Running Head: NICOTINE AND ADHD 1

Transcription

1 Running Head: NICOTINE AND ADHD 1 Effects of Acute Nicotine on a Response-Withholding Task in an Animal Model of Attention Deficit Hyperactivity Disorder: Implications for Sustained Attention Abstract

2 NICOTINE AND ADHD 2 The present study examined nicotine effects on Attention Deficit Hyperactivity Disorder (ADHD) related behavior in the leading animal model of ADHD (Spontaneously Hypertensive Rat; SHR). Specifically, the study examined two behaviors: impulsivity (Response Inhibition Capacity, RIC) and sustained attention. Nicotine has been shown to manage impulsivity (RIC) in humans with ADHD, but has inconsistent effects on RIC in non-adhd subjects. The use of nicotine has also contributed to improvement in sustained attention in both ADHD and non- ADHD subjects, suggesting the hypothesis that individuals smoke in order to alleviate symptoms of their disorder (self-medication hypothesis). In the present study, I administered acute nicotine injections to SHR preceding a response-withholding task, as a means to measure nicotine effects on RIC and sustained attention. Because of data suggesting nicotine-induced sustained attention improvement in ADHD and non-adhd subjects alike, I expected nicotine to improve sustained attention non-differentially for SHR and the Wistar-Kyoto Rat (WKY). Additionally, RIC has been improved in ADHD subjects, but showed inconsistent improvement in non-adhd subjects. Therefore, I expected nicotine to improve RIC in SHR significantly more than in WKY. As expected, I found that nicotine improved sustained attention in both strains, but that a lower dose led to a greater improvement in SHR than in WKY. This finding suggests that ADHD subjects may be more sensitive to lower doses of nicotine. Contrary to predictions, RIC was decreased similarly in both strains, with a greater dose of nicotine leading to a higher deficit. A decrease in RIC parallels existing data in animal research with non-adhd models suggesting that nicotine non-differentially decreases RIC. The results validate the SHR as an animal model of nicotine effects on sustained attention. These results can also lead to further understanding of neural and behavioral mechanisms of this pattern.

3 NICOTINE AND ADHD 3 Introduction ADHD and Smoking Attention Deficit Hyperactivity Disorder (ADHD) is a behavioral disorder characterized by inordinate levels of impulsivity, inattentiveness, and hyperactivity (Sagvolden, 2000). Individuals diagnosed with ADHD typically display deficiencies in response inhibition capacity, i.e., difficulties withholding responses, delaying responding to a stimulus, terminating ongoing responses, and avoiding distraction from competing stimuli (Barkley, 1999). They also exhibit impairments in sustained attention, i.e., task oriented persistence, and engagement in a task over a sustained period of time (Barkley, 1997). ADHD is a highly prevalent disorder diagnosed in 8-12% of school aged children (Biederman & Faraone, 2005). In addition, it is often comorbid with several other disorders (anxiety, conduct, oppositional defiant, tic and mood disorders) (Singh, 2008). There is a significant correlation between ADHD diagnosis in adolescence and smoking (Milberger, Biederman, Faraone, & Jones, 2010). Twice as many people diagnosed with ADHD by age 17 report smoking regularly than those without the disorder (Lambert & Hartsough, 1998). Inattention is associated with increased likelihood of smoking (Kollins, McClernon, & Fuemmeler, 2005). ADHD is also linked to early initiation of smoking, primarily at midadolescence (Lando et al., 1999). This finding is of particular significance because earlier onset of smoking has been correlated with lower likelihood of quitting (Khuder, Dayal, & Mutgi, 1999). The behavioral and psychological factors causing early initiation are therefore all the more important to discover. The most studied hypothesis linking smoking to ADHD is known as the self-medication hypothesis. It suggests that individuals with ADHD smoke to mitigate deficits in impulse control

