes et al., 2018; Mendez et al., 2012), the number of lever presses on the large, risky lever was used in this study due to the impact of most of the cholinergic receptor ligands on omissions in the free choice trials. A greater number of trial omissions could obfuscate an accurate representation of preference for the large, risky reward. For example, the use of percent choice would equate risk preference between a rat that pressed the large lever three times (and omitted the remaining 7) with a rat that pressed the large lever 10 times (e.g., 100% risk preference). Although this analysis approach may not reflect choice per se, it provides a more accurate representation of lever preference that is uncontaminated by failure to nosepoke or lever press (i.e., different forms of omissions). Nevertheless, graphs of the effects of each cholinergic receptor ligand on percent choice of the large, risky reward (Experiments 1 and 2) or large reward (Experiment 3) are presented in the Supplemental Material.
Behavioral stability was determined with a repeated-measures analysis of variance (RMANOVA), using lever presses in each trial block across three consecutive days. Stability for the RDT was defined as a main effect of trial block but an absence of a main effect of day and an absence of a significant day X trial block interaction. Stability for RD (Experiment 3) was defined as an absence of a main effect of trial block and day and no significant interaction between day and trial block. Stability analyses included sex as a between-subjects factor to ensure that performance was stable for both males and females.
Effects of cholinergic receptor ligands on behavior were determined using a RMANOVA, with dose and trial block as within-subjects factors and sex as the between-subjects factor. Only if these analyses yielded a significant effect of dose or dose X trial block for the RDT, additional trial-by-trial analyses were conducted to determine how changes in sensitivity to outcomes contributed to the changes in choice behavior. Specifically, these analyses were used to identify the extent to which the outcome of the previous trial affected choice in the subsequent trial. Win-stay trials, or trials on which a rat chose the large, risky lever after receipt of the large reward without footshock (i.e., large, unpunished reward), were used as a proxy for reward sensitivity. The proportion of win-stay trials was calculated by dividing the number of free choice trials on which a rat chose the large, risky lever after receipt of the large, unpunished reward by the total number of free choice trials on which a rat received a large, unpunished reward. In contrast, lose-shift trials, or trials on which a rat chose the small, safe lever after receipt of the large reward with footshock delivery (i.e., large, punished reward), were used as a proxy as sensitivity to negative feedback. The proportion of lose-shift trials were calculated by dividing the number of free choice trials on which a rat chose the small, safe lever after receiving a large, punished reward by the total number of free choice trials on which the rat received large, punished rewards. An RMANOVA was then used to assess the effects of drug doses on the proportion of win-stay and lose-shift trials. If there were no effects of the pharmacological manipulation on lever pressing, win-stay and lose-shift analyses were not conducted.
Latencies to press the small vs. large levers during the forced choice trials were averaged across trial blocks and analyzed using an RMANOVA, with dose and lever identity (small vs. large lever) as the within-subjects factors and sex as the between-subjects factor. In addition to latencies to press levers, other ancillary behavioral measures were extracted from the data and compared between doses. These measures included baseline locomotor activity, locomotor activity during shock delivery and the percentage of omissions during free choice trials. Each measure was compared between doses using a RMANOVA, with dose as the within-subjects factor and sex as the between-subjects factor.
For all data analyses, a p-value equal to or less than 0.05 was considered statistically significant. A p-value between 0.05 and 0.1 was considered a trend toward statistical significance. If parent ANOVAs yielded a main effect of dose or significant interaction between dose and another variable, additional ANOVAs or t-tests were used to determine the source of significance. Bonferroni’s corrections were applied to correct for multiple comparisons. Effect sizes are reported as ƞp^2^ for ANOVAs and the absolute value of Cohen’s d for t-tests.
Upon reaching stability, one male was excluded from all subsequent experiments as he demonstrated an almost exclusive preference (>90%) for the small, safe lever in the first trial block in which the risk of shock delivery was 0%. To determine the effects of a nicotine receptor agonist on risk taking, nicotine was systemically administered prior to testing in the RDT. An RMANOVA yielded a main effect of trial block [F (4, 76) = 63.18, p < 0.01, ƞp^2^ = 0.77], indicating that rats decreased their preference for the large, risky lever as risk of punishment increased (Figure 2A). Unless otherwise noted, all analyses of RDT behavior resulted in a main effect of trial block and will not be reported further. Although there was no main effect of dose [F (3, 57) = 0.37, p = 0.78, ƞp^2^ = 0.02], there was a significant dose X trial block interaction [F (12, 228) = 1.79, p = 0.05, ƞp^2^ = 0.09], suggesting that nicotine dose-dependently altered performance differently across the 5 trial blocks (Figure 2). Post-hoc analyses of the dose X trial block interaction were therefore conducted, comparing each dose with vehicle across trial blocks. After correcting for multiple comparisons, however, there were no differences in lever pressing on the large, risky lever between vehicle and any dose of nicotine [vehicle vs. dose, F (1, 4) = 0.01, p = 0.91, ƞp^2^ < 0.01; dose X trial block, F (4, 80) = 2.26, p = 0.07, ƞp^2^ = 0.10; vehicle vs. dose, F (1, 4) = 0.14, p = 072, ƞp^2^ < 0.01; dose X trial block, F (4, 80) = 0.70, p = 0.59, ƞp^2^ = 0.03; vehicle vs. high, dose, F (1, 4) = 0.99, p = 0.33, ƞp^2^ = 0.05; dose X trial block, F (4, 80) = 2.68, p = 0.04, ƞp^2^ = 0.12]. There was no main effect of sex [F (1, 19) = 1.85, p = 0.19, ƞp^2^ = 0.09], although there were trends toward significant sex X trial block [F (4, 76) = 2.28, p = 0.07, ƞp^2^ = 0.12] and sex X dose X trial block [F (12, 228) = 1.67, p = 0.07, ƞp^2^ = 0.08] interactions. While the former suggests that there were sex differences in discounting of the large reward, the latter interaction suggests that nicotine may have affected lever preference differently between sexes. Because this interaction did not reach significance, however, additional post-hoc analyses were not performed, and subsequent analyses were collapsed across sexes.
