Antagonism of nucleus accumbens M2 muscarinic receptors disrupts operant responding for sucrose under a progressive ratio reinforcement schedule

Behavioural Brain Research 181 (2007) 127–135

Research report

Antagonism of nucleus accumbens M2 muscarinic receptors disrupts operant responding for sucrose under a progressive ratio reinforcement schedule Graham A. Cousens ∗,1 , Jacob T. Beckley Department of Psychology and Program in Cognitive and Neuroscience Studies, Macalester College, 1600 Grand Avenue, Saint Paul, Minnesota 55105, United States Received 17 September 2006; received in revised form 15 March 2007; accepted 30 March 2007 Available online 5 April 2007

Abstract Diverse cholinergic signaling mechanisms regulate the excitability of striatal principal neurons and modulate striatal-dependent behavior. These effects are mediated, in part, by action at muscarinic receptors (mAChR), subtypes of which exhibit distinct patterns of expression across striatal neuronal populations. Non-selective mAChR blockade within the nucleus accumbens (NAc) has been shown to disrupt operant responding for food and to inhibit food consumption. However, the specific receptor subtypes mediating these effects are not known. Thus, we evaluated effects of intra-NAc infusions of pirenzepine and methoctramine, mAChR antagonisits with distinct binding affinity profiles, on operant responding for sucrose reward under a progressive ratio (PR) reinforcement schedule. Moderate to high doses of methoctramine disrupted operant responding and reduced behavioral breakpoint. In contrast, pirenzepine failed to impact operant performance at any dose tested. Methoctramine failed to affect latencies to complete appetitive-consummatory response sequences or to impact measures of acoustic startle, suggesting that its’ disruptive effects on operant behavior were not consequent to gross motor impairment. Since methoctramine has a greater affinity for M2 receptors compared to pirenzepine, which has a greater relative affinity for M1 and M3 receptors, these findings suggest that M2 mAChRs within the NAc regulate behavioral processes underling the acquisition of reward. © 2007 Elsevier B.V. All rights reserved. Keywords: Muscarinic receptors; Nucleus accumbens; Ventral striatum

1. Introduction The ventral striatum is thought to comprise an integral component of neural circuitry underlying reward-related motivational processes [2,15,29,31]. This region has been conceptualized as an interface between limbic forebrain areas implicated in sensory, mnemonic, and affective processes and pallidal and hypothalamic areas involved in the initiation and modulation of goal-directed behavior [21]. Understanding how intrinsic ventral striatal circuitry modulates information flow

∗

Corresponding author. Tel.: +1 651 696 6109; fax: +1 651 696 6348. E-mail addresses: [email protected], [email protected] (G.A. Cousens). 1 Department of Psychology, Drew University, 36 Madison Avenue, Madison, NJ 07940, United States. 0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.03.036

has broad relevance to our understanding of both basic and clinically-related motivational processes. Anatomical studies have provided a detailed account of the organization of extrinsic inputs to ventral striatal territories, including the nucleus accumbens (NAc), and have further revealed a complex intrinsic organization [11,20]. Fundamental to this circuitry are local circuit interneurons, which prominently include large aspiny cholinergic cells. Though these cells comprise only 1–2% of all striatal neurons, they ramify extensively and provide a rich source of acetycholine (ACh) [14,34]. Cholinergic markers, cholineacetyltransferase (ChAT) and acetylcholinesterase (AChE), are expressed at high levels throughout the striatum [36], suggesting that cholinergic signaling mechanisms have a strong modulatory influence on striatal output by directly or indirectly influencing the activity of projection neurons. Consistent with this notion, diverse nicotinic and muscarinic cholinergic receptor-mediated mechanisms have