4 NICOTINE AND ADHD 4 and concentration (Wilens et al., 2007). The overarching goal of the present study is to further investigate the self-medication hypothesis in relation to response inhibition capacity and sustained attention. This hypothesis claims that exposure to nicotine improves these variables. Therefore, we examined whether nicotine improved response inhibition capacity and sustained attention in the Spontaneously Hypertensive Rat (SHR), an animal model of ADHD (Sagvolden, 2000). SHR The Spontaneously Hypertensive Rat (SHR) is the most commonly used model of ADHD (Sagvolden & Johansen, 2012). The SHR was developed by selectively inbreeding Wistar rats exhibiting persistently higher blood pressure (Okamoto & Aoki, 1974). SHR displays most behaviors that are characteristic of human ADHD in tasks that involve hyperactivity, inattention, and impulsivity (Johansen, Killeen, & Sagvolden, 2007). Inattention has been measured in SHR in temporal discrimination tasks, which are tasks that require timing (Multiple FI-120 s EXT; DRL) as well as tasks requiring sustained attentional engagement (5-CSRTT). Examples of temporal discrimination tasks include pressing a lever only after a certain amount of time and then stopping pressing after reinforcement is no longer given (Fixed Interval/Extinction; FI/Ext), or pressing the lever and waiting the time interval before making another press (Differential Reinforcement of Low Rates; DRL). An example of a task measuring engagement would be choosing to make a head-entry only into the one briefly light- illuminated hole of a 9-hole panel (5 Choice Serial Reaction Time Task; 5-CSRTT). Impulsivity has been measured in tasks testing the ability to withhold responses. Typical tasks used to measure the ability to withhold responses are the aforementioned DRL and

5 NICOTINE AND ADHD 5 5-CSRTT, because they involve attentional as well as impulsivity measures. Additionally, the Stop-Signal Task is used, which involves pressing the right lever and subsequently the left, as well as withholding a lever press at the sound of a stop tone. Results in SHR are similar to those obtained from humans, providing additional support for SHR as a model of ADHD (Sagvolden, 2000). As an attentional measure, the same visual discrimination task was used for ADHD children, with the same schedule as SHR (FI/Ext), the same interface and computer program, but with different manipulations and reinforcers. Both SHR and children with ADHD showed the same increase in number of responses during the extinction component, significantly more than the control groups. Much like the 5- CRSST, the Continuous Performance Test (CPT) requires participants to maintain attention while ignoring distractors when selecting for target stimuli. Responses made when no target is present are categorized as impulsive (errors of commission). When the target is displayed and no response is made, the lack of response is categorized as an attentional deficit (errors of omission). In a meta-analytic study of ADHD and CPT, Losier, McGrath, and Klein (2006) discovered that ADHD subjects make significantly more errors of omission and commission than non-adhd subjects. Compared to control strains, SHR also have significantly impaired performance on 5-CSRTT (De Bruin, Kiliaan, De Wilde, & Broersen, 2003). The similar results of Losier and DeBruin support the SHR as a valid model of attentional deficits of ADHD children. In tasks measuring impulsivity, SHR have also displayed behaviors similar to children with ADHD (Johansen & Sagvolden, 2005). Children with ADHD display short and rapid sequences of activity. In a task that operationalized this by measuring burst responses with short intervals in between each response, SHR exhibit significantly more of these than control strains

6 NICOTINE AND ADHD 6 (Sagvolden, 2000). Lastly, SHR also display similar hyperactivity to that of children with ADHD by making significantly more overall responses in the FI/Ext task. These similarities validate SHR as a model of ADHD, with useful applications for studies of this disorder. Nicotine and Response Inhibition Nicotine has been linked to improvements in response inhibition capacity (RIC) in humans with ADHD. Several assessments have been utilized to evaluate this: the Continuous Performance Test (CPT), Stop-signal, and Delay Discounting Tasks (Nichols & Waschbusch, 2004). Surprisingly, in data that analyzed effects of acute and prenatal exposure to nicotine on RIC in non-adhd subjects, nicotine has largely been shown to have no effect in Stop-signal tasks and CPT (Galván, Poldrack, McGlennen, & London, 2011; Rezvani & Levin, 2001). Contrastingly, acute nicotine has been shown to increase RIC in ADHD adolescents in a Stopsignal task (Potter & Newhouse, 2004). The display of decreased impulsivity in ADHD smokers, but not in non-adhd smokers may be due to a neurological predisposition of ADHD individuals to effects of nicotine. Nicotine may be a trigger that accentuates these differences. Effects of nicotine on RIC in rat models of ADHD have not been investigated (Vendruscolo, Izídio, & Takahashi, 2009), but its effects on normal rats have largely suggested decreases in RIC. This decrease in RIC has been exhibited in 5-CSRTT testing (Schneider, Bizarro, Asherson, & Stolerman, 2012), DRL (Kirshenbaum, Johnson, Schwarz, & Jackson, 2010), and Go/No-Go Tasks (Kolokotroni, Rodgers, & Harrison, 2011). This finding is different from human subjects in which nicotine shows no effect. Though this finding may raise challenges to the validity of rat strains in portraying human behavioral effects of nicotine, it doesn t raise issues in the realm of the present study.