Although latencies to press levers were longer in females than males [F (1, 17) = 7.45, p = 0.01, ƞp^2^ = 0.31] and longer for the large, risky lever relative to the small, safe lever [F (1, 17) = 17.28, p < 0.01, ƞp^2^ = 0.50], nicotine did not affect latencies to press either lever in males or females [dose, F (3, 51) = 0.74, p = 0.53, ƞp^2^ = 0.04; dose X sex, F (3, 51) = 0.21, p = 0.89, ƞp^2^ = 0.01; dose X lever identity, F (3, 51) = 0.24, p = 0.87, ƞp^2^ = 0.01; dose X sex X lever identity, F (3, 51) = 0.14, p = 0.91, ƞp^2^ < 0.01]. Similarly, there was no effect of nicotine on baseline locomotor activity [dose, F (3, 57) = 1.40, p = 0.25, ƞp^2^ = 0.07; dose X sex, F (3, 57) = 0.21, p = 0.89, ƞp^2^ = 0.01; sex, F (1, 19) = 1.30; p = 0.27, ƞp^2^ = 0.06] or locomotor activity during shock delivery [dose, F (3, 30) = 1.91, p = 0.15, ƞp^2^ = 0.16; dose X sex, F (3, 30) = 1.96, p = 0.14, ƞp^2^ = 0.16; sex, F (1, 10) = 0.92; p = 0.36, ƞp^2^ = 0.08]. Although females made significantly more omissions than males [F (1, 19) = 19.45, p < 0.01, ƞp^2^ = 0.51], nicotine did not affect omissions in either sex [dose, F (3, 57) = 1.05, p = 0.38, ƞp^2^ = 0.05; dose X sex, F (3, 57) = 1.49, p = 0.23, ƞp^2^ = 0.07]. Graphical depictions of the effects of nicotine on latencies, locomotor activity and omissions can be found in Supplemental Figure 1.
Collectively, these data suggest that activation of nAchRs with nicotine is not effective in altering risk taking in males or females.
To determine the effects of a nicotinic receptor antagonist on risk taking, mecamylamine was systemically administered before testing the rats in the RDT. One female was excluded from data analysis as she was under veterinary care during the injection period. Although Figure 2B suggests that mecamylamine may have decreased the number of lever presses on the large risky lever, the results of a RMANOVA showed that mecamylamine had no effect on performance in the RDT in either sex [dose, F (3, 54) = 1.80, p = 0.16, ƞp^2^ = 0.09; dose X trial block, F (12, 216) = 1.29, p = 0.23, ƞp^2^ = 0.07; dose X sex, F (3, 54) = 1.31, p = 0.28, ƞp^2^ = 0.07; dose X sex X trial block, F (12, 216) = 1.38, p = 0.18, ƞp^2^ = 0.07]. Given the absence of effects of mecamylamine on risk taking, the proportion of win-stay and lose-shift trials was not analyzed.
In contrast to lever presses, mecamylamine did affect latencies to press levers, with a main effect of dose [F (3, 54) = 6.01, p < 0.01, ƞp^2^ = 0.25] and a significant dose X lever identity interaction [F (3, 54) = 3.36, p = 0.03, ƞp^2^ = 0.16]. Although there was a trend toward a significant dose X sex interaction [F (3, 54) = 2.66, p = 0.06, ƞp^2^ = 0.13] and a main effect of sex [F (1, 18) = 12.71, p < 0.01, ƞp^2^ = 0.41], there was no significant dose X lever identity X sex interaction [F (3, 54) = 1.81, p = 0.16, ƞp^2^ = 0.41]. To identify the nature of the effects of mecamylamine on latencies to press the small safe vs. large risky levers, separate RMANOVAs were used to compare latencies across doses for each lever separately. Because the effect of mecamylamine on latencies was not sex-dependent (i.e., there was no dose X lever identity X sex interaction), data analyses did not include sex as a between-subjects factor. Mecamylamine had no effect on latencies to press the small safe lever [F (3, 57) = 2.18, p = 0.10, ƞp^2^ = 0.10], but did increase the latencies to press the large, risky lever [F (3, 57) = 5.33, p < 0.01, ƞp^2^ = 0.22], with the highest dose driving this effect (p < 0.01). Analysis of locomotor activity revealed that mecamylamine did not alter baseline locomotor activity [dose, F (3, 54) = 1.43, p = 0.24, ƞp^2^ = 0.07; dose X sex, F (3, 54) = 0.82, p = 0.49, ƞp^2^ = 0.04; sex, F (1, 18) = 1.36, p = 0.26, ƞp^2^ = 0.07] or locomotor activity during shock delivery [dose, F (3, 36) = 1.40, p = 0.26, ƞp^2^ = 0.11; dose X sex, F (3, 36) = 0.89, p = 0.46, ƞp^2^ = 0.07; sex, F (1, 12) = 0.19, p = 0.67, ƞp^2^ = 0.02]. Mecamylamine did, however, increase the percentage of omitted free choice trials to a greater extent in females compared with males [Figure 2C; dose, F (3, 54) = 3.07, p = 0.04, ƞp^2^ = 0.15; dose X sex, F (3, 54) = 3.11, p = 0.03, ƞp^2^ = 0.15; sex, F (1, 18) = 19.54, p < 0.01, ƞp^2^ = 0.52]. Post-hoc analyses confirmed that this effect was specific to females [males: dose, F (3, 27) = 0.61, p = 0.62, ƞp^2^ = 0.06; dose, F (3, 27) = 3.23, p = 0.02, ƞp^2^ = 0.26]. Additional post-hoc analyses were used to determine which dose significantly differed from vehicle, but these analyses did not survive corrections for multiple comparisons (ps > 0.0167). Graphical depictions of the effects of mecamylamine on latencies and locomotor activity can be found in Supplemental Figure 2.