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been shown to potently regulate the excitability of striatal neurons [5,10,16,33]. Pharmacological studies have demonstrated that behavioral effects of NAc ACh are mediated in part by action at muscarinic receptors (mAChR). Thus, for example, intra-NAc infusion of the non-selective mAChr antagonist, scopolamine, has been shown to modulate the performance of tasks that involve both appetitive and aversive stimuli [7,26,27,30,37]. Consistent with the notion that NAc mAChR in part regulate reward-related motivational processes, intra-NAc infusion of scopolamine has been shown to disrupt appetitive responding for food and food consumption [25,26]. Of the five cloned mAChR subtypes (M1 –M5 ) [3,4,17,24], M1 –M4 are highly expressed in the striatum [12]. These receptors show distinct patterns of expression across striatal cell types [1,12], so knowledge of the involvement of particular receptor subtypes in reward-related behaviors would help to elucidate the functional organization of NAc intrinsic circuitry. However, behavioral neuropharmacological studies have been hampered by a dearth of subtype-selective mAChR ligands. Subtypeselective agonists have not been identified, and though some antagonists do exhibit a degree of subtype selectivity, pharmacologically dissecting the contributions of particular receptors requires comparison of multiple compounds [6]. In order to gain insight to the contribution of specific mAChR subtypes to NAc-mediated motivational processes, we compared effects of local infusions of pirenzepine and methoctramine, antagonisits with distinct binding affinity profiles, on operant responding for sucrose reward under a progressive ratio reinforcement schedule. Under this schedule, Ss are required to make an increasing number of responses in order to gain access to successive rewards, and the behavioral ‘breakpoint’ at which responses cease is taken as a measure of motivation to obtain reward. Operant tasks involving PR schedules have been used widely to assess the reinforcing properties of natural rewards, such as food [13], and a range of addictive drugs [28]. Since methoctramine has a greater affinity for M2 receptors compared to pirenzepine, which has a greater relative affinity for M1 and M3 receptors [6], we reasoned that comparison of the behavioral effects of these drugs on PR performance would help to elucidate the relative contribution of mAChR subtypes to reward-related behavioral processes. 2. Materials and methods Three experiments were conducted to examine the behavioral effects of intraNAc infusions of pirenzepine and methoctramine. In Experiment 1, behavioral dose–response functions were determined for these drugs on operant responding for sucrose solution under a PR schedule of reinforcement in sessions of fixed 60 min duration. In Experiment 2, the effects of nM methoctramine (but not pirenzepine) were compared to the minimum effective dose of the drug observed in Experiment 1 using a behavioral breakpoint procedure in which sessions were terminated after a 10 min lapse in responding. In Experiment 3, effects of methoctramine were evaluated on the acoustic startle reflex and prepulse inhibition (PPI) of this reflex.

2.1. Subjects Forty-two male Sprague–Dawley rats bred at Macalester College and weighing between 250 and 350 g at study onset served as Ss. Ss were housed

individually in Plexiglas cages in a humidity- and temperature-controlled vivarium and were maintained on a 12-h light:12-h dark cycle with lights on at 07:00 h. All behavioral sessions were conducted at the same time each day during the light phase of the cycle. In Experiments 1 (n = 18) and 2 (n = 12), Ss were afforded free access to water but were maintained at 85% of free-feeding weight by restricting access to food in the home cage, except as noted below. In Experiment 3 (n = 12), Ss were afforded free access to food and water continuously. All experimental procedures were approved by the Macalester College Institutional Animal Care and Use Committee and are in accordance with US Public Heath Service guidelines.

2.2. Apparatus 2.2.1. Operant behavior Operant behavior was assessed using six standard operant chambers (MedAssociates, St. Albans, VT) measuring 30.5 cm × 24.1 cm × 21.0 cm, each housed within a sound-attenuating cubicle equipped with a high-output ventilation pole fan blower. A single fluid cup was situated on the center of one wall of each chamber 5 cm from the grid floor, and a 6 W stimulus light was situated 3 cm from the ceiling directly above the fluid cup. Each chamber contained two identical nose-poke portals situated 3 cm from the chamber floor on either side of the fluid cup. Aqueous sucrose solution (10%, m/v) was delivered to the fluid cup using a syringe pump. Nose-poke responses and fluid cup head-entries were continuously monitored using photoelectric beams, and all paradigmatic events were controlled by a microcomputer running Med PC IV software (Med-Associates). 2.2.2. Acoustic startle PPI of the acoustic startle response was assessed using a single acoustic startle system (Hamilton-Kinder, Poway, CA) comprised of a Plexiglas startle chamber measuring 17.0 cm × 9.0 cm × 14.0 cm and housed within a ventilated sound-attenuating cubicle. A single speaker, used to provide background white noise, startle stimuli, and prepulse stimuli, was mounted adjacent to a 6 W chamber light in the cubicle ceiling 9.0 cm above the chamber ceiling. Vertical force associated with the startle reflex was measured continuously for 250 ms after the onset of each startle stimulus, and all paradigmatic events were controlled by a microcomputer running Hamilton-Kinder software.

