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Nicotine does not improve recovery from learned nonuse nor enhance constraint-induced therapy after motor cortex stroke in the rat
Behavioural Brain Research 198 (2009) 411–419
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Research report
Nicotine does not improve recovery from learned nonuse nor enhance constraint-induced therapy after motor cortex stroke in the rat Diana H. Lim ∗ , Mariam Alaverdashvili, Ian Q. Whishaw Department of Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4
a r t i c l e
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Article history: Received 13 August 2008 Received in revised form 10 November 2008 Accepted 18 November 2008 Available online 30 November 2008 Keywords: Nicotine and recovery Learned nonuse Motor cortex stroke Constraint-induced therapy Skilled reaching Rehabilitation after stroke
a b s t r a c t Nicotine, a cholinergic agonist, rapidly crosses the blood–brain barrier, promotes neuronal plasticity and has been suggested to enhance behavior in a variety of neurological conditions. Nicotine has also been suggested to benefit functional recovery in rodent models of stroke. At present there has been no systematic investigation of the potential benefits of nicotine therapy in both the acute and chronic post-stroke period. This was the objective of the present study and to that end, the effects of nicotine administration prior to and following motor cortex stroke were examined in a skilled reaching task. The task provides a thorough assessment of learned nonuse and constraint-induced recovery of behavior as determined by both end-point and movement element analysis. Nicotine (0.3 mg/kg PO) was administered twice daily during reach training and following motor cortex stroke. Rats were divided into four groups based on their pre/post-stroke treatment: nicotine/nicotine, nicotine/vehicle, vehicle/nicotine, vehicle/vehicle. After stroke, nicotine did not counteract learned nonuse, facilitate constraint-induced therapy, or improve long-term recovery as measured by end-point analysis and movement element analysis. The results are discussed in relation to the problem of identifying pharmacotherapeutic agents that augment rehabilitation following stroke. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nicotine is a cholinergic agonist that indirectly activates other neural systems including the dopaminergic and adrenergic systems [44,61]. Nicotine has been suggested to play a protective role with respect to some neurodegenerative diseases including Alzheimer’s disease [39,42], schizophrenia [11,41], and Parkinson’s disease [43,53]. In human studies, nicotine has been shown to enhance vigilance [31] and performance on skilled motor tasks [50]. In animal studies, nicotine has been shown to enhance attention [38,45] and memory [27], increase motor activity [13], and overcome depression [49] and learned helplessness e.g. in the forced swim test [46]. It is also suggested that nicotine is a possible treatment for promoting recovery after brain damage [6,7,24,29,52,53], including brain damage induced in a rodent model of motor cortex stroke [23]. At present there has been no exhaustive examination of the effects of nicotine after stroke; the specific improvements, the time points at which it is effective or the relation of putative beneficial effects on post-stroke rehabilitation have yet to be addressed. The examination of these factors was the objective of the present study. Rats received nicotine in association with pretraining and
∗ Corresponding author. Tel.: +1 403 329 2637; fax: +1 403 329 2775. E-mail address: [email protected] (D.H. Lim). 0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.11.038
post-stroke rehabilitation. The experiment used a skilled-reachingfor-food task that assesses forelimb use in reaching for a small piece of food [54,55,59], a motoric act that is frequently impaired by stroke in humans [19]. The task leads itself to the examination of both the learned and innate motoric effects of motor cortex stroke and has the strength of providing multiple measures of performance. Stroke was induced via pial stripping. This model of stroke is advantageous because behavioral assessment can be made in the acute post-stroke period, during rehabilitation, and after recovery [56]. In the acute period following motor cortex stroke, rats display learned nonuse of their affected limb in that they show a decline in reaching attempts [16]. When limb use is forced by constraint-induced therapy, that is, by limiting use of the nonaffected limb, there is a gradual recovery/compensation associated with rehabilitation [33,40,58]. In the chronic post-stroke period there may be regression of performance in the absence of rehabilitation. These learning-related aspects of performance are assessed by three end-point measures, first attempt success, total success, and total attempts, while the effects of stroke on movement are assessed by a detailed analysis of the thirteen movement elements of the reaching act [3]. The model of stroke used in the present study allows a comprehensive assessment of the phases of poststroke recovery and is a good model of human stroke [56]. As such it provides a strong assessment tool for evaluating drug-related facilitation of recovery in association with rehabilitation after stroke.
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For the present study, rats were trained on the single pellet reaching task and received a daily regime of nicotine prior to and/or following motor cortex stroke targeting the forelimb area. Skilled reaching performance was assessed in the acute and chronic poststroke period using end-point measures of the reach action as well as a rating of the movement elements required to perform a reach [59]. 2. Materials and methods 2.1. Subjects Thirty-nine female Long-Evans hooded rats (approximately 100 days old and weighing 250–300 g at the beginning of the experiment) from the University of Lethbridge animal colony were used. Rats were housed in Plexiglas cages in groups of two or three. The colony room was maintained on a 12:12 h light:dark cycle and was controlled for temperature and humidity. The experiment was conducted with approval from the University of Lethbridge animal care committee and in compliance with the guidelines set by the Canadian Council on Animal Care.
of the box. These indentations ensure that the food target is placed such that the rat cannot lap up the food with its tongue and must use the paw contralateral to the indentation to grasp the target; this ensures the use of a single paw for reaching. 2.6. Bracelets for limb constraint To prevent rats from reaching with both paws, a bracelet made of Elastoplast adhesive tape (Smith & Nephew, Lachine, Quebec, Canada) was wrapped around the non-preferred wrist. The bracelet enlarges the paw so that it cannot be inserted through the slot of the reaching box, thus inducing constraint, but does not change the rat’s performance during the testing session [58]. Bracelets were applied pre- and post-stoke only if the rat attempted to reach with the non-preferred (ipsilesional) paw for two or more trials. 2.7. Video recording Reaching performance was video recorded during each reaching session. A Sony 3CCD camcorder (1/1000 s shutter speed) and a cold light source were used. Subsequent frame-by-frame analysis was completed using a Sony videocassette recorder (DSR-11).
2.2. Food restriction
2.8. Reach training
Three weeks prior to behavioral testing, rats were gradually food-deprived to 95–98% of their body weight. This was achieved by once-a-day feeding of Purina Rat Chow (20 g per rat per day). Prior to training, rats also received ∼900 mg/day of dustless precision pellets (Bioserve Inc.) which would serve as the reaching targets in the single pellet reaching task. One week prior to behavioral testing rats also received ∼1 g of Kraft Smooth Peanut Butter (Kraft Canada) in the morning before training, and in the evening, prior to daily feeding. The peanut butter would later serve as the vehicle for drug administration (nicotine solution was given in the peanut butter). The rats were maintained at 95–98% of their body weight until the completion of behavioral testing.
The first week of reach training consisted of daily 10 min sessions for each rat. The objective was to introduce the rat to the reaching cage with pellets on the shelf and to allow the rat to retrieve the pellet by paw or tongue. Once a rat was successfully retrieving pellets, pellets on the shelf were moved further away in order to encourage the use of a paw. After a rat demonstrated a preference for one paw by making most reaching attempts with it, individual pellets were placed into the indentation contralateral to that paw. Rats continued to receive daily training sessions, consisting of 20 discrete trials with inter-trial intervals during which they were shaped to leave the slot, walk to the rear wall of the cage, turn and approach the slot again for the next pellet. In addition, by withholding food on semi-randomly selected trials, rats were taught to sniff the shelf for a pellet and to reach only if a pellet was present. Thus, each rat eventually learned to orient to the food pellet, transport its limb through the slot, grasp the food pellet, and retract its paw through the slot to release the food into its mouth [20]. Training took place over a period of three weeks.