Hence, nAchR blockade had no effect on preference for the large, risky lever. It did, however, alter other behavioral measures of the RDT, including latencies to press levers and omissions, with the latter affected to a greater extent in females than males.
To identify the effects of a mAchR agonist on risk taking, oxotremorine was systemically administered to rats prior to testing in the RDT. One female was excluded from data analysis as she was under veterinary care during the injection period (the same female that was excluded from the mecamylamine experiment). A RMANOVA revealed a main effect of dose [F (3, 54) = 6.69, p < 0.01, ƞp^2^ = 0.27] and a significant interaction between dose and trial block [F (12, 216) = 2.84, p < 0.01, ƞp^2^ = 0.14] such that oxotremorine decreased presses on the large, risky lever, with the greatest effects occurring during the initial blocks (Figure 3A). Effects of oxotremorine did not, however, differ between males and females [dose X sex, F (3, 54) = 1.90, p =1.90, ƞp^2^ = 0.10; dose X sex X trial block, F (12, 216) = 1.69, p =0.08, ƞp^2^ = 0.09; sex, F (1, 18) < 0.01, p = 0.95, ƞp^2^ < 0.01]. Because there were no sex differences in the effects of oxotremorine on performance in the RDT, post-hoc analyses were conducted on data collapsed across sexes. These analyses revealed that only the high dose of oxotremorine was effective in decreasing lever pressing on the large, risky lever (vehicle vs. low and medium ps > 0.05; vehicle vs. high dose, F (1, 19) = 7.69, p = 0.01, ƞp^2^ = 0.29; dose X trial block, F (4, 76) = 3.85, p < 0.01, ƞp^2^ = 0.17]. Although analyses of the proportion of win-stay and lose-shift trials did not yield main effects of dose [F (3, 33) = 0.29, p = 0.83, ƞp^2^ = 0.03] or sex [F (1, 11) = 0.96, p = 0.35, ƞp^2^ = 0.08] or reveal significant dose X sex [F (3, 33) = 1.22, p = 0.32, ƞp^2^ = 0.10] or dose X trial type [F (3, 33) = 0.09, p = 0.97, ƞp^2^ < 0.01] interactions, there was a significant dose X trial type X sex interaction [F (3, 33) = 3.35, p = 0.03, ƞp^2^ = 0.23], which suggested that oxotremorine altered win-stay vs. lose-shift performance differently in males and females. To determine the source of this significance, another RMANOVA was conducted separately in males and females, with dose and trial type as the within-subjects factors. This analysis, however, did not reveal significant effects of oxotremorine on win-stay or lose-shift performance in either males [dose, F (3, 21) = 0.44, p = 0.73, ƞp^2^ = 0.06; trial type, F (1, 7) = 0.40, p = 0.55, ƞp^2^ = 0.05; dose X trial type, F (3, 21) = 1.45, p = 0.26, ƞp^2^ = 0.17] or females [dose, F (3, 12) = 1.09, p = 0.39, ƞp^2^ = 0.21; trial type, F (1, 4) = 4.33, p = 0.11, ƞp^2^ = 0.52; dose X trial type, F (3, 12) = 2.06, p = 0.16, ƞp^2^ = 0.34].
When latencies to press levers were analyzed, there were main effects of dose [F (3, 54) = 6.16, p < 0.01, ƞp^2^ = 0.26], lever identity [F (1, 18) = 23.25, p < 0.01, ƞp^2^ = 0.56] and sex [F (1, 18) = 6.86, p = 0.02, ƞp^2^ = 0.28]. The latter two effects reflected the overall longer latencies to press the large, risky lever compared with the small, safe lever and that latencies to press levers in general were longer for females relative to males. Oxotremorine did not, however, alter latencies to press levers in a sex-dependent manner; hence, data were collapsed across sexes for subsequent post-hoc analyses conducted to determine the source of the main effect of dose. These analyses revealed that only the highest dose of oxotremorine increased latencies to press both levers (i.e., not specific to the small vs. large lever; dose, F (1, 19) = 7.56, p = 0.01, ƞp^2^ = 0.29; dose X lever identity, F (1, 19) = 0.89, p = 0.36, ƞp^2^ = 0.05; vehicle vs. low and medium doses, ps > 0.05]. Oxotremorine also increased the percentage of omitted free choice [F (3, 54) = 5.21, p < 0.01, ƞp^2^ = 0.22] trials and this effect was greater in females than males [Figure 3B; sex, F (1, 18) = 9.09, p < 0.01, ƞp^2^ = 0.34; dose X sex, F (3, 54) = 4.49, p = 0.01, ƞp^2^ = 0.20]. Additional analyses indicated omissions in females were significantly greater under the medium dose relative to vehicle [t (9) = −3.12, p = 0.01, d = 0.99; comparisons between vehicle and other doses, ps > 0.05]. After correcting for multiple comparisons, none of the oxotremorine doses affected omissions in males (ps > 0.05). Finally, oxotremorine decreased baseline locomotor activity in both males and females [dose, F (3, 54) = 8.75, p < 0.01, ƞp^2^ = 0.33; sex, F (1, 18) = 0.39, p = 0.54, ƞp^2^ = 0.02; dose X sex, F (3, 54) = 0.57, p = 0.64, ƞp^2^ = 0.03]. Post-hoc analyses indicated that the highest dose contributed to this main effect of dose [vehicle vs. high t (19) = 3.96, p < 0.01, d = 0.89; vehicle vs. low and medium doses, ps > 0.05]. There was, however, no effect of oxotremorine on locomotor activity during the delivery of footshock in males or females [dose, F (3, 39) = 0.24, p = 0.87, ƞp^2^ = 0.02; sex, F (1, 13) = 1.50, p = 0.24, ƞp^2^ = 0.10; dose X sex, F (3, 39) = 1.33, p = 0.28, ƞp^2^ = 0.09]. Graphical depictions of the effects of oxotremorine on latencies and locomotor activity can be found in Supplemental Figure 3.