2.3. Procedures 2.3.1. Operant behavior At the onset of Experiments 1 and 2, access to home cage water was restricted for 24 h prior to a single operant response shaping session. During this session, ‘active’ nose-poke responses directed to the right-hand portal were rewarded with delivery of 0.163 ml sucrose solution under a fixed-ratio 1 (FR1) schedule. Fluid was delivered over a 5 s interval, during which additional nose-pokes were recorded but had no programmed consequences. ‘Inactive’ responses directed to the left-hand portal were continuously recorded but had no programmed consequences at any point during the session. After 100 reinforced responses, the stimulus light was extinguished and the session was terminated. Next, Ss received free access to water and were gradually reduced to 85% free-feeding weight (determined prior to water restriction) over 3 days by limiting access to food in the home cage. Ss were administered daily 1 h operant training sessions across which they were exposed to variable ratio (VR) and progressive ratio (PR) reinforcement schedules. In Experiment 1, Ss received seven pre-surgical sessions according to the following sequence: FR1; VR5 (three sessions); PR5 (three sessions; increasing within-session response requirements of 5, 10, 15, and so on). In Experiment 2, the three pre-surgical PR sessions were deemed unnecessary and were not conducted. Then, Ss were prepared for surgery as described below. Following recovery from surgery, Ss were weighed and gradually reduced to 85% free-feeding weight. Then, they were administered daily 1 h operant sessions under an exponential PR schedule. This schedule required an increasing number of nose-poke responses in order to gain access to successive rewards during a session and was determined according to the function Required responses = 5 eik − 5

G.A. Cousens, J.T. Beckley / Behavioural Brain Research 181 (2007) 127–135 where i is the reward number and k is a constant [28,38]. In our case, i was set to 0.22, which yielded the following sequence of required nose-poke responses: 1, 3, 7, 12, 19, 29, 44, 65, 95, 137, 198, 284, 406, and 581. In each of the first two experiments, Ss were administered seven PR sessions prior to the first drug infusion. 2.3.2. Prepulse inhibition of the acoustic startle response PPI of the acoustic startle response was assessed in Experiment 3 only. Following recovery from surgery, Ss received three initial PPI sessions followed by sessions on 2 consecutive days per week over a 3-week interval, yielding nine sessions total. Session parameters were modeled after those shown previously to be sensitive to intra-NAc infusions of quinpirole, a dopamine D2 receptor agonists with some affinity for D3 sites [9]. During each session, subjects were exposed to continuous background white noise (70 dB). Following an initial 5 min acclimatization period, 30 white noise startle stimuli (120 dB; 40 ms duration) were presented under a 15 s variable time schedule (interstimulus interval range 10–20 s). Half of these presentations were preceded by 100 ms by a prepulse stimulus (75, 80, or 85 dB, five trials each; 20 ms duration). Both the order of prepulse/pulse-only trials and the order of prepulse intensities were determined according to a pseudorandom sequence. 2.3.3. Surgery All Ss received surgery to implant a bilateral 27 ga guide cannula (Plastics One, Roanoke, VA) immediately dorsal to the NAc medial shell (bregma: 3.0 mm anterior; 1.2 mm lateral; 6.5 mm ventral). Surgery was conducted following initial response shaping in Experiments 1 and 2 but prior to any behavioral procedures in Experiment 3. In preparation for surgery, Ss were afforded free access to food for at least 3 days. They were anesthetized with isoflurane vapor in 100% O2 and mounted in a stereotaxic frame with the tooth bar positioned 5 mm dorsal to the interaural line. Throughout the surgery, the plane of anesthesia was regularly monitored by verifying the absence of hind-limb retraction. The cranium was exposed, and two small burr holes were drilled in the frontal bones overlying the NAc. The cannula was slowly lowered into position, and the base was affixed to the skull with dental acrylic and skull screws placed in the nasal, parietal, and occipital bones. A stylet was inserted in order to maintain cannula patency. Following surgery, Ss were administered a topical antibiotic along with 4% lidocaine solution and were placed under a heat lamp where they remained until they were alert and could be returned to the colony. One subject in Experiment 1 expired during surgery. Ss were afforded a 10–14 days post-surgical recovery period before the resumption or initiation of behavioral procedures. 2.3.4. Drug infusions and experimental designs In order to acclimatize Ss to the infusion procedure in all experiments, the first drug infusion session was preceded by two restraint sessions, during which Ss were gently hand-restrained for 5 min immediately prior to being placed into the behavioral chamber, and one sham infusion session. During the sham infusion session, the stylet was removed, a bilateral 33 ga infusion cannula was inserted with the tip positioned 9.5 mm ventral to skull surface (3 mm beyond the guide cannula tip) and then removed, and the stylet was replaced. During subsequent drug infusion sessions, these procedures were conducted again, and drug was delivered at a rate of 0.25 ␮l/min over a 2 min period. The infusion cannula was held in place for 30 s prior to drug infusion and, to facilitate drug diffusion, for 1 min after drug infusion. These procedures were identical for all experiments, and following their completion subjects were placed immediately into the behavioral chamber. In Experiment 1, 17Ss were assigned to one of two drug treatment groups slated to receive intra-NAc infusions (vehicle, 0.2, 2.0, 10.0, and 20.0 ␮g per hemisphere) of either pirenzepine dihydrochloride (Sigma–Aldrich Co., St. Louis, MO; group PIR; n = 9) or methoctramine tetrahydrochloride (Sigma–Aldrich Co.; group MET; n = 8) dissolved in 0.5 ␮l artificial cerebrospinal fluid. These groups were run in separate batches, but with the exception of the drug administered, all experimental procedures were identical. Restraint procedures were conducted prior to post-surgical Sessions 5 and 6, and sham infusions were conducted prior to Session 7. Infusions were administered according to a randomized, within-subject design such that each subject received each dose of either drug across five drug infusion sessions with consecutive infusions

