β2* nAChR sensitivity modulates acquisition of cocaine self-administration in male rats

Abstract

Signaling through nicotinic acetylcholine receptors (nAChRs) plays a role in cocaine reward and reinforcement, suggesting that the cholinergic system could be manipulated with therapeutics to modulate aspects of cocaine use disorder (CUD). We examined the interaction between nAChRs and cocaine reinforcement by expressing a hypersensitive β2 nAChR subunit (β2Leu9′Ser) in the ventral tegmental area of male Sprague Dawley rats. Compared to control rats, β2Leu9′Ser rats acquired (fixed ratio) intravenous cocaine self-administration faster and with greater likelihood. By contrast, β2Leu9′Ser rats were approximately equivalent to controls in their intake of cocaine on a progressive ratio schedule of reinforcement, suggesting differential effects of cholinergic signaling depending on experimental parameters. Like progressive ratio cocaine SA, β2Leu9′Ser rats and controls did not differ significantly in food SA assays, including acquisition on a fixed ratio schedule or in progressive ratio sessions. These results highlight the specific role of high-affinity, heteropentameric β2* (β2-containing) nAChRs in acquisition of cocaine SA, suggesting that mesolimbic acetylcholine signaling is active during this process.

Introduction

Cocaine use disorder (CUD) impacts more than 1 million people in the United States, contributing to increased health care consumption, decreased economic productivity, and overdose-related deaths [1–3]. Tobacco and/or nicotine is a key modulator of cocaine’s reinforcing properties, as 75% of cocaine-dependent individuals reported regular cigarette use compared to only 22% in the general population [4]. Nicotine, the key psychoactive molecule in tobacco, is known to enhance cue-mediated cocaine craving, whereas blocking nicotine’s action reduces such craving [5, 6]. Similarly, treatment with the nicotinic acetylcholine receptor (nAChR) partial agonist varenicline is sufficient to reduce cocaine reward [7]. Despite decades of preclinical research on cocaine addiction, there are no suitable pharmacological treatments available for CUD. The nicotinic cholinergic system presents an opportunity to target components of the dopamine reward pathway to modulate cocaine reinforcement.

Brain nAChRs bind acetylcholine (ACh) and nicotine with high affinity. ACh is membrane impermeant and evanescent, susceptible to degradation by acetylcholinesterase within milliseconds of its release from an axon terminal. On the other hand, nicotine crosses in and out of cellular organelles [8] and can persistently activate and desensitize nAChRs for minutes to hours. β2 subunits are key components of heteropentameric, high-affinity brain nAChRs through their ability to assemble with α4, α5, β3, and α6 subunits [9–11]. β2* nAChRs are strongly expressed in circuits involved in motivated behavior. The most pertinent of these is the ventral tegmental area (VTA), comprising intra-VTA circuitry [12] and forward dopaminergic, GABAergic, and glutamatergic projections [13] to forebrain targets such as nucleus accumbens (NAc). VTA β2 nAChR subunits assemble into functional pentameric receptors with α4 and/or α6 subunits, with additional β3 and α5 subunit incorporation playing a modulatory role [14–18].

Preclinical research has repeatedly identified midbrain nAChRs for their ability to modulate cocaine reward and reinforcement. Cocaine self-administration (SA) induces a sustained rise in VTA and NAc ACh levels [19, 20], where it potently activates nAChRs and muscarinic AChRs on VTA neurons and presynaptic terminals that terminate in VTA. Co-administration of the non-selective nAChR antagonist mecamylamine reduced cocaine intake [21, 22] and prevented escalation of cocaine intake during 6 h SA sessions [23]. Sazetidine-A, a partial agonist at β2* nAChRs that antagonizes nAChR function via its desensitizing property, also reduced cocaine SA [24]. Conversely, addition of nicotine to a cocaine solution enhanced SA compared to cocaine alone [25]. These preclinical data largely mirror results in humans reviewed above; acute nAChR activation potentiates cocaine reinforcement whereas reducing nAChR activity attenuates it.