7 NICOTINE AND ADHD 7 The desired outcome in this study is that SHR rats will respond similarly to human ADHD subjects, rather than mirror the response of previously tested non-adhd model rat strains. Because SHR has typically mirrored ADHD humans on behavioral tasks, the anticipation is for SHR to display a significantly higher increase in RIC with nicotine exposure than the control subjects (Wistar Kyoto; WKY), a response similar to humans with ADHD. Nicotine and Sustained Attention Nicotine has also been strongly linked to improvement in sustained attention in rats and humans. In humans both ADHD and non-adhd diagnosed, attentional performance on CPT is significantly improved with nicotine exposure (Levin et al., 1998; Poltavski & Petros, 2006; Myers, Taylor, Salmeron, Waters, & Heishman, 2012). Nicotine has also been shown to improve sustained attention in rats as well (Day et al., 2007; Koffarnus & Katz, 2011). Because of the lack of studies on behavioral effects of nicotine on SHR, nicotine effects on sustained attention in SHR have not been investigated. SHR do display baseline deficiencies in sustained attention compared to WKY (Sagvolden, Dasbanerjee, Zhang-James, Middleton, & Faraone, 2008) but because of the substantial improvement in sustained attention in humans and rats both ADHD and non-adhd, we anticipate both strains (SHR and WKY) to display a similar improvement with nicotine exposure. Fixed Minimum Interval Task For this study, the primary task utilized to measure RIC and sustained attention is the Fixed Minimum Interval (FMI) task, as illustrated in figure 1. To begin, a retractable lever is inserted into an operant chamber. The rat is then required to press it to begin a six second timer.

8 NICOTINE AND ADHD 8 Once these six seconds have elapsed, the rat s subsequent head entry into the food hopper is reinforced with a sugar pellet. A head entry made prior to the required time lapse doesn t receive reinforcement. The six second interval is the crucial point of interest for analyzing response inhibition and sustained attention. Notably, this task separately examines response inhibition and motivational factors (Mika et al., 2012). Separate examination of response inhibition and motivational factors is important because response inhibition is often confounded by incentive motivation in many responsewithholding tasks such as DRL (Doughty & Richards, 2002; Hill, Covarrubias, Terry, & Sanabria, 2012). They can be separated in FMI because the task-initiating response (initial lever press) differs from the task-terminating response (head-entry) (Mika et al., 2012). The intervals between lever presentation and initial lever-press are recorded as latencies; these intervals are utilized to measure incentive motivation. Intervals between initial lever press and head entries are recorded as inter-response times (IRTs); these intervals are used as a measure of impulsivity and sustained attention, the critical areas of interest for the present study. Temporal Regulation Model In order to understand the distribution of IRTs, the Temporal Regulation model was used (Sanabria & Killeen, 2008). In this model, every trial begins with the rat entering either a timing state (with probability of P) or a non-timing state (with a probability of 1-P) (Mika et al., 2012). In a timing state, IRTs are normally distributed along a gamma distribution and are concentrated around the FMI timing interval (six seconds). When a rat is in a timing state, its responses most frequently occur around the aimed-for time, which explains the IRT distribution. In a non-timing state, IRTs are emitted at a constant average rate along an exponential distribution. Because each

9 NICOTINE AND ADHD 9 head entry is not timed, it is random and therefore as likely to occur at one time as at any other time. This model proposes that throughout the duration of the procedure, a mixture of gamma (timed responses) and exponential (non-timed responses) distributions contribute to the distribution of IRTs (Mika et al., 2012). As illustrated in figure 2, a rat that is more likely to enter a timing state (increase in P) exhibits an increase in attention toward the task, because timing requires engaged cognition. Therefore, P was utilized to measure sustained attention. We expected nicotine to increase sustained attention similarly for SHR and WKY. In addition, to measure response inhibition capacity (RIC) parameter θ was obtained by dividing the mean IRT by the target time, six seconds. If the rat waits longer for the reinforcer, the waiting time causes an increase in θ and indicates an increase in RIC (Hill, Herbst, & Sanabria, 2012). We expected that nicotine would increase θ and therefore improve RIC greater for SHR than WKY, attributable to improvements in managing impulsivity. Specific Research Questions 1. Does acute administration of nicotine at doses of 0.1, 0.3, 0.6 mg/kg in adolescent and young adult SHR dose-dependently increase P and θ in an FMI task intended to measure ADHD-related impulsivity and sustained attention? 2. How do nicotine-induced changes in SHR performance compare to analogous changes in the performance of a normo-active control, WKY? If question 1 receives a positive answer, then it will support the SHR as a legitimate model of nicotine-reduced impulsivity and sustained attention. A comparison against WKY performance

10 NICOTINE AND ADHD 10 (question 2) will determine the extent to which nicotine effects on ADHD-related impulsivity and sustained attention are specific to an animal model of ADHD.

