Original Article Dose-Response: An International Journal Buspirone Dose-Response on Facilitating January-March 2021:1-10 ª The Author(s) 2021 Article reuse guidelines: Forelimb Functional Recovery in Cervical sagepub.com/journals-permissions DOI: 10.1177/1559325821998136 Spinal Cord Injured Rats journals.sagepub.com/home/dos Rakib Uddin Ahmed1, V. Reggie Edgerton2,3,4,5,6, Shuai Li1 , Yong-Ping Zheng1, and Monzurul Alam1 Abstract Buspirone, widely used as a neuropsychiatric drug, has also shown potentials for motor function recovery of injured spinal cord. However, the optimum dosages of such treatment remain unclear. In this study, we investigated the dose-response of Buspirone treatment on reaching and grasping function in cervical cord injured rats. Seventeen adult Sprague-Dawley rats were trained to reach and grasp sugar pellets before a C4 bilateral dorsal column crush injury. After 1 week post-injury, the rats were divided into 3 groups to receive 1 of 3 different dosages of Buspirone (i.p., 1 dose/day: 1.5, n ¼ 5; 2.5, n ¼ 6 and 3.5 mg/kg b.w., n ¼ 6). Forelimb reaching and grip strength test were recorded once per week, within 1 hour of Buspirone administration for 11 weeks post-injury. Different dose groups began to exhibit differences in reaching scores from 4 weeks post-injury. From 4-11 weeks post-injury, the reaching scores were highest in the lowest-dose group rats compared to the other 2 dose groups rats. Average grip strength was also found higher in the lowest-dose rats. Our results demonstrate a significant dose-dependence of Buspirone on the recovery of forelimb motor functions after cervical cord injury with the best performance occurring at the lowest dose tested. Keywords serotonin, cervical cord injury, neuromodulation, functional rehabilitation Introduction improved functional recovery in standing and stepping in SCI paraplegics, limited progress has been made in improving arm Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine neu- and hand functions using such neurochemical modulation.12 rotransmitter. Almost all 5-HT axons found within the mam- Moreover, 5HT2 receptor agonists alone can facilitate the malian spinal cord supraspinally originate from the brainstem.1 Serotonin plays a vital role in modulating the activity of the spinal network for the gain control of volitional limb move- ments2 and inducing partial recovery of plantar stepping.3 1 Department of Biomedical Engineering, The Hong Kong Polytechnic Disruption of serotonin pathways following spinal cord injury University, Hung Hom, Kowloon, Hong Kong SAR, China 2 Department of Neurobiology, University of California, Los Angeles, CA, USA (SCI) results in depletion of 5-HT in the spinal neural network, 3 Department of Neurosurgery, University of California, Los Angeles, CA, USA dysregulation of 5-HT transporters as well as elevated expres- 4 Brain Research Institute, University of California, Los Angeles, CA, USA 5 sion and sensitivity of specific 5-HT receptors below the spinal Institut Guttmann, Hospital de Neurorehabilitació, Institut Universitari lesion.4 6 adscrit a la Universitat Autònoma de Barcelona, Barcelona, Badalona, Spain These changes in the serotonergic system of the spinal cord The Centre for Neuroscience and Regenerative Medicine, Faculty of Science, University of Technology Sydney, Ultimo, New South Wales, Australia can produce varying degrees of functional complications after paralysis. To date, various neuropharmacologies have been Received 26 January 2021; received revised 26 January 2021; accepted 05 tested to restore hindlimb functions after SCI.5-9 5-HT receptor February 2021 agonists, when combined with electrical stimulation restores Corresponding Author: locomotor function in SCI rats primarily by neuromodulating Monzurul Alam, Department of Biomedical Engineering, The Hong Kong the physiological states of spinal networks that generate step- Polytechnic University, Hong Kong 999077, China. ping.10,11 Although serotonin agonists have demonstrated Email: md.malam@connect.polyu.hk Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 2 Dose-Response: An International Journal Figure 1. Experimental procedure. Seventeen Sprague-Dawley rats were trained to reach and grasp the pellets for 6 weeks. After mastering the task, all rats received a dorsal funiculus crush injury at the C4 level and subsequent implantation of EMG electrodes in the preferred forearm muscles (extensor digitorum and flexor digitorum). After 1 week of recovering from surgery, the rats were ranked and divided into 3 Buspirone dosages groups: Group A, B and C; low, 1.5, n ¼ 5; medium, 2.5; n ¼ 6 and, high-dose, 3.5 mg/kg b.w., n ¼ 6; respectively. Weekly forelimb reaching task and grip strength tests were conducted for weeks 1-6 continuously