We tested the mechanistic hypothesis that VTA β2* nAChRs potentiate cocaine reinforcement by expressing a gain-of-function β2 subunit in the VTA of male rats prior to cocaine SA. This approach sensitizes the VTA to cholinergic tone [26–28], allowing us to assess acquisition of cocaine SA in the context of enhanced nicotinic signaling. We examined cocaine and food SA acquisition (fixed ratio) and motivated responding (progressive ratio).

Materials and Methods

Materials –

Cocaine HCl was obtained from the National Institute on Drug Abuse Drug Supply Program. Injectable heparin sodium (catalog #00409-2720-02) was obtained from Pfizer, Inc. (via Medline). Injectable meloxicam (catalog #07-893-7565) and isoflurane (catalog #07-894-8943) were obtained from Patterson Veterinary Supply.

Molecular Biology –

We previously [29] conceived of and designed the AAV5.2-hSyn-DIO-Chrnb2Leu273Ser-P2A-GFP (β2 Leu9′Ser) vector, which was then prepared by Virovek, Inc. Briefly, a linear DNA open reading frame containing Chrnb2Leu273Ser-P2A-GFP was ligated into a shuttle vector, followed by production of a recombinant baculovirus which was used to infect insect Sf9 cells for production and purification of AAV particles (2 × 10¹³ vg/mL). AAV5-hSy-nmCherry-Cre was obtained from the University of North Carolina Vector Core Facility.

Animals –

All experimental protocols involving vertebrates were reviewed and approved by the Wake Forest University School of Medicine Animal Care and Use Committee. Procedures also followed the guidelines for the care and use of animals provided by the National Institutes of Health Office of Laboratory Animal Welfare. All efforts were made to minimize animal distress and suffering during experimental procedures, including during the use of anesthesia. A total of n=86 male SD rats (Envigo) were used. SD rats were ~300 g (approximately eight weeks old) when they arrived at our facility. Rats were housed at 22°C on a reverse 12/12 h light/dark cycle (4 P.M. lights on, 4 A.M. lights off).

Behavioral Apparatus –

Rats were trained in Med Associates operant chambers (interior dimensions, in inches: 11.9 × 9.4 × 11.3, catalog #MED-007-CT-B1) located within sound-attenuating cabinets. The SA system was housed in a dedicated room within the same laboratory suite as the rat’s housing room. A PC computer was used to control the SA system via Med PC IV software. Each chamber had transparent plastic walls, a stainless-steel grid floor, and was equipped on the right-side wall with two levers which flanked a pellet receptacle coupled to a pellet dispenser. A white stimulus light was located above each lever, and a house light was located at the top of the chamber on the left-side wall. During food and drug SA sessions, responses on the active lever activated either the pellet dispenser or an infusion pump, respectively. Responses on the inactive lever had no consequence. For intravenous drug infusions, each rat’s catheter was connected to a liquid swivel via polyethylene tubing protected by a metal spring. The liquid swivel was connected to a 10-mL syringe loaded onto the syringe pump.

Stereotaxic Surgery –

After arrival at our facility, rats were anesthetized with isoflurane (3% induction, 2–5% maintenance) and were introduced into a stereotaxic rat frame. Rats received a bilateral injection into the VTA using coordinates from bregma (AP: −5.40, ML: ±0.70, DV: −8.20). Coordinates were derived from “Brain Maps 4.0” (Larry Swanson; University of Southern California) (Swanson, 2018). Virus injection volumes were as follows: 1.6 μL (0.8 μL per hemisphere, 0.4 μL β2Leu9′Ser and 0.4 μL mCherry-Cre) for β2Leu9′Ser +Cre rats and 0.8 μL (0.4 μL per hemisphere of β2Leu9′Ser only) for β2Leu9′Ser −Cre rats. Virus was infused using a 22-gauge Hamilton injection syringe at a rate of 0.1 μL/min and the injection needle was left in place for 5 minutes after each injection before gradually being retracted. Rats were then given 6–7 days for recovery.

