This work was supported by Interdisziplin&#228;res Zentrum f&#252;r Klinische Forschung (IZKF), University Clinics W&#252;rzburg, Germany (project N-215).

Animal experiments were approved by the University of Wuerzburg and the legal state authorities of Lower Franconia in accordance with the animal protection guidelines and European Communities Council guidelines (approval number: 55.2-2531.01 76/11). All efforts were made to minimize pain or discomfort of the animals used.
Note: Electrode implantation was performed as described elsewhere18.
1. Cylinder Test (Figure 2)
<ol>
	<li>Prepare a clear plastic glass cylinder (height: 40 cm, diameter: 19 cm) by cleaning the cylinder with a 0.1% acetic acid solution.</li>
	<li>Prepare cards with date of experiment and each rat&#39;s identification number.</li>
	<li>Place two mirrors in 90&#176; angle behind the cylinder.</li>
	<li>Place the camera in front of the cylinder such that the distance between the camera and the cylinder allows a good view of the paws.</li>
	<li>Place the rat in the transport box. 
	Note: The animals should be handled by the experimenter prior to the test to avoid stress.</li>
	<li>Transport the rat from home cage to the cylinder using the transport box.</li>
	<li>Place the rat in the cylinder (Figure 3).
	<ol>
		<li>Always perform all behavioral tests at the same time of the day to avoid circadian differences in activity. If the animal is connected to the stimulus generator by cable make sure the cable is not twisted during the experiment.</li>
	</ol>
	</li>
	<li>Press the &#34;record&#34; button on camera. Show the card with the actual date of experiment and rat&#39;s identification number to the camera. Start recording.</li>
	<li>After five minutes, remove the animal from the cylinder and put it back into the home cage using the transport box.</li>
	<li>Clean the cylinder with a 0.1% acetic acid solution.</li>
	<li>Evaluate the paw use from recorded video by counting the left and right paw wall contacts (paw use in percent) as well as rearings (standing on hind paws with or without supporting on the cylinder wall). The cylinder test can be also evaluated automatically by an appropriate software. 
	Note: A healthy rat uses both paws equally. The hemiparkinsonian rat uses the paw affected due to lesion a lesser extent.</li>
</ol>
2. Staircase Test (Figure 4)
<ol>
	<li>Acquisition Phase
	<ol>
		<li>One day prior to the training familiarize the animals with the pellets used in the Staircase test.
		<ol>
			<li>Optional: To increase the animal&#39;s motivation use a dietary restriction (10-15 g of standard laboratory chow to maintain body weight at 90% of the free feeding level16). However, this is not obligatory to achieve a positive training effect. This study was conducted without food restriction.</li>
		</ol>
		</li>
		<li>Prepare a clear plastic glass staircase box (height: 34.5 cm, length: 35.5 cm, width: 12 cm and narrow compartment 6 cm) by cleaning the box with a 0.1% acetic acid solution. Note: The staircase box is a two-compartment-box with a raised platform and two stairs in the narrow compartment. The left steps on the stairs in the narrow compartment can be reached only with the left paw, the right steps only with the right paw. 
		Note: The standard staircase boxes consist of two compartments with a lid, if it is to be used for experiments with rats stimulated via cable, use a high box without lid.</li>
		<li>Remove the staircase and fill the wells on each step with eight 45 mg pellets.</li>
		<li>Insert the staircase and put eight additional pellets on the heightened platform.</li>
		<li>Place the rat in the transport box.</li>
		<li>Transport the rat from home cage to the staircase box using the transport box.</li>
		<li>Place the rat in the staircase box (Figure 5).</li>
		<li>After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box.</li>
		<li>Note how many pellets were eaten from platform and (eventually) from right and left staircase.</li>
		<li>Refill the staircase by filling the wells on each step with eight 45 mg pellets.</li>
		<li>Clean the staircase box with a 0.1% acetic acid solution and place the additional pellets on the platform.</li>
		<li>Repeat this procedure (acquisition phase) three days in a row. 