This experiment revealed that mAchR activation reduced preference for the large, risky lever in both male and female rats. In addition to the effects on lever preference, oxotremorine increased latencies to press levers, irrespective of lever identity, and increased the percentage of omissions, with the latter effect greater in females than males. Finally, mAchR activation with the highest dose of oxotremorine decreased overall locomotor activity in both sexes.
Because of the behavioral effects of administration of the muscarinic receptor agonist oxotremorine on RDT performance, rats were then administered the muscarinic receptor antagonist scopolamine. Surprisingly, scopolamine caused a decrease in the number of large, risky lever presses in males and females [Figure 4A; dose, F (3, 57) = 3.97, p = 0.01, ƞp^2^ = 0.17; sex, F (1, 19) = 0.32, p = 0.58, ƞp^2^ = 0.02; dose X sex, F (3, 57) = 0.33, p = 0.80, ƞp^2^ = 0.02; dose X trial block, F (12, 228) = 5.49, p < 0.01, ƞp^2^ = 0.22; dose X sex X trial block, F (12, 228) = 5.49, p = 0.28, ƞp^2^ = 0.06] similar to that observed after oxotremorine administration. Post-hoc analyses revealed that both the medium and high doses of scopolamine decreased the number of large lever presses relative to vehicle administration [vehicle vs. dose, F (1, 20) = 11.90, p < 0.01, ƞp^2^ = 0.37; vehicle vs. dose, F (1, 20) = 8.31, p < 0.01, ƞp^2^ = 0.29; dose X trial block, F (4, 80) = 9.77, p < 0.01, ƞp^2^ = 0.33; vehicle vs. low, ps > 0.05]. Because there were no sex differences in the effects of scopolamine on performance in the RDT and the fact that there was a significant number of omissions, analyses of win-stay and lose-shift performance were collapsed across sexes. Although there was no effect of scopolamine on lose-shift behavior [F (3, 26) = 1.61, p = 0.21], there was a main effect of scopolamine dose on win-stay behavior [F (3, 34) = 3.18, p = 0.04]. Additional analyses indicated that this effect was driven specifically by the medium and high dose of scopolamine; after correcting for multiple comparisons, however, post-hoc analyses did not confirm that these doses significantly affected win-stay behavior (ps > 0.05).
Scopolamine also increased latencies to press both the small, safe and large, risky lever [dose, F (3, 36) = 4.03, p = 0.01, ƞp^2^ = 0.25; dose X lever identity, F (3, 36) = 1.10, p = 0.36, ƞp^2^ = 0.08] and these effects did not differ between males and females [sex, F (1, 12) = 2.69, p = 0.13, ƞp^2^ = 0.18; dose X sex, F (3, 36) = 1.26, p = 0.30, ƞp^2^ = 0.10; dose X lever identity X sex, F (3, 36) = 1.09, p = 0.36, ƞp^2^ = 0.08]. Because there was no dose X lever identity interaction, post-hoc analyses were conducted on latency data collapsed across lever identity. These analyses revealed that the medium [t (17) = −2.63, p = 0.02, d = 0.62] and high [t (15) = −2.94, p = 0.01, d = 0.74] doses of scopolamine increased latencies to press levers. Scopolamine significantly increased the percentage of omitted free choice trials [Figure 4B; F (3, 57) = 17.49, p < 0.01, ƞp^2^ = 0.48] and appeared to affect one sex more than the other [F (3, 57) = 6.18, p < 0.01, ƞp^2^ = 0.25]. To determine the source of these effects, separate RMANOVAs were conducted for males and females, with dose as the between-subjects factor. In both sexes, there was a main effect of dose [male: F (3, 27) = 21.95, p < 0.01, ƞp^2^ = 0.71; F (3, 30) = 5.93, p < 0.01, ƞp^2^ = 0.37], which prompted additional post-hoc analyses in which omissions at each dose were compared with omissions after vehicle administration. Only the comparisons between vehicle and the high dose survived corrections for multiple comparisons in males [t (9) = −6.55, p < 0.01, d = 2.07]. In females, comparisons between vehicle and both the medium [t (10) = −3.31, p < 0.01, d = 1.00] and high [t (10) = −2.76, p = 0.01, d = 0.83] doses survived corrections for multiple comparisons. These results indicate that scopolamine increased omissions in both sexes, but perhaps had a greater effect in females than males. In contrast, there were no effects of scopolamine on baseline locomotor activity [dose, F (3, 57) = 0.26, p = 0.85, ƞp^2^ = 0.01; dose X sex, F (3, 57) = 0.22, p = 0.88, ƞp^2^ = 0.01; sex, F (1, 19) = 2,57, p = 0.13, ƞp^2^ = 0.12]. Due to the significant number of omissions, analyses of locomotor activity during shock delivery were collapsed across males and females. Similar to baseline locomotor activity, there was no effect of scopolamine on locomotor activity during shock delivery [F (3, 15) = 0.15, p = 0.93, ƞp^2^ = 0.03]. Graphical depictions of the effects of scopolamine on latencies and locomotor activity can be found in Supplemental Figure 4.
Collectively, these data show that, like activation of mAchRs, blockade of mAchRs with an antagonist reduces preference for the large, risky lever in males and females. In addition to altering lever preference, scopolamine increased latencies to press levers, irrespective of their identity. Finally, scopolamine increased the percentage of omissions, and this effect was greater in females than males.