11 NICOTINE AND ADHD 11 Literature Review No previous research utilizing the FMI task was found, but nicotine has been investigated in several other response-withholding tasks in rats. In the 5-Choice Serial Reaction Time Task rats are presented with a light stimulus in one of five holes and are required to make a head-entry corresponding to the illumination to receive food reinforcement (Bari et al. 2008). In this task, anticipatory responses before the light stimulus are regarded as a measure of impulsivity. Acute nicotine has been shown to increase anticipatory responses in rats (Schneider et al., 2012). It has also caused significant increases in anticipatory responding at a small dose (Tsutsui-Kimura et al., 2009), as well as small increases in anticipatory responding (Semenova, Stolerman, & Markou, 2007). It has also had no effect on anticipatory responses (Day et al., 2007). Effects of nicotine on rodent performance in the Differential Reinforcement of Low Rates (DRL), Go/No-Go, and Delay Discounting tasks have produced similar reduction of RIC. DRL tasks require animal subjects to withhold responses for a certain amount of time between lever presses to obtain reward (Kirshenbaum, Brown, Hughes, & Doughty, 2008). Nicotine exposure impairs RIC in DRL (Kirshenbaum et al., 2010). Furthermore, acute nicotine reduces RIC (increases in Go responses) in a Go/No-Go task, which requires discrimination between a Go stimulus signifying reinforcement and a No-Go stimulus signifying no reinforcement (Kolokotroni, Rodgers, & Harrison, 2012). In Delay Discounting tasks, i.e., choice of smaller, sooner vs. larger, later rewards (Shamosh & Gray, 2008), nicotine has been shown to increase preference for smaller-immediate rewards, signifying increased impulsivity (Locey & Dallery, 2011). Contrastingly, Anderson and Diller (2010) demonstrated that a typically impulsive rat strain (Lewis rats) displayed significantly decreased choice of smaller-immediate reward following a low dose of nicotine than the normotensive strain (Fischer 344); this was shown in

12 NICOTINE AND ADHD 12 acute, but not in repeated administration. These experiments were limited, however, because they enlisted only normal rats. Therefore, none of these results pertained to ADHD rats. Results in humans have been more inconsistent with effects on the Continuous Performance Test (CPT), Stop-signal, and Delay Discounting Tasks. In CPT, subjects are required to respond as quickly as possible to target stimuli (i.e. an X on the screen) while withholding responses to non-target stimuli (any other letter) (Levin & Rezvani, 2000). Nicotine administration has resulted in an increase in errors of commission (impulsive response when no stimulus is present) (Spinalla et al., 2002). On the other hand, in groups selected for low attention, nicotine has produced fewer errors of commission (Poltavski & Petros, 2005). Nicotine has also demonstrated no effects (Levin et al., 1996; Rezvani & Levin, 2001). In Stop-Signal tasks, in which response-withholding speed on previously reinforced behavior is utilized as a measure of impulsivity (Perry & Carroll, 2008), nicotine improves Stop- Signal Reaction Time in subjects with ADHD (Potter & Newhouse, 2008). Administration of nicotine has also led to no effect on Stop Signal Reaction Time performance in adolescent smokers (Galván et al., 2011). In Delay Discounting tasks, smokers have demonstrated significantly steeper delay discounting than non-smokers (Baker, Johnson, & Bickel, 2003). Subjects with high levels of nicotine intake display greater discounting of delayed monetary reinforcement (Ohmura, Takahashi, & Kitamura, 2005). Sustained attention in individuals with or without ADHD has been studied extensively, and was reported as sensitive to nicotine administration (Rezvani & Levin, 2001). To explain the differential effects of nicotine on attention in SHR, it is useful to consider the neurotransmitters involved in this function. Sustained attention involves the operation of acetylcholine (ACh) (McGaughy & Sarter, 1999), norepinephrine (NE) (Beane & Marrocco, 2004), and dopamine