		Note: All experiments described were performed on male Sprague Dawley rats. The duration of the different training modules can differ in rats of different strain, sex and vender.</li>
	</ol>
	</li>
	<li>Free Choice Test
	<ol>
		<li>Clean the staircase box with a 0.1% acetic acid solution.</li>
		<li>Remove the staircase and fill the wells on each step with eight 45 mg pellets.</li>
		<li>Place the rat in the transport box.</li>
		<li>Transport the rat from home cage to the staircase box using the transport box.</li>
		<li>Place the rat in the staircase box.</li>
		<li>After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box. 
		Note how many pellets were eaten from right and left staircase.</li>
		<li>Note: If the animals still have problems with grasping pellet, add some more on the platform where they can be easily reached.</li>
		<li>Refill the staircase by filling the wells on each step with eight 45 mg pellets.</li>
		<li>Clean the staircase box with a 0.1% acetic acid solution for the next animal.</li>
		<li>Repeat this procedure (free choice phase) three days in a row. 
		Note: The presented results were obtained by training conducted without a resting period between modules. Some groups prefer a resting day for consolidation, to support the training process.</li>
	</ol>
	</li>
	<li>Forced Choice Test
	<ol>
		<li>Clean the staircase box with a 0.1% acetic acid solution.</li>
		<li>Remove the staircase and fill the wells on each step on the left staircase with eight (first three days of the module) or four (consecutive three days of the module) 45 mg pellets.
		<ol>
			<li>Perform the forced choice test on the side, where the impairment will occur. 
			Note: We perform the Parkinson lesion on the right hemisphere and therefore train selectively the left paw.</li>
		</ol>
		</li>
		<li>Place the rat in the transport box.</li>
		<li>Transport the rat from home cage to the staircase box using the transport box.</li>
		<li>Place the rat in the staircase box.</li>
		<li>After five minutes, remove the animal from the staircase box and put it back into the home cage using the transport box.</li>
		<li>Note how many pellets were eaten from left staircase.</li>
		<li>Refill the staircase by filling the wells on each step with eight or four 45 mg pellets (number of pellets depends on training day).</li>
		<li>Clean the staircase box with a 0.1% acetic acid solution for the next animal.</li>
		<li>Repeat this procedure (forced choice phase) six days in a row.</li>
	</ol>
	</li>
	<li>Data Acquisition
	<ol>
		<li>Perform the experiment as described for the forced choice module (four pellets in each well on the left staircase) on two consecutive days. Calculate the success rate (number of pellets eaten) as mean of the two days.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Fasano, A., Lozano, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+for+movement+disorders:+2015+and+beyond.">Deep brain stimulation for movement disorders: 2015 and beyond.</a> Current opinion in neurology. , (2015).</li><li>McIntyre, C. C., Savasta, M., Kerkerian-Le Goff, L., Vitek, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+the+mechanism(s)+of+action+of+deep+brain+stimulation:+activation,+inhibition,+or+both.">Uncovering the mechanism(s) of action of deep brain stimulation: activation, inhibition, or both.</a> Clinical neurophysiology : official journal of the International Federation of Clinical Neurophysiology. 115, 1239-1248 (2004).</li><li>Deniau, J. M., Degos, B., Bosch, C., Maurice, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+mechanisms:+beyond+the+concept+of+local+functional+inhibition.">Deep brain stimulation mechanisms: beyond the concept of local functional inhibition.</a> The European journal of neuroscience. 32, 1080-1091 (2010).</li><li>Modolo, J., Legros, A., Thomas, A. W., Beuter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-driven+therapeutic+treatment+of+neurological+disorders:+reshaping+brain+rhythms+with+neuromodulation.">Model-driven therapeutic treatment of neurological disorders: reshaping brain rhythms with neuromodulation.</a> Interface focus. 1, 61-74 (2011).