Experiment 1 - NMDA receptors gate the magnitude and valence of the effects of nicotine on brain reward systems
Rats (n=9) were prepared with ICSS electrodes and trained in the ICSS threshold procedure until stable thresholds were established, see above. Rats were then pre-treated with saline or LY235959 (1 mg/kg) 30 min prior to testing, and received a nicotine injection (0, 0.125, 0.25 or 0.5 mg/kg; within-subjects Latin-square design) 10 min prior to being tested in the ICSS procedure. Next, the same rats were injected with saline or LY235959 (2.5 or 5 mg/kg; I.P.) 30 min prior to testing, and also a saline or nicotine (0.25 mg/kg; S.C.) injection 10 min prior to reward threshold assessment (Latin-square design). This dose of nicotine (0.25 mg/kg) was chosen because it induced maximal lowering of reward thresholds when tested alone or in combination with the lower dose (1 mg/kg) of LY235959 (see ). There were at least 48 h between each drug injection.
Fig. 1 NMDA receptor antagonism switched the effects of experimenter-administered nicotine on brain reward systems from stimulatory to inhibitory. (a) Rats (n=9) were pretreated with saline or LY235959 (1 mg/kg), and subsequently received saline or nicotine (more ...)
Mean (± SEM) baseline ICSS threshold prior to drug treatment was 128.8 ± 13.9 μA. Two-way repeated-measures ANOVA on the data involving the 1 mg/kg LY235959 dose demonstrated a significant main effect of Nicotine (F(3,24)=6.3, p<0.01), no effect of LY235959 (1 mg/kg) (F(1,8)=0.18), nor a significant LY235959 × Nicotine interaction: (F (3,24)=1.1). Further analysis of the main effect of Nicotine demonstrated that thresholds were significantly lowered in rats pre-treated with saline or LY235959 at nicotine doses 0.125–0.25 mg/kg (p<0.05; ).
Two-way repeated-measures ANOVA on the data involving the two higher doses of LY235959 (2.5–5 mg/kg) and the threshold-lowering effects of a single dose of nicotine (0.25 m/kg) demonstrated a main effect of LY235959 (F(1,8)=6.4, p<0.01), and a significant Nicotine × LY235959 interaction (F(1,8)=6.3, p<0.01), and no statistically significant main effect of nicotine (F(1,8)=0.02, p=0.9). Further analysis of the main effect of LY235959 demonstrated that reward thresholds were significantly elevated in rats pre-treated with the highest does of LY235959 (5 mg/kg) (p<0.01; ).
Experiment 2 - Reward-enhancing and reinforcing effects of self-administered nicotine require NMDA receptors
Rats (n=16) were prepared with ICSS electrodes and trained in the ICSS threshold procedure until stable reward thresholds were achieved. All rats were then prepared with intravenous catheters. One group of rats (nicotine rats; n=9) was trained in the nicotine self-administration procedure, and reward thresholds were assessed 1-h before (pre-thresholds) and 15 min after (post-thresholds) each self-administration session. The remaining rats (control rats; n=7) were tested in the ICSS procedure at the same times each day as the self-administering rats, but were returned to their home-cages during the self-administration sessions, and thus remained nicotine-naïve throughout the experiment.