and week 9, after which the drug was withdrawn and the rats were tested again in week 11. persistent sodium inward current which increases the excitation Methods of motorneurons in chronic spinal cord injured rats.13 The All experimental procedures were carried out according to the above findings indicate the importance of serotonin receptor guidelines and approval of the Animal Subjects Ethics Sub- agonists in spinal cord injuries. committee of The Hong Kong Polytechnic University. Buspirone, a serotonin 5-HT1A receptor partial agonist acts as an antagonist for dopamine D2 autoreceptors. There is some evidence of a weak affinity for 5-HT2 receptors, com- Animal Subjects monly used as an anxiolytic drug, and has the potential to Seventeen adult female Sprague-Dawley rats (230 + 20 grams improve brain functions after injuries or diseases.14 In recent b.w.) were used in this study. Acclimation of the animals was studies on SCI patients, it has been suggested that electrical done for 1 week prior to the behavioral training. Adlibitum stimulation of the spinal cord, when combined with regular food and water were provided before starting the forelimb Buspirone administration can restore voluntary control of the training. Body weights were monitored after the surgery hand12 and locomotor function.15,16 Previous reports have weekly. The room temperature (24 C) and humidity (40%) shown that low doses of Buspirone can stimulate the soma- were carefully maintained. todendritic 5-HT1A receptors in an ex-vivo bovine brain17 and can trigger motor activity in rats.18 However, high doses of Buspirone can block the postsynaptic terminals on dopami- Reaching and Grasping Task nergic neurons and induce behavioral changes.19 In Parkin- All the rats were trained (thrice/week) to reach, grasp and eat son’s diseases, a dose of 2 mg/kg b.w. of Buspirone has also the 45 mg dustless sugar pellets (Bio-serv®, Flemington, USA) been reported to reduce locomotor activity. Similar effects from a pit flatform (3 cm  2 cm) of a specialized box (18 cm have also been reported in other neurological disorders.20  15 cm  31 cm) as described before.21,22 Food restriction Likewise, high doses of a 5HT2 receptor agonist may activate was provided before each task to master the technique. During 5HT1A receptor agonists and modulate the interneuronal each test, 30 pellets were provided to grasp and eat. After 6 activity to fire repetitively in chronic conditions.13 The opti- weeks of training, the rats demonstrated at least a 60% success mal dose response of Buspirone for improving arm and hand rate in their reaching and grasping task. Beginning at 1-week functions in cervical SCI is unclear. The lack of clarity in the post-injury, weekly reaching and grasping scores were mea- dose-response relationships among the experiments noted sured for 11 weeks (Figure 1). above demonstrate that to understand the efficacy of a given dose of Buspirone, in spite of its long history of clinical use, care must be taken to control the dosage, physiological sys- Grip Strength Test tem, the timing of the response and the species of interest. In Prior to the surgery, forelimb grip strength tests were con- the current study, we investigated the dose-responses of Bus- ducted to acclimatize the animal to the test apparatus using a pirone treatment on reaching and grasping function after a custom-made grip strength meter as described previously.23 By cervical spinal cord injury in rats. holding the base of the tail, the rat was gently pulled away from Ahmed et al 3 the grid which was connected to a force sensor. From 1-week The analgesic, Buprenorphine HCL, was administered for post-injury, weekly maximum grip strength was measured for 3 days post-surgery. At the same time an antibiotic, Enrofloxacin each rat (Figure 1). (Baytril®, 0.5mg/kg, s.c) was administered and continued twice daily for 3 days to prevent any infection. The rats were then moved to a temperature and humidity-controlled incubator Cervical Cord Injury (AEOLUS Incubator, ICU-1801, USA) to recover from the anesthesia. After recovering, the animals were transferred to All surgical procedures were carried out under aseptic condi- their individual home cages. Fresh fruit and juice were provided tions. To induce anesthesia, at first the rats were anesthetized for a quick recovery. The rat’s condition was monitored con- with 5% isoflurane gas which was maintained at 1.5-2% tinuously for at least 1 week. throughout the surgery via a face mask. To prevent hypother- mia, the body temperature was maintained at 37 C by placing the rat on an automated heating pad (ThermoStar Homeother- Drug Treatment mic Monitoring System, RWD Life Science Co. Ltd., China). After recovering from the surgery at day 7, the animals’ ability