Because both oxotremorine and scopolamine affected performance in the first block in which rats chose between the small and large reward with no punishment, it is possible that targeting mAchRs affects reward discrimination or reward motivation rather than risk taking per se. To address this, a separate experiment was performed in which rats were trained on the Reward Discrimination (RD) task and then received oxotremorine or scopolamine prior to testing in the RD task.
An RMANOVA revealed main effects of dose [F (3, 24) = 7.20, p < 0.01, ƞp^2^ = 0.47] and sex [F (1, 8) = 44.73, p < 0.01, ƞp^2^ = 0.85] and a significant dose X trial block interaction [F (12, 96) = 2.71, p < 0.01, ƞp^2^ = 0.25]. There was a trend toward a significant dose X sex interaction [F (3, 24) = 2.81, p = 0.06, ƞp^2^ = 0.26], but there was no significant interaction between dose X sex X trial block [F (12, 96) = 0.93, p = 0.52, ƞp^2^ = 0.19]. Upon inspection of Figure 5A, it appears that the highest dose of oxotremorine, particularly within the second block of trials, drove the effect of oxotremorine on large lever presses. Given the main effect of sex, post-hoc analyses were conducted separately for males and females. In males, there was a significant dose X trial block interaction [F (12, 48) = 1.99, p = 0.05, ƞp^2^ = 0.33]; however, post-hoc analyses in which each dose of oxotremorine was compared with vehicle did not survive corrections for multiple comparisons (ps > 0.05). In females, there was a main effect of dose [F (3, 12) = 5.56, p = 0.01, ƞp^2^ = 0.58], but no dose X trial interaction [F (12, 48) = 1.79, p = 0.08, ƞp^2^ = 0.31]. Post-hoc analyses revealed that, relative to vehicle, only the highest dose of oxotremorine decreased the number of lever presses for the large reward [t (12) = 3.52, p = 0.02]. Comparisons between the vehicle and the other doses (i.e., low and medium) did not yield any significant effects of oxotremorine on performance in females (ps < 0.05).
Oxotremorine increased latencies to press levers [dose, F (3, 24) = 5.17, p < 0.01, ƞp^2^ = 0.39], but this did not differ between the small and large lever [dose X lever identity, F (3, 24) = 0.58, p = 0.63, ƞp^2^ = 0.07]. Although females took longer to press levers relative to males [F (1, 8) = 10.12, p = 0.01, ƞp^2^ = 0.56], oxotremorine did not affect latencies in a sex-dependent manner [dose X sex, F (3, 24) = 0.50, p = 0.68, ƞp^2^ = 0.06; dose X sex X lever identity, F (3, 24) = 0.12, p = 0.95, ƞp^2^ = 0.01]. Post-hoc analyses revealed that compared with vehicle, the highest dose of oxotremorine increased latencies to press levers [F (1, 8) = 7.45, p = 0.02, ƞp^2^ = 0.48]; latencies under other doses were no different than those under vehicle conditions (all ps < 0.05). Locomotor activity was higher in males compared with females [F (1, 8) = 5.78, p = 0.04, ƞp^2^ = 0.42] but was unaffected by oxotremorine [dose, F (3, 24) = 2.78, p = 0.06, ƞp^2^ = 0.26; dose X sex, F (3, 24) = 1.25, p = 0.31, ƞp^2^ = 0.14]. In contrast, oxotremorine increased the percentage of omitted free choice trials [F (3, 24) = 5.36, p < 0.01, ƞp^2^ = 0.40] and did so to a greater extent in females relative to males [sex X dose, F (3, 24) = 3.80, p = 0.02, ƞp^2^ = 0.32; sex, F (1, 8) = 52.72, p < 0.01, ƞp^2^ = 0.87]. Graphical depictions of the effects of oxotremorine on latencies, locomotor activity and omissions in the RD can be found in Supplemental Figure 5.
These data show that, similar to the effects observed in the RDT, mAchR activation reduced preference for the large reward. In addition to the effects on lever preference, oxotremorine increased latencies to press levers, irrespective of their identity, and increased the percentage of omissions.
Similar to the effects of oxotremorine, scopolamine decreased the number of lever presses for the large reward in both males and females [Figure 5B; dose, F (3, 24) = 10.93, p < 0.01, ƞp^2^ = 0.58; dose X sex, F (3, 24) = 0.05, p = 0.98, ƞp^2^ < 0.01; dose X trial block, F (12, 96) = 2.07, p = 0.03, ƞp^2^ = 0.21; dose X sex X trial block, F (12, 96) = 0.60, p = 0.84, ƞp^2^ = 0.07; Figure 5B]. Post-hoc analyses were used to determine which of the three doses led to this decrease relative to vehicle. Although the low dose was ineffective in altering behavior [dose*, F* (1, 9) = 3.55, p = 0.09, ƞp^2^ = 0.28; dose X trial block, F (4, 36) = 1.68, p = 0.18, ƞp^2^ = 0.16], both the medium and high dose of scopolamine decreased the number of lever presses for the large reward [medium: dose, F (1, 9) = 5.75, p = 0.04, ƞp^2^ = 0.39; dose X trial block, F (4, 36) = 5.32, p < 0.01, ƞp^2^ = 0.37; dose, F (1, 9) = 98.54, p < 0.01, ƞp^2^ = 0.92; dose X trial block, F (4, 36) = 4.03, p < 0.01, ƞp^2^ = 0.31].
