Effects of AZD3480, a neuronal nicotinic acetylcholine receptor agonist, and donepezil on dizocilpine-induced attentional impairment in rats
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- Rezvani, A.H., Cauley, M.C., Johnson, E.C. et al. Psychopharmacology (2012) 223: 251. doi:10.1007/s00213-012-2712-2
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Background and rationale
Nicotinic acetylcholine systems play major roles in cognitive function. Nicotine and a variety of nicotinic agonists improve attention, and nicotinic antagonist exposure impairs it. This study was conducted to investigate the effect of a novel nicotinic receptor agonist at α4β2 nicotinic receptors (AZD3480) on attention and reversal of pharmacologically induced attentional impairment produced by the NMDA glutamate antagonist dizocilpine (MK-801).
Adult female Sprague-Dawley rats were trained to perform an operant visual signal detection task to a stable baseline of accuracy. The rats were then injected subcutaneously following a repeated measures, counter-balanced design with saline, AZD3480 (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations 30 min before the test. The effect of donepezil on the same pharmacologically induced attentional impairment was also tested. A separate group of rats was injected with donepezil (0.01, 0.1, and 1 mg/kg), dizocilpine (0.05 mg/kg), or their combinations, and their attention were assessed. Saline was the vehicle control.
Dizocilpine caused a significant (p < 0.0005) impairment in percent correct performance. This attentional impairment was significantly (p < 0.0005) reversed by 0.01 and 0.1 mg/kg of AZD3480. AZD3480 by itself did not alter the already high baseline control performance. Donepezil (0.01–1.0 mg/kg) also significantly (p < 0.005) attenuated the dizocilpine-induced attentional impairment.
AZD3480, similar to donepezil, showed significant efficacy for counteracting the attentional impairment caused by the NMDA glutamate antagonist dizocilpine. Low doses of AZD3480 may provide therapeutic benefit for reversing attentional impairment in patients suffering from cognitive impairment due to glutamatergic dysregulation and likely other attentional disorders.
KeywordsNicotinic receptorsCognitionNicotinic agonistsDizocilpineMK-801NMDAα4β2 nicotinic receptorsAricept®
The role of the neuronal nicotinic system in attention has been documented in both preclinical and clinical studies. Nicotine has been shown to improve attentional performance in experimental animals (Grilly 2000; Mirza and Bright 2001; Mirza and Stolerman 1998; Muir et al. 1995; Rezvani and Levin 2003; Stolerman et al. 2000). Nicotine has also been demonstrated to improve attentional impairment such as that seen in Alzheimer's disease, schizophrenia, and attention deficit hyperactivity disorder (ADHD) (Levin et al. 1996; McEvoy and Allen 2002; White and Levin 1999). Using an operant visual signal detection task, we have demonstrated that systemic administration of nicotine can improve attention in rats (Rezvani and Levin 2003). In addition, chronic nicotine infusion has been shown to significantly diminish the impairing effects of both typical and atypical antipsychotic drugs on attention in rats (Rezvani et al. 2004, 2008). Nicotinic analogs have also been shown to improve attention. Terry et al. (2002) found that the nicotinic agonist SIB-1553A significantly improves performance of rats on a five-choice attentional task when performance accuracy was reduced pharmacologically by NMDA antagonist dizocilpine (MK-801). Nicotine agonist ABT-418 has been found to improve attentional performance in rats (McGaughy et al. 1999) and reduces symptoms of ADHD in humans (Wilens et al. 1999). Conversely, the nicotinic antagonist mecamylamine has been shown to impair attentional performance in rats (Grottick and Higgins 2000; Mirza and Stolerman 1998). These findings suggest the involvement of the nicotinic cholinergic system in cognitive function.