</li><li>Groppa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+anatomical+decomposition+of+subthalamic+neurostimulation+effects+in+essential+tremor.">Physiological and anatomical decomposition of subthalamic neurostimulation effects in essential tremor.</a> Brain : a journal of neurology. 137, 109-121 (2014).</li><li>Reich, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+pulse+width+widens+the+therapeutic+window+of+subthalamic+neurostimulation.">Short pulse width widens the therapeutic window of subthalamic neurostimulation.</a> Annals of clinical and translational neurology. 2, 427-432 (2015).</li><li>Blandini, F., Armentero, M. T., Martignoni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+6-hydroxydopamine+model:+news+from+the+past.">The 6-hydroxydopamine model: news from the past.</a> Parkinsonism &amp; related disorders. 14, 124-129 (2008).</li><li>Bove, J., Perier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxin-based+models+of+Parkinson's+disease.">Neurotoxin-based models of Parkinson's disease.</a> Neuroscience. 211, 51-76 (2012).</li><li>Metz, G. A., Tse, A., Ballermann, M., Smith, L. K., Fouad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unilateral+6-OHDA+rat+model+of+Parkinson's+disease+revisited:+an+electromyographic+and+behavioural+analysis.">The unilateral 6-OHDA rat model of Parkinson's disease revisited: an electromyographic and behavioural analysis.</a> The European journal of neuroscience. 22, 735-744 (2005).</li><li>Miklyaeva, E. I., Castaneda, E., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+deficits+in+unilateral+dopamine-depleted+rats:+impairments+in+movement+and+posture+and+compensatory+adjustments.">Skilled reaching deficits in unilateral dopamine-depleted rats: impairments in movement and posture and compensatory adjustments.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 14, 7148-7158 (1994).</li><li>Li, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+stimulation+of+the+subthalamic+nucleus+restores+neural+and+behavioral+functions+during+reaction+time+task+in+a+rat+model+of+Parkinson's+disease.">High-frequency stimulation of the subthalamic nucleus restores neural and behavioral functions during reaction time task in a rat model of Parkinson's disease.</a> Journal of neuroscience research. 88, 1510-1521 (2010).</li><li>Darbaky, Y., Forni, C., Amalric, M., Baunez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+stimulation+of+the+subthalamic+nucleus+has+beneficial+antiparkinsonian+effects+on+motor+functions+in+rats,+but+less+efficiency+in+a+choice+reaction+time+task.">High frequency stimulation of the subthalamic nucleus has beneficial antiparkinsonian effects on motor functions in rats, but less efficiency in a choice reaction time task.</a> The European journal of neuroscience. 18, 951-956 (2003).</li><li>Fang, X., Sugiyama, K., Akamine, S., Namba, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+motor+behavioral+tests+during+deep+brain+stimulation+of+the+subthalamic+nucleus+in+rats+with+different+degrees+of+unilateral+parkinsonism.">Improvements in motor behavioral tests during deep brain stimulation of the subthalamic nucleus in rats with different degrees of unilateral parkinsonism.</a> Brain research. 1120, 202-210 (2006).</li><li>Lindemann, C., Krauss, J. K., Schwabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+of+the+subthalamic+nucleus+in+the+6-hydroxydopamine+rat+model+of+Parkinson's+disease:+effects+on+sensorimotor+gating.">Deep brain stimulation of the subthalamic nucleus in the 6-hydroxydopamine rat model of Parkinson's disease: effects on sensorimotor gating.</a> Behavioural brain research. 230, 243-250 (2012).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Montoya, C. P., Campbell-Hope, L. J., Pemberton, K. D., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'staircase+test':+a+measure+of+independent+forelimb+reaching+and+grasping+abilities+in+rats.">The 'staircase test': a measure of independent forelimb reaching and grasping abilities in rats.</a> Journal of neuroscience. 36, 219-228 (1991).</li><li>Kloth, V., Klein, A., Loettrich, D., Nikkhah, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colour-coded+pellets+increase+the+sensitivity+of+the+staircase+test+to+differentiate+skilled+forelimb+performances+of+control+and+6-hydroxydopamine+lesioned+rats.">Colour-coded pellets increase the sensitivity of the staircase test to differentiate skilled forelimb performances of control and 6-hydroxydopamine lesioned rats.