Mean (± SEM) baseline ICSS thresholds prior to the start of nicotine self-administration were 92.1 ± 15.2 μA and 75.3 ± 5.2 μA in the nicotine and control rats, respectively. Mean (± SEM) baseline number of nicotine infusions earned by nicotine rats was 10.5 ± 1.0. As expected, pre-thresholds and post-thresholds remained stable and unaltered in the control rats (). However, in replication of our recently published data (Kenny and Markou, 2006
; Paterson et al, 2007
), daily post-thresholds were lowered after each self-administration session in the nicotine rats compared with pre-thresholds during the initial 7-day access to nicotine, prior to any LY235959 treatment (). In addition, a downward drift was observed in pre- and post-thresholds in nicotine rats () similar to that previously reported (Kenny and Markou, 2006
), reflecting a long-lasting nicotine-induced increase in the baseline sensitivity of brain reward systems. Accordingly, two-way repeated-measures ANOVA over this 7-day period on threshold data from control and nicotine rats demonstrated a significant main effect of Drug (nicotine or control group) (F(1,13)
=5.7, p<0.05), a main effect of Session (pre-thresholds or post-thresholds) (F(1,13)
=6.3, p<0.05), and a significant Drug × Session interaction (F(1,13)
=7.9, p<0.05). Two-way ANOVA of the threshold data obtained only from the nicotine rats over this time period revealed significant main effects of Days (F(6,48)
=2.3, p<0.05) and Session (F(1,8)
Fig. 2 NMDA receptor antagonism switched the effects of self-administered nicotine on brain reward systems from stimulatory to inhibitory and thereby decreased nicotine self-administration in rats. (a) Pre-thresholds and post-thresholds for control rats (n=7), (more ...)
After 7 consecutive days of nicotine self-administration, the effects of LY235959 on reward thresholds were examined in nicotine and control rats. First, the effects of LY235959 on pre-thresholds, nicotine self-administration, and post-thresholds were assessed. That is, both groups of rats were pre-treated with LY235959 (0, 0.5 1 or 2.5 mg/kg; Latin-square design; minimum of 7 days between injections), and 15 min later pre-thresholds were assessed. Rats were then permitted to self-administer nicotine or were returned to their home-cages (control rats), and post-thresholds were subsequently assessed. Administration of LY235959 before pre-threshold assessment did not alter pre-thresholds or post-thresholds in control rats (). However, LY235959 blocked the lowering of post-nicotine reward thresholds typically observed in nicotine rats (). Three-way repeated-measures ANOVA on the reward threshold data confirmed a significant main effect of LY235959 (F(3,42)=4.5, p<0.01), and a significant Drug (nicotine or control group) × LY235959 interaction (F(3,42)=3.8, p<0.05). Two-way ANOVA on the reward threshold data from the nicotine rats demonstrated a main effect of LY235959 (F(3,24)=6.5, p<0.01).
Because no effect of Session was observed in the above analysis, data from the two Sessions (pre-thresholds and post-thresholds) were combined for each dose and then compared with the 0 mg/kg treatment condition for the nicotine rats. This analyses indicated that there was a main effect of LY235959 (F(3,24)=10.3, p<0.001), and that thresholds tended to be elevated at the 0.5 mg/kg dose (p=0.06), and were significantly elevated at the 1 mg/kg (p<0.05) and 2.5 mg/kg (p<0.001) LY235959 doses compared with thresholds observed after saline treatment ().
In addition to reversing the lowering effects of self-administered nicotine on ICSS thresholds, LY235959 also decreased nicotine intake. Mean (± SEM) baseline number of nicotine reinforcers earned by the nicotine rats prior to LY235959 treatment was 12.0 ± 1.5. LY235959 dose-dependently decreased nicotine intake in the nicotine rats (), reflected in a significant effect of LY235959 (F(3,24)=41.0, p<0.001). Post-hoc analysis demonstrated that nicotine intake was significantly decreased by the 0.5 mg/kg (p<0.05) and the 1–2.5 mg/kg (p<0.01) LY235959 doses ().