Scopolamine also indiscriminately increased latencies to press levers in both males and females [dose, F (3, 24) = 5.62, p < 0.01, ƞp^2^ = 0.41; dose X sex, F (3, 24) = 1.29, p = 0.30, ƞp^2^ = 0.14; dose X lever identity, F (3, 24) = 0.13, p = 0.94, ƞp^2^ = 0.02; dose X lever identity X sex, F (3, 24) = 0.42, p = 0.70, ƞp^2^ = 0.05]. Post-hoc analyses revealed that this effect of scopolamine on latencies was driven by the highest dose [F (1, 9) = 9.54, p = 0.01, ƞp^2^ = 0.51]; neither the low nor medium dose significantly affected this behavioral measure (all ps < 0.05). In contrast to oxotremorine, scopolamine dose-dependently decreased locomotor activity in both males and females [dose, F (3, 24) = 5.84, p < 0.04, ƞp^2^ = 0.42; dose X sex, F (3, 24) = 2.34, p = 0.10, ƞp^2^ = 0.23]. Subsequent analyses showed that although the low dose did not alter locomotor activity relative to vehicle [t (9) = 1.76, p = 0.07, d = 0.56, both the medium and high doses decreased locomotor activity (medium: t (9) = 4.93, p < 0.01, d = 1.65; t (9) = 3.51, p = 0.01, d = 1.11]. Finally, scopolamine increased the percentage of omitted free choice trials in both males and females [dose, F (3, 24) = 18.06, p < 0.01, ƞp^2^ = 0.69; dose X sex, F (3, 24) = 0.99, p = 0.42, ƞp^2^ = 0.11]. Like the effect on lever pressing, however, this effect was specific to the high dose of scopolamine [low: t (9) = −1.83, p = 0.10, d = 0.58; t (9) = −1.73, p = 0.12, d = 0.55; high = t (9) = −7.12, p < 0.01, d = 2.25]. Graphical depictions of the effects of scopolamine on latencies, locomotor activity and omissions in the RD can be found in Supplemental Figure 6.
Like mAchR activation with an agonist, blockade of mAchRs with an antagonist reduced preference for the large reward, increased latencies to press levers and increased omissions in males and females. Finally, scopolamine decreased locomotor activity in a dose-dependent manner.
Based on findings from this experiment, the changes in risk taking observed in Experiment 2 are likely unrelated to altered processing of risk-related information. Although decreased preference for the large reward would indicate that impaired reward discrimination could account for the effects of oxotremorine and scopolamine on performance in the RDT, it is more likely that these drugs are impairing general reward-related behavior. In support of this, both muscarinic receptor ligands indiscriminately affected latencies to press levers and increased the number of omissions. Hence, activation or blockade of muscarinic receptors lead to interference with general reward-based motivated behavior.
The primary objective of the current study was to determine the role of nAchRs and mAchRs in decision making involving risk of explicit punishment. A secondary objective was to identify whether these roles differed as a function of biological sex. In contrast to manipulations of dopaminergic neurotransmission (Blaes et al., 2018; Simon et al., 2011), the current study suggests that manipulations of cholinergic receptors have only a moderate impact on decision making involving risk of explicit punishment. Neither nAchR activation nor nAchR blockade affected lever pressing for the large, risky reward; nAchR antagonism did, however, alter other behavioral measures that are considered to be proxies for outcome evaluation and decision-making strategies. Manipulations of mAchRs did disrupt decision making, with both an agonist as well as an antagonist causing a reduction in lever pressing for the large, risky reward. While this suggests that mAchRs are necessary for punishment-based risky decision making, control experiments in which these receptors were manipulated during a reward discrimination task (i.e., choice between a small reward and a large reward without the element of risk of punishment) revealed that changes in lever pressing for the large, risky reward may merely be due to impairments in reward discrimination or reward motivation. Our findings are incongruent with those of other studies that showed clear roles of nAchRs vs. mAchRs in cost/benefit decision making (Betts et al., 2021; Mendez et al., 2012; Silveira et al., 2015); such a discrepancy once again highlights the idea that the neurobiological mechanisms that contribute to decision making may differ depending on the risk involved in the decision (risk of reward omission vs. risk of explicit punishment).
Prior work reported that acute administration of the nAchR agonist nicotine increased choice of the large, risky reward in the RDT (Mitchell et al., 2011). In the current study, however, nicotine had no effect on risk taking. These results suggest that cholinergic tone on nAchRs does not influence preference for a large, risky reward. Despite the inconsistency between our findings and those of Mitchell et al. (2011), they are congruent with work showing that nicotine has no effect on other forms of risk-based decision making (Betts et al., 2021; Mendez et al., 2012; Silveira et al., 2015). The reason for the inconsistency between our findings and those reported by Mitchell et al. (2012) is not clear, but possible explanations include differences in housing conditions [e.g., light cycle (current study, lights off at 0800; Mitchell study, lights on at 0800), food composition (current study, soy-free; Mitchell soy-based) and vendor source location (current study, Kingston NY or bred in-house; Mitchell study, Raleigh NC). Given that most other studies have not found an effect of nicotine on risk taking, the most parsimonious conclusion is that activation of nAchRs is ineffective in modulating risk-based decision making.
Similar to the absence of an effect of an nAchR agonist on risk taking, the nAchR antagonist mecamylamine did not affect lever pressing for the large, risky reward in either sex. Despite an absence of effects on lever preference, mecamylamine did selectively increase latencies to press the large risky lever, suggesting that blocking nAchRs affects the deliberative process that occurs prior to execution of a choice. During this pre-choice period, each option is evaluated with respect to the salience of possible rewards and risks in that given moment. An increase in latency to press the large, risky lever may therefore indicate that blocking nAchRs augmented the salience of the risk of punishment, causing rats to take more time before deciding to press the lever. In addition to affecting latencies to press the large, risky lever, mecamylamine also increased the percentage of omitted free choice trials. This effect, however, was specific to females. The most parsimonious explanation for mecamylamine’s effects on latencies and omissions, at least in females, is that the blockade of nAchRs led to a general decrease in overall motor output, which would consequently affect these behavioral measures. Indeed, others have reported that mecamylamine has similar effects on choice latencies and omissions in other decision-making tasks (Betts et al., 2021; Silveira et al., 2015). Arguing against this interpretation, however, is the fact that in the current study, mecamylamine had no effect on locomotor activity, either at baseline or during shock delivery. Further, rather than serving as a measure of reward motivation or motor activity, trial omissions have been suggested to be another measure of risk aversion, particularly in females (Orsini & Setlow, 2017; Truckenbrod, Cooper, & Orsini, 2023). Consequently, increased omissions in females after mecamylamine administration may be a manifestation of increased risk aversion, which would be consistent with the longer latencies to press the large, risky lever. Considered together, it is therefore likely that, even in the absence of overt effects on lever preference, the blockade of nAchRs with mecamylamine affected cognitive processing necessary for evaluating risks inherent in decision making.