To minimize the pain, an analgesic, Buprenorphine HCl to reach and grasp the sugar pellets was tested. One of the rats (Buprenex®, 0.5mg/kg, s.c.) was administered before the sur- died during the recovery period from the surgery and hence one gery. In deep anesthetic condition, an incomplete spinal cord group was slightly imbalanced. Based on the scores, the injury at the C4 level was carried out as described previously.24 animals were than ranked and divided into 3 balanced groups In brief, after a 3-4 cm skin incision was made using a sharp to be administered with different dosages of Buspirone (i.p., scalpel blade, the skin and fascia were retracted by a forceps to 1 dose/day): Group A with a low dose (1.5 mg/kg b.w.; n ¼ 5), reveal the muscles beneath. After retracting the paravertebral Group B with a medium dose (2.5 mg/kg b.w.; n ¼ 6) and muscles a laminectomy was performed at the C3-C4 vertebrae Group C with a high dose (3.5 mg/kg b.w.; n ¼ 6). The drug, to reveal the spinal cord. The tips of fine forceps were used to Buspirone (Tocris®, UK), was previously prepared by dissol- crush the dorsal funiculi of the spinal cord (2 mm apart, 2 mm ving 1 mg/1 ml of ultrapure distilled water (Invitrogen®, USA). depth). A thin 2-mm length of a stainless steel sterile rod was The drug was administered intraperitoneally into different dose used to ensure the consistency of the lesion size during each groups of SCI rats up to 9 weeks post-injury. Behavior tests and surgery at the C4 level. The SCI resulted in significant loss of electrophysiological recordings were conducted from 0.5 to supraspinal control with moderate motor and sensory impair- 1 hour of Buspirone administration. Each animal was video- ments.25,26 Saline water was used to moisturize the skin area taped with a camera27 while retrieving food pellets. After each and the muscles were sutured by using 4.0 Vicryl sutures. forelimb reaching task and electrophysiological recording, a grip strength test for each rat was conducted once per week, for 9 weeks. To see the effects of the drug, after 9 weeks, the Electrode Implantation drug administration was ceased, and the behavior tests and To record the EMG signals from the preferred paw, intramus- electrophysiological recording were done only at week-11 cular EMG electrodes were implanted into the forelimb flexor post-injury. At the end of all the experiments, the rats were and extensor digitorum muscles. A skin incision was made on euthanized with a high dose of Ketamine and Xylazine mixture the skull to place a head-plug. The skin and connective tissues and confirmed by a surgeon according to the guideline of the were retracted from the skull. Cotton gauze was soaked and Animal Subjects Ethics Sub-Committee of The Hong Kong placed on the skull to prevent dryness. Two longitudinal skin Polytechnic University. incisions around 2 cm were carried out to place the Teflon- coated stainless steel wires (AS631, Cooner Wire, USA) in the flexor and extensor digitorum muscles. The fascia of the mus- Data Acquisition and Statistical Analysis cles was retracted to reveal the belly of the desired muscle. By Videotape footage of the forelimb reaching task of each rat was using forceps the wires were then passed to the muscles examined frame by frame in windows media player to identify subcutaneously. A 27-gauge needle was then inserted in the the components of the grasping task. The EMG signals were muscle belly to implant the electrode wires. After insertion, a bandpass filtered at 10-1000 Hz and amplified (1,000 times) by part of the Teflon (*0.5 mm) from the wire was removed to an analog amplifier (Model 1700 Differential AC Amplifier, make a recording electrode. The wire electrodes were then AM Systems, USA). The signals were then digitized by a data anchored by using 4.0 Ethilon sutures and stimulation was acquisition system (Power1401-3A, Cambridge Electronics given through the connector to verify the position of the elec- Design Ltd., UK). The data were then visualized and recorded trode. The EMG wires were coiled to relieve the stress and the on a computer for further analysis via a software interface skin incisions were closed by using 4.0 Nylon sutures. Finally, (Signal, Cambridge Electronics Design Ltd., UK). Based on to anchor the head-plug, 4 screws were firmly placed into the the synchronization of the video, extensor and flexor muscle skull after drilling and thoroughly dried. Dental cement was EMGs for every trial were plotted and calculated by a custom then applied to immerge the screws to support the head plug script written in MATLAB (MathWorks Inc., USA). From the after drying. EMG signals of extensor digitorum and flexor digitorum 4 Dose-Response: An International Journal muscles, area-under-the curve (AUC) values were calculated as Group B rats started to decay from 5 weeks post-injury and described previously.24 The normalized AUC values for differ- never recovered. At the end of the study, Group A low-dose ent dose groups were then tested for statistical significance rats exhibited an average of 69% improvement of reaching and using 2-way analysis of variance (ANOVA) followed by grasping function over Group B and C medium- and high-dose Tukey’s multiple comparison post-hoc test using 2-way anal- rats. Although the low-dose rats displayed noticeable improve- ysis of variance (ANOVA) followed by Tukey’s multiple com- ments in later weeks after treatment compared to the higher- parison post-hoc test. Pre- and post-injury reaching scores were dose rats, no statistical significance was found between their compared for significance of difference by using a paired t-test. normalized reaching scores (at 11 weeks post-injury: Group A The success rates of forelimb reaching of the 3 dose groups vs. Group B, p ¼ 0.8786; Group A vs. Group C, p ¼ 0.6715). were normalized to their pre-injury success rate using the fol- lowing equation: Low- to Medium-Dose BUSPIRONE Treatment Improved   Original success rate Distal Muscle Co-Ordination Normalized reaching score ¼  100: Preinjury success rate Examples of raw EMG signals from the extensor digitorum and The scores were then evaluated for significant differences flexor digitorum muscles during reaching and grasping for dif- by 2-way ANOVA. One-way ANOVA was used to analyze the ferent dose groups are presented in Figure 3. At 1 week post- dose responses in each group. All the statistical measurements injury, the amplitude and bursting properties during the sugar were carried out using software (GraphPad Software Inc., pellet reaching task were relatively low compared to 9 weeks USA) with significance levels set at p < 0.05 for all the post-injury. comparisons. The EMG signals of the extensor digitorum and flexor digi- torum muscles were normalized for each rat to calculate the area-under-the-curve (AUC). The normalized AUC values of Results different dose groups were analyzed for statistical significance. In each group of rats the normalized AUC of the extensor A Low Dosage of Buspirone Facilitates Forelimb Reaching muscle tended to increase gradually after week 6 and even after and Grasping Function withdrawal of the drug (Figure 4a). However, the EMG from Following cervical cord injury, after a few attempts the animals the flexor digitorum muscle did not show any consistent trend could place their forelimbs on the food platform; however, over the 11 weeks of post-injury measurements, presumably in mostly failed to grasp the pellets. The post-injury forelimb large part due to the low number of rats, particularly for Group grasping success rates were compared with the pre-injury C. In Group C, only 2 rats were found to have good EMG scores. Forelimb reaching scores dropped significantly 1 week signals and were included in Figure 4 for analysis. In the other after the cervical cord injury (68.82 + 2.33 vs. 2.05 + 1.25; rats, the EMG signals were noisy and were not reliable for ***p < 0.001, paired t-test). analysis. In Group A, the flexor muscle activity dropped at All the rats were administered Buspirone, according to their week 9 and dramatically increased at week 11 after withdrawal dose group, till 9 weeks post-injury and the grasping success of the drug, while in Group C the opposite phenomenon hap- rate was measured. The animals started to increase their grasp- pened (Figure 4b). The medium-dose group, Group B, how- ing function noticeably after 3 weeks post-injury. The success ever, showed a similar increasing trend like the extensor rates of each dose group were compared with 1-week post- digitorum muscle. This may imply that a low- to medium- injury scores. In Group A, significant improvements of reach- dose of Buspirone may have more consistent effects on the ing function were found at weeks 4 and 5, respectively (40.33 forelimb muscles on reaching and grasping function. Although + 6.33 and 41.99 + 4.89, **p < 0.01, 1-way ANOVA). The no statistically significant differences between the dose groups reaching score remained significantly high till week 9, and were found, the similarity of the relative patterns of differences even at week 11 after cessation of the drug treatment (respec- across time in the extensor muscle and a consistently different tively 46.33 + 5.22 and 45.33 + 4.54;**p < 0.01, ANOVA) pattern observed in the flexor, regardless of group identity, (Figure 2a). suggest that the dosage-time effect was different between the The medium-dose rats, Group B, significantly improved extensor and flexor muscles. their forelimb grasping function at and after 4 weeks