2.3. Drug administration and dose Subjects were assigned to four different groups based on drug administration before and after surgery (pre-/post-stroke). Groups were: nicotine/nicotine (N/N; n = 9), nicotine/vehicle (N/V; n = 10), vehicle/nicotine (V/N; n = 10), and vehicle/vehicle (V/V; n = 10). Nicotine hydrogen tartrate salt (Sigma–Aldrich) was dissolved in distilled water to a concentration of 1 mg/mL [48]. Rats were administered 0.3 mg/kg of nicotine solution twice per day, in accordance with previous work [23,24]. Administration occurred in the morning, half an hour before training or testing (9:00 AM) and in the evening, after training or testing but prior to daily feeding (4:00 PM). Administration of drug or vehicle took place for 21 days, during which rats were tested daily. Oral administration was used throughout, with peanut butter (Kraft Smooth Peanut Butter, Kraft Canada) being used as the vehicle. A pilot study justified the per oral route of administration by examining the activation effects of nicotine between per oral (PO) and subcutaneous (SC) administration using a VersaMax Animal Activity Monitoring System (AccuScan Instruments, Inc. [email protected]). The locomotor activity following nicotine administration was used as an index of nicotinic activation. Rats received a dose of 0.3 mg/kg via SC or PO administration. Both routes of administration resulted in enhanced motor activity but there was no difference in motor activity between groups over an 8-day testing period (F(1, 6) = 0.11, p > 0.05). Onset of drug-induced locomotion following SC administration was slightly more rapid, but within 15 min of administration, activity in the two groups was equivalent. 2.4. Stroke All rats received stroke to the forelimb area of motor cortex, as defined by previous anatomical and electrophysiological studies [2,14,26]. Rats were anesthetized with sodium pentobarbital (45 mg/kg, i.p.; Sigma–Aldrich) and were given an analgesic (0.3 mg/kg Buprenorphine, i.p.; Sigma–Aldrich) and an injection of atropine (0.1 mg/kg, i.p.; Sigma–Aldrich) to facilitate respiration throughout the surgery. Rats were placed in a stereotaxic device and then a portion of the skull was removed in the hemisphere contralateral to the preferred reaching paw (1 mm posterior and 4 mm anterior to bregma, and 1–4 mm lateral to midline). Exposed tissue was devascularized by gently wiping away the blood vessels with a saline-soaked cotton swab [28,47]. The incision was sutured, and animals were kept in individual cages for 24 h to monitor recovery. 2.5. Reaching boxes Rats were trained on the single pellet reaching task [59]. Reaching boxes were made of clear Plexiglas and had the following dimensions: 45 cm high × 13 cm wide × 35 cm long. A 1 cm-wide vertical slot on the front is located 2–15 cm above the ground. In front of the slot there is a 2 cm-wide shelf mounted 3 cm above the ground. The shelf has two small indentations where food targets can be placed. These indentations are on either edge of the slot, and are 2 cm away from the inside wall
2.9. Quantitative analysis of reaching Rats were tested on 20 trials of the reaching task per testing day. Behavior was analyzed on the following measures: (1) First attempt success. A first attempt success was recorded when the rat grasped the pellet on the first limb advance, transported it toward the mouth, and released it into the mouth for eating. Success scores were calculated as: %success = (number of food pellets obtained on first attempt/20 trials) × 100%. (2) Total success. A reach was defined as ‘successful’ when the rat grasped the pellet from the shelf, transported it toward the mouth, and released it into the mouth for eating. Rats were allowed as many limb advances/grasps at the pellet as required, as long as the pellet remained in the indentation on the shelf. Total success scores were calculated as: %success = (number of food pellets obtained/20 trials) × 100%. (3) Total attempts. The total number of attempts was recorded for each testing session. Attempts were recorded during offline analysis and were defined as any movement of the paw towards the food pellet. 2.10. Movement element analysis of reaching Successful reaches were analyzed during offline, frame-by-frame video analysis (30 frames/s). Movement element scores for the first three successful reaches of each rat were recorded on the following testing sessions: before nicotine treatment (pre-nicotine), during nicotine treatment prior to motor cortex stroke (Day −2), and during nicotine treatment after motor cortex stroke (Day 14). Individual movements were scored based on a framework derived from Eshkol-Wachmann Movement Notation (EWMN) [17,59]. In this movement analysis, the movements of the rat are scored in relation to the food pellet. Each movement was given a score according to level of impairment (0, normal; 0.5 slightly abnormal; 1.0 severely abnormal or absent). The following movements were scored: (1) Orient. The head and snout are directed to the food pellet and the rat sniffs the food pellet to locate it. (2) Lift. The preferred reaching forepaw is lifted off of the floor of the reaching box. (3) Digits close. The digits of the reaching forepaw are semiflexed. The paw is turned 90◦ and oriented towards the midline of the body. (4) Aim. The elbow is brought in towards the midline of the body while the digits remain in position at the midline. (5) Advance. The forepaw is advanced through the slot towards the food pellet. (6) Digits open. The digits of the reaching paw are extended over the food pellet.
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Fig. 1. (A) Left hemisphere forelimb motor cortex lesion in a representative brain. The location of the forelimb motor cortex map is shown on the right hemisphere (CFA, caudal forelimb area; RFA, rostral forelimb area). (B) Cresyl violet coronal sections (measurements in mm relative to bregma) of a representative brain with a right hemisphere forelimb motor cortex lesion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(7) Pronation. The digits of the reaching paw are placed over the pellet in an arpeggio movement [57]. (8) Grasp. The arm remains still while the digits close around the food pellet. (9) Supination 1. The reaching paw is turned 90◦ as the forepaw is withdrawn from the shelf. (10) Supination 2. The reaching paw is turned a further 90◦ as the forepaw is brought to the mouth. (11) Release. The digits open and the food pellet is released into the mouth. (12) Place. The reaching paw is lowered to the floor of the reaching box. (13) Posture. The rat maintains an orientation toward the food pellet, with the limbs in a position such that they straddle the slot, and no lateral stepping movements take place during the reach. 2.11. Histological analysis At the completion of the experiment, rats were given an overdose of sodium pentobarbital and were intracardially perfused with saline (0.9%) followed by paraformaldehyde (4%). The brains were removed and postfixed with a cryoprotectant (30% sucrose and 4% paraformaldehyde). Tissue was sectioned at 40 m and stained with cresyl violet and an acetylcholinesterase stain. Fig. 1A shows the lesion in a representative brain. Cresyl sections were analyzed to determine infarct size. Sections from the following planes, through the area of stroke, from bregma were measured: +3.7, +2.7, +1.7, +0.7, and −0.3 mm (as shown in Fig. 1B). Lesion size was estimated by transposing landmarks in the lesion hemisphere to the intact hemisphere and measuring the enclosed area (this was to avoid the tissue distortion present in the lesion hemisphere, which is a result of the skull being removed during surgery). Measurements were completed using Image J (http://rsb.info.nih.gov/ij/). Tissue stained for acetylcholinesterase was first exposed to tetraisopropylpyrophosphoramide and then to a reaction mixture. Densitometry was completed on sections stained for acetylcholinesterase using NIH image (http://rsb.info.nih.gov/nih-image/download.html). The gray value measure of the corpus callosum was considered baseline. Density was calculated for lesion vs. nonlesion cortex. Four cortical areas (medial, dorsal, lateral, and ventral) were measured per hemisphere, per section to assess acetylcholinesterase levels in the whole cor-
tex. The gray values of the cortical areas were averaged together and subtracted from baseline. The following planes from bregma were measured for acetylcholinesterase density: +3.7, +2.7, +1.7, and +0.7 mm (as shown in Fig. 1B). Acetylcholinesterase measures were directed toward assessing whether the nicotine produced enduring effects on acetylcholine activity in the neocortex. 2.12. Statistical analysis Statistical analyses were performed using SPSS13. Data were treated as interval data according to the definition by Field and Hole [18]. Results for end-point variables (first attempt success, total success and total attempts) and movement elements were analyzed using a mixed design repeated measures analysis of variance (RANOVA), with “Days” or “Elements” as the within-subjects variables and “Groups” as the between-subjects factor. Mauchly’s test and Greenhouse-Geisser corrections were used to assess and correct sphericity of data, respectively. Multiple comparisons between groups were performed by follow-up Bonferronni tests. Dependent t-tests were used to compare pre- and post-stroke reaching performance. Results for learned nonuse (reaching attempts in the acute post-stroke period) within and between sessions were analyzed using Mixed Design RANOVA; “Days” as the within-subjects variable and “Groups” as the between-subjects factor. Spearman Rank Order Correlations (rs ) were computed with the “Trials” as the independent measure, and “Number of reaching attempts” as the dependent measure. Statistical tests for lesion size and acetylcholinesterase density among groups were completed using an analysis of variance (ANOVA). For all measures p < 0.05 was considered significant. 2.13. Procedure and time-line A time-line for the treatments administered in the course of the study is illustrated in Fig. 2. Rats were pre-trained on the single pellet reaching task and were then tested and scored for 7 days (Day −7 to −1), at 20 trials per day. During testing days rats received either nicotine or vehicle (depending on which group they were in) twice per day. All rats received a motor cortex stroke via pial stripping on Day 0. Twenty-four hours after surgery, rats resumed daily testing and drug or vehicle
Fig. 2. Experimental timeline. Rats were administered 0.3 mg/kg nicotine or vehicle twice daily on Days −7 to 14.
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administration for 14 days. Following a 2-week interval, rats were re-tested at POD28 (post-operative day 28), POD35, and POD42 with no drug administration.
3. Results 3.1. Lesions Lesions were located in the sensorimotor cortex in the region of the forelimb area of motor cortex, as it has been defined by previous studies [14,26]. Lesion size and location was similar to that described previously [4]. A dorsal view of a representative lesion is shown in Fig. 1A. Coronal sections through the rostro-caudal extent of the same lesion are shown in Fig. 1B. An ANOVA revealed that there was no significant difference between groups with respect to lesion size (F(3, 31) = 0.57, p > 0.05). Fig. 3 illustrates the average gray value per section found in the lesion and nonlesion hemispheres on measures of the density of acetylcholinesterase. A RANOVA revealed that there was a main effect of hemisphere, with the lesion hemisphere consistently showing a higher average gray value than the nonlesion hemisphere (F(1, 34) = 41.90, p < 0.001). There was no difference in average gray value between groups (F(3, 34) = 0.407, p > 0.05). 3.2. Pre-stroke scores Pre-stroke end-point measures indicated that rats receiving nicotine had depressed total success and first reach success scores relative to vehicle treated rats (Fig. 4). A RANOVA for first attempt success (Fig. 4A) gave a main effect of group (F(1, 38) = 6.95, p < 0.05), but no Day by Group interaction (F(4.90, 186.04) = 1.33, p > 0.05), and no effect of Days (F(4.90, 186.04) = 1.23, p > 0.05). The RANOVA for total success (Fig. 4B) gave a main effect of Group (F(1, 38) = 12.99, p < 0.01), a Day by Group interaction (F(6, 228) = 4.21, p < 0.001), but no effect of Days (F(6, 228) = 0.79, p > 0.05). The RANOVA for number of reaching attempts (Fig. 4C) gave no difference between Groups (F(1, 38) = 2.20, p > 0.05), and no Day by Group interaction (F(6, 228) = 0.30, p > 0.05), but did show an effect of Days (F(6, 228) = 5.00, p < 0.001). 3.3. Post-stroke scores Performance on first attempt success, total success, and reach attempts was significantly depressed in the acute post-stroke period but recovered toward preoperative levels. Nicotine treat-
Fig. 4. Pre-stroke scores (mean ± S.E.M.) on the single pellet reaching task for (A) first attempt success; (B) total success; and (C) total number of attempts. Success percent is the mean score for that day based on twenty trials. Asterisks denote a significant difference (p < 0.05) between groups.