13 NICOTINE AND ADHD 13 (DA) (Jucaite, Fernell, Halldin, Forssberg, & Farde, 2005). SHR have less DA activity in the prefrontal cortex (PFC) (Sullivan & Brake, 2003) than WKY. Expression of DA is higher in SHR than WKY, whereas DA transporter activity is low, which leads to reduced DA uptake (Viggiano, Vallone, & Sadile, 2004). SHR also display decreased DA release (Russell, de Villiers, Sagvolden, Lamm, & Taljaard, 1995). A proposed reason for poor attention in SHR is an imbalance of DA and NE (Russell, 2002). Unlike DA release that is decreased, NE concentration is elevated (Reja, Goodchild, & Pilowsky, 2002) as is NE uptake (Myers et al. 1981). Lastly, SHR also appear to have cholinergic deficits involving reduced ACh receptors sites (Hohnadel, Hernandez, Gearhart, & Terry, 2005). Nicotine has been shown to increase release of ACh, NE, and DA (Singer et al., 2004; Rossi, Singer, Shearman, Sershen, & Lajtha, 2005). Many researchers have published findings based on 5-CSRTT. For example, errors of omission (i.e., insensitivity to reinforcement contingencies) decrease with low doses of nicotine (Day et al., 2007). This effect of nicotine on 5-CSRTT performance is analogous to nicotineinduced reductions in errors of omission in human CPT performance (Myers et al., 2012), which are interpreted as high levels of engagement in the task or improvements in sustained attention. Several papers examined the relation between nicotine and NE. Nicotine increases NE release and SHR display greater NE uptake than WKY (Myers et al., 1981). However, high rates of NE discharge decreased attentional performance were also reported (Usher, Cohen, Servan- Schreiber, Rajkowski, & Aston-Jones, 1999). This finding was consistent with the administration of larger doses (increased NE release). In SHR, larger doses led to a decline in performance compared with smaller dose performance. Lastly, DA activity and release was decreased in SHR, but lesions of DA led to cognitive deficits (Nieoullon, 2002).

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15 NICOTINE AND ADHD 15 Methods Subjects This study involves 23 male rats: 12 Spontaneously Hypertensive Rats (SHR) and 11 Wistar-Kyoto Rats (WKY). Initially, 20 rats arrived at post-natal day twenty-four, but three WKY-strain rats died within the first week, due to poor health. Therefore, the experiment was run in two parts. In the first trial, 17 rats (10 SHR, 7 WKY) were run and six rats (2 SHR, 4 WKY) were run in the second trial. This procedure ensured equal number of rats in each group. Apparatus Experiments were conducted in 10 MED Associates modular test chambers (305 mm x 241 MM x 210 mm). The floor was made of thin metal bars with a catch pan of sanitary chip litter. The ceiling, front and rear walls were made of transparent plastic with the front wall serving as a latched door. Each box contained a ventilation fan, which provided masking noise of around 60 db. All were equipped with a square food aperture (51 mm sides, 15 mm from the chamber floor) for dispensing 45-mg sucrose pellets. Retractable levers were on either side of the food dispenser (the inside edge of each lever was 8 mm from the closest vertical edge of the receptacle), yet only the right lever was used in the course of the experiment. Three-color stimuli lights (ENV 222M) were located directly above each lever and could be illuminated red, green, or yellow. A speaker located at the top of the side wall contained the house light that emitted 75 db tones from a generator. A dimly lit house light was located behind the left wall of the chamber.

16 NICOTINE AND ADHD 16 Timeline Rats were immediately pair-housed with same strain upon arrival on post-natal day 24 in the first trial and 26 in the second trial. Rats were initially fed with moist rat chow pellets for digestion inside their cages, as well as hard food pellets on the lid ad libitum. Access to food was gradually reduced from 24 hours/day to 1 hour/day. Each rat was weighed prior to running and fed 1 hour after running. In order to ensure motivation in the task, rats were not fed before running. The rats' running weights were based on 85% of their expected free-feeding weights, as estimated from a logistic function fitted to the provider's growth curves (Sanabria & Killeen, 2008). Rats were acclimated to rat chambers and to the dispensing of sugar pellets starting at post natal day 30 in the first trial and 29 in the second. Prior to chamber acclimation, ten sugar pellets were dropped into rat cages in order to familiarize the rats with the reinforcer. In the chambers, pressing the lever illuminated the three LED lights above the levers. When the detector within the food receptacle was activated (head-entry) sugar pellets were dispensed along with a 0.5-s 3 khz tone. Sugar pellets were initially given every 45 seconds in the acclimation stage. Pellets were counted after every session to evaluate progress of each rat. Once consistent pellet eating was displayed, lever press shaping began; lever presses produced reward pellets. FMI Procedure Following lever press shaping, fixed minimum interval (FMI) procedure began at postnatal day 40 in the first trial and 38 in the second. A lever was inserted into the operant chamber (see figure 2). Rats were required to press the lever and withhold a head-entry response until after the designated interval (six seconds). A successfully accomplished task was reinforced by