Indwelling Jugular Catheter Surgery –

After recovering from stereotaxic surgery, rats were anesthetized with isoflurane (3% induction, 2–3% maintenance) and implanted with indwelling jugular catheters (Instech, catalog #C30PU-RJV1402). Meloxicam (2 mg/kg) was administered postoperatively to relieve pain and reduce inflammation. Rats were singly housed following surgery and throughout all SA procedures. Rats were allowed 7 d for recovery from surgery, and catheters were flushed several times during this recovery period with heparin sodium dissolved in sterile saline.

Intravenous Drug Self-Administration –

After recovery from catheter surgery, rats were allowed to self-administer cocaine (0.375 mg/kg/inf) during 2-h SA sessions, Monday through Friday (no SA sessions occurred on weekends). Cocaine was dissolved in sterile saline. Infusions, delivered by an infusion pump, were triggered by one response on the active lever. Infusions were simultaneously paired with illumination of the stimulus light over the active lever for 3 s. An active lever response that resulted in an infusion caused active and inactive lever retraction and extinguished the house light for a 20-s timeout period (TO-20). Responses on the inactive lever were recorded but had no scheduled consequence. At the end of the session, the house light was extinguished and responding had no consequences. Rats were removed from the training chambers as soon as possible after the end of the 2-h session (or earlier if the session maximum was achieved) and were rationed to 20 g of standard lab chow at least 1 h after finishing their sessions. Modified chow availability was used throughout SA and a range of 85–90% of free-feeding body weight was maintained. SD rats were allowed to self-administer cocaine for 2 to 21 days on an FR1/TO-20s schedule of reinforcement (20 infusion cap). Rats were deemed to have acquired cocaine SA after achieving 2 consecutive sessions achieving the 20-infusion cap by the 21^st session. Rats were then transitioned to additional FR1/TO-20s training with a 40-infusion cap (2-h session duration). Once rats achieved 3 sessions meeting the 40-infusion cap, they were transitioned to 2-h progressive ratio (PR) SA sessions. In PR sessions, infusions were contingent on an increasing number of responses, incremented as follows: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, etc. [30] (formula: response ratio = [5e^{(injection number × 0.2)}] – 5). Breakpoint was defined as the last completed ratio before the end of the 2-h session. For example, if the last response ratio completed was 178, the rat received a total of 18 infusions, and this corresponded to a breakpoint of 178. Rats participated in 7 cocaine PR sessions. For cocaine SA, a total of n=31 rats were injected with both β2 Leu9′Ser and mCherry-Cre vectors and n = 23 were injected with the β2Leu9′Ser vector without mCherry-Cre co-injection. N = 26 β2Leu9′Ser +Cre and n = 14 β2Leu9′Ser −Cre acquired cocaine SA, respectively. N = 3 of 26 β2Leu9′Ser +Cre rats that acquired cocaine SA failed to advance to PR due to health issues related to catheter patency. All n = 14 β2Leu9′Ser −Cre rats that acquired SA moved on to PR.

Food Self-Administration –

Food SA was conducted in a different cohort of rats that was never exposed to cocaine and which did not receive indwelling catheters. Approximately 3 weeks after stereotaxic surgery, rats were allowed to self-administer food. Food SA was conducted in the same operant chambers used for cocaine SA. Lever operation and visual cues (cue light and house light) were the same except that an active lever response resulted in deposition of a food pellet (45 mg standard chow; Bio-Serv, cat# F0021) into the food hopper located between the active and inactive lever. Rats self-administered food (2-h session duration) for 7 sessions on an FR1 schedule with a session cap of 100 pellets. After FR1 SA, rats were transitioned to PR food SA. Food rewards were contingent on an increasing number of responses, incremented as described above for cocaine SA.

Fluorescence Microscopy –

Rats were deeply anesthetized with isoflurane and perfused transcardially with 50 mL of PBS followed by 250 mL of 4% paraformaldehyde (PFA) in PBS. Brains were removed and stored at 4°C overnight in PBS containing 4% PFA and 4% sucrose (to dehydrate tissue). Coronal sections (12 μm) were cut in triplicate on a microtome and collected into 12-well tissue culture plates filled with PBS and 0.1% sodium azide. Sections were immediately mounted and coverslipped with sodium bicarbonate and native GFP and mCherry fluorescence was imaged on an Olympus IX83 inverted microscope equipped with a 20X UPLXAPO objective (0.8 NA) and a 16-bit (2048 × 2048 pixel frame size) Hamamatsu ORCA-Flash 4.0 camera. Tiled GFP and mCherry images were stitched together in Olympus cellSens Dimension software.