</a> Brain research bulletin. 70, 68-80 (2006).</li><li>Fluri, F., Volkmann, J., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+guided+implantation+of+electrodes+into+the+subthalamic+nucleus+of+rats+for+long-term+deep+brain+stimulation.">Microelectrode guided implantation of electrodes into the subthalamic nucleus of rats for long-term deep brain stimulation.</a> JoVE. , (2015).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotactic coordinates. , (2008).</li><li>Nikkhah, G., Rosenthal, C., Hedrich, H. J., Samii, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+acquisition+and+full+performance+in+skilled+forelimb+use+as+measured+by+the+'staircase+test'+in+five+rat+strains.">Differences in acquisition and full performance in skilled forelimb use as measured by the 'staircase test' in five rat strains.</a> Behavioural brain research. 92, 85-95 (1998).</li><li>Angelov, S. D., Dietrich, C., Krauss, J. K., Schwabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Deep+Brain+Stimulation+in+Rats+Selectively+Bred+for+Reduced+Prepulse+Inhibition.">Effect of Deep Brain Stimulation in Rats Selectively Bred for Reduced Prepulse Inhibition.</a> Brain stimulation. , (2014).</li><li>de Haas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+implantable+micro-stimulation+device+for+high+frequency+bilateral+deep+brain+stimulation+in+freely+moving+mice.">Wireless implantable micro-stimulation device for high frequency bilateral deep brain stimulation in freely moving mice.</a> Journal of neuroscience methods. 209, 113-119 (2012).</li><li>Heo, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+Implantable+Deep+Brain+Stimulation+System+with+Wireless+Power+Transmission+for+Long-term+Use+in+Rodent+Models+of+Parkinson's+Disease.">Fully Implantable Deep Brain Stimulation System with Wireless Power Transmission for Long-term Use in Rodent Models of Parkinson's Disease.</a> Journal of Korean Neurosurgical Society. 57, 152-158 (2015).</li><li>Gut, N. K., Winn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+brain+stimulation+of+different+pedunculopontine+targets+in+a+novel+rodent+model+of+parkinsonism.">Deep brain stimulation of different pedunculopontine targets in a novel rodent model of parkinsonism.</a> J. Neurosci. 35, 4792-4803 (2015).</li><li>Whishaw, I. Q., Gorny, B., Foroud, A., Kleim, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Evans+and+Sprague-Dawley+rats+have+similar+skilled+reaching+success+and+limb+representations+in+motor+cortex+but+different+movements:+some+cautionary+insights+into+the+selection+of+rat+strains+for+neurobiological+motor+research.">Long-Evans and Sprague-Dawley rats have similar skilled reaching success and limb representations in motor cortex but different movements: some cautionary insights into the selection of rat strains for neurobiological motor research.</a> Behavioural brain research. 145, 221-232 (2003).</li><li>Honndorf, S., Lindemann, C., Tollner, K., Gernert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+Wistar+rats+obtained+from+different+breeders+vary+in+anxiety-like+behavior+and+epileptogenesis.">Female Wistar rats obtained from different breeders vary in anxiety-like behavior and epileptogenesis.</a> Epilepsy research. 94, 26-38 (2011).</li><li>Jadavji, N. M., Metz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+skilled+movement+in+response+to+restraint+stress+and+recovery+from+stress.">Sex differences in skilled movement in response to restraint stress and recovery from stress.</a> Behavioural brain research. 195, 251-259 (2008).</li><li>Kucker, S., Tollner, K., Piechotta, M., Gernert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kindling+as+a+model+of+temporal+lobe+epilepsy+induces+bilateral+changes+in+spontaneous+striatal+activity.">Kindling as a model of temporal lobe epilepsy induces bilateral changes in spontaneous striatal activity.</a> Neurobiology of disease. 37, 661-672 (2010).</li><li>Smith, L. K., Metz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+alters+fine+motor+function+in+rats.">Dietary restriction alters fine motor function in rats.</a> Physiology &amp; behavior. 85, 581-592 (2005).</li></ol>