17 NICOTINE AND ADHD 17 sugar pellet dispensing. Head entry prior to the designated interval (anticipatory responses) resulted in neither pellet nor tone. Time between lever press and head entry was recorded as inter-response times (IRTs). FMI began at 0.5 seconds and gradually increased as IRTs began to occur consistently around the designated interval until the six second interval was achieved. Once this interval was achieved, a Variable Interval-30 (VI-30) schedule was introduced, which meant that once the timer which was running the entire session hit 30 seconds, the following correct IRT would be reinforced. Reponses within 30 seconds of each other received no reinforcement. This procedure equalized sugar pellet reinforcement across subjects, because not every correct IRT was reinforced. After reinforcement, the timer reset. In training, rats were required to accomplish progressing VI schedules until all were at VI-30. All head-entries (reinforced and non-reinforced) turned off the LED lights and resulted in a 10 second inter-trial interval, signified by lever retraction and house light illumination. Nicotine Administration Rats were acclimated to injections by receiving subcutaneous saline injections on PND 32 and 33 before testing. Nicotine hydrogen tartrate was dissolved in sterile saline. Injections were given twice weekly (Saturday and Tuesday) immediately before testing beginning on post natal day 57 in both trials. Each rat received two subcutaneous injections of each dose as well as two pre-feeding trials (saline, pre-feeding, 0.1, 0.3, and 0.6 mg/kg) on a counterbalanced schedule. Pre-feeding trials were executed in order to control for appetite suppressing effects of nicotine (Dandekar, Nakhate, Kokare, & Subhedar, 2011). Following the completion of the injection schedule, rats went through extinction procedures on PND 83 (young adulthood) with

18 NICOTINE AND ADHD 18 the final injection on postnatal day 88 in the first trial and 89 in the second. Figure 3 displays one example of a counterbalanced schedule with post-natal day indicated.

19 NICOTINE AND ADHD 19 Data Analysis Temporal Regulation Model IRTs (times between first lever press and head entry) were collected for analysis on every injection day, on the day preceding each injection (pre-injection days), and on the last two experimental days (FMI 0.5-s on vehicle and on 0.6 mg/kg nicotine). Parameters of the Temporal Regulation (TR) model (Hill, Herbst, & Sanabria, 2012) were estimated from the distribution of IRTs of each individual rat on each injection day. Response inhibition capacity (RIC) is indexed by θ, and responsiveness to schedule of reinforcement is indexed by P. Log-transformed TR parameter estimates served as dependent measures; parameter P was transformed into log odds of timed IRTs = ln(p / 1 P). Both transformations follow suggestions on the estimation of population parameters in a similar model (Brackney, Cheung, Neisewander, & Sanabria, 2011). All dependent measures are reported as back-transformed mean ± SEM. TR parameters were estimated for each rat using the method of maximum likelihood (Myung, 2003). Analysis of variance (ANOVA) ANOVA was implemented to establish the effects of nicotine on log-transformed Temporal Regulation parameters, using a significance threshold of α = When sphericity was violated according to Mauchly s test, a Huynh-Feldt correction was implemented. Significant interaction effects were followed by 2-tailed t-tests. No post hoc tests were conducted in the absence of a significant main or interaction effect. Only significant effects are reported.

20 NICOTINE AND ADHD 20 Nicotine effects A separate (strain; cycle; dose) mixed-design ANOVA was conducted on each dependent measure to establish the dose effects of nicotine on Temporal Regulation parameters, and whether those effects were modulated by strain. Significant dose effects were followed by 2- tailed paired-samples t-tests between vehicle and each other dose. No post hoc tests were conducted in the absence of a significant main or interaction effect.