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separated by two drug-free sessions. Thus, drug infusions were conducted prior to Sessions 8, 11, 14, 17, and 20. In Experiment 2, 12Ss received intra-NAc infusions of methoctramine. (vehicle, 200 pg, or 20.0 ␮g per hemisphere) following a protocol similar to that of Experiment 1. Restraint procedures were conducted prior to Sessions 5 and 6 and sham infusions were conducted prior to Session 7. In order to reduce potential effects of acute drug-induced alterations in home cage feeding on task performance, drug infusion sessions were separated by six drug-free sessions. Thus, drug infusions were conducted prior to Sessions 8, 15, 22, and 29. As in Experiment 1, drug infusions were administered according to a randomized, within-subject design. In addition, in order to evaluate the effect of drug dose on the behavioral ‘breakpoint’ (the number of responses after which responding ceased), each drug infusion session continued until a lapse in responding of 10 min was observed. In Experiment 3, 12Ss received intra-NAc infusions of methoctramine at the doses administered in Experiment 2. Restraint procedures were conducted prior to Sessions 1 and 2, and sham infusions were conducted prior to Session 3. Drug infusions commenced 1 week later and were conducted prior to Sessions 5, 7, and 9. Infusion sessions were conducted 1 week apart, and each was preceded by 1 day by a single drug free session. 2.3.5. Histology Following the completion of behavioral testing, Ss were deeply anesthetized with isoflurane, and brains were extracted and submerged in 10% formalin solution. Brains were post-fixed in a 10% formalin solution with 30% sucrose (m/v) for at least 2 days. Brains were frozen and sectioned coronally at 40 ␮m, and alternate sections were mounted on gelatin-subbed microscope slides and stained with 0.2% natural red. Cannula tracks and infusion sites were reconstructed with the aide of a light microscope.

3. Results 3.1. Cannula placement Infusion cannulae were targeted at the medial shell region of NAc (bregma: 3.0 mm anterior; 1.2 mm lateral; 9.5 mm ventral; the tooth bar positioned 5 mm dorsal to the interaural line). Fig. 1 shows a composite schematic of infusion sites for Ss across all three experiments. In some cases, infusion sites could be identified as localized areas of tissue damage ventral to the guide cannula track. However, in other cases, infusion sites were not visible and were estimated based on the ventral-most position of the guide cannula damage and the infusion cannula protrusion distance (3 mm). Infusion sites were generally localized to the NAc medial shell or core medial to the anterior commissure. Since the intercannula distance was fixed (2.4 mm), medial displacement in one hemisphere was typically associated with lateral displacement in the other hemisphere. Thus, bilateral NAc core infusion sites were not identified in any Ss, and direct comparison between core and shell infusions was not possible. One subject from Experiment 2 and one from Experiment 3 were excluded from the analyses described below due to cannula placement outside the NAc. 3.2. Experiment 1 Following recovery from surgery, all Ss readily responded under the PR reinforcement schedule. During the sham infusion session conducted 1 day prior to the first drug infusion, Ss earned on average 7.59 ± 0.33 (mean ± S.E.M.) rewards and made 259.76 ± 24.13 water cup head-entries. A depen-

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Fig. 1. Locations of infusion cannula tips directed at the nucleus accumbens medial shell. Cannula placement was estimated from Nissl-stained coronal sections. Adapted with permission [22]; Experiment 1, triangles; Experiment 2, squares; Experiment 3, circles; methoctramine, closed figures; pirenzepine, open figures.