post- injury compared to week 1 (Figure 2b). At week 11 after the Low-Dosage of Buspirone Treatment Improved Forelimb cessation of Buspirone administration, the reaching scores dropped but still remained significantly high compared to the Grip Strength week 1 post-injury score. Group C did not exhibit any signif- Following the forelimb reaching task, the maximum value of icant improvement of reaching function (Figure 2c). The aver- the forelimb grip strength test was carried out weekly for 6 age normalized reaching scores of all groups were compared weeks and finally at week 9. After the withdrawal of Buspir- with each other (Figure 2d), showing that Group B and C one, the tests were carried out at week 11. The forelimb grip scores increased up to 3 weeks post-injury, while Group A strength of all 3 groups exhibited continuous improvement up continued to improve up to 6 weeks. The reaching scores of to week 4 (Figure 5). Ahmed et al 5 Figure 2. Reaching and grasping scores of 3 different dose groups after Buspirone administration. a) Mean (+SEM) success rates of Group A rats (n ¼ 30 trials/rat/test session, 5 rats). 1-week vs. 4 and 5-week post-injury scores (**p < 0.01). 1-week vs. 6, 9 and 11-week post-injury scores (***p < 0.001). b) Mean (+SEM) success rates of Group B rats (n ¼ 30 trials/rat/test session, 6 rats). 1-week vs. 4 and 5-week post-injury scores (***p < 0.001). 1-week vs. 6 and 9-week post-injury score (**p < 0.01). 1-week vs. 11-week post-injury score (*p < 0.05). c) No significant difference was found in the mean (+SEM) success rates of Group C (n ¼ 30 trials/rat/test session, 6 rats). d) The progression of the mean (+SEM) normalized reaching scores of all 3 groups. The dotted line is the score of a representative rat for each group. A similar effect of Buspirone was also found in Group B optimum level, but dropped afterward. The results indicate that and C. For Group B, the force was boosted from 6.67 N (weeks each week the motor task had an influence on the functional 1&2 post-injury) to 13.42 N (weeks 6&9 post-injury). For recovery. Unlike Group A and B rats, high-dose Group C rats Group C, the maximum grip force increased from 5.60 N did not undergo any improvement of fine motor control. Over- (weeks 1&2 post-injury) to 12.16 N (week 4 post-injury) and all, a higher success rate and grip strength were found for the dropped to 11.09 N at weeks 6&9 post-injury. After withdrawal low-dose rats (Figure 6). The results indicate that the lowest of the drug, at 11 weeks post-injury, the average grip force dose of buspirone tested provided the most consistent pattern of increased slightly to about 11.24 N for Groups A and B. Sig- functional improvements compared to the medium and high nificantly higher grip strength (**p < 0.01 and *p < 0.05 dosages, but also suggest that maximum grip force was entirely ANOVA) in Group A, compared to Group C was found at unrelated to performance success. It also suggests that regard- 5 and 11 weeks post-injury (Figure 5d). less of group identity, the mean highest grip force occurred at No significant relation was found between the forelimb the 11th week, i.e. grip strength increased with time, but with- reaching score and the forelimb grip force in the early post- out Buspirone. injury weeks (data not shown). From 6 weeks of drug treat- ment, both reaching success and grip strength values increased in almost all the dose groups (Figure 6). Group A exhibited the Discussion most consistent functional recovery as assessed for both fine As a serotonin agonist, Buspirone, primarily known as an anxio- motor control of reaching and for gross forelimb grip strengths. lytic drug, has high affinity for 5HT1A receptors and weak affi- These improvements were also sustained after withdrawal of nity for 5HT2A receptors. In contrast, the drug has antagonistic the low dosage of the drug. The success rate and grip strength properties against dopamine D2 receptors.14 A recent study has of Group B rats increased at 6 weeks post-injury to an shown the potential of the drug to improve forearm grip strength 6 Dose-Response: An International Journal Figure 3. Raw EMG signals (n ¼ 30 trials) during forelimb reaching and grasping task at 1, 6, 9 and 11 weeks post-injury from one representative rat (marked as a dotted line in Figure 2) from each treatment group. Only at 6 and 9 weeks post-injury was the EMG recorded during the Buspirone administration period, while week 1 was before, and week 11 was 2 weeks after the drug treatment. function along with electric stimulation in tetraplegic patients, can improve forelimb reaching function in rats with cervical but these effects do not provide strong evidence for significant cord injury.29 The spontaneous recovery of forelimb function of functional effects