21 NICOTINE AND ADHD 21 Results A significant main effect of dose on θ estimates was observed, F(3, 63) = 4.64, p =.005 (see figure 4). Post hoc paired-sample t-tests revealed that 0.1, 0.3, and 0.6 mg/kg of nicotine significantly reduced θ, t(22) = 3.31, 2.58, and 2.81, p =.003,.017, and.010, respectively. Significant strain dose interaction effects on P estimates were observed [P: F(3, 63) = 5.05, p =.003] (figure 5). Post hoc t-tests on P revealed that it was substantially higher for SHR than WKY on 0.1 mg/kg nicotine, t(21) = 2.33, p =.030, was higher for SHR on 0.1 and 0.3 mg/kg nicotine than saline [t(11) = 4.41, p =.001, and t(11) = 4.05, p =.002, respectively], and was higher for WKY on 0.3 and 0.6 than saline [t(10) = 2.79, p =.019, and t(10) = 2.64, p =.024]. Overall, these results suggest that nicotine reduced response inhibition capacity, but dosedependently increased performance on the timing aspect of the task, requiring a lower dose for the improvement in SHR.

22 NICOTINE AND ADHD 22 Discussion Research Questions My research set out to answer two questions. First, does acute administration of nicotine increase impulsivity and sustained attention in the SHR rat? Second, were these changes significantly different than the comparative control strain WKY? I expected that nicotine would dose dependently manage both impulsivity and sustained attention in SHR. I also hypothesized that sustained attention would be similarly increased in both strains, but SHR would have significantly greater improvements in RIC then WKY. Research results were surprising. Contrary to the first hypothesis, ability to manage impulsivity gradually worsened in both strains. Supportive of the first hypothesis, however, there was a general improvement in sustained attention in both strains. Contrary to the second hypothesis, there were no inter-strain differences in effects on impulsivity. Also contrary to the second hypothesis, however, a low nicotine dose caused a significantly greater increase in SHR sustained attention. Nicotine Reduced RIC Both WKY and SHR strains displayed a statistically significant decrease in θ, which demonstrated that nicotine reduced RIC; higher doses resulted in greater impulsive responding. There is no prior published data for this FMI task, but much of the research has displayed that nicotine increases impulsivity in rats. Because nicotine did produce decreases in RIC, my results are consistent with the literature. Those studies differ in their investigation of an animal model of ADHD versus normal rats, but my results indicate that nicotine similarly affects normal rats and ADHD-model rats.

23 NICOTINE AND ADHD 23 In humans, literature has shown that nicotine has been shown to increase, decrease and have no effect on RIC in subjects without ADHD. On the other hand, nicotine has been shown to improve RIC in adolescent and young adult subjects with ADHD in a Stop-signal task (Potter & Newhouse 2004; 2008). Research on nicotinic effects appears to differ greatly between humans and animal models. In animals, RIC is generally decreased with nicotine exposure, but in humans without ADHD RIC has been increased, decreased, or has had no effect. In humans with ADHD however, acute nicotine has shown an increase in RIC. Therefore, my data align well with animal model and non-adhd related human findings, but not with enhanced RIC displayed in ADHD humans. Because the data display a non-differential effect of nicotine on RIC, generalizations to the human population are difficult to determine, as are inferences of the effects nicotine has on RIC and ADHD. The general difference in nicotine effects between animal models and humans calls for further investigation. I expected that because SHR behavior has aligned well with ADHD human behavior that nicotine would similarly affect SHR and ADHD subjects. Our results did not align with this because SHR display similar decreases in impulsivity as normal rats as previous literature has shown. There are several potential reasons why this difference occurred. In animal studies, the dosage is dependent on weight (mg/kg) whereas in humans all subjects receive a uniform dosage (i.e. 7mg). Another reason is that the research in humans with ADHD is in a Stop-signal task and this experiment was conducted with the FMI task, which is quite different in its methodology. In future studies, I could possibly remove this limitation by utilizing the Stop-signal task, which has been utilized in animal studies to measure response inhibition (Eagle et al., 2008). With similar