dent samples t-test revealed a significantly greater number of active nose-poke responses (222 ± 31.04) compared to inactive responses (2.76 ± 0.68; t16 = 7.078, p < 0.001) during this session. An independent samples t-tests comparing the two treatment groups confirmed no significant between-group differences in active responses (t15 = 0.442, p = 0.664), inactive responses (t15 = 0.485, p = 0.635), rewards earned (t15 = 0.101, p = 0.921), or head-entries (t15 = 0.510, p = 0.617) during this session. Fig. 2 shows the effects of intra-NAc pirenzepine and methoctramine on performance of the PR task. Pirenzepine infusion had no significant impact on task performance. One-way repeated measures ANOVA failed to reveal main effects of pirenzepine dose on active nose-pokes (F4,32 = 0.584, p = 0.676), rewards earned (F4,32 = 1.038, p = 0.403), or fluid-cup head-entries (F4,32 = 0.805, p = 0.531). Pirenzepine significantly elevated the number of inactive responses (F4,32 = 3.70, p = 0.014), and post hoc analyses (Fisher LSD) revealed that this effect was due to enhanced responding following the 0.2 and 20 ␮g doses, relative to that observed following vehicle infusion (data not shown). However, the mean number of inactive nose-pokes was low and variable across doses (range 2.00 ± 1.20–8.42 ± 3.01). One subject in the MET expired prior to the completion of behavioral testing and was excluded from the following anal-

Fig. 2. Progressive ratio performance following intra-accumbens infusion of vehicle, 0.2, 2.0, 20.0, or 40 ␮g/0.5 ␮l/hemisphere methoctramine or pirenzepine. (A) Active nose-pokes; (B) rewards earned; (C) fluid-cup head-entries; * indicates significant difference from vehicle condition, Fisher LSD.

yses. As shown in Fig. 2, methoctramine infusion tended to disrupt operant performance under the PR schedule. Separate one-way repeated measures ANOVA revealed significant effects of methoctramine dose on active nose-pokes (F4,24 = 3.311, p = 0.027) and rewards earned (F4,24 = 4.468, p = 0.008), and subsequent post hoc analyses (Fisher LSD) revealed that these effects were due to a reduction in both of these measures following the two highest doses (20 and 40 ␮g per hemisphere), relative to vehicle infusion. A trend toward fewer fluid-cup headentries was also observed with increasing methoctramine dose; however, this trend was not significant (F4,24 = 2.353, p = 0.083). Planned contrasts (Fisher LSD) separately comparing the number of head-entries following the two highest doses to that observed following vehicle infusion yielded significant differences (p = 0.018 and 0.014 for 20 and 40 ␮g doses, respectively). Similar contrasts conducted for the two highest pirenzepine

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doses failed to reveal any significant differences (p’s > 0.17). The basal level of inactive nose-pokes was low (3.86 ± 1.52), and no effect of dose was observed (F4,24 = 1.379, p = 0.271). 3.3. Experiment 2 Findings from Experiment 1 suggested that intra-NAc methoctramine, but not pirenzepine, disrupts operant responding for sucrose reward under a PR schedule during behavioral sessions of fixed 60 min duration. Experiment 2 was conducted to build on these findings in several ways. First, in order to explore whether methoctramine alters behavioral breakpoint, sessions were allowed to continue until a lapse in responding of 10 min was observed. Examination of response patterns for MET Ss in Experiment 1 revealed that only 2Ss would have met this breakpoint criterion following saline infusion and that responding during the final 10 min of these sessions was robust (mean: 40.86 ± 29.34). Second, in order to reduce the possibility that task performance could be influenced by potential acute druginduced changes in homecage feeding, infusions were separated by six drug-free sessions, rather than the two. Finally, since nM concentrations of methoctramine (lower than the range of doses tested in Experiment 1) have been shown to modulate synaptic plasticity at corticosriatal synapses following tissue perfusion in vitro [5], we compared 542 nM (200 pg/0.5 ␮l) methoctramine to the minimal effective dose concentration (20 ␮g/0.5 ␮l or 54.22 mM) observed in Experiment 1. As shown in Fig. 3, methoctramine significantly disrupted task performance. Separate one-way repeated measures ANOVA revealed significant effects of dose on active nose-pokes