Neuronal nicotinic acetylcholine receptors constitute a variety of receptor subtypes, which are important for a variety of neurobehavioral functions including cognitive function (Changeux et al. 1998). Nicotinic α4β2 receptors have been shown to be critically involved in cognitive function. Agonists of α4β2 nicotinic receptors produce significant long-lasting improvement in memory function (Levin and Christopher 2002; Lippiello et al. 1996; Papke et al. 2000) and attentional performance in rats (Grottick and Higgins 2000). Both selective α7 and α4β2 nicotinic receptor agonists have been shown to play a role in attention, learning, and working memory (Buccafusco and Terry 2009; Cincotta et al. 2008).
In the clinic, AZD3480 produced statistically significant enhancement of attention in healthy young volunteers and attention and age-associated memory impairment in elderly volunteers (Dunbar et al. 2007) and showed beneficial effects on cognitive deficiencies (e.g., working memory, recognition memory, and attention) in adult ADHD (Locke et al. 2010).
The goal of the current experiment was to build upon previous preclinical studies that focused solely on cognitive endpoints to an animal model of impaired attention using an operant visual signal detection task. This task has been shown in our laboratory and others to be a reliable and valid assessment of attentional function (Bushnell 1998; McGaughy et al. 1999; Rezvani and Levin 2003; Sarter et al. 2009), useful indicator of attentional impairment, and sensitive way to assess the efficacy of novel therapeutic treatments.
Based on existing literature, cholinergic hypothesis of cognition and pharmacological properties of AZD3480 and donepezil, it was hypothesized that this drug will reverse the attentional impairment induced by dizocilpine, an NMDA glutamate antagonist, in rats. Demonstrating that AZD3480 could reverse the attentional impairing effects of NMDA receptor blockade in rats would help back translate the clinical data to an animal model for further profiling of novel nicotinic drugs for treating attentional impairment.
Materials and methods
Adult female Sprague-Dawley rats (Taconic Farms, Germantown, NY, USA) were used in the proposed experiments (N = 13–15 for each group). Rats were housed in groups of three in plastic cages with wood shavings in a vivarium with 12L:12D reversed light schedule (light on at 7:00 p.m.). The rats had unrestricted access to drinking water but were fed daily after testing such that their weights were kept at 80–85 % of free-feeding values. The treatment and care of the animals was carried out under an approved protocol of the Animal Care and Use Committee of Duke University in an AAALAC-approved facility.
In one group of rats, the acute effects of AZD3480, dizocilpine, and the combination of AZD3480 and dizocilpine on attention were examined. In another group of rats, the acute effects of donepezil and dizocilpine and their combination were tested. First, rats were trained to perform a visual signal detection task. After establishing a stable baseline of performance, one group of rats were injected SC with saline, AZD3480 (0.01, 0.1, and 1.0 mg/kg), dizocilpine (0.05 mg/kg), or a combination of AZD3480 (0.01, 0.1, and 1.0 mg/kg) with dizocilpine (0.05 mg/kg). Another group of rats were injected with donepezil (0.01, 0.1, and 1.0 mg/kg), dizocilpine (0.05 mg/kg), or a combination of donepezil (0.01, 0.1, and 1.0 mg/kg) with dizocilpine (0.05 mg/kg) 30 min before the testing for attention. All animals in each group received all treatments following a crossover design with random assignment. At least 3 days were allowed between injections.
Apparatus for signal detection task
Each chamber was equipped with a signal light, a house light, two retractable levers, a food cup (Coulbourn Instruments, Lehigh Valley, PA, USA), and a white noise generator (Med Associates Inc., Georgia, VT, USA). The two retractable levers were located on both sides of the food cup 13 cm apart and 2.5 cm above the floor of the chamber. The levers were inserted simultaneously horizontally 2.5 cm into the chamber. The signal, or cue light, was located above the food cup at the center of the front panel 28 cm above the floor of the chamber. A signal consisted of 500 ms increase in the brightness of the signal light to levels of 0.027, 0.269, and 1.22 lux above a background illumination of 1.2 lux (Rezvani et al. 2011).