24 NICOTINE AND ADHD 24 testing methodologies, I expect that SHR would have similar improvements in impulsivity to humans with ADHD. Nicotine Improved Sustained Attention The present study revealed that nicotine dose-dependently improved sensitivity to timing contingencies, as indicated by parameter P (high responsiveness to schedule of reinforcement), and that SHR required a lower dose to display this effect than WKY. The increase of P by nicotine is consistent with the findings based on 5-CSRTT: the decrease of errors of omission corresponding to low doses of nicotine. In turn, reduced errors are connected to improvements in sustained attention. Thus, changes in P appear to be linked with nicotine effects on sustained attention. It is yet unclear why sustained attention is more sensitive to nicotine in SHR than in WKY. Sustained attention in individuals with or without ADHD appears to be similarly sensitive to nicotine administration (Rezvani & Levin, 2001). To explain the differential effects of nicotine on attention in SHR, it is useful to consider the neurotransmitters involved in this function. The reduced density of ACh receptor sites in SHR relative to WKY may explain the improvement in P following the administration of a lower dose of nicotine in SHR, but does not explain the dose effect differences between SHR and WKY. However, the smaller dose needed to improve attention in SHR than WKY is consistent with the published result that nicotine increases NE release and SHR display greater NE uptake than WKY (Myers, Whittemore, & Hendley, 1981). Additionally, the larger doses (increased NE release) leading to a decline in performance compared to smaller doses in SHR is consistent with the published result that high

25 NICOTINE AND ADHD 25 rates of NE discharge decrease attentional performance (Usher et al., 1999). Lastly, the effects on P may be explained by the effects of nicotine on SHR attentional performance in the ACh system, as described in published literature (Nieoullon, 2002). However, only strain differences in NE function may explain the heightened responsiveness of SHR to nicotine, relative to WKY.

26 NICOTINE AND ADHD 26 Conclusion The results are consistent with the notion that nicotine has a non-specific disruptive effect on Response Inhibition Capacity. Results also revealed an SHR-specific cognitive-enhancement effect of a low dose of nicotine to the response-withholding requirement of FMI, possibly related to nicotine effects on sustained attention. This finding supports the self-medication hypothesis for attention enhancement from nicotine. The results also validate the SHR as a rat model of nicotine effects on ADHD. The absence of baseline differences between strains challenges the validity of SHR as an animal model of ADHD, but the differential responsiveness of this strain to nicotine supports its use in the investigation of genetic vulnerability to tobacco dependence related to cognitive impairment.

27 NICOTINE AND ADHD 27 Figures Figure 1. Flowchart of FMI procedure. Every sequence began with an acclimation period in a dark chamber. The right lever was inserted into the box to initiate the sequence. This was followed by a lever press that turned on the cue lights above it and started the timer. A subsequent head-entry that exceeded the 6-second requirement was reinforced with a sucrose pellet on a variable interval 30 second (VI-30) schedule and a tone. Therefore only some correct responses were reinforced with a sucrose pellet, but all were followed by a tone. Incorrect responses were followed by a blackout with no tone or sucrose reinforcement. Times between lever presentation and lever press (latencies) were categorized as motivation to initiate the trial, whereas times between lever press and head-entry (inter-response times, IRTs) were categorized as response inhibition capacity (RIC) and sustained attention.

28 NICOTINE AND ADHD 28 Figure 2. TR model parameters of IRTs in an FMI procedure. P denotes probability of rats engaging in the timing of the task. Nicotine administration was expected to increase P similarly for SHR and WKY. θ is the mean IRT divided by the target time (6 seconds). Nicotine was expected to increase θ more for SHR than WKY indicating improved impulsivity. The dashed line denotes a reduction in P and θ. The solid line denotes a normal distribution.

29 NICOTINE AND ADHD 29 Figure 3. Nicotine administration schedule. This example of a counterbalanced schedule for one rat indicates at what post-natal day injections were received and at which dose (saline, prefeeding, 0.1, 0.3, and 0.6). The numbers under injection type indicate post natal day. Each rat received two administrations of each dosage in two identical cycles. Every rat was given a different schedule.

30 NICOTINE AND ADHD 30 Figure 4. Strain and dose effects on response threshold (θ). These results reveal that θ was significantly reduced at doses of 0.1, 0.3, and 0.6 respectively compared to vehicle. There were no significant strain differences. This suggests that nicotine caused a general reduction in response inhibition capacity.

31 NICOTINE AND ADHD 31 SHR WKY Figure 5. Effect of strain and dose on engagement probability (P). P was significantly higher for SHR than WKY at 0.1 mg/kg of nicotine. This suggests a greater improvement in sustained attention for SHR compared to WKY at a low dose. P was also higher at 0.1 and 0.3 mg/kg of nicotine than saline for SHR and higher at 0.3 and 0.6 mg/kg for WKY than saline. This suggests an overall improvement in sustained attention by nicotine, but SHR requiring a lower dose to display this.

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