Fig. 3. Measures of densitometry based on gray value scores (mean ± S.E.M.). Four sections per brain were stained and measured for acetylcholinesterase. Note: Absence of differences between nicotine (N) and vehicle (V) groups.
ment did not affect end-point measures of reaching performance (Fig. 5). RANOVAs for first attempt success showed no effect of Group (F(3, 35) = 0.89, p > 0.05), no Day by Group interaction (F(39, 455) = 0.91, p > 0.05), and a significant effect of Days (F(13, 455) = 23.01, p < 0.001). The RANOVA for total success gave no effect of Group (F(3, 35) = 0.18, p > 0.05), no Day by Group interaction (F(39, 455) = 0.48, p > 0.05) and an effect of Days (F(13, 455) = 45.66, p < 0.001). The RANOVA for reaching attempts gave no effect of Group (F(3, 35) = 2.27, p > 0.05), no Day by Group interaction (F(39, 455) = 1.40, p > 0.05), but showed an effect for Days (F(13, 455) = 49.58, p < 0.001).
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3.5. Learned nonuse Rats in all groups made reach attempts when first returned to the test box following stroke. However, reach attempts in all groups was similar in that they declined with successive approaches to the food over the first three days following stroke before beginning to recover on POD4 through POD6 (Fig. 6). The RANOVA on reach attempts during the six days immediately after stroke gave no effect of Group (F(3, 35) = 0.04, p > 0.05), and no Day by Group interaction (F(15, 175) = 0.84, p > 0.05), but did yield a significant effect of Days (F(5, 175) = 29.43, p < 0.001). The decline in reach attempts across the testing session for the first three days post-stroke was significant for all groups: N/N rs = −0.67, N/V rs = −0.60, V/N rs = −0.89, V/V rs = −0.47 (p < 0.05). 3.6. Movement element analysis of reaching
Fig. 5. Post-stroke scores (mean ± S.E.M.) on the single pellet reaching task for (A) first attempt success; (B) total success; and (C) total number of attempts. Days 28, 35, and 42 were completed without drug administration. Note: A reduction in success and attempts after stroke, and an absence of a nicotine effect (N, nicotine; V, vehicle).
The movement element analysis indicated that stroke impaired limb use, but nicotine treatment before and/or after stroke did not improve stroke-associated movement impairments. Rather, nicotine impaired movement elements before stroke. Following stroke, rats in all groups were similarly impaired on a number of elements, especially aim, pronation and supination. Separate analyses were preformed for three scoring days (baseline, pre-nicotine; Day −2, nicotine pre-stroke; POD14, post-stroke). There was no significant group difference on the pre-nicotine test (F(3, 35) = 0.52, p > 0.05). There was a significant Group effect on the nicotine pre-stroke test (F(1, 37) = 8.75, p < 0.01), no significant Element effect, (F(5.40, 199.67) = 0.98, p > 0.05), but there was a Group by Element interaction (F(5.40, 199.68) = 14.30, p < 0.001), as the nicotine treated group were impaired in pronation and supination. Following stroke (POD14) there was a significant effect of Element (F(4.86, 165.22) = 16.57, p < 0.001), but no Group effect (F(3, 34) = 2.26, p > 0.05) and no Group by Element interaction (F(14.58, 165.22) = 1.23, p > 0.05). Following stroke all groups were impaired on aim, pronation and supination. In order to determine whether there was a treatment effect on the reaching elements, pre-nicotine performance was compared to post-stroke performance. Fig. 7 indicates that the pattern of impairment on POD14 was similar in all of the lesion groups. There was no effect of Groups (F(3, 34) = 2.64, p > 0.05), but a significant effect of Days (F(1, 34) = 33.10, p < 0.001), and Elements (F(5.90, 200.51) = 23.97, p < 0.001). The Element by Group (F(17.69, 200.51) = 1.26, p > 0.05), Day by Group (F(3, 34) = 1.28, p > 0.05), and Element by Day by Group interactions (F(15.80, 179.12) = 0.99, p > 0.05) were not significant. There was a significant Element by Day interaction (F(5.27, 179.12) = 9.25, p < 0.001). Paired t-tests were used to compare the elements in each group pre-stroke (Day −2) and post-stroke (POD14), as illustrated by Fig. 7. 4. Discussion
3.4. Measures during the chronic post-stroke period Chronic post-stroke days (POD28, POD35, and POD42), on which the rats received neither nicotine nor vehicle treatment, did not give differences on any end-point measures of performance (Fig. 5). ANOVAs revealed no difference for groups on first attempt success (F(3, 35) = 0.83, p > 0.05), success (F(3, 35) = 0.19, p > 0.05), or number of attempts (F(3, 35) = 1.53, p > 0.05). There was a main effect of Days for first attempt success (F(2, 70) = 6.77, p < 0.01), and success (F(1.92, 67.03) = 7.30, p < 0.01), but not for attempts (F(1.82, 63.54) = 0.96, p > 0.05). There were no Day by Group interactions for first attempt success (F(6, 70) = 0.47, p > 0.05), total success (F(5.75, 67.03) = 1.53, p > 0.05), or attempts (F(5.5, 63.54) = 0.77, p > 0.05).
The objective of the present study was to examine whether nicotine could improve post-stroke motor performance at different phases of post-stroke recovery. Nicotine administered per oral prior to stroke, post-stoke, or both prior to and post-stroke, was not beneficial on any measure of post-stroke reaching behavior. First, learned nonuse, in which rats decrease reaching attempts in the acute post-stroke period, was not changed. Second, the reacquisition of reaching in a constraint-induced paradigm, in which rats were required to use their affected paw, was not improved. Third, retention of the reacquired skilled reaching act after a rehabilitation and treatment holiday was not improved. The present results suggest that neither the activating effects of nicotine nor its potential effects on brain plasticity promote the
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Fig. 6. Regression of the number of reaching attempts in the single pellet reaching task across each session during the acute post-stroke period. Note: Nicotine failed to prevent the development of learned nonuse and failed to enhance recovery from learned nonuse. (N, nicotine; V, vehicle; POD, post-operative day).
recovery of the learned or innate motoric aspects of skilled reaching. Although the results of the present study are negative with respect to a putative role for nicotine in promoting recovery of function after stroke, the study is nonetheless instructive. Preclinical studies of functional recovery after stroke have been difficult to translate into clinical treatments, mainly because preclinical studies are seldom replicated [10,21]. In this respect, the present study has a number of strengths, including a stroke model that has proved useful for the study of functional recovery in previous research [4], the use of a nicotine regime that falls within the range of that used in previous studies [23,24], and an improved route of drug administration. Furthermore, the behavioral analysis that was used is suitable for both the acute and chronic phases of recovery from stroke. Nevertheless, the study contains a number of potential weaknesses, including the use of a single drug treatment dose, a single stroke size, and an assessment of a single behavior. Strengths and caveats of the present approach to investigating drug-induced potentiation of recovery of function from stroke are discussed with respect to the idea that nicotine is useful in promoting recovery of function only in limited circumstances. The search for drug treatments for enhancing recovery of function after brain injury is made difficult by many potential treatment variables associated with administration. For the present study a procedure was developed in which nicotine could be administered orally rather than via subcutaneous injection. The drug dose selected for use in the present study was based on previous work [23,24], and was confirmed by behavioral assessment. Nicotine in solution was found to be rejected either alone or in a number of foods, but when administered in conjunction with a dab of peanut butter it was quickly and reliably consumed at a prescribed treatment time. The advantage of per oral administration is that it is less stressful than systemic administration, especially when repeated injections are given, and is less likely to cause infection or cutaneous trauma [12]. Although absorption is slightly more rapid with subcutaneous administration vs. per oral administration [5], subcutaneous and per oral administration of nicotine have similar dose–effect pharmacokinetics [9]. It should be noted that the use of peanut butter in nicotine administration, the timing of drug administration, or the food-deprivation schedule may have affected
the absorption of nicotine, however, a pilot study in which rats on a food-deprivation schedule were administered nicotine per oral in peanut butter vs. subcutaneously indicated that the activation effect of the drug, as measured by an increase in locomotor activity, was similar for both routes across an 8-day testing period. Finally, nicotine as administered here had a similar effect of somewhat depressing the pre-stroke reaching success, as has been reported following subcutaneous injections [23,24]. Thus differences in the present findings vs. the findings in previous works are not easily ascribed to drug absorption and action. Although the drug dose used here was selected with the intention of replicating previous work, it would have been ideal to have used a range nicotine doses. Nevertheless, there is a trade-off between the detail in which a behavior is analyzed and the number or size of experimental groups. The use of only a single dose of the drug for the present analysis was made necessary by the exhaustive behavioral assessment. Additionally, were future investigations to evaluate different administrative procedures, such as administration through cutaneous patches or via a mini pump, a single dose would likely be administered. Possibly, an investigation of a more appropriate dose of nicotine could use the behavioral screening methods previously used (for a review of dose selection in the rat see [34]). However, the objective of the present study was not to find an ideal dose, but to complete an in-depth analysis of a purported beneficial dose. Furthermore, although it is possible that a nicotine treatment with a drug dose range smaller or larger than that used here could be effective, there were no statistical trends in the present results to encourage such speculation. The way in which a stroke occurs could potentially affect the outcome of a treatment, but three lines of evidence suggest that this consideration is unlikely to account for the present findings. First, it is unlikely that the present results are specific to the form of motor cortex stroke that was used. The selected stroke method – devascularization via pial stripping – was similar to that used in previous studies [23,28], which produces a behavioral syndrome comparable to an internal capsule human stroke. Second, an explicit comparison of different methods of inducing stroke to motor cortex finds that behavioral deficits are very similar across all methods of rodent stroke [22]. Third, it could be argued that the lesion was too large, leaving little residual tissue to mediate recovery; it is reported
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Fig. 7. Movement element measures of reaching on POD14. Asterisks denote significance between scores on testing Day −2 (pre-stroke) and POD14. Rats without motor cortex stroke would achieve scores close to 0 on all movements. Note: Similar impairment in all groups (N, nicotine; V, vehicle; POD, post-operative day).