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(F2,22 = 11.484, p < 0.001) and rewards earned (F2,22 = 17.488, p < 0.001). These effects were accompanied by a significant reduction in behavioral breakpoint (F2,22 = 11.484, p < 0.001). Post hoc analyses (Fisher LSD) revealed that main effects for each of these three measures were due to significant reductions following the 20 ␮g dose but not the 200 pg dose, relative to vehicle. As in Experiment 1, ANOVA failed to reveal a main effect of methoctramine dose on fluid-cup head-entries, despite a negative trend following the 20 ␮g dose (F2,22 = 2.823, p = 0.081). However, a planned contrast (Fisher LSD) comparing the number of head-entries following the 20 ␮g dose to that observed following vehicle infusion revealed a significant difference (p = 0.043). The number of inactive responses following vehicle infusion (8.91 ± 2.49) was slightly higher than that observed in Experiment 1, and no main effect of dose was observed (F2,22 = 1.756, p = 0.196). In order to determine whether methoctramine administration altered the pattern of operant responding, in addition to the observed reduction in breakpoint, we examined cumulative records in a subset of 7Ss for which behavioral timestamp data had been collected across each of the three drug conditions. The number of active nose-pokes across the three drug doses (vehicle: 359.00 ± 79.69; 200 pg: 386.57 ± 108.86; 20 ␮g: 76.29 ± 24.99) was similar in this subset to that of the larger sample, and a repeated measures ANOVA conducted on active nose-pokes confirmed a significant effects of dose on this measure (F2,12 = 11.740, p = 0.001). As shown in representative records in Fig. 4, reductions in the number of responses following 20 ␮g methoctramine was typically associated with decreased latency to cease responding

Fig. 3. Progressive ratio performance following intra-accumbens infusion of vehicle, 200 pg, or 20 ␮g/0.5 ␮l/hemisphere methoctramine. (A) Active nose-pokes; (B) rewards earned; (C) breakpoint; (D) fluid-cup head-entries. The highest dose disrupted task performance; n = 11; * indicates significant difference from vehicle condition, Fisher LSD.

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Fig. 4. Active nose-poke response profiles for two representative Ss (A and B). Figure shows cumulative number of responses following intra-accumbens infusion of vehicle, 200 pg, or 20 ␮g/0.5 ␮l/hemisphere methoctramine. Traces cease following the final nose-poke, but sessions continued for an additional 10 min as per the breakpoint procedure; n = 7.

(vehicle: 77.86±18.43 min; 200 pg: 69.86 ± 17.85 min; 20 ␮g: 33.86 ± 10.15 min). However, a repeated measures ANOVA conducted on latency to final nose-poke response failed to reveal a significant effect of dose (F2,12 = 2.910, p > 0.05). We attributed this negative finding to one subject that ceased responding after only 6 min following vehicle infusion, and when this subject was removed from the sample, the same analysis revealed a significant effect of dose (F2,10 = 4.99, p = 0.031). Post hoc analyses (Fisher LSD) revealed that this effect was due to a significant reduction in latency following the 20 ␮g dose relative to vehicle. Thus, these data suggest that 20 ␮g methoctramine decreased both the number of active nose-poke responses prior to breakpoint and the latency to reach the final response. A methoctramine-induced disruption in instrumental responding could be the result of gross motor impairment. In order to investigate this possibility, we measured water-cup head-entry latencies following active nose-pokes in the subset of 7Ss identified above. Latencies were determined only when nose-pokes were followed by a head-entry within 5 s, and in cases where multiple nose-pokes were made prior to a head-entry, only the last nose-poke in the train of responses was counted. Greater than 95% of all latencies were less than 2 s under each drug condition, and group median response latencies were similar: 0.8, 0.7, and 0.9 s, following vehicle, 200 pg, and 20 ␮g doses, respectively. These findings are reflected in Fig. 5, which shows that functions describing the group cumulative proportion of latencies under each drug condition were nearly identical.

repeated measures ANOVA were conducted on each measure. As shown in Table 1, methoctramine dose had no significant effect on average startle amplitude (F2,20 = 0.357, p = 0.704), peak startle amplitude (F2,20 = 0.586, p = 0.567), or latency to peak amplitude (F2,20 = 0.197, p = 0.823). The effects of methoctramine were then assessed across trials preceded by a prepulse stimulus, with prepulse values expressed as a percentage of non-prepulse baseline values. As shown in Table 1, prepulse intensities were directly related to percent inhibition of startle amplitude and indirectly related to startle peak latency. Methoctramine had no effect on startle amplitude or latency measures at any of the three prepulse intensities (all p’s > 0.31; see Table 1). In light of the mild trend toward weaker inhibition of average and peak startle amplitude following 80 and 85 dB prepulse stimuli observed in the 20 ␮g condition, separate planned contrasts (Fisher LSD) were conducted to compare these values directly to corresponding vehicle control values. None of these comparisons yielded significant differences (all p’s > 0.20).