Next, we examined the effects of LY235959 (0–2.5 mg/kg; Latin-square design) administered immediately after the self-administration session on post-thresholds assessed 15 min later. In this manner, we could directly assess the influence of LY235959 on threshold lowering induced by self-administered nicotine whilst avoiding the confounding inhibitory effects of LY235959 on nicotine self-administration. LY235959 administered in this manner reversed the lowering of post-thresholds observed in the nicotine rats, but did not alter thresholds in control rats (). Two-way repeated-measures ANOVA demonstrated a significant main effect of LY235959 (F(3,42)=6.5, p<0.01), and a significant Drug (nicotine or control group) × LY235959 interaction (F(3,42)=8.3, p<0.001), and no effect of Drug (F(1,14)=1.7, p=0.2). One-way repeated-measures ANOVA of threshold data from nicotine rats demonstrated a significant effect of LY235959 (F(3,24)=13.5, p<0.001). Post-hoc analysis on the reward thresholds in nicotine rats demonstrated that thresholds were significantly elevated after the 0.5 mg/kg (p<0.05), 1 mg/kg (p<0.01) and 2.5 mg/kg (p<0.001) LY235959 doses compared with thresholds after saline administration ().
To determine if the inhibitory effects of LY235959 on nicotine self-administration observed in the nicotine rats above () were secondary to a disruptive effect on operant performance, we again assessed the effects of LY235959 (0.1–5 mg/kg) on nicotine self-administration in a new cohort of rats, and compared these effects with the actions of LY235959 on responding for food reinforcement. Nicotine self-administering rats (n=7) were injected with LY235959 (0, 0.1, 0.5, 1, 2.5 or 5 mg/kg; Latin-square design), and nicotine intake was evaluated 30 min later. The effects of LY235959 were also assessed in rats (n=8) trained to respond for food reinforcement (45 mg pellet) under a FR5TO20 sec schedule, and in rats (n=7) trained under a FR5TO210 sec schedule. This adjusted schedule incorporated a longer time-out period (210 s vs. of 20 s) that better equated rates of responding for nicotine and food.
Mean (± SEM) baseline number of nicotine infusions earned by this second cohort of rats prior to treatment with LY235959 was 10.8 ± 0.7. The mean (± SEM) baseline number of food rewards earned by rats under the FR5TO20 sec and the FR5TO 210 sec schedules of reinforcement prior to LY235959 treatment were 109.5 ± 9.6 and 16.3 ± 0.3, respectively. Two-way repeated-measures ANOVA demonstrated a significant effect of Reinforcer (F(2,19)=17.2, p<0.001), a significant effect of LY235959 (F(5,10)=19.4, p<0.001), and no Reinforcer × LY235959 interaction (F(10,95)=1.4). Pre-planned comparisons demonstrated that LY235959 decreased nicotine intake at doses ≥ 0.5 mg/kg (; confirming the results reported above in a separate cohort of rats shown in ), but decreased food intake under FR5TO20 sec or FR5TO210 sec reinforcement schedules only at the highest doses tested (2.5–5 mg/kg; ). Further analyses of the above data demonstrated that LY235959 decreased nicotine intake with an IC50 of 0.53 mg/kg, and decreased food responding under FR5TO20 and FR5TO210 sec schedules with IC50values of 1.88 and 2.2 mg/kg, respectively.
Next, to determine whether glutamate transmission in general, or if NMDA receptor-mediated glutamate transmission selectively impacts nicotine reward, the effects of the AMPA receptor antagonist NBQX (0, 0.1, 0.5, 1 and 5 mg/kg; Latin-square design) on nicotine self-administration (n=8) and on food responding (n=5) were assessed under FR5TO20 sec reinforcement schedules. Mean (± SEM) baseline number of nicotine infusions and food reinforcers earned prior to NBQX treatment was 17.1 ± 1.8 and 141.1 ± 8.3, respectively. Two-way repeated-measures ANOVA demonstrated that NBQX did not alter responding for nicotine or food reinforcement at any dose tested [Reinforcer: (F(1,11)=0.5); NBQX, (F(4,4)=0.08); Reinforcer × NBQX (F(4,44)=1.5)] ().