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that greater recovery can be expected from lesions that increasingly spare greater portions of functional areas [25]. Nevertheless, the present study did find substantial recovery/compensation with rehabilitation along with some enduring deficits, suggesting that there was considerable room for improvement with pharmacological assisted treatment. Although a smaller stroke size would likely produce smaller deficits it would allow less room for demonstrating treatment benefits. The major strength of the present study is the scope and the detail of the behavioral analysis that encompassed three phases of recovery; an acute post-stroke period (POD1–6), a sub-acute poststroke period (POD7–14), and a chronic post-stroke period (POD28, 35, and 42). Previous work has shown that in the immediate days following stroke rats display a trial-by-trial decrease in the number of reach attempts, a behavior termed learned nonuse [4,16], and this finding was confirmed in all of the treatment groups in the present study. It was expected that rats given nicotine would demonstrate a less severe trend of learned nonuse, because nicotine is motorically activating [8] and has an antidepressant-like effect in learned helplessness paradigms [46]. Despite any such effects that nicotine has on general motor behavior, the rats exposed to nicotine displayed the same nonuse trend as vehicle treated rats. Furthermore, all groups showed a similar recovery pattern from learned nonuse. Thus, nicotine did not prevent the occurrence of learned nonuse, and did not expedite recovery from learned nonuse. Chronic post-stroke improvement in skilled reaching is dependent upon using a constraint-induced recovery method [32,33]. Constraint-induced therapy here was accomplished through braceletting the non-affected limb and placing the food pellet contralateral to the affected limb [58,59]. Based on previous studies citing beneficial effects of nicotine on motor skills, it was expected that nicotine might improve the rate of recovery/compensation and improve eventual levels of success. Nicotine failed to improve the progress of recovery as measured by end-point measures of success on first reach attempts, success on total attempts, or number of reaching attempts. To evaluate the effects of nicotine treatment on motoric retention of skilled reaching in the chronic post-stroke period, the rats were reevaluated after a nicotine and rehabilitation holiday, after which performance typically declines [56]. Again, performance was similar in all groups, despite differences in nicotine treatment. Thus, as in the acute post-stroke period, in the chronic post-stroke period there were no beneficial effects of nicotine on any end-point measure of performance. In addition to end-point measures of success, performance measures evaluated in the present study included movement element analysis of reaching. It has been reported that nicotine improves the motor act of reaching after stroke on the tray reach task, although surprisingly, this improvement was not associated with improved success on single pellet reaching [23,24]. To evaluate whether nicotine could encourage the use of more normal movements following stroke, movement element analysis was performed at a number of different time points in the study. It was found that there were no differences in reaching elements between rats administered nicotine vs. vehicle. After stroke all groups were equally impaired in performance of rotatory movements of the limb, including aiming, pronation, and supination. The present results indicate that nicotine does not improve movement scores, which is inconsistent with previous studies [23], however, studies using multiple measures risk Type I errors, which speaks to the necessity for replication of positive findings such as that conducted here. In conclusion, despite the activating effects of nicotine and its effects on brain plasticity [35], nicotine did not promote recovery of skilled reaching after stroke. The present study does not represent an exhaustive measure of the effects of nicotine on behavior or the effects of nicotine on recovery after motor cortex stroke. It does, however, show that single pellet reaching resists nicotine
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pharmacotherapy, and in doing so is consistent with a number of previous lines of investigation concerning the resistance of single pellet reaching to pharmacotherapy. Amphetamine treatment has been widely reported to improve post-stroke motor behavior, but it does not improve performance on skilled reaching [4]. Brain transplants of fetal dopamine tissue given to animals depleted of dopamine can improve a number of aspects of motor behavior but skilled reaching is not improved [15]. In nonhuman animals and humans with reduced levels of dopamine, levodopa treatments improve a number of aspects of motor performance after the loss of dopamine, but such treatment does not improve performance on skilled reaching [36,37]. A similar absence of improvement has been reported using fluoxetine following ischemia [60], despite the fact that this drug, like nicotine, has positive effects on brain activity and growth factors. Current research using antibodies for neuritegrowth inhibitors during post-stroke recovery are promising [51], and indicate that post-stroke skilled reaching may not be entirely resistant to treatment. For facilitation of sparing or recovery of function to occur on skilled reaching it may be necessary to spare some portion of the forelimb area [1,25,30], use larger or smaller drug doses, or prolong treatment. Nevertheless, it should also be considered that whereas nicotine may be effective following brain injury on some aspects of behavior [6,7,23,24,29,51], skilled reaching is not among them. Acknowledgements The authors would like to thank Jessica Cummins for help with histological analysis, and Dr. Seong-Keun Moon for help with surgery. This research was supported by grants from the Alberta Heritage Foundation for Medical Research (D.H.L.) and the Natural Sciences and Engineering Research Council of Canada (I.Q.W.). References [1] Adkins-Muir DL, Jones TA. Cortical electrical stimulation combined with rehabilitative training: enhanced functional recovery and dendritic plasticity following focal cortical ischemia in rats. Neurol Res 2003;25(8):780–8. [2] Alaverdashvili M, Foroud A, Lim DH, Whishaw IQ. “Learned baduse” limits recovery of skilled reaching for food after forelimb motor cortex stroke in rats: a new analysis of the effect of gestures on success. Behav Brain Res 2008;188(2):281–90. [3] Alaverdashvili M, Leblond H, Rossignol S, Whishaw IQ. Cineradiographic (video X-ray) analysis of skilled reaching in a single pellet reaching task provides insight into relative contribution of body, head, oral, and forelimb movement in rats. Behav Brain Res 2008;192(2):232–47. [4] Alaverdashvili M, Lim DH, Whishaw IQ. No improvement by amphetamine on learned non-use, attempts, success or movement in skilled reaching by the rat after motor cortex stroke. Eur J Neurosci 2007;25(11):3442–52. [5] Barnes CD, Eltherington LG. Drug dosage in laboratory animals: a handbook. 2nd rev. enlarged ed. California: University of California Press; 1973. p. 171. [6] Brown RW, Gonzalez CLR, Kolb B. Nicotine improves Morris water task performance in rats given medial frontal cortex lesions. Pharmacol Biochem Behav 2000;67(3):483–8. [7] Brown RW, Gonzalez CLR, Whishaw IQ, Kolb B. Nicotine improvement of Morris water task performance after fimbria-fornix lesion is blocked by mecamlyamine. Behav Brain Res 2001;119(2):185–92. [8] Clarke PB, Kumar R. The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br J Pharmacol 1983;78(2):329–37. [9] Craft RM, Howard JL. Cue properties of oral and transdermal nicotine in the rat. Psychopharmacology (Berl) 1988;96(3):281–4. [10] Cramer SC. Repairing the human brain after stroke. II. Restorative therapies. Ann Neurol 2008;63(5):549–60. [11] Dalack GW, Healy DJ, Meador-Woodruff JH. Nicotine dependence in schizophrenia: clinical phenomena and laboratory findings. Am J Psychiatry 1998;155(11): 1490–501. [12] Dilsaver SC, Majchrzak MJ. Effects of placebo (saline) injections on core temperature in the rat. Prog Neuropsychopharmacol Biol Psychiatry 1990;14(3): 417–22. [13] Djuric VJ, Dunn E, Overstreet DH, Dragomir A, Steiner M. Antidepressant effect of ingested nicotine in female rats of Flinders resistant and sensitive lines. Physiol Behav 1999;67(4):533–7. [14] Donoghue JP, Wise SP. The motor cortex of the rat: cytoarchitecture and microstimulation mapping. J Comp Neurol 1992;212(1):76–88.