Although targeting nAchRs had more nuanced effects on behavior in the RDT, targeting mAchRs with an agonist (oxotremorine) or antagonist (scopolamine) had a significant impact on lever preference. Oxotremorine decreased preference for the large, risky lever, with a trend toward a greater effect in males relative to females. Oxotremorine also increased latencies to press levers and increased the percentage of omitted free choice trials, with females exhibiting greater sensitivity to this latter effect of oxotremorine compared with males. Finally, the highest dose of this muscarinic agonist decreased baseline locomotor activity in both sexes. Although the decrease in locomotor activity is consistent with prior work (Mendez et al., 2012), the reduction in preference for the large, risky reward upon oxotremorine administration has not been previously observed. These effects of oxotremorine initially suggest that activation of mAchRs can selectively influence decision making involving risk of punishment; however, the comparable effects of scopolamine, a mAchR antagonist, implies that manipulating mAchRs in general may instead lead to impairments in other aspects of the task unrelated to risk of punishment. Indeed, not only did scopolamine decrease pressing for the large, risky lever, it also indiscriminately increased latencies to press levers, increased omissions, and decreased locomotor activity. Because both oxotremorine and scopolamine reduced pressing for the large lever in the first trial block in which there was no risk of punishment (i.e., choice between a small and large reward), one interpretation of our findings is that manipulating mAchRs with either an agonist or antagonist reduces the ability to discriminate between rewards of different magnitudes. Alternatively, and consistent with work showing that mAchR antagonists interfere with motivation for food and/or consummatory behavior (Sharf & Ranaldi, 2006; Yousuf, Girardi, Crouse, & Picciotto, 2023), oxotremorine and/or scopolamine administration may have reduced general reward motivation.
To address these alternative explanations for the effects of mAchR ligands on risk taking, additional experiments were therefore conducted in which the effects of oxotremorine or scopolamine on reward magnitude discrimination were examined. As depicted in Figure 5, both drugs reduced lever pressing for the large reward. It is notable that the largest effect of oxotremorine was in the first two blocks, which could reflect changes in the pharmacodynamics of the drug over the course of the test session. The highest dose of scopolamine decreased lever pressing for the large reward across all trial blocks, although the reduction was greatest in later trial blocks. Rather, the effects of these muscarinic ligands on behavior may be due to disrupted motivation for food. Indeed, infusions of scopolamine directly into the nucleus accumbens decreases lever pressing for rewards (Pratt & Kelley, 2004, 2005). Additionally, Mendez et al. (2012) found that scopolamine decreased choice of the large reward in a probability discounting task, even when delivery of the large reward was guaranteed. In this same study, scopolamine increased trial omissions, consistent with the current findings. Recent work, however, suggests that scopolamine’s effects on appetitive behavior cannot solely be due to reduced motivation for food. Systemic scopolamine administration at doses higher than that used in the current study decreased sign-tracking behavior, or behavioral responding directed towards cues predictive of a reward, and increased goal-directed behavior, or behavioral responding directed toward the location of subsequent reward delivery (Gheidi, Fitzpatrick, Gregory, & Morrow, 2023). This behavioral shift was thought to reflect a reduction in the incentive salience attributed to the cues predictive of the reward. Given these findings, rather than impaired food motivation, the effects of scopolamine on behavior in the current study could be due to a reduction in the incentive value attributed to the large reward (with or without risk). Anecdotal observations that rats consumed all food pellets during the test session in addition to the chow provided in their homecage support the contention that scopolamine’s effects on behavior may be due to aspects other than or in addition to changes in food motivation.
Cholinergic receptors are found throughout the central and peripheral nervous system (Winek, Soreq, & Meisel, 2021). Consequently, the effects of systemic administration of mAchR ligands could also be due their effects on physiological function (e.g., altered autonomic function, which could impact task performance) or on basic motor operation. Indeed, scopolamine slowed locomotor activity at the highest dose in the current study, consistent with findings from other groups (Betts et al., 2021; Mendez et al., 2012; Silveira et al., 2015). This effect could account for the decreased number of lever presses, increased latencies to press levers and increased omissions. Recent work, however, has shown that despite a reduction in motor activity, systemic administration of the same doses of scopolamine improves decision making in a cued rodent gambling task by increasing preference of more advantageous options (Betts et al., 2021). Furthermore, administration of atropine, another muscarinic receptor antagonist, has effects similar to that of scopolamine on choice behavior in a probability discounting task without reducing locomotor activity (Mendez et al., 2012). Finally, others have repeatedly shown that rather than slowing motor activity, scopolamine induces hypermotility (Anagnostaras, Maren, Sage, Goodrich, & Fanselow, 1999; Bauer, 1982; Brambilla, Ghiorzi, Pitsikas, & Borsini, 1993). Hence, although blocking mAchRs systemically may affect overall motor function, the impact on choice behavior is unlikely to be secondary to the decreased locomotor activity observed after scopolamine administration. Nevertheless, additional studies can specifically address this issue with injections of scopolamine (or other mAchR antagonists) directly into brain regions known to be involved in risk-based decision making, such as the nucleus accumbens and ventral tegmental area.