Membrane Preparation –

Following euthanasia, rats were decapitated and their brains were rapidly removed. The nucleus accumbens was then dissected. The tissue was homogenized in 20 vol ice-cold Tris-HCl buffer (50 mM Tris-HCl, pH 7.6) using a motor-driven Teflon-glass homogenizer. The homogenate was centrifuged at 48,000 × g for 15 min. The pellet was resuspended in fresh Tris-HCl buffer and was centrifuged again at 48,000 × g for 15 min. The resulting supernatant was discarded, and the pellet was resuspended in assay buffer and stored in aliquots at −80°C.

[³H]Epibatidine Binding Assay –

Membranes (80 μg protein) were incubated in the presence of 800 pM [³H]epibatidine in polypropylene tubes containing assay buffer (144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM HEPES, pH 7.5) for 4 h in a total volume of 500 μL at 25°C. This duration has been demonstrated to be sufficient for binding to reach equilibrium [31]. Non-specific binding was determined in the presence of 300 μM (–)-nicotine. For competition binding assays, membranes were incubated in the same conditions as above with the addition of 150 nM cytisine. Incubations were terminated by vacuum filtration through Whatman GF/B filters pre-soaked in 0.1% polyethyleneimine using a Hoefer FH225V 10-place filter manifold (Hoefer Inc., San Francisco, CA). The filters were washed three times with 1.5 mL aliquots of assay buffer. The radioactivity retained in the samples was counted in CytoScint Liquid Scintillation Cocktail (MP Biomedicals, SKU: 01882453-CF) using a Beckman LS 5801 liquid scintillation counter (Beckman Coulter, Fullerton, CA). All assays were carried out in triplicate.

Experimental Design and Statistical Analysis –

A parametric statistical test (two-tailed unpaired student’s t-test) was used to analyze [³H]epibatidine binding data (Fig. 1E–G) and cocaine SA acquisition data (Fig. 3B,C). A Log-rank (Mantel-Cox) test was used for survival analysis of cocaine acquisition data (Fig. 2D). SA data files, produced by Med Associates MedPC IV software, were analyzed with custom scripts written in MATLAB and/or GraphPad Prism 9. Rat brain anatomy graphics were derived from “Brain Maps 4.0” (Larry Swanson; University of Southern California) [32].

Figure 1 –

Expression of β2Leu9′Ser nAChR subunits in rat VTA. A. Adeno associated virus for Cre-dependent expression of β2Leu9′Ser subunits. B. β2Leu9′Ser subunits incorporate into natively-expressed β2-containing nAChRs such as α4β2 receptors. C. Fluorescence microscopy image of GFP reporter and mCherry-Cre expression in a rat microinjected in VTA. The corresponding anatomical location from the rat brain atlas is shown at right. D. High-magnification image of GFP & Cre expression in VTA from the image in C. E-G. Epibatidine binding to membranes from control rats and rats expressing β2Leu9′Ser nAChR subunits. Total epibatidine (E) was determined, followed by competition with 150 nM cytisine, revealing cytosine-sensitive (F) and cytisine-resistant (G) components.

Figure 3 –

Analysis of cocaine SA acquisition in β2Leu9′Ser +Cre and −Cre rats. A. For each rat in the indicated group that acquired cocaine SA, infusions per day were aligned such that day 0 represents the first day when each rat earned 20 infusions. B. For rats in the indicated group that acquired cocaine SA, summary data is shown for infusions on the day immediately before the first day when each rat earned 20 infusions. C. For rats that did not acquire cocaine SA, the mean number of infusions per day across all training sessions is shown. D-E. For each rat in the +Cre (D) and −Cre (E) group that acquired cocaine SA, active and inactive responses per day were aligned such that day 0 represents the first day when each rat earned 20 infusions. F. Mean (± SEM) active and inactive responses are shown for each +Cre and −Cre rat for FR1 SA sessions with an infusion cap of 40.