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Cereb cortex 2006;16:1106– 15. [36] Melvin KG, Doan J, Pellis SM, Brown L, Whishaw IQ, Suchowersky O. Pallidal deep brain stimulation and L-dopa do not improve qualitative aspects of skilled reaching in Parkinson’s disease. Behav Brain Res 2005;160(1):188–94. [37] Metz GA, Farr T, Ballermann M, Whishaw IQ. Chronic levodopa therapy does not improve skilled reach accuracy or reach range on a pasta matrix reaching task in 6-OHDA dopamine-depleted (hemi-Parkinson analogue) rats. Eur J Neurosci 2001;14(1):27–37. [38] Mirza NR, Stolerman IP. Nicotine enhances sustained attention in the rat under specific task conditions. Psychopharmacology (Berl) 1998;148(3–4):266–74. [39] Nordberg A. Nicotinic receptor abnormalities of Alzheimer’s Disease: therapeutic implications. Biol Psychiatry 2001;49(3):200–10. [40] Nudo RJ. Postinfarct cortical plasticity and behavioral recovery. Stroke 2007;38(2 Suppl.):840–5. [41] Paterson D, Nordberg A. Neuronal nicotinic receptors in the human brain. 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Research report
Nicotine does not improve recovery from learned nonuse nor enhance constraint-induced therapy after motor cortex stroke in the rat Diana H. Lim ∗ , Mariam Alaverdashvili, Ian Q. Whishaw Department of Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4
a r t i c l e
i n f o
Article history: Received 13 August 2008 Received in revised form 10 November 2008 Accepted 18 November 2008 Available online 30 November 2008 Keywords: Nicotine and recovery Learned nonuse Motor cortex stroke Constraint-induced therapy Skilled reaching Rehabilitation after stroke
a b s t r a c t Nicotine, a cholinergic agonist, rapidly crosses the blood–brain barrier, promotes neuronal plasticity and has been suggested to enhance behavior in a variety of neurological conditions. Nicotine has also been suggested to benefit functional recovery in rodent models of stroke. At present there has been no systematic investigation of the potential benefits of nicotine therapy in both the acute and chronic post-stroke period. This was the objective of the present study and to that end, the effects of nicotine administration prior to and following motor cortex stroke were examined in a skilled reaching task. The task provides a thorough assessment of learned nonuse and constraint-induced recovery of behavior as determined by both end-point and movement element analysis. Nicotine (0.3 mg/kg PO) was administered twice daily during reach training and following motor cortex stroke. Rats were divided into four groups based on their pre/post-stroke treatment: nicotine/nicotine, nicotine/vehicle, vehicle/nicotine, vehicle/vehicle. After stroke, nicotine did not counteract learned nonuse, facilitate constraint-induced therapy, or improve long-term recovery as measured by end-point analysis and movement element analysis. The results are discussed in relation to the problem of identifying pharmacotherapeutic agents that augment rehabilitation following stroke. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Nicotine is a cholinergic agonist that indirectly activates other neural systems including the dopaminergic and adrenergic systems [44,61]. Nicotine has been suggested to play a protective role with respect to some neurodegenerative diseases including Alzheimer’s disease [39,42], schizophrenia [11,41], and Parkinson’s disease [43,53]. In human studies, nicotine has been shown to enhance vigilance [31] and performance on skilled motor tasks [50]. In animal studies, nicotine has been shown to enhance attention [38,45] and memory [27], increase motor activity [13], and overcome depression [49] and learned helplessness e.g. in the forced swim test [46]. It is also suggested that nicotine is a possible treatment for promoting recovery after brain damage [6,7,24,29,52,53], including brain damage induced in a rodent model of motor cortex stroke [23]. At present there has been no exhaustive examination of the effects of nicotine after stroke; the specific improvements, the time points at which it is effective or the relation of putative beneficial effects on post-stroke rehabilitation have yet to be addressed. The examination of these factors was the objective of the present study. Rats received nicotine in association with pretraining and
∗ Corresponding author. Tel.: +1 403 329 2637; fax: +1 403 329 2775. E-mail address: [email protected] (D.H. Lim). 0166-4328/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.11.038
post-stroke rehabilitation. The experiment used a skilled-reachingfor-food task that assesses forelimb use in reaching for a small piece of food [54,55,59], a motoric act that is frequently impaired by stroke in humans [19]. The task leads itself to the examination of both the learned and innate motoric effects of motor cortex stroke and has the strength of providing multiple measures of performance. Stroke was induced via pial stripping. This model of stroke is advantageous because behavioral assessment can be made in the acute post-stroke period, during rehabilitation, and after recovery [56]. In the acute period following motor cortex stroke, rats display learned nonuse of their affected limb in that they show a decline in reaching attempts [16]. When limb use is forced by constraint-induced therapy, that is, by limiting use of the nonaffected limb, there is a gradual recovery/compensation associated with rehabilitation [33,40,58]. In the chronic post-stroke period there may be regression of performance in the absence of rehabilitation. These learning-related aspects of performance are assessed by three end-point measures, first attempt success, total success, and total attempts, while the effects of stroke on movement are assessed by a detailed analysis of the thirteen movement elements of the reaching act [3]. The model of stroke used in the present study allows a comprehensive assessment of the phases of poststroke recovery and is a good model of human stroke [56]. As such it provides a strong assessment tool for evaluating drug-related facilitation of recovery in association with rehabilitation after stroke.
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For the present study, rats were trained on the single pellet reaching task and received a daily regime of nicotine prior to and/or following motor cortex stroke targeting the forelimb area. Skilled reaching performance was assessed in the acute and chronic poststroke period using end-point measures of the reach action as well as a rating of the movement elements required to perform a reach [59]. 2. Materials and methods 2.1. Subjects Thirty-nine female Long-Evans hooded rats (approximately 100 days old and weighing 250–300 g at the beginning of the experiment) from the University of Lethbridge animal colony were used. Rats were housed in Plexiglas cages in groups of two or three. The colony room was maintained on a 12:12 h light:dark cycle and was controlled for temperature and humidity. The experiment was conducted with approval from the University of Lethbridge animal care committee and in compliance with the guidelines set by the Canadian Council on Animal Care.
of the box. These indentations ensure that the food target is placed such that the rat cannot lap up the food with its tongue and must use the paw contralateral to the indentation to grasp the target; this ensures the use of a single paw for reaching. 2.6. Bracelets for limb constraint To prevent rats from reaching with both paws, a bracelet made of Elastoplast adhesive tape (Smith & Nephew, Lachine, Quebec, Canada) was wrapped around the non-preferred wrist. The bracelet enlarges the paw so that it cannot be inserted through the slot of the reaching box, thus inducing constraint, but does not change the rat’s performance during the testing session [58]. Bracelets were applied pre- and post-stoke only if the rat attempted to reach with the non-preferred (ipsilesional) paw for two or more trials. 2.7. Video recording Reaching performance was video recorded during each reaching session. A Sony 3CCD camcorder (1/1000 s shutter speed) and a cold light source were used. Subsequent frame-by-frame analysis was completed using a Sony videocassette recorder (DSR-11).
2.2. Food restriction
2.8. Reach training
Three weeks prior to behavioral testing, rats were gradually food-deprived to 95–98% of their body weight. This was achieved by once-a-day feeding of Purina Rat Chow (20 g per rat per day). Prior to training, rats also received ∼900 mg/day of dustless precision pellets (Bioserve Inc.) which would serve as the reaching targets in the single pellet reaching task. One week prior to behavioral testing rats also received ∼1 g of Kraft Smooth Peanut Butter (Kraft Canada) in the morning before training, and in the evening, prior to daily feeding. The peanut butter would later serve as the vehicle for drug administration (nicotine solution was given in the peanut butter). The rats were maintained at 95–98% of their body weight until the completion of behavioral testing.
The first week of reach training consisted of daily 10 min sessions for each rat. The objective was to introduce the rat to the reaching cage with pellets on the shelf and to allow the rat to retrieve the pellet by paw or tongue. Once a rat was successfully retrieving pellets, pellets on the shelf were moved further away in order to encourage the use of a paw. After a rat demonstrated a preference for one paw by making most reaching attempts with it, individual pellets were placed into the indentation contralateral to that paw. Rats continued to receive daily training sessions, consisting of 20 discrete trials with inter-trial intervals during which they were shaped to leave the slot, walk to the rear wall of the cage, turn and approach the slot again for the next pellet. In addition, by withholding food on semi-randomly selected trials, rats were taught to sniff the shelf for a pellet and to reach only if a pellet was present. Thus, each rat eventually learned to orient to the food pellet, transport its limb through the slot, grasp the food pellet, and retract its paw through the slot to release the food into its mouth [20]. Training took place over a period of three weeks.