Another objective of the current study was to determine whether there were sex differences in cholinergic regulation of risk taking. Not only are there robust sex differences in decision making involving risk of explicit punishment (Orsini et al., 2016; Truckenbrod et al., 2023), but there are also sex differences in the sensitivity of risk taking to dopaminergic manipulations, with dopamine receptor agonists inducing greater behavioral effects in females relative to males (Georgiou et al., 2018; Hynes et al., 2020; Islas-Preciado et al., 2020; Orsini et al., 2016). With respect to the cholinergic system, there are sex differences in sensitivity of nicotinic receptors to nAchR agonists (Barrett, Geary, Steiner, & Bevins, 2017, 2018; Damaj, 2001; Moen & Lee, 2021) as well as in the role of muscarinic receptors in the ventral tegmental area in other forms of decision making (Nunes et al., 2023). Importantly, cholinergic signaling in the mesolimbic system is a critical regulator of dopamine neurotransmission (Cachope & Cheer, 2014; Forster & Blaha, 2003; Miller & Blaha, 2004; Yeomans, 1995). Hence, sex differences in dopamine-dependent behavior, such as risk taking, may arise from differential cholinergic modulation of dopamine signaling between sexes. Despite these known sex differences, there were no sex-dependent effects of any cholinergic receptor ligand on choice preference in the current study. The only sex difference present across both nAchR and mAchR manipulations was a greater effect of mecamylamine and oxotremorine on the percentage of omitted free choice trials in females relative to males. Because omissions can be considered another risk-averse decision-making strategy (Orsini & Setlow, 2017; Orsini et al., 2016; Truckenbrod et al., 2023), these effects might suggest that nAchRs and mAchRs may contribute to greater risk aversion in females. Additional experiments, however, are needed to fully examine whether these sex differences are relevant to decision-making processes (i.e., augmenting the salience of the risky option, leading to omissions) or are a product of greater effects of these ligands on other behavioral processes (e.g., motivation, food intake) in females compared with males.
One notable difference between the current study and other similarly designed pharmacology studies was the dependent variable used in the analyses. Rather than the number of lever presses on the large lever, other studies predominantly use the percentage of free choice trials on which a rat pressed the large lever as the main metric for risk taking (e.g., Blaes et al., 2018; Mendez, et al., 2012; Simon et al., 2011). Many of the cholinergic receptor agonists, however, increased the percentage of omissions in the free choice trials, which obscured an accurate representation of the effects of these drugs on risk preference. This is illustrated in the comparison between the effects of oxotremorine or scopolamine on the number of large lever presses (Figure 5) vs. on the percentage of large lever presses (Supplemental Material) in the Reward Discrimination task (Experiment 3). Both drugs decreased preference for the large lever, but the extent of this effect is more apparent when the number of large lever presses are used as the dependent variable. Although the use of the number of large lever presses as the metric for risk taking precludes the ability to assess effects on choice per se (risky vs. safe; large vs. small), it does provide a more accurate representation of preference for the larger reward that is not contaminated by trial omissions. Nevertheless, it is important to highlight the distinct approaches to data analysis as each dependent variable (i.e., lever presses vs. percent choice) provides slightly different insight into how cholinergic neurotransmission affects decision making.
The findings of the current study do demonstrate that cholinergic transmission via muscarinic receptors is involved in aspects of decision making involving risk of explicit punishment, but additional experiments are necessary to better understand how this modulatory neurotransmitter system influences risk taking. For example, the mAchR ligands used in the current study affected multiple aspects of behavior, rendering it challenging to unequivocally identify their specific role in this form of decision making. Accordingly, additional control experiments in which the effects of oxotremorine and/or scopolamine on specific measures of motivation to work for food (e.g., progressive ratio schedule of reinforcement), attentional processing (e.g., 5-choice serial reaction time task) and consummatory behavior (e.g., assessment of food intake over 24 hours) would be helpful to address this limitation of the current study. Brain region-specific injections of mAchR ligands may also circumvent some of the general behavioral impairments that were observed, particularly those involving locomotor activity. Indeed, previous work in which scopolamine was administered directly into the ventral tegmental area showed that this mAchR antagonist had selective effects on operant learning without altering general task performance (Sharf & Ranaldi, 2006). Moreover, intra-NAc injections of scopolamine decrease lever pressing for food while increasing locomotor activity (Pratt & Kelley, 2004, 2005). Future studies will therefore employ site-specific microinjections of mAchR ligands to identify the specific behavioral mechanisms in risk-based decision making that are mediated by mAchRs.
Certain neuropsychiatric disorders associated with altered cholinergic signaling are characterized by cognitive impairments that include deficits in decision making. For example, altered risk-based decision making is commonly observed in individuals with substance use disorder or attention deficit hyperactivity disorder (DeVito et al., 2008; Ersche et al., 2005; Gowin et al., 2013; Potter et al., 2006). Previous work has shown that many of these diseases are linked to altered cholinergic receptor function or expression (Breese et al., 2000; Court et al., 1998; Leonard et al., 2000; Mukhin et al., 2008). Furthermore, targeting the cholinergic system holds promise for treating these conditions (Nakamura et al., 2007; Potter & Newhouse, 2008; Wilens & Decker, 2007). Indeed, scopolamine decreases opioid self-administration in rats (Li et al., 2010) and reduces heroin craving in individuals with opioid use disorder (Jensen et al., 2018; Liu et al., 2013; Yang, Xu, & Luo, 1996). The results of the current study therefore suggest that administration of agents selective for the cholinergic system may be useful in ameliorating the dysfunctional risk-based decision making that accompany certain psychiatric conditions. In support of this, recent work shows that treatment with cholinergic receptor agonists attenuated methamphetamine-induced impairments in decision making in a rat gambling task (Mizoguchi, Wang, Kusaba, Fukumoto, & Yamada, 2019). Future studies designed to identify the specific cholinergic receptor subtypes involved in risk taking are therefore necessary to guide the development of cholinergic-based treatment strategies that could mitigate pathological choice behavior.