Drugs were prepared in saline solution. AZD3480 and donepezil were provided by AstraZeneca Pharmaceuticals (Wilmington, VT, USA), and dizocilpine was purchased from Sigma (St. Louis, MO, USA). Rats were injected with drugs and the control vehicle subcutaneously in a volume of 1 ml/kg body weight. Rats began testing 30 min after drug administration. All experiments were carried out during the dark phase of the dark–light cycle.
Visual signal detection task
Rats were trained to perform a visual signal detection task (Bushnell 1998; Rezvani et al. 2009a). Animals were tested every day except weekends and holidays. The task was conducted in daily 240-trial sessions approximately 45 min in duration. Two trial types, “signal” and “blank,” were presented in equal number in each session in groups of four (two signal and two blank, in random order) at each of the three signal intensities. Each signal trial included a pre-signal interval, the signal (cue light), and a post-signal interval. Following the signal, a post-signal interval of 2, 3, or 4 s (selected randomly) occurred. Blank trials were presented identically, except the signal light was not present (Bushnell 1998; Rezvani et al. 2011).
A trial began with both levers retracted from the chamber; then, both levers were inserted into the chamber simultaneously at the end of the post-signal interval. The levers were both retracted simultaneously when one was pressed or if 5 s passed without a press. Every correct response (i.e., a press on the signal lever in a signal trial or a press on the blank lever in a blank trial) was followed by the illumination of the food cup and delivery of one 20-mg food pellet. After each incorrect response (i.e., a press on the signal lever in a blank trial or a press on the blank lever in a signal trial) or response failure, the rat received a 2-s period of darkness (time out). If no press occurred, a response failure was recorded, and the trial was not repeated.
Behavioral measures and statistical analysis
There were two measures of choice accuracy. “Hits” were defined as correct responses on signal trials, while “correct rejections” were counted as correct responses on blank trials. Both hit and correct rejection lead to delivery of a pellet. Percent hit = (number of hits/total number of responses on signal trials) × 100, and percent correct rejection = (number of correct rejections/total number of responses on blank trials) × 100. Response latency was defined as the time elapsed between insertion of the levers and the first lever press by the rat. A response omission was recorded if the rat did not press a lever within 5 s after insertion of the levers. Increase in hit and/or correct rejection was an indicative of enhanced attention, and increase in response omission suggested the opposite. Each dependent variable was subjected to an independent analysis of variance (Superanova/Statview, SAS, Cary, NC, USA). Significant interactions were followed by tests of simple main effects. The threshold for significance was set at p < 0.05.
Dizocilpine significantly increased response latency, an effect which was reversed by AZD3480. Response latency was significantly affected by AZD3480 (F(3,36) = 7.08, p < 0.005) and dizocilpine (F(1,12) = 13.99, p < 0.005), and there was a significant interaction as well (F(3,36) = 9.76, p < 0.0005). Tests of the simple main effects showed that there was a significant (p < 0.0005) increase in response latency (i.e., slowing of response) caused by dizocilpine (224 ± 24 ms) vs. vehicle (104 ± 9 ms). The lower doses of 0.01 mg/kg (125 ± 14 ms) and 0.1 mg/kg (143 ± 20 ms) of AZD3480 caused significant (p < 0.0005) reversal of the dizocilpine-induced increase in response latency. However, the higher 1.0-mg/kg dose of AZD3480 (203 ± 23 ms) was not effective.
Dizocilpine caused a significant increase in response omissions, an effect which was reversed by AZD3480. With response omissions, there was an interaction of dizocilpine × AZD3480 (F(3,36) = 2.76, p < 0.06), which was followed up by tests of the simple main effects. These showed that dizocilpine (19.0 ± 5.1) caused a significant (p < 0.005) increase in response omissions relative to performance after vehicle (4.3 ± 2.6). As with response latency, the dizocilpine-induced increase on response omissions was significantly (p < 0.01) reversed by 0.01 mg/kg (5.2 ± 2.2) and 0.1 mg/kg (4.8 ± 3.2) of AZD3480, but not by the higher 1.0-mg/kg dose (13.9 ± 6.5).