2.3. Drug administration and dose Subjects were assigned to four different groups based on drug administration before and after surgery (pre-/post-stroke). Groups were: nicotine/nicotine (N/N; n = 9), nicotine/vehicle (N/V; n = 10), vehicle/nicotine (V/N; n = 10), and vehicle/vehicle (V/V; n = 10). Nicotine hydrogen tartrate salt (Sigma–Aldrich) was dissolved in distilled water to a concentration of 1 mg/mL [48]. Rats were administered 0.3 mg/kg of nicotine solution twice per day, in accordance with previous work [23,24]. Administration occurred in the morning, half an hour before training or testing (9:00 AM) and in the evening, after training or testing but prior to daily feeding (4:00 PM). Administration of drug or vehicle took place for 21 days, during which rats were tested daily. Oral administration was used throughout, with peanut butter (Kraft Smooth Peanut Butter, Kraft Canada) being used as the vehicle. A pilot study justified the per oral route of administration by examining the activation effects of nicotine between per oral (PO) and subcutaneous (SC) administration using a VersaMax Animal Activity Monitoring System (AccuScan Instruments, Inc. [email protected]). The locomotor activity following nicotine administration was used as an index of nicotinic activation. Rats received a dose of 0.3 mg/kg via SC or PO administration. Both routes of administration resulted in enhanced motor activity but there was no difference in motor activity between groups over an 8-day testing period (F(1, 6) = 0.11, p > 0.05). Onset of drug-induced locomotion following SC administration was slightly more rapid, but within 15 min of administration, activity in the two groups was equivalent. 2.4. Stroke All rats received stroke to the forelimb area of motor cortex, as defined by previous anatomical and electrophysiological studies [2,14,26]. Rats were anesthetized with sodium pentobarbital (45 mg/kg, i.p.; Sigma–Aldrich) and were given an analgesic (0.3 mg/kg Buprenorphine, i.p.; Sigma–Aldrich) and an injection of atropine (0.1 mg/kg, i.p.; Sigma–Aldrich) to facilitate respiration throughout the surgery. Rats were placed in a stereotaxic device and then a portion of the skull was removed in the hemisphere contralateral to the preferred reaching paw (1 mm posterior and 4 mm anterior to bregma, and 1–4 mm lateral to midline). Exposed tissue was devascularized by gently wiping away the blood vessels with a saline-soaked cotton swab [28,47]. The incision was sutured, and animals were kept in individual cages for 24 h to monitor recovery. 2.5. Reaching boxes Rats were trained on the single pellet reaching task [59]. Reaching boxes were made of clear Plexiglas and had the following dimensions: 45 cm high × 13 cm wide × 35 cm long. A 1 cm-wide vertical slot on the front is located 2–15 cm above the ground. In front of the slot there is a 2 cm-wide shelf mounted 3 cm above the ground. The shelf has two small indentations where food targets can be placed. These indentations are on either edge of the slot, and are 2 cm away from the inside wall
2.9. Quantitative analysis of reaching Rats were tested on 20 trials of the reaching task per testing day. Behavior was analyzed on the following measures: (1) First attempt success. A first attempt success was recorded when the rat grasped the pellet on the first limb advance, transported it toward the mouth, and released it into the mouth for eating. Success scores were calculated as: %success = (number of food pellets obtained on first attempt/20 trials) × 100%. (2) Total success. A reach was defined as ‘successful’ when the rat grasped the pellet from the shelf, transported it toward the mouth, and released it into the mouth for eating. Rats were allowed as many limb advances/grasps at the pellet as required, as long as the pellet remained in the indentation on the shelf. Total success scores were calculated as: %success = (number of food pellets obtained/20 trials) × 100%. (3) Total attempts. The total number of attempts was recorded for each testing session. Attempts were recorded during offline analysis and were defined as any movement of the paw towards the food pellet. 2.10. Movement element analysis of reaching Successful reaches were analyzed during offline, frame-by-frame video analysis (30 frames/s). Movement element scores for the first three successful reaches of each rat were recorded on the following testing sessions: before nicotine treatment (pre-nicotine), during nicotine treatment prior to motor cortex stroke (Day −2), and during nicotine treatment after motor cortex stroke (Day 14). Individual movements were scored based on a framework derived from Eshkol-Wachmann Movement Notation (EWMN) [17,59]. In this movement analysis, the movements of the rat are scored in relation to the food pellet. Each movement was given a score according to level of impairment (0, normal; 0.5 slightly abnormal; 1.0 severely abnormal or absent). The following movements were scored: (1) Orient. The head and snout are directed to the food pellet and the rat sniffs the food pellet to locate it. (2) Lift. The preferred reaching forepaw is lifted off of the floor of the reaching box. (3) Digits close. The digits of the reaching forepaw are semiflexed. The paw is turned 90◦ and oriented towards the midline of the body. (4) Aim. The elbow is brought in towards the midline of the body while the digits remain in position at the midline. (5) Advance. The forepaw is advanced through the slot towards the food pellet. (6) Digits open. The digits of the reaching paw are extended over the food pellet.
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Fig. 1. (A) Left hemisphere forelimb motor cortex lesion in a representative brain. The location of the forelimb motor cortex map is shown on the right hemisphere (CFA, caudal forelimb area; RFA, rostral forelimb area). (B) Cresyl violet coronal sections (measurements in mm relative to bregma) of a representative brain with a right hemisphere forelimb motor cortex lesion. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
(7) Pronation. The digits of the reaching paw are placed over the pellet in an arpeggio movement [57]. (8) Grasp. The arm remains still while the digits close around the food pellet. (9) Supination 1. The reaching paw is turned 90◦ as the forepaw is withdrawn from the shelf. (10) Supination 2. The reaching paw is turned a further 90◦ as the forepaw is brought to the mouth. (11) Release. The digits open and the food pellet is released into the mouth. (12) Place. The reaching paw is lowered to the floor of the reaching box. (13) Posture. The rat maintains an orientation toward the food pellet, with the limbs in a position such that they straddle the slot, and no lateral stepping movements take place during the reach. 2.11. Histological analysis At the completion of the experiment, rats were given an overdose of sodium pentobarbital and were intracardially perfused with saline (0.9%) followed by paraformaldehyde (4%). The brains were removed and postfixed with a cryoprotectant (30% sucrose and 4% paraformaldehyde). Tissue was sectioned at 40 m and stained with cresyl violet and an acetylcholinesterase stain. Fig. 1A shows the lesion in a representative brain. Cresyl sections were analyzed to determine infarct size. Sections from the following planes, through the area of stroke, from bregma were measured: +3.7, +2.7, +1.7, +0.7, and −0.3 mm (as shown in Fig. 1B). Lesion size was estimated by transposing landmarks in the lesion hemisphere to the intact hemisphere and measuring the enclosed area (this was to avoid the tissue distortion present in the lesion hemisphere, which is a result of the skull being removed during surgery). Measurements were completed using Image J (http://rsb.info.nih.gov/ij/). Tissue stained for acetylcholinesterase was first exposed to tetraisopropylpyrophosphoramide and then to a reaction mixture. Densitometry was completed on sections stained for acetylcholinesterase using NIH image (http://rsb.info.nih.gov/nih-image/download.html). The gray value measure of the corpus callosum was considered baseline. Density was calculated for lesion vs. nonlesion cortex. Four cortical areas (medial, dorsal, lateral, and ventral) were measured per hemisphere, per section to assess acetylcholinesterase levels in the whole cor-
tex. The gray values of the cortical areas were averaged together and subtracted from baseline. The following planes from bregma were measured for acetylcholinesterase density: +3.7, +2.7, +1.7, and +0.7 mm (as shown in Fig. 1B). Acetylcholinesterase measures were directed toward assessing whether the nicotine produced enduring effects on acetylcholine activity in the neocortex. 2.12. Statistical analysis Statistical analyses were performed using SPSS13. Data were treated as interval data according to the definition by Field and Hole [18]. Results for end-point variables (first attempt success, total success and total attempts) and movement elements were analyzed using a mixed design repeated measures analysis of variance (RANOVA), with “Days” or “Elements” as the within-subjects variables and “Groups” as the between-subjects factor. Mauchly’s test and Greenhouse-Geisser corrections were used to assess and correct sphericity of data, respectively. Multiple comparisons between groups were performed by follow-up Bonferronni tests. Dependent t-tests were used to compare pre- and post-stroke reaching performance. Results for learned nonuse (reaching attempts in the acute post-stroke period) within and between sessions were analyzed using Mixed Design RANOVA; “Days” as the within-subjects variable and “Groups” as the between-subjects factor. Spearman Rank Order Correlations (rs ) were computed with the “Trials” as the independent measure, and “Number of reaching attempts” as the dependent measure. Statistical tests for lesion size and acetylcholinesterase density among groups were completed using an analysis of variance (ANOVA). For all measures p < 0.05 was considered significant. 2.13. Procedure and time-line A time-line for the treatments administered in the course of the study is illustrated in Fig. 2. Rats were pre-trained on the single pellet reaching task and were then tested and scored for 7 days (Day −7 to −1), at 20 trials per day. During testing days rats received either nicotine or vehicle (depending on which group they were in) twice per day. All rats received a motor cortex stroke via pial stripping on Day 0. Twenty-four hours after surgery, rats resumed daily testing and drug or vehicle
Fig. 2. Experimental timeline. Rats were administered 0.3 mg/kg nicotine or vehicle twice daily on Days −7 to 14.
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administration for 14 days. Following a 2-week interval, rats were re-tested at POD28 (post-operative day 28), POD35, and POD42 with no drug administration.