in performing daily motor tasks.12 rats without any drug intervention was found to be less than Skilled forelimb function such as forelimb reaching and 20% while the rats which were administered Buspirone reached grasping task is commonly used to examine the functional a success rate of over 60% by 6 weeks post-injury. Thus, ser- recovery after cervical cord injury in rats.28 In a previous study, otonergic modulation enabled over 3-fold improvements in we showed that an incremental dose of Buspirone (1 to 2 mg/kg) forelimb reaching and grasping after post-injury Buspirone Ahmed et al 7 Figure 4. Average area-under-the-curve (AUC) of normalized EMG signals for a) extensor digitorum and b) flexor digitorum muscles during the single pellet reaching task in 3 different Buspirone dose groups (Group A, n ¼ 5; Group B, n ¼ 5; and Group C, n ¼ 2). administration. In the present study, we demonstrated that dif- performance by 25% and 20%, respectively.32 A low dose of ferent dosages of Buspirone have different effects on forelimb Buspirone can significantly increase the synthesis and availabil- functional recovery after a dorsal funiculus injury in rats. Our ity of dopamine in the pre-synaptic terminal, whereas a higher main finding is that the low dose (1.5 mg/kg b.w.) of Buspirone dose blocks postsynaptic dopaminergic receptors.19 markedly improved forelimb fine motor control and grip Generally, these findings demonstrate some of the complex- strength compared to a higher dose. Furthermore, a low to ities of Buspirone given the multiple autoreceptors in the ner- medium dose (1.5-2.5 mg/kg b.w.) of Buspirone improved the vous system, which could contribute to the dose dependencies forelimb muscle synergies of the distal muscles. of its physiological responses. The present findings suggest that When considering the possibility of using Buspirone as a the functional window of efficacy for Buspirone, and probably pharmacological way to enhance the recovery of motor func- other potential pharmacological candidates with limited speci- tions, it is important to consider the dose-response characteris- ficity for spinal neuromodulation, can be narrow. These data, tics of other serotonergic systemic responses that have been for example, show that the efficacy of Buspirone not only is a observed that may be detrimental or beneficial either acutely function of relatively narrow ranges of dose; the timeframe or chronically. Based on the history of its development the dose over which functional changes are likely to occur gives a clue regimen for the treatment of behavioral effects will be of obvi- as to the persistence of the responses. ous importance. The effects on cerebral glucose metabolism in Other variables that need to be examined are the potential response to doses ranging from 0.4 mg/kg to 40 mg/kg have interactive effects associated with activity-dependent mechan- shown reduced cerebral metabolic rates for glucose in rats. A isms that might be in play. Regarding this specific interaction dose of 0.4 mg/kg Buspirone can preferentially activate the of a pharmacological and activity-dependent interaction, there 5-HT1A autoreceptors.30 Pain sensation has been studied in rats have been clear examples of these 2 interventional modes hav- using dosages ranging from 0.1-2.0 mg/kg.31 Such dose can ing antagonist or synergistic effects depending on whether they also improve the acquisition and retention of memory in a were presented sequentially or simultaneously. Finally, it is water maze.31 Given the distinctly dose-dependent differences logical to assume that the highly complex serotonergic receptor observed in the present study, each of the very few cases noted types within the spinal cord and the fact that almost all of the here emphasizes the criticalness of dosage in defining its effec- serotonin is derived from supraspinal nuclei, and their axonal tiveness for a specific function. projections rapidly degenerate post injury. Thus, it seems likely From neurochemical evidence, it was found that a low that the serotonergic spinal network response to a serotonergic (1 mg/kg) but not high dose (5 mg/kg) of Buspirone stimulates acting drug will be significantly different when tested in acute 5-HT1A receptors and decreases striatal metabolism. The finding phases, as in this study, compared to the more frequently avail- also suggests that a low dose could help to release dopaminergic able chronic state in humans with a spinal injury. In the present neurons that could be useful in reducing Parkinsonian-like study, it was expected that the higher dose could activate the effects.18 Furthermore, a model of L-DOPA-induced dyskinesia cervical spinal network as occurs among the lumbosacral net- in Parkinson’s disease 1 mg/kg b.w. of Buspirone did not works.33 The present results showed a robust effect of a low to impair the rotarod performances in rats, while higher doses medium dosage effect on forelimb grasping function in the (2 and 4 mg/kg b.w.) significantly reduced the rotarod acute phase of adaptation to the injury imposed. It appears that 8 Dose-Response: An International Journal Figure 5. Maximum grip force of the 3 different dose groups after Buspirone administration. a) Mean (+SEM) maximum grip force of Group A (n ¼ 3 trials/rat/test session, 5 rats). 2 weeks vs. 11 weeks and 9 weeks vs. 11 weeks post-injury (*p < 0.05) b) Mean (+SEM) maximum grip force of Group B (n ¼ 3 trials/rat/test session, 6 rats). No significant difference was found. c) Mean (+SEM) maximum grip force of Group C (n ¼ 3 trials/rat/test session, 6 rats). 1 week vs. 6 weeks and 11 weeks post-injury (*p < 0.05); and 1 week vs. 9 weeks post-injury (**p < 0.01). d) Post-injury mean (+SEM) maximum grip force of 3 dose groups. Group A showed significantly higher grip strength at week 5 (**p < 0.01) and at week 11 post-injury (*p < 0.05) compared to Group C. an intervention after a spinal injury is highly dependent on the timing at which a biochemical and an activity-dependent inter- vention is presented.34,35 Conclusions Buspirone had clear effects on the recovery of forelimb grasp- ing and grip strength following administration at a dose of 1.5 mg/kg b.w. after an incomplete cervical cord injury. The magnitude of these effects were highly dose- and post treat- ment time-dependent. At the longest post-treatment time stud- ied among all 3 dosage groups there was a direct relationship between grip strength and reach and grasp success rate. How- ever, a larger sample size and more functional tests may con- Figure 6. Overall forelimb functions of each drug dose group. firm the significance and synergy of the drug dosage. Based on Normalized success rate vs. normalized grip force at 6, 9, and the EMG data represented as the total EMG activity of a pri- 11 weeks post-injury. Data are presented as the mean (+SEM). The mary extensor and flexor muscle per reach and grasp effort, no marker sizes (smaller to larger) represents 6, 9 and 11 weeks post- clear pattern of changes was found to be closely linked to injury, respectively. successful performances, an issue that needs comprehensive assessment to understand the subject-specific functional neural a critical question now is, what would the dose responses be for reorganization adopted strategies that shaped the patterns of motor tasks in the acute vs. chronic stage of a spinal injury. We recovery. From the EMG responses, it can be observed that the have demonstrated previously, for example, that the efficacy of low-dose rats had most consistent patterns of forelimb function Ahmed et al 9 compared to the medium- and high-dose rats. The two beha- 8. Antri M, Mouffle C, Orsal D, Barthe J-Y. 5-HT1A receptors are vioral motor tests and forelimb electrophysiology combined involved in short- and long-term processes responsible for 5-HT- may suggest that a low dose of Buspirone has better influence induced locomotor function recovery in chronic spinal rat. Eur J on forelimb functional recovery than a higher dose. Neurosci. 2003;18(7):1963-1972. doi:10.1046/j.1460-9568.2003. 02916.x Declaration of Conflicting Interests 9. Landry ES, Lapointe NP, Rouillard C, Levesque D, Hedlund PB, The author declared the following potential conflicts of interest with Guertin PA. Contribution of spinal 5-HT1A and 5-HT7 receptors respect to the research, authorship, and/or publication of this article. to locomotor-like movement induced by 8-OH-DPAT in spinal V.R.E. holds shareholder interests in NeuroRecovery Technologies cord-transected mice. Eur J Neurosci. 2006;24(2):535-546. and holds certain inventorship rights over intellectual property doi:10.1111/j.1460-9568.2006.04917.x licensed by the Regents of the University of California to NeuroRe- 10. Courtine G, Gerasimenko Y, van den Brand R, et al. Transforma- covery Technologies. V.R.E holds shareholder interests in spineX Inc. tion of nonfunctional spinal circuits into functional states after and holds certain inventorship rights over intellectual property the loss of brain input. Nat Neurosci. 2009;12(10):1333-1342. licensed by the Regents of the University of California to spineX Inc. doi:10.1038/nn.2401 V.R.E. serves on the scientific advisory board of in vivo Therapeutics 11. Gerasimenko YP, Ichiyama RM, Lavrov IA, et al. Epidural spinal and ArianRF and serves as the Chair of the Scientific Advisory board cord stimulation plus quipazine administration enable stepping in at spineX. complete spinal adult rats. J neurophysiol. 2007;98(5):2525-2536. 12. 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