3.4. Experiment 3 Experiment 3 was conducted to evaluate the effects of intraNAc methoctramine on acoustic startle and PPI of acoustic startle. Three indices of startle were evaluated: average startle amplitude across a 250 ms period following the startle stimulus; peak startle amplitude during this interval; and latency to peak amplitude. These measures were averaged across the 15 non-prepulse trials for each subject, and separate one-way

Fig. 5. Effect of methoctramine on fluid-cup head-entry latencies relative to onset of active nose-poke reponses. Figure shows cumulative proportion of latencies following intra-accumbens infusion of vehicle, 200 pg, or 20 ␮g/0.5 ␮l/hemisphere methoctramine; n = 7.

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Table 1 Effects of methoctramine on accoustic startle and prepulse inhibition Vehicle

200 pg methoctramine

20 ␮g methoctramine

ANOVA

Accoustic startle Average amplitude (N) Peak amplitude (N) Latency to peak (ms)

0.07 ± 0.01 0.65 ± 0.19 34.48 ± 4.05

0.08 ± 0.02 0.78 ± 0.21 32.06 ± 3.07

0.07 ± 0.02 0.63 ± 0.21 33.73 ± 3.85

F = 0.357, p = 0.704 F = 0.586, p = 0.567 F = 0.197, p = 0.823

Prepulse inhibition Average amplitude (% inhibition) 75 dB 80 dB 85 dB

37.55 ± 9.04 47.60 ± 6.07 58.44 ± 6.09

33.03 ± 7.75 49.58 ± 9.65 57.63 ± 5.57

30.96 ± 11.10 32.31 ± 12.28 48.86 ± 8.40

F = 0.049, p = 0.619 F = 0.181, p = 0.835 F = 0.213, p = 0.809

Peak amplitude (% inhibition) 75 dB 80 dB 85 dB

35.67 ± 11.47 52.93 ± 5.72 63.74 ± 6.09

34.62 ± 8.88 55.83 ± 7.42 65.74 ± 4.48

30.17 ± 14.05 30.23 ± 14.60 48.82 ± 11.48

F = 0.649, p = 0.533 F = 0.203, p = 0.619 F = 0.202, p = 0.818

Latency to peak (ms) 75 dB 80 dB 85 dB

125.20 ± 3.19 121.01 ± 3.12 110.29 ± 7.98

125.15 ± 2.66 129.2 ± 1.11 113.36 ± 3.77

120.87 ± 4.88 123.36 ± 7.07 113.06 ± 7.52

F = 0.481, p = 0.625 F = 1.209, p = 0.319 F = 0.103, p = 0.902

Top: startle amplitude and peak latency to a 120 dB pulse stimulus following intra-accumbens infusion of vehicle, 200 pg, or 20 ␮g/0.5 ␮l/hemisphere methoctramine. Bottom: startle amplitude and peak latency to a 120 dB pulse stimulus preceded by 75, 80, and 85 dB prepulse stimului. Amplitude measures represent percent inhibition relative to non-prepulse values. All values represent mean ± S.E.M. F-values and p-values are reported for one-way repeated measures ANOVA with 2,20 degrees of freedom; n = 11.

4. Conclusions and discussion Intra-NAc infusion of methoctramine disrupted performance of an operant nose-poke task under a PR schedule of reinforcement. In Experiment 1, methoctramine decreased both the number of appetitive nose-poke responses and the number of fluid-cup head-entries associated with reward consumption. In Experiment 2, these effects were again observed and were accompanied by a decrease in behavioral breakpoint. The effects of methoctramine were dose dependent as only the higher doses of 20 and 40 ␮g/hemisphere disrupted behavior. In contrast, intra-NAc infusion of pirenzepine failed to significantly affect the number of active nose-poke responses, rewards earned, or fluid-cup head-entries at any dose tested. Although it is tempting to speculate that the disruptive effects of methoctramine on PR task performance are consequent to modulation of underlying motivational processes, other explanations should be entertained. Indeed, the NAc has been implicated in diverse motivational, motor, affective, and learning-related functions (reviewed in Ref. [23]) which could viably explain the present findings. However, the present evidence suggests that the disruptive effects of methoctramine were likely not due to a gross impairment in motor activity. First, although 20 ␮g methoctramine significantly reduced the number of nose-pokes and head-entries exhibited, this dose had no effect on the latency between these responses. It might reasonably be expected that gross motor impairment would prolong this latency. Second, in Experiment 3, this dose failed to affect either latency or amplitude measures of the acoustic startle response, indicating that motor coordination necessary for this reflexive response was intact. It should be stated that since potential motor effects of 40 ␮g/hemisphere methoctramine were not assessed, the possibility that the disruptive effects of this dose on operant behavior