3. Results 3.1. Lesions Lesions were located in the sensorimotor cortex in the region of the forelimb area of motor cortex, as it has been defined by previous studies [14,26]. Lesion size and location was similar to that described previously [4]. A dorsal view of a representative lesion is shown in Fig. 1A. Coronal sections through the rostro-caudal extent of the same lesion are shown in Fig. 1B. An ANOVA revealed that there was no significant difference between groups with respect to lesion size (F(3, 31) = 0.57, p > 0.05). Fig. 3 illustrates the average gray value per section found in the lesion and nonlesion hemispheres on measures of the density of acetylcholinesterase. A RANOVA revealed that there was a main effect of hemisphere, with the lesion hemisphere consistently showing a higher average gray value than the nonlesion hemisphere (F(1, 34) = 41.90, p < 0.001). There was no difference in average gray value between groups (F(3, 34) = 0.407, p > 0.05). 3.2. Pre-stroke scores Pre-stroke end-point measures indicated that rats receiving nicotine had depressed total success and first reach success scores relative to vehicle treated rats (Fig. 4). A RANOVA for first attempt success (Fig. 4A) gave a main effect of group (F(1, 38) = 6.95, p < 0.05), but no Day by Group interaction (F(4.90, 186.04) = 1.33, p > 0.05), and no effect of Days (F(4.90, 186.04) = 1.23, p > 0.05). The RANOVA for total success (Fig. 4B) gave a main effect of Group (F(1, 38) = 12.99, p < 0.01), a Day by Group interaction (F(6, 228) = 4.21, p < 0.001), but no effect of Days (F(6, 228) = 0.79, p > 0.05). The RANOVA for number of reaching attempts (Fig. 4C) gave no difference between Groups (F(1, 38) = 2.20, p > 0.05), and no Day by Group interaction (F(6, 228) = 0.30, p > 0.05), but did show an effect of Days (F(6, 228) = 5.00, p < 0.001). 3.3. Post-stroke scores Performance on first attempt success, total success, and reach attempts was significantly depressed in the acute post-stroke period but recovered toward preoperative levels. Nicotine treat-
Fig. 4. Pre-stroke scores (mean ± S.E.M.) on the single pellet reaching task for (A) first attempt success; (B) total success; and (C) total number of attempts. Success percent is the mean score for that day based on twenty trials. Asterisks denote a significant difference (p < 0.05) between groups.
Fig. 3. Measures of densitometry based on gray value scores (mean ± S.E.M.). Four sections per brain were stained and measured for acetylcholinesterase. Note: Absence of differences between nicotine (N) and vehicle (V) groups.
ment did not affect end-point measures of reaching performance (Fig. 5). RANOVAs for first attempt success showed no effect of Group (F(3, 35) = 0.89, p > 0.05), no Day by Group interaction (F(39, 455) = 0.91, p > 0.05), and a significant effect of Days (F(13, 455) = 23.01, p < 0.001). The RANOVA for total success gave no effect of Group (F(3, 35) = 0.18, p > 0.05), no Day by Group interaction (F(39, 455) = 0.48, p > 0.05) and an effect of Days (F(13, 455) = 45.66, p < 0.001). The RANOVA for reaching attempts gave no effect of Group (F(3, 35) = 2.27, p > 0.05), no Day by Group interaction (F(39, 455) = 1.40, p > 0.05), but showed an effect for Days (F(13, 455) = 49.58, p < 0.001).
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3.5. Learned nonuse Rats in all groups made reach attempts when first returned to the test box following stroke. However, reach attempts in all groups was similar in that they declined with successive approaches to the food over the first three days following stroke before beginning to recover on POD4 through POD6 (Fig. 6). The RANOVA on reach attempts during the six days immediately after stroke gave no effect of Group (F(3, 35) = 0.04, p > 0.05), and no Day by Group interaction (F(15, 175) = 0.84, p > 0.05), but did yield a significant effect of Days (F(5, 175) = 29.43, p < 0.001). The decline in reach attempts across the testing session for the first three days post-stroke was significant for all groups: N/N rs = −0.67, N/V rs = −0.60, V/N rs = −0.89, V/V rs = −0.47 (p < 0.05). 3.6. Movement element analysis of reaching
Fig. 5. Post-stroke scores (mean ± S.E.M.) on the single pellet reaching task for (A) first attempt success; (B) total success; and (C) total number of attempts. Days 28, 35, and 42 were completed without drug administration. Note: A reduction in success and attempts after stroke, and an absence of a nicotine effect (N, nicotine; V, vehicle).
The movement element analysis indicated that stroke impaired limb use, but nicotine treatment before and/or after stroke did not improve stroke-associated movement impairments. Rather, nicotine impaired movement elements before stroke. Following stroke, rats in all groups were similarly impaired on a number of elements, especially aim, pronation and supination. Separate analyses were preformed for three scoring days (baseline, pre-nicotine; Day −2, nicotine pre-stroke; POD14, post-stroke). There was no significant group difference on the pre-nicotine test (F(3, 35) = 0.52, p > 0.05). There was a significant Group effect on the nicotine pre-stroke test (F(1, 37) = 8.75, p < 0.01), no significant Element effect, (F(5.40, 199.67) = 0.98, p > 0.05), but there was a Group by Element interaction (F(5.40, 199.68) = 14.30, p < 0.001), as the nicotine treated group were impaired in pronation and supination. Following stroke (POD14) there was a significant effect of Element (F(4.86, 165.22) = 16.57, p < 0.001), but no Group effect (F(3, 34) = 2.26, p > 0.05) and no Group by Element interaction (F(14.58, 165.22) = 1.23, p > 0.05). Following stroke all groups were impaired on aim, pronation and supination. In order to determine whether there was a treatment effect on the reaching elements, pre-nicotine performance was compared to post-stroke performance. Fig. 7 indicates that the pattern of impairment on POD14 was similar in all of the lesion groups. There was no effect of Groups (F(3, 34) = 2.64, p > 0.05), but a significant effect of Days (F(1, 34) = 33.10, p < 0.001), and Elements (F(5.90, 200.51) = 23.97, p < 0.001). The Element by Group (F(17.69, 200.51) = 1.26, p > 0.05), Day by Group (F(3, 34) = 1.28, p > 0.05), and Element by Day by Group interactions (F(15.80, 179.12) = 0.99, p > 0.05) were not significant. There was a significant Element by Day interaction (F(5.27, 179.12) = 9.25, p < 0.001). Paired t-tests were used to compare the elements in each group pre-stroke (Day −2) and post-stroke (POD14), as illustrated by Fig. 7. 4. Discussion
3.4. Measures during the chronic post-stroke period Chronic post-stroke days (POD28, POD35, and POD42), on which the rats received neither nicotine nor vehicle treatment, did not give differences on any end-point measures of performance (Fig. 5). ANOVAs revealed no difference for groups on first attempt success (F(3, 35) = 0.83, p > 0.05), success (F(3, 35) = 0.19, p > 0.05), or number of attempts (F(3, 35) = 1.53, p > 0.05). There was a main effect of Days for first attempt success (F(2, 70) = 6.77, p < 0.01), and success (F(1.92, 67.03) = 7.30, p < 0.01), but not for attempts (F(1.82, 63.54) = 0.96, p > 0.05). There were no Day by Group interactions for first attempt success (F(6, 70) = 0.47, p > 0.05), total success (F(5.75, 67.03) = 1.53, p > 0.05), or attempts (F(5.5, 63.54) = 0.77, p > 0.05).
The objective of the present study was to examine whether nicotine could improve post-stroke motor performance at different phases of post-stroke recovery. Nicotine administered per oral prior to stroke, post-stoke, or both prior to and post-stroke, was not beneficial on any measure of post-stroke reaching behavior. First, learned nonuse, in which rats decrease reaching attempts in the acute post-stroke period, was not changed. Second, the reacquisition of reaching in a constraint-induced paradigm, in which rats were required to use their affected paw, was not improved. Third, retention of the reacquired skilled reaching act after a rehabilitation and treatment holiday was not improved. The present results suggest that neither the activating effects of nicotine nor its potential effects on brain plasticity promote the
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Fig. 6. Regression of the number of reaching attempts in the single pellet reaching task across each session during the acute post-stroke period. Note: Nicotine failed to prevent the development of learned nonuse and failed to enhance recovery from learned nonuse. (N, nicotine; V, vehicle; POD, post-operative day).
recovery of the learned or innate motoric aspects of skilled reaching. Although the results of the present study are negative with respect to a putative role for nicotine in promoting recovery of function after stroke, the study is nonetheless instructive. Preclinical studies of functional recovery after stroke have been difficult to translate into clinical treatments, mainly because preclinical studies are seldom replicated [10,21]. In this respect, the present study has a number of strengths, including a stroke model that has proved useful for the study of functional recovery in previous research [4], the use of a nicotine regime that falls within the range of that used in previous studies [23,24], and an improved route of drug administration. Furthermore, the behavioral analysis that was used is suitable for both the acute and chronic phases of recovery from stroke. Nevertheless, the study contains a number of potential weaknesses, including the use of a single drug treatment dose, a single stroke size, and an assessment of a single behavior. Strengths and caveats of the present approach to investigating drug-induced potentiation of recovery of function from stroke are discussed with respect to the idea that nicotine is useful in promoting recovery of function only in limited circumstances. The search for drug treatments for enhancing recovery of function after brain injury is made difficult by many potential treatment variables associated with administration. For the present study a procedure was developed in which nicotine could be administered orally rather than via subcutaneous injection. The drug dose selected for use in the present study was based on previous work [23,24], and was confirmed by behavioral assessment. Nicotine in solution was found to be rejected either alone or in a number of foods, but when administered in conjunction with a dab of peanut butter it was quickly and reliably consumed at a prescribed treatment time. The advantage of per oral administration is that it is less stressful than systemic administration, especially when repeated injections are given, and is less likely to cause infection or cutaneous trauma [12]. Although absorption is slightly more rapid with subcutaneous administration vs. per oral administration [5], subcutaneous and per oral administration of nicotine have similar dose–effect pharmacokinetics [9]. It should be noted that the use of peanut butter in nicotine administration, the timing of drug administration, or the food-deprivation schedule may have affected
the absorption of nicotine, however, a pilot study in which rats on a food-deprivation schedule were administered nicotine per oral in peanut butter vs. subcutaneously indicated that the activation effect of the drug, as measured by an increase in locomotor activity, was similar for both routes across an 8-day testing period. Finally, nicotine as administered here had a similar effect of somewhat depressing the pre-stroke reaching success, as has been reported following subcutaneous injections [23,24]. Thus differences in the present findings vs. the findings in previous works are not easily ascribed to drug absorption and action. Although the drug dose used here was selected with the intention of replicating previous work, it would have been ideal to have used a range nicotine doses. Nevertheless, there is a trade-off between the detail in which a behavior is analyzed and the number or size of experimental groups. The use of only a single dose of the drug for the present analysis was made necessary by the exhaustive behavioral assessment. Additionally, were future investigations to evaluate different administrative procedures, such as administration through cutaneous patches or via a mini pump, a single dose would likely be administered. Possibly, an investigation of a more appropriate dose of nicotine could use the behavioral screening methods previously used (for a review of dose selection in the rat see [34]). However, the objective of the present study was not to find an ideal dose, but to complete an in-depth analysis of a purported beneficial dose. Furthermore, although it is possible that a nicotine treatment with a drug dose range smaller or larger than that used here could be effective, there were no statistical trends in the present results to encourage such speculation. The way in which a stroke occurs could potentially affect the outcome of a treatment, but three lines of evidence suggest that this consideration is unlikely to account for the present findings. First, it is unlikely that the present results are specific to the form of motor cortex stroke that was used. The selected stroke method – devascularization via pial stripping – was similar to that used in previous studies [23,28], which produces a behavioral syndrome comparable to an internal capsule human stroke. Second, an explicit comparison of different methods of inducing stroke to motor cortex finds that behavioral deficits are very similar across all methods of rodent stroke [22]. Third, it could be argued that the lesion was too large, leaving little residual tissue to mediate recovery; it is reported
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Fig. 7. Movement element measures of reaching on POD14. Asterisks denote significance between scores on testing Day −2 (pre-stroke) and POD14. Rats without motor cortex stroke would achieve scores close to 0 on all movements. Note: Similar impairment in all groups (N, nicotine; V, vehicle; POD, post-operative day).