None of the AZD3480 doses had significant effects when given alone when compared with vehicle treatment on hit, response latency, or omissions.
Response latency was significantly affected by donepezil (F(3,42) = 4.78, p < 0.01) and dizocilpine (F(1,14) = 30.00, p < 0.0005), and there was a significant interaction as well (F(3,42) = 5.01, p < 0.005). Tests of the simple main effects showed that there was a significant (p < 0.0005) slowing of response caused by dizocilpine (222 ± 19 ms) vs. vehicle (124 ± 12 ms). The lowest 0.01-mg/kg dose (152 ± 18 ms) significantly (p < 0.0005) reversed the dizocilpine-induced increase in latency. However, the middle dose 0.1 mg/kg (212 ± 25 ms) was not effective. Interestingly, the 1.0-mg/kg (193 ± 16 ms) did significantly (p < 0.05) attenuate the dizocilpine-induced slowing.
With response omissions, there was significant main effects of dizocilpine (F(1,14) = 9.16, p < 0.01) and donepezil (F(3,42) = 3.56, p < 0.05), and there was a significant interaction (F(3,42) = 4.89, p < 0.01), which was followed up by tests of the simple main effects. These showed that dizocilpine (26.1 ± 6.5) caused a significant (p < 0.0005) increase in omissions relative to performance after vehicle (4.8 ± 1.0). As with response latency, the lowest 0.01-mg/kg dose (8.7 ± 3.7) significantly (p < 0.005) reversed the dizocilpine-induced increase in latency. However, the middle dose 0.1 mg/kg (23.2 ± 5.9 ms) was not effective. Interestingly, the 1.0-mg/kg (9.9 ± 3.7 ms) did significantly (p < 0.005) attenuate the dizocilpine-induced slowing.
None of the donepezil doses had significant effects when given alone when compared with vehicle treatment on hit, correct rejection, response latency, or omissions.
Acute subcutaneous administration of AZD3480 and donepezil showed significant efficacy for counteracting the attentional impairment caused by the NMDA glutamate antagonist dizocilpine. Both drugs have been shown to increase acetylcholine in the brain, although through different mechanisms: AZD3480 by direct nicotinic acetylcholinergic receptor agonist action and donepezil by inhibition of acetylcholine degradation via inhibition of acetylcholinesterase. Both drugs caused significant improvement with both hit and correct rejection measures of accuracy, demonstrating the robust nature of their effects. Like acetylcholinesterase inhibitors, which are indirect cholinergic agonists, direct nicotinic cholinergic agonist drugs may be clinically effective treatments of attentional impairment.
Our findings are consistent with the repeated demonstration that nicotine and other nicotinic agonists enhance cognitive performance in experimental animals (Buccafusco and Terry 2009; Hahn and Stolerman 2002; Hahn et al. 2011; Howe et al. 2010; Levin et al. 2006; Mirza and Stolerman 2000; Rezvani et al. 2011; Sarter et al. 2009; Young et al. 2004) and humans (Levin et al. 1998; Newhouse et al. 2004). Nicotine also has been shown to diminish pharmacologically induced attentional impairment in rats. In an earlier study, we found that nicotine effectively attenuated the attentional impairment caused by dizocilpine (Rezvani and Levin 2004). Furthermore, it was shown that chronic nicotine administration diminished the impairing effects of haloperidol on sustained attention in rats (Rezvani et al. 2004). Pretreatment with an α7 nicotinic receptor agonist GTS-21 has also been shown to produce a dose-dependent improvement in a computer-assisted delayed response task in non-human primates (Buccafusco and Terry 2009).
Demonstrating clinical relevance, a variety of studies has shown that nicotine effectively improves cognitive function including attention in humans, including those with diminished function with Alzheimer's disease and ADHD (Lawrence et al. 2002; Levin et al. 1998; Newhouse et al. 2004).