Experiment 3 - Nicotine self-administration alters glutamate receptor subunit expression levels in reward circuits
In Experiments 1–2, we found that NMDA receptors regulate nicotine-induced lowering of ICSS thresholds and nicotine self-administration in rats. Next, we hypothesized that populations of NMDA receptors that regulate the nicotine reinforcement may undergo adaptations, such as altered expression profiles, upon repeated exposure to self-administered nicotine. Hence, we examined the expression levels of NMDA receptor subunits NR1, NR2A and NR2B in the brains of nicotine self-administering rats (n=9; 21 consecutive 1-h daily sessions) and control saline-self-administering rats (n=8) that were also prepared with intravenous catheters and had as many exposures to the operant testing chambers as the nicotine self-administering rats. Rats were decapitated without anesthesia 24 h after their last self-administration session (at the time when the next self-administration session would have occurred) and their brains were rapidly frozen. This treatment regimen was chosen because it corresponds closely with the total number of days of access to nicotine that rats had in Experiment 2 above. In addition, we examined the expression levels of AMPA receptor subunits GluR1 and GluR2. Tissue samples were processed for Western blotting as described in the Methods section.
Mean (± SEM) baseline number of nicotine and saline infusions earned prior to tissue harvesting was 10.6 ± 0.9 and 3.3 ± 0.9, respectively. Three-way repeated measures ANOVA revealed a statistically significant Brain Region × Treatment × Protein interaction (F(20,192)=2.4344, p=0.001). Post-hoc comparisons among expression levels in discrete brain regions revealed the following: In the VTA, there was a significant increase of NR2A subunit expression in nicotine rats compared with controls (p<0.05; ); in the CeA there were statistically significant increases in the expression of NR2A, NR2B, NR1, GluR1 and GluR2 subunits in nicotine rats compared with controls (p<0.05 in each case; ); in the NAcc there was a significant increase in GluR1 expression in nicotine rats compared with controls (p<0.05; ). Conversely, in the PFC, there were statistically significant decreases in the expression of NR2A, NR2B and GluR2 receptor subunits in nicotine rats compared with controls (p<0.05 in each case; ). There were no differences in expression levels between nicotine rats and controls in CPu or BLA (data not shown).
Fig. 3 Nicotine self-administration alters the expression of glutamate receptor subunits in reward-related cortical and limbic brain regions. Rats were permitted to self-administer nicotine for 21 days, or remained nicotine-naive over the same time-period and (more ...)
Experiment 4 - Nicotine increases NMDA receptor-mediated transmission in rat CeA slices
In Experiment 3, we found that chronic nicotine self-administration increased NMDA receptor subunit expression in the CeA and VTA (see ). Based on these data we hypothesized that nicotine may act in the CeA to increase NMDA receptor-mediated transmission similar to its effects in the VTA (see Introduction), an action that could potentially contribute to its reinforcing and reward-enhancing properties. To test this hypothesis, we examined the effects of nicotine on NMDA receptor-mediated transmission in rat CeA neurons.
Recordings were made from seven CeA neurons (from n=7 rats) that had a mean resting membrane potential of −76 ± 2 mV and a mean input resistance of 108 ± 6 MΩ. After obtaining a stable baseline of evoked, pharmacologically isolated NMDA-EPSCs (10–15 min), nicotine (1 μM) was superfused for 7–10 mins. Nicotine applied for 7–10 min increased (~25 %; assessed at ~2–3 min of nicotine application; F(4,14) = 7.05; p<0.05) NMDA-EPSCs in rat CeA slices (, upper and lower panels). Nicotine had no effects on basic membrane properties such as membrane potential, input resistance, or spike amplitude (data not shown). To investigate whether the stimulatory effects of nicotine on NMDA-EPSCs were regulated by a presynaptic action on excitatory glutamate inputs, the paired-pulse-facilitation (PPF) of NMDA-EPSC in CeA neurons was examined before, during, and after nicotine application. Nicotine (1 μM) decreased (F(5,5) = 10.2; p<0.05) the PPF ratio of NMDA-EPSCs (; upper and lower panels). Similarly, nicotine also decreased (p<0.05) the PPF of compound glutamate (NMDA and non-NMDA) EPSCs (isolated by bicuculline and CGP-55845A to block GABAA and GABAB receptors, respectively; n=6) and also non-NMDA EPCSs (isolated by aminophosphonovaleric acid [APV; NMDA receptor blocker], bicuculline and CGP-55845A; n=5), by approximately 15% in CeA (data not shown). Thus, nicotine likely increased NMDA receptor-mediated transmission in the CeA by activating presynaptic nicotinic receptors to facilitate the release of glutamate, which subsequently activated postsynaptic NMDA receptors.
Fig. 4 Nicotine increases NMDA receptor-mediated transmission in rat CeA slices. (a) Nicotine (1 μM) increased the amplitude of evoked, pharmacologically isolated NMDA receptor EPSCs in CeA neurons. Upper panel, current records of evoked NMDA-EPSCs in (more ...)
Experiment 5 - NMDA receptors in the VTA and CeA regulate nicotine self-administration
Based on the protein expression and electrophysiological data obtained in Experiments 3 and 4, we hypothesized that NMDA receptors in the CeA and VTA may regulate nicotine reinforcement. To test this hypothesis, we next examined the effects of intra-VTA and intra-CeA infusions of LY235959 on nicotine self-administration. Rats with bilateral cannulae directed toward the CeA or VTA were trained in the nicotine self-administration procedure until stable nicotine intake was achieved. Rats were then injected directly into the CeA or VTA with LY235959 (0, 0.1, 0.5, 1 or 10 ng/0.5 μl/side; n=10 VTA, n=6 CeA; Latin-square design; minimum of 7 days between injections), and nicotine self-administration was evaluated 10–15 min later. Next, rats received a control injection of LY235959 (1 ng/0.5 μl/side) 2 mm above the CeA or VTA. At the conclusion of the experiments, all animals were anaesthetized and perfused with 10% formalin solution, and their brains removed. The brains were cut in 50 μm sections, stained by cresyl violet staining, and placements of the injectors and the cannulae were examined. Only those rats with injection tips located within the CeA or VTA were included in statistical analyses.
Mean (± SEM) baseline numbers of nicotine reinforcers earned prior to intra-VTA or intra-CeA LY235959 infusions were 11.5 ± 1.3 and 11.22 ± 2.1, respectively. Injection of LY235959 (0.1–10 ng/0.5 μl/side) into the VTA dose-dependently decreased nicotine self-administration (F(4,28)=7.3, p<0.001, ; see for a graphical representation of the injection sites after histological examination. Post-hoc analysis revealed that nicotine intake was significantly decreased by doses of LY235959 ≥ 1 ng/0.5 μl/side (). Further analyses of the above data demonstrated that LY235959 decreased nicotine intake with an IC50 of 0.87 ng/0.5 μl (per side), and by approximately 40% at the IC50 value. A dose of LY235959 (1 ng/0.5 μl/side) that significantly decreased nicotine self-administration after intra-VTA administration did not alter nicotine intake when infused 2 mm above the VTA (). Similarly, injection of LY235959 (0.1–10 ng/0.5 μl/side) into the CeA dose-dependently decreased nicotine self-administration (F(5,15)=7.5, p<0.01, ; see for a graphical representation of the injection sites. Post-hoc analysis revealed that nicotine intake was significantly decreased by doses of LY235959 ≥ 1 ng/0.5 μl/side (). LY235959 (1 ng/0.5 μl/side) infused 2 mm above the CeA did not alter nicotine intake (). LY235959 administered into the CeA decreased nicotine self-administration with an IC50 value of 0.97 ng/0.5 μl (per side), and reduced intake by approximately 20% at the IC50value.
Fig. 5 NMDA receptors in the VTA and CeA regulate nicotine self-administration (a) Diagrammatic representation of coronal sections from the rat brain showing histological reconstruction of the injection sites in the VTA. Black lines indicate locations of cannulae (more ...)