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that greater recovery can be expected from lesions that increasingly spare greater portions of functional areas [25]. Nevertheless, the present study did find substantial recovery/compensation with rehabilitation along with some enduring deficits, suggesting that there was considerable room for improvement with pharmacological assisted treatment. Although a smaller stroke size would likely produce smaller deficits it would allow less room for demonstrating treatment benefits. The major strength of the present study is the scope and the detail of the behavioral analysis that encompassed three phases of recovery; an acute post-stroke period (POD1–6), a sub-acute poststroke period (POD7–14), and a chronic post-stroke period (POD28, 35, and 42). Previous work has shown that in the immediate days following stroke rats display a trial-by-trial decrease in the number of reach attempts, a behavior termed learned nonuse [4,16], and this finding was confirmed in all of the treatment groups in the present study. It was expected that rats given nicotine would demonstrate a less severe trend of learned nonuse, because nicotine is motorically activating [8] and has an antidepressant-like effect in learned helplessness paradigms [46]. Despite any such effects that nicotine has on general motor behavior, the rats exposed to nicotine displayed the same nonuse trend as vehicle treated rats. Furthermore, all groups showed a similar recovery pattern from learned nonuse. Thus, nicotine did not prevent the occurrence of learned nonuse, and did not expedite recovery from learned nonuse. Chronic post-stroke improvement in skilled reaching is dependent upon using a constraint-induced recovery method [32,33]. Constraint-induced therapy here was accomplished through braceletting the non-affected limb and placing the food pellet contralateral to the affected limb [58,59]. Based on previous studies citing beneficial effects of nicotine on motor skills, it was expected that nicotine might improve the rate of recovery/compensation and improve eventual levels of success. Nicotine failed to improve the progress of recovery as measured by end-point measures of success on first reach attempts, success on total attempts, or number of reaching attempts. To evaluate the effects of nicotine treatment on motoric retention of skilled reaching in the chronic post-stroke period, the rats were reevaluated after a nicotine and rehabilitation holiday, after which performance typically declines [56]. Again, performance was similar in all groups, despite differences in nicotine treatment. Thus, as in the acute post-stroke period, in the chronic post-stroke period there were no beneficial effects of nicotine on any end-point measure of performance. In addition to end-point measures of success, performance measures evaluated in the present study included movement element analysis of reaching. It has been reported that nicotine improves the motor act of reaching after stroke on the tray reach task, although surprisingly, this improvement was not associated with improved success on single pellet reaching [23,24]. To evaluate whether nicotine could encourage the use of more normal movements following stroke, movement element analysis was performed at a number of different time points in the study. It was found that there were no differences in reaching elements between rats administered nicotine vs. vehicle. After stroke all groups were equally impaired in performance of rotatory movements of the limb, including aiming, pronation, and supination. The present results indicate that nicotine does not improve movement scores, which is inconsistent with previous studies [23], however, studies using multiple measures risk Type I errors, which speaks to the necessity for replication of positive findings such as that conducted here. In conclusion, despite the activating effects of nicotine and its effects on brain plasticity [35], nicotine did not promote recovery of skilled reaching after stroke. The present study does not represent an exhaustive measure of the effects of nicotine on behavior or the effects of nicotine on recovery after motor cortex stroke. It does, however, show that single pellet reaching resists nicotine
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pharmacotherapy, and in doing so is consistent with a number of previous lines of investigation concerning the resistance of single pellet reaching to pharmacotherapy. Amphetamine treatment has been widely reported to improve post-stroke motor behavior, but it does not improve performance on skilled reaching [4]. Brain transplants of fetal dopamine tissue given to animals depleted of dopamine can improve a number of aspects of motor behavior but skilled reaching is not improved [15]. In nonhuman animals and humans with reduced levels of dopamine, levodopa treatments improve a number of aspects of motor performance after the loss of dopamine, but such treatment does not improve performance on skilled reaching [36,37]. A similar absence of improvement has been reported using fluoxetine following ischemia [60], despite the fact that this drug, like nicotine, has positive effects on brain activity and growth factors. Current research using antibodies for neuritegrowth inhibitors during post-stroke recovery are promising [51], and indicate that post-stroke skilled reaching may not be entirely resistant to treatment. For facilitation of sparing or recovery of function to occur on skilled reaching it may be necessary to spare some portion of the forelimb area [1,25,30], use larger or smaller drug doses, or prolong treatment. Nevertheless, it should also be considered that whereas nicotine may be effective following brain injury on some aspects of behavior [6,7,23,24,29,51], skilled reaching is not among them. Acknowledgements The authors would like to thank Jessica Cummins for help with histological analysis, and Dr. Seong-Keun Moon for help with surgery. This research was supported by grants from the Alberta Heritage Foundation for Medical Research (D.H.L.) and the Natural Sciences and Engineering Research Council of Canada (I.Q.W.). References [1] Adkins-Muir DL, Jones TA. Cortical electrical stimulation combined with rehabilitative training: enhanced functional recovery and dendritic plasticity following focal cortical ischemia in rats. Neurol Res 2003;25(8):780–8. [2] Alaverdashvili M, Foroud A, Lim DH, Whishaw IQ. “Learned baduse” limits recovery of skilled reaching for food after forelimb motor cortex stroke in rats: a new analysis of the effect of gestures on success. Behav Brain Res 2008;188(2):281–90. [3] Alaverdashvili M, Leblond H, Rossignol S, Whishaw IQ. Cineradiographic (video X-ray) analysis of skilled reaching in a single pellet reaching task provides insight into relative contribution of body, head, oral, and forelimb movement in rats. Behav Brain Res 2008;192(2):232–47. [4] Alaverdashvili M, Lim DH, Whishaw IQ. No improvement by amphetamine on learned non-use, attempts, success or movement in skilled reaching by the rat after motor cortex stroke. Eur J Neurosci 2007;25(11):3442–52. [5] Barnes CD, Eltherington LG. Drug dosage in laboratory animals: a handbook. 2nd rev. enlarged ed. California: University of California Press; 1973. p. 171. [6] Brown RW, Gonzalez CLR, Kolb B. Nicotine improves Morris water task performance in rats given medial frontal cortex lesions. Pharmacol Biochem Behav 2000;67(3):483–8. [7] Brown RW, Gonzalez CLR, Whishaw IQ, Kolb B. Nicotine improvement of Morris water task performance after fimbria-fornix lesion is blocked by mecamlyamine. Behav Brain Res 2001;119(2):185–92. [8] Clarke PB, Kumar R. The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br J Pharmacol 1983;78(2):329–37. [9] Craft RM, Howard JL. Cue properties of oral and transdermal nicotine in the rat. Psychopharmacology (Berl) 1988;96(3):281–4. [10] Cramer SC. Repairing the human brain after stroke. II. Restorative therapies. Ann Neurol 2008;63(5):549–60. [11] Dalack GW, Healy DJ, Meador-Woodruff JH. Nicotine dependence in schizophrenia: clinical phenomena and laboratory findings. Am J Psychiatry 1998;155(11): 1490–501. [12] Dilsaver SC, Majchrzak MJ. Effects of placebo (saline) injections on core temperature in the rat. Prog Neuropsychopharmacol Biol Psychiatry 1990;14(3): 417–22. [13] Djuric VJ, Dunn E, Overstreet DH, Dragomir A, Steiner M. Antidepressant effect of ingested nicotine in female rats of Flinders resistant and sensitive lines. Physiol Behav 1999;67(4):533–7. [14] Donoghue JP, Wise SP. The motor cortex of the rat: cytoarchitecture and microstimulation mapping. J Comp Neurol 1992;212(1):76–88.
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