Figure 2 –

Modulation of Cocaine self-administration acquisition by VTA expression of β2Leu9′Ser nAChR subunits. A. Cocaine SA paradigm. Stereotaxic AAV injection surgery and jugular catheter surgery were conducted, then rats were allowed to acquire cocaine SA (FR1) at a dose of 0.375 mg/kg/inf. PR sessions were run for 5 days after SA acquisition. B. Raster plot of cocaine infusions showing the acquisition history of a representative +Cre rat during FR1 (20 infusion cap) training. C. Summary of cocaine SA acquisition in β2Leu9′Ser +Cre and −Cre rats. D. Kaplan-Meier curve plotting percent of +Cre and −Cre rats acquiring cocaine SA as a function of SA session #. E-F. Histogram of days to acquire (bin size = 2 days) in +Cre (E) and −Cre (F) rats.

Results

To assess the impact of hypersensitive VTA nAChRs on cocaine reinforcement, we microinjected a custom Cre-dependent AAV for expressing β2Leu9′Ser subunits (Fig. 1A) bilaterally into the VTA of male Sprague Dawley rats. mCherry-Cre AAVs were co-injected or omitted, respectively, in our two experimental groups. As previously described [29], this expresses β2Leu9′Ser subunits irrespective of other nAChR subunit expression. In cells already expressing nAChR subunits, such as VTA neurons that nearly all express α4 and β2 [13], the ectopic β2Leu9′Ser subunits likely displace one or more WT β2 subunits in α4β2 receptors during assembly in the endoplasmic reticulum (Fig. 1B). Depending on viral transduction and expression levels of β2Leu9′Ser subunits, the pool of α4β2* nAChRs in infected cells is probably a mixture of receptors containing zero, one, two, or even three hypersensitive subunits. We validated efficient viral transduction of VTA neurons via fluorescence microscopy, which confirmed GFP marker expression in cells co-infected with mCherry-Cre AAVs (Fig. 1C,D). Although this viral approach may cause overexpression of β2 subunits, we expect biogenesis of pentameric α4β2 receptors to be limited by α4 subunit availability. To determine if this is true, we studied assembled receptors by measuring cytisine-sensitive epibatidine binding to neuronal membranes from striatal tissue from control rats and rats injected with β2Leu9′Ser AAVs. This reports the approximate number of ligand binding sites on presynaptic β2-containing nAChRs [33–35]. Although total epibatidine binding was reduced in striatum from rats injected with β2Leu9′Ser AAVs (Fig. 1E), cytisine-sensitive (Fig. 1F) and cytisine-resistant (Fig. 1G) epibatidine binding was not significantly reduced in these samples. Thus, there was insufficient statistical support for the hypothesis that β2-containing receptors are overexpressed in rats transduced with β2Leu9′Ser AAVs. Our recent work confirmed that this AAV system results in hypersensitive nAChRs in infected VTA neurons [29].