The authors declare that they have no competing financial interests.

This article describes a detailed training protocol for the cylinder and staircase test. The latter is designed to assess complex grasping behavior and fine motor movement due to skilled reaching in rats16,17. The outcome measurement is expressed as number of pellets eaten during the test, which is an objective measurement. The protocol can be used in rat models for Parkinson&#39;s disease and other motor disease models. The cylinder test involves a simple approach to evaluate paw use in rats. It requires no training and can be used in a highly standardized way, by blinded evaluation from a videotape. We choose these tests among others (open field, single pellet reaching test, and gait analysis) for several reasons. Both tests reliably show the paw impairment created in our Parkinson&#39;s model10. They are relatively robust in outcome and the test results can be obtained in a highly objective manner. Both tests can be used repeatedly in short time intervals. In the staircase test, pellet grasping increases during the learning phase and stabilizes on an individual plateau level, so after reaching plateau, it can be used without having to consider training-dependent improvement over time16,20.
Whereas the cylinder test is easy to apply, rats can became bored and inactive. The animals can be motivated by darkness (performing the test with red light) or late in the light-dark-cycle. The measurement of rearings is helpful to monitor the rat&#39;s normal activity. For successful staircase training, it is crucial to motivate the animals during the acquisition phase. It is important to ensure that the rat is able to grab some pellets at the first grasping approach. The immediate positive reward results in good training effects in later phases. Another critical step occurs when the rat is not interested in the task. In this case it may be necessary to apply a mild food restriction until the proper motivation level is reached.
Another important issue for our study design was a good predictive validity of the established procedure. The design was planned to be used as a screening platform for novel stimulation protocols. Although deep brain stimulation is a treatment option not only for Parkinson&#39;s disease, but also for tremor, depression, compulsive disorders, dystonia and many other conditions, the mechanisms lying behind it effectiveness remains poorly understood1,21. To address novel, rational based stimulation approaches a good animal model is required.
A more pragmatic approach forced us to choose a behavioral setup which could be performed by animals connected via cable to the stimulation device. The cylinder was appropriate for this purpose. The commercially available staircase box has a lid, therefore we designed a copy of the original box, which is higher and without a lid. This allows to test performance during deep brain stimulation. The problem of cable driven stimulation is common in preclinical research. There are some devices enabling stimulation without a cable but their use is still limited22-24. For our research we have to set different stimulation patterns and also perform long term stimulation. At present this can be done only by cable driven stimulation because it allows a quick reprogramming of various stimulation features without touching the animal. In this respect this study design is suited for most research groups dealing with motor outcome in deep brain stimulated rats.
In summary, this manuscript presents a full length protocol for studying motor outcome in hemiparkinsonian rats under different experimental DBS conditions. It describes a detailed protocol for training in the staircase box, as well as the use of the cylinder test. Problems associated with the described training method may occur when rats of different strain, sex or vender are used. Rats differ according to sex and strain in their performance of behavioral tests20,25,26. In female rats the estrus cycle has also an impact on their daily performance27. To deal with this limitation, female rats can be housed without males which desynchronizes the estrus cycle28. It can also be necessary to adjust the duration of the training phase according to individual learning curves in outbreed rat strains. Food restriction often used in behavioral tests should be applied carefully. Food deprivation may increase motivation but on the other hand decreases the accuracy of grasping20,29. The described behavioral battery, in combination with the hemiparkinsonian model, can be used to study different treatment options and their impact on motor outcome. For deep brain stimulation of the subthalamic nucleus, this study design has a high predictive validity.