Recent research suggests that drugs activating nicotinic acetylcholine receptors in the brain might be promising therapy in cognitive decline seen in the elderly and Alzheimer's disease. AZD3480, a highly selective α4β2 nicotine acetylcholine receptor partial agonist, has shown memory-enhancing properties in rodents and humans with good tolerability profile (Gatto et al. 2004; Dunbar et al. 2007). Recently, we showed that sazetidine-A, a selective potent agent and partial agonist at β2-containing nAChRs, when give acutely can significantly reduce the impairing effects of scopolamine and dizocilpine on attention in rats (Rezvani et al. 2011). These current findings along with our discovery with AZD3480 provide more support for the important role that α4β2 nAChR subtypes play in attention.
As mentioned in the “Introduction” section, AZD3480, similar to sazetidine-A, represents a suitable clinical candidate with a selective partial agonist activity at the α4β2 nAChR receptor subtype with little to no binding or functional activity at α7, α3β4, or α1β1δγ nicotinic receptors. Furthermore, it is orally active and displays additively or synergy with donepezil (Gatto et al. 2004). With this selective receptor subtype profile, AZD3480 may have efficacy with fewer side effects seen with an indirect cholinergic agonist such as donepezil or a nonselective nicotinic agonist like nicotine.
Based on the cholinergic hypothesis of cognition (Janowsky et al. 1994), decline in cognitive function is predominantly related to a decrease in cholinergic function in the brain. The fact that AZD3480 has been shown to release acetylcholine in the cortex of rats (Gatto et al. 2004) may explain its improving effects on cognition. Donepezil is a highly selective, reversible inhibitor of acetylcholinesterase and been shown to increase acetylcholine level in the extracellular space of rat cerebral cortex, and in the rat models of cholinergic hypofunction (Sugimoto et al. 2002). Thus, it may exert its improving effect on attention by increasing the level of acetylcholine in the brain.
Regarding the AZD3480 effect, there was evidence for inverted U-shaped dose–effect functions with lower doses being more effective than higher doses. This is not surprising since, as a general rule, this is the way most cognitive-enhancing drugs act. It may be the case that at higher doses, more off-target effects become expressed, which diminished the effectiveness of the drug. Alternatively, there may be an optimal level of nicotinic receptor activation for cognitive improvement that if exceeded diminishes effectiveness. It also may be the case that at higher doses, agonists and even partial agonists like AZD3480 can decrease the fidelity of moment-to-moment phasic shifts in cholinergic transmission that carries information regarding attentional processing. In the current experiment, only acute effect of AZD3480 was studied. Thus, further research should examine the chronic effects of AZD3480 particularly at low doses to determine if there is a development of tolerance to its therapeutic effect. However, Dunbar et al. (2007) have demonstrated lack of tolerance with 3 weeks of treatment in humans. Also, its efficacy should be tested in another model of attentional impairment such as the impairment with the muscarinic cholinergic antagonist scopolamine to determine the generalizability of the effect. Using the same signal attention task, we have previously shown that methylphenidate effectively reversed the attentional impairment caused by either dizocilpine or scopolamine (Rezvani et al. 2009b). It also might be useful to test the effect of this compound on attentional performance of unimpaired rats with lower baseline to rule out the ceiling effect.
In conclusion, these results demonstrated the efficacy of low-dose AZD3480 and donepezil treatment for reversing attentional impairment caused by the NMDA glutamate antagonist dizocilpine in rats. The efficacy of this drug suggests that drugs which act as agonists at α4β2 nicotinic receptors and/or which inhibit acetylcholinesterase to increase acetylcholine levels in the brain may provide a path forward for the treatment of cognitive impairments in general and attentional deficits specifically.
This research was funded by AstraZeneca Pharmaceuticals in collaboration with Targacept, Inc. Dr. Edwin Johnson is an employee of AstraZeneca Pharmaceuticals. Dr. Gregory J. Gatto is an employee of Targacept, Inc.