Next, we designed a self-administration paradigm to assess the impact of VTA β2Leu9′Ser subunit on cocaine reinforcement. Male SD rats were microinjected with 1) β2Leu9′Ser + Cre AAVs or 2) β2Leu9′Ser without Cre, followed 10–14 days later by jugular catheter implantation. After recovery from catheter surgery, rats were allowed to self-administer cocaine (0.375 mg/kg/inf) on an FR1 schedule with an initial cap of 20 infusions, followed by additional FR1 sessions with a 40-infusion cap, followed finally by progressive ratio SA for 7 sessions (Fig. 2A). The dose of cocaine was selected based on previous work demonstrating that 0.375 mg/kg/inf resulted in acquisition in only ~50% of rats [36]. We reasoned that this was a useful dose for our examination of the impact of hypersensitive β2 nAChRs on cocaine reinforcement, as it may allow us to detect attenuated or enhanced SA acquisition. A raster plot from a representative β2Leu9′Ser +Cre rat is provided, showing its infusion history through all FR1 sessions (Fig. 2B). Responding for this animal was typical of those that acquired cocaine SA, as there was a sudden increase in reward frequency on the first day when the rat earned the maximum (20) infusions, and this frequency tended to increase on the second 20-infusion day (sessions #5 and #6, respectively in Fig. 2B). Rats successfully acquired cocaine SA after having achieved the 20-infusion cap for two sessions. Based on this criterion, n=26 of 31 β2Leu9′Ser +Cre rats acquired cocaine SA, and n=14 of 23 β2Leu9′Ser −Cre rats acquired (Fig. 2C). Acquisition (two sessions meeting the 20-infusion cap) in the β2Leu9′Ser +Cre and −Cre groups was subjected to survival analysis, revealing a significant difference between the two groups (Log-rank test, Chi square = 7.81; p = 0.0052) (Fig. 2D). Two aspects were noted: 1) β2Leu9′Ser +Cre rats appeared to acquire faster than −Cre rats, and 2) a greater fraction of β2Leu9′Ser +Cre rats acquired compared to −Cre rats. These features in the data are easily visualized in frequency distributions (Fig. 2E,F). Moreover, we found a significant difference in the number of days to acquire comparing β2Leu9′Ser +Cre and −Cre rats that successfully acquired SA (students t-test, t = 2.12, df = 38; p = 0.041, not shown).

For rats that acquired cocaine SA, we aligned their infusion history such that the first day rats earned 20 infusions was designated as “day 0”. This allowed us to examine their responses and infusions in the days immediately preceding acquisition. Based on this analysis, we observed that both groups demonstrated a sudden increase in infusions to the 20-infusion maximum (Fig. 3A). On the day immediately preceding day 0, however, the β2Leu9′Ser +Cre group earned more infusions than β2Leu9′Ser −Cre rats (Fig. 3B). Among the minority of rats that did not acquire cocaine SA, we examined their infusion records to determine how close or far they were from achieving the acquisition criteria. The mean number of infusions earned for each rat that failed to acquire was not statistically different for the β2Leu9′Ser +Cre vs −Cre group (Fig. 3C). This argues against the possibility that the β2Leu9′Ser −Cre group “barely” failed to acquire; by contrast, rats that failed to acquire did so convincingly in both groups. Similar to infusions (Fig. 3A), we aligned active and inactive responses such that day 0 was the first day each rat earned 20 infusions. As a group, +Cre rats did not appear to differentiate the active and inactive lever until day 0 (Fig. 3D). The −Cre group appeared to differentiate the active and inactive levers one or two days before the first day they earned 20 infusions (Fig. 3E). After FR1 sessions with an infusion cap of 20 were completed, three or more FR1 sessions were conducted with an infusion cap of 40. The mean number of active and inactive responses for each rat, in each group, is shown for these 40-infusion cap sessions (Fig. 3F). There was no significant difference in active or inactive responses between +Cre and −Cre groups (Fig. 3F).

After acquisition, rats were allowed to self-administer cocaine on FR1 for additional sessions but with an infusion cap of 40. After having achieved 3 sessions with 40 infusions, rats were allowed to self-administer cocaine on a progressive ratio schedule for 7 consecutive sessions. A representative infusion raster plot and cumulative response plot (Fig. 4A; showing infusions and active/inactive responses) for a cocaine PR SA session is shown for the same rat whose data is depicted in Fig. 2F. Summary data for infusions (Fig. 4B) during PR and breakpoint achieved (Fig. 4C) revealed no notable difference between cocaine intake in β2Leu9′Ser +Cre and −Cre rats.

Figure 4 –

Cocaine PR self-administration in rats expressing β2Leu9′Ser nAChR subunits in VTA. A. Raster plot (top) and cumulative response plot for the final PR session is shown for the same rat whose data is depicted in Fig. 2F. Red vertical tick marks indicate infusions, blue vertical ticks indicate unrewarded responses on the active lever, and black vertical tick marks indicate responses on the inactive lever. Inset: a magnified view of active responses and infusions is shown for an early part of the session. B. Mean (± SEM) number of infusions achieved during each PR session for the indicated groups. C. Geometric mean (± 95% CI) breakpoint achieved during each PR session for the indicated groups.