Deep brain stimulation of the subthalamic nucleus (STN) is an effective treatment option for Parkinson&#39;s disease1 and other movement disorders. The underlying mechanisms are still poorly understood and multifactorial, but a key feature is the modulation of neuronal network activity by repetitive depolarization of axons in the vicinity of the stimulating electrode2-4. High-frequency (&#62;100 Hz) stimulation is required for a beneficial effect in most brain targets and for most indications of DBS. Side effects of deep brain stimulation result from inadvertent coactivation of other fibers, which are covered by the stimulation volume and which subserve different functions, such as the pyramidal tract. Hence, it would be desirable to develop stimulation parameters, which preferentially activate beneficial neural elements, while avoiding the coactivation of side effect elements5,6. Although neurophysiology may offer such fine tuning options of DBS, the scientific progress has been minimal during the past two decades, because programming strategies have primarily been assessed by &#34;trial and error&#34; in patients and restricted by the limited programming options of commercially available DBS devices, rather than using neurophysiological insight and defined experimental settings to systematically explore the full parameter space.
To overcome the translational roadblock in DBS research we are proposing a protocol to screen alternative stimulation parameters in rodent models of Parkinsonism prior to clinical exploration. Unilateral Parkinson&#39;s disease in rats is modeled using 6-hydroxydopamine injections into the right medial forebrain bundle7,8. The resulting lesion, described further as hemiparkinsonian, is assessed in the apomorphine test by evaluation of rotation score after low dose apomorphine injection and confirmed post mortem by tyrosine hydroxylase immunohistochemistry. The method is easy to apply and highly reproducible, while bearing a low mortality and morbidity. The resulting motor deficits are very discrete7,8; the animals exhibit a slight impairment of the contralateral left paw during both spontaneous exploration and complex grasping behavior9,10.
To assess the effectiveness of deep brain stimulation protocols tests are required which allow measuring a quick and reliable change in motor performance and can be repeated over time with different neurostimulation settings. Several groups have proposed different stimulation approaches and different tests to assess the motor functions in rats11 with highly variable and inconsistent outcomes11-14. This forced us to choose a set of tests with high predict validity and complementarity. Additionally, for assessment of motor outcome under deep brain stimulation conditions, tests were favored which could be performed by animals connected via cable to the stimulus generator. For these purposes we established our test battery consisting of one test for paw use asymmetry and one test for skilled reaching. The study design is illustrated in Figure 1.
For spontaneous paw use we performed the cylinder test described by Shallert15, which is a widely used test for paw use during vertical exploration. No training of the animal is required. For the assessment of more complex grasping behavior we established the staircase test according to Montoya16. Our protocol is modified according to Kloth17. The rats are trained for a period of twelve days in reaching pellets from the test box. After the training period the test can be applied to measure the complex grasping behavior by counting the success rate described as number of pellets eaten. The article presents the detailed training in the staircase box as well as the performance of both behavioral tests under na&#239;ve, hemiparkinsonian and deep brain stimulation conditions.