are due to motor impairment cannot be excluded. However, this explanation is unlikely due to the similar operant response levels observed following the two higher doses of pirenzepine in Experiment 1. Although methoctramine failed to affect specific coordinated motor responses, additional non-motivational accounts should be considered. Thus, for example, enhancement of ventrolateral neostriatal ACh (dorsolateral to the present infusion sites) has been shown to produce tremulous motor activity in Ref. [8] potentially incompatible with operant behavior. Although informal observation of the present Ss failed to reveal obvious signs of tremulous motor activity or focused stereotypy, the possibility that methoctramine elicited covert behavior that interfered with the initiation of operant behavior cannot be excluded. Nevertheless, a motivational account of the present data would be consistent with the finding that NAc ACh is elevated toward the end of feeding bouts [19]. Such an account would also be consistent with the finding that Ss exhibited slightly reduced body weight 24 h after high doses of methoctramine in the present study (data not shown) and that intra-NAc scopolamine decreased time spent feeding and volume of food consumed in published reports [25,26]. Thus, the present findings are accounted for, but not exclusively explained by, decreased motivation to obtain reward. Since pirenzepine and methoctramine exhibit different binding affinity profiles across mAChR subtypes, comparison of the effects of these drugs could help to distinguish the relative contribution to task performance of these receptor subtypes. Review of literature examining binding affinity constants suggests that methoctramine has a greater affinity for M2 receptors compared to pirenzepine, which has a greater relative affinity for M1 and M3 receptors [6]. This same review suggests that affinity of

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these drugs for M4 receptors is roughly equivalent. Thus, the selective effects of methoctramine in the present study are most easily explained by the drug’s action M2 receptors. In the striatum, these receptors are predominantly, though not exclusively, localized to ChAT-positive interneurons, where they are present in somatodendritic and axon terminal compartments [1,12,32] and are thought to play a role in regulating ACh release by modulating N- and P-type Ca2+ channels [35]. Thus, it is tempting to speculate that presynaptic regulation of ACh release from NAc local-circuit cholinergic cells is in part responsible for modulating behavioral processes underlying performance of the PR task. Though methoctramine is a potent antagonist of M2 receptors in nM concentrations in vitro, in ␮M concentrations it has been shown both to stimulate the formation of inositol phosphate and to inhibit the formation of cAMP in a mAChR-independent fashion in dissociated rat striatum [18]. Thus, the behavioral effects observed of the minimum effective dose in the present study (54.22 mM or 20 ␮g/0.5 ␮l) could be consequent to direct modulation of secondary binding sites that directly affect second messenger signaling. This notion is consistent with the finding that infusion of 542 nM (200 pg/0.5 ␮l) methoctramine, a concentration that likely selectively binds mAChRs, failed to exert behavioral effects in the present study. However, processes of diffusion and metabolism would be expected to significantly reduce the actual concentration of drug at receptors in non-steady-state conditions, such as following a single in vivo infusion. Since drug concentrations in brain were not measured in the present study, it is not possible to directly determine whether methoctramine was at a sufficient concentration to exert non-receptor-mediated effects. The disruptive effects of methoctramine on PR performance are largely similar to those previously reported for intra-NAc infusions of 1 or 10 ␮g/hemisphere scopolamine, a non-selective mAChR antagonist [26]. Intra-NAc scopolamine retarded acquisition of operant responding for sucrose reward, decreased behavioral breakpoint under a PR reinforcement schedule, and reduced reward consumption without altering latencies to complete appetitive-consumatory behavioral sequences [25,26]. The authors concluded that these behavioral effects were due to muscarinic receptor blockade. In the present study, intraNAc pirenzepine failed to influence operant performance, even though this manipulation within the range of doses examined has been shown to exert other behavioral effects, such as enhancing forced swimming [7]. The findings that intra-NAc infusions of either scopolamine or methoctramine, but not pirenzepine, disrupt operant performance provide indirect evidence that M2 receptors are involved in behavioral processes underlying PR performance. In conclusion, the present findings confirm the notion that NAc mAChRs contribute to behavioral processes underlying the acquisition of food reward. Further, they suggest that modulation of ACh release from cholinergic neurons through M2 receptormediated mechanisms may play a more significant role in such processes than the activation of MSNs and other interneurons through M1 and M3 receptors.

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