Given that β2Leu9′Ser +Cre rats were more likely to acquire cocaine SA @ 0.375 mg/kg/inf, we asked whether β2Leu9′Ser +Cre rats demonstrated similarly enhanced acquisition of food SA. Male SD rats were injected in VTA with β2Leu9′Ser AAVs with or without AAV-mCherry-Cre, as in Fig. 1A. After ~21 days for viral expression, β2Leu9′Ser +Cre and −Cre rats were allowed to acquire self-administration of food pellets using the same cue conditions as used for cocaine. Rats self-administered food pellets for 7 sessions at FR1 with a 100-pellet cap per 2-h session. This was followed by 5 sessions of PR food SA where there was no cap on the number of available rewards (Fig. 5A). β2Leu9′Ser +Cre and −Cre rats rapidly achieved the maximum (100) number of available food rewards during FR1 SA sessions, and there was no important difference in the rate of acquisition (Fig. 5B). Similarly, both groups performed similarly in PR food SA sessions (Fig. 5C).

Figure 5 –

Food self-administration in rats expressing β2Leu9′Ser nAChR subunits in VTA. A. Food SA paradigm. Stereotaxic AAV injection surgery was conducted, then rats were allowed to self-administer food for 7 days (FR1). PR sessions were run for 5 days after FR1 food SA. B. Mean (± SEM) number of food rewards earned during each FR1 session for the indicated groups. C. Mean (± SEM) number of food rewards earned during each PR session for the indicated groups.

Discussion

In this study, we asked whether expression of hypersensitive β2-containing nAChRs in VTA is sufficient to alter cocaine reinforcement. We used an AAV approach, expressing an ectopic β2 nAChR subunit bearing a leucine to serine mutation in a conserved residue in the pore-forming α helix. Incorporation of this subunit into native β2* receptors in VTA neurons was associated with a faster rate and greater overall probability of acquiring cocaine SA. β2 hypersensitivity was not associated with differences in cocaine SA under a PR schedule of reinforcement. Further, we found no important effect of β2 hypersensitivity in food SA (FR or PR).

Our system for generating rats with hypersensitive nAChRs in VTA employs AAV-mediated β2 nAChR gene transfer. This may lead to greater levels of β2 expression than are ideal. However, we did not find evidence for overexpression of ligand binding sites for β2-containing nAChRs in DAergic presynaptic terminals. This is consistent with previous studies. For example, presumptive overexpression of α6Leu9′Ser nAChR subunits via a bacterial artificial chromosome transgenic platform did not result in overexpression of binding sites for α6-containing receptors [26]. β2-containing receptor binding sites were also not upregulated in knockin mice homozygous for a mutant α4Leu9′Ala subunit gene [37]. In fact, cytisine-sensitive binding sites were reduced in many brain areas in α4Leu9′Ala knockin mice [37]. Binding sites – assembled receptors by inference – are not upregulated because heteromeric receptors are limited by the availability of other obligate subunits beyond the one that may be overexpressed. In our case, the number of β2 subunits may be greater than normal due to the viral approach, but because α4 and/or α6 subunits are expressed at endogenous levels, assembled receptor levels are normal.

Our key result is that β2* nAChR hypersensitivity in VTA neurons causes a low dose of cocaine to be more likely to promote acquisition of cocaine SA behavior and/or to do so more quickly. Importantly, cocaine SA is known to induce sustained ACh release in both the VTA [19] and NAc [20] in rats. Cocaine also stimulates ACh release in hippocampus and the caudate nucleus, an effect that is blocked by a D1 DA receptor antagonist [38]. Assuming ACh release occurred in our rat model and in response to the dose of cocaine we tested, this mechanism provides a plausible means by which cocaine engages with hypersensitive nAChRs to promote DAergic activity and enhanced reinforcement behavior. ACh molecules released following cocaine exposure would be expected to encounter somatodendritic and/or presynaptic β2-containing nAChRs [13, 39]. If those β2* nAChRs harbored a Leu9′Ser mutation, low levels of ACh (low because the dose of cocaine we used is also low) would be much more capable of activating nAChRs, leading to depolarization of VTA neuron cell bodies and/or presynaptic terminals in NAc [26, 28]. Our recent study on nicotine SA in β2Leu9′Ser +Cre rats supports this mechanism, as we demonstrated that nAChR-mediated inward currents in VTA neurons from these rats are more sensitive to ACh [29]. Likewise, DA release from β2Leu9′Ser +Cre striatal slices is enhanced in the presence of nicotine [29]. Other studies support our finding that cholinergic signaling enhances cocaine SA acquisition, including that ACh release induced by cocaine was greater in rats self-administering the drug compared to those receiving it non-contingently [20]. The amount of ACh release in NAc was also correlated with the speed at which rats moved to obtain cocaine [40], and cocaine SA was strongly correlated with activation of striatal cholinergic interneurons [41].

Interestingly, we did not find β2Leu9′Ser expression in VTA to induce a differential sensitivity to cocaine in the progressive ratio paradigm. As rats self-titrate their cocaine intake, cocaine levels in the brain are maintained at a relatively constant concentration that is thought to maximize the rewarding aspect of cocaine and minimize its aversive effects [42, 43]. Constant cocaine levels likely lead to constant or sustained ACh release, as previously suggested [19, 20]. Sustained ACh levels may be required to engage with hypersensitive β2 nAChRs to promote reinforcement behavior in our fixed ratio sessions (Fig. 2). The key difference between FR and PR SA is the increasing response requirement for each subsequent drug infusion in the PR paradigm. Since cocaine levels in the brain are expected to fall during PR sessions [42], cocaine-mediated ACh release may also fall and therefore not engage with nAChRs as efficiently. This could explain why β2Leu9′Ser +Cre rats did not show a significant difference from controls in PR responding for cocaine. An alternative reason could simply be that the hypersensitive β2 nAChRs facilitate the neuroadaptations (i.e. glutamatergic transmission, etc.) supporting acquisition of cocaine SA, and are not required for ongoing maintenance of SA or for motivated responding as seen in PR sessions.

Although we have now reported that VTA nAChR hypersensitivity impacts acquisition of operant responding for both cocaine (this study) and nicotine [29], our food SA data suggest that this is not a generalized enhancement of reinforcement behavior. Acquisition of food SA in FR sessions was not different in controls vs. β2Leu9′Ser +Cre rats, nor was responding in PR food SA sessions. Consistent with this, and with our cocaine SA data, is a report showing that blockade of nAChRs by mecamylamine is sufficient to attenuate cocaine SA but not food SA [21].

We note that our study has some limitations. First, we employed infusion restrictions during FR sessions for two reasons: 1) to prevent accidental overdose, and 2) to roughly equalize cocaine exposure within each group. This prevented us from learning about the upper limit of cocaine intake in β2Leu9′Ser +Cre rats. Second, this study is limited by our selection of a single dose of cocaine (0.375 mg/kg/inf) and by our selection of a 2-h SA session duration. These limitations point to the need for further studies to examine the mechanisms by which cocaine enhances endogenous cholinergic transmission to promote self-administration of drugs of abuse. Given the number of available pharmacological agents that impact the cholinergic system, this mechanism may be a viable route for development of therapeutics for cocaine use disorder.

Highlights.

• Rats with hypersensitive β2 nicotinic acetylcholine receptors in ventral tegmental area were made using viral vectors.

• β2Leu9′Ser rats do not overexpress nAChRs in VTA.

• β2Leu9′Ser rats acquire intravenous cocaine self-administration faster and/or more frequently than control rats.

• β2Leu9′Ser rats do not differ from controls in progressive ratio cocaine self-administration.

• β2Leu9′Ser rats do not differ from controls in fixed ratio or progressive ratio food self-administration.