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			<td height="42" style="height:42px;width:216px;">Staircase box witout lid</td>
			<td style="width:71px;">Glas Keil, Germany</td>
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			<td style="width:167px;">custom made</td>
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			<td height="42" style="height:42px;width:216px;">Cylinder box</td>
			<td style="width:71px;">Glas Keil, Germany</td>
			<td style="width:119px;"></td>
			<td>custom made</td>
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			<td height="42" style="height:42px;width:216px;">Dustless precision pellets, 45 mg</td>
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			<td>F0021</td>
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a novel approach to assess motor outcome of deep brain stimulation effects in the hemiparkinsonian rat: staircase and cylinder test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

All animals underwent a post mortem histological verification of both the dopaminergic lesion and the electrode location. Only animals with correct electrode placement inside the STN (Figure 6) and complete dopaminergic lesion (&#62;90% loss of dopaminergic neurons in substantia nigra) were included into the results section (Figure 7).
The cylinder test performed under lesioned condition showed that the lesioned left paw use decreased from approximately 50% (na&#239;ve, healthy rat) to 15.11% (mean). Under regular 130 Hz stimulation following a 24 hr stimulation period (pulse width 60 &#181;sec), which is the standard protocol in Parkinson&#39;s patients, the paw use increased to 21.9% (Figure 8). Due to a high standard deviation this difference is not statistically significant. The number of rearings showed no differences between sham and stimulated condition, but this parameter will be included in further studies as measurement of the rat&#39;s activity. The performance was apparently not disturbed by the stimulation cable (Figure 9).
All animals used in our deep brain stimulation study were able to learn the pellet reaching within the described timeline of twelve days. The mean success rate before lesioning was 13.5 out of 28 pellets (48.2%). After lesioning the mean success rate decreased significantly to 2.4 pellets (8.6%). It increased again to 7 pellets (25%) under regular 130 Hz stimulation at the end of a 24 hr stimulation period (Figure 10). The performance was not disturbed by the stimulation cable (Figure 7). The two motor tests were complementary in assessing motor deficits in the hemiparkinsonian rat and had a good predict validity based on the improvement under default therapeutic conditions. The stimulation cable seemed to have no impact on the test performance (Figure 11).
<img alt="Figure 1" src="/files/ftp_upload/53951/53951fig1.jpg" /> 
Figure 1: Study design. The single steps to conduct an experiment on different stimulation protocols in hemiparkinsonian rats. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53951/53951fig2.jpg" /> 
Figure 2: Cylinder test setup. The cylinder box arranged with mirrors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53951/53951fig3.jpg" /> 
Figure 3: Cylinder test. A hemiparkinsonian rat during cylinder test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53951/53951fig4.jpg" /> 
Figure 4: Staircase test setup. The staircase box arranged with pellets on left staircase. (A) side view, (B) view from above. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53951/53951fig5.jpg" /> 
Figure 5: Staircase test. A hemiparkinsonian rat during staircase test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53951/53951fig6.jpg" /> 
Figure 6: Stimulation site. Coronary rat brain section (thionine staining) with right STN indicated by black circle and the corresponding spike signal recorded during implantation surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53951/53951fig7.jpg" /> 
Figure 7: Documentation of the 6-OHDA lesion. Immunohistochemistry for tyrosine hydroxylase, a marker enzyme of dopaminergic neurons. Coronary section of the rat brain after unilateral 6-OHDA lesion. Comparison of the left healthy side (Le) and the lesioned right side (Ri). Unilateral loss of dopaminergic fibers in the Striatum (a) and dopaminergic neurons in the Substantia nigra pars compacta (SNc), (c). Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53951/53951fig8.jpg" /> 
Figure 8: Paw use cylinder test. The results of the cylinder test expressed as contralateral (affected due to lesion) paw use in percent (100%&#160;&#8212; ipsilateral paw use [%]), under various conditions (lesioned vs. 130 Hz stimulation). The data are given as mean &#177; SEM, n = 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/53951/53951fig9.jpg" /> 
Figure 9: Cylinder test with stimulation cable. A stimulated hemiparkinsonian rat during cylinder test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/53951/53951fig10.jpg" /> 
Figure 10: Success rate in the staircase test. The results of the staircase test expressed as number of grasped pellets under various conditions (healthy, lesioned and 130 Hz stimulation). The data are given as mean &#177; SEM, n = 7. P &#60;0.05 (*) was considered statistically significant (one way ANOVA + t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/53951/53951fig11.jpg" /> 
Figure 11: Staircase test with stimulation cable. A stimulated hemiparkinsonian rat during staircase test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53951/53951fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test at JoVE.com

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

a-novel-approach-to-assess-motor-outcome-deep-brain-stimulation

Nanyang Technological University

Research

JoVE Journal

Behavior

14.6K Views.  University Hospital Wuerzburg. Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Video: A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as &#34;inchworming,&#34; was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.

Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal ...

Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore by rearing and touching the walls of the cylinder with their forelimb paws for postural support. Following ischemic injury to the forelimb sensorimotor cortex, rats rely more heavily on their unaffected forelimb paw for postural support resulting in fewer touches with their affected paw which is termed forelimb asymmetry. In contrast, focal ischemic damage in the mouse brain fails to result in comparable consistent deficits in forelimb asymmetry. While forelimb asymmetry deficits are infrequently observed, mice do demonstrate a novel behaviour post stroke termed &#8220;paw-dragging&#8221;. Paw-dragging is the tendency for a mouse to drag its affected paw along the cylinder wall rather than directly push off from the wall when dismounting from a rear to a four-legged stance. We have previously demonstrated that paw-dragging behaviour is highly sensitive to small cortical ischemic injuries to the forelimb motor cortex. Here we provide a detailed protocol for paw-dragging analysis. We define what a paw-drag is and demonstrate how to quantify paw-dragging behaviour. The cylinder test is a simple and inexpensive test to administer and does not require pre-training or food deprivation strategies. In using paw-dragging analysis with the cylinder test, it fills a niche for predicting cortical ischemic injuries such as photothrombosis and Endothelin-1 (ET-1)-induced ischemia &#8211; two models that are ever-increasing in popularity and produce smaller focal injuries than middle cerebral artery occlusion. Finally, measuring paw-dragging behaviour in the cylinder test will allow studies of functional recovery after cortical injury using a wide cohort of transgenic mouse strains where previous forelimb asymmetry analysis has failed to detect consistent deficits.

Classical forelimb asymmetry analysis of the cylinder test is routinely used to assess behavioural deficits in rats following brain injury or stroke; however, it fails to detect consistent deficits in mice. This study demonstrates that quantifying paw-dragging behaviour is a more sensitive analysis of brain injury in mice.

The cylinder test is routinely used to predict focal ischemic damage to the forelimb motor cortex in rodents. When placed in the cylinder, rodents explore ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Sit-to-stand-and-walk from 120% Knee Height: A Novel Approach to Assess Dynamic Postural Control Independent of Lead-limb

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: STW). Thus, in clinical rehabilitation separation of sit-to-stand and gait initiation - termed sit-to-stand-and-walk (STSW) - is usual. However, a standardized STSW protocol with a clearly defined analytical approach suitable for pathological assessment has yet to be defined.
Hence, a goal-orientated protocol is defined that is suitable for healthy and compromised individuals by requiring the rising phase to be initiated from 120% knee height with a wide base of support independent of lead limb. Optical capture of three-dimensional (3D) segmental movement trajectories, and force platforms to yield two-dimensional (2D) center-of-pressure (COP) trajectories permit tracking of the horizontal distance between COP and whole-body-center-of-mass (BCOM), the decrease of which increases positional stability but is proposed to represent poor dynamic postural control.
BCOM-COP distance is expressed with and without normalization to subjects&#39; leg length. Whilst COP-BCOM distances vary through STSW, normalized data at the key movement events of seat-off and initial toe-off (TO1) during steps 1 and 2 have low intra and inter subject variability in 5 repeated trials performed by 10 young healthy individuals. Thus, comparing COP-BCOM distance at key events during performance of an STSW paradigm between patients with upper motor neuron injury, or other compromised patient groups, and normative data in young healthy individuals is a novel methodology for evaluation of dynamic postural stability.

Here, we present a novel protocol to measure positional stability at key events during the sit-to-stand-to-walk using the center-of-pressure to the whole-body-center-of-mass distance. This was derived from the force platform and three-dimensional motion-capture technology. The paradigm is reliable and can be utilized for the assessment of neurologically compromised individuals.

Individuals with sensorimotor pathology e.g., stroke have difficulty executing the common task of rising from sitting and initiating gait (sit-to-walk: ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, the traditional ways to monitor behavioral changes in dogs are complicated and not efficient. In the current clinical evaluation, a new, simple monitoring system was used to assess the effectiveness of a specific diet in positively modulating the intense and restless activity of 24 dogs of different ages and breeds. This protocol describes how to easily and rapidly evaluate improvement in a set of symptoms related to generalized anxiety by using a specific sensor, a mobile phone app, a wireless router, and a computer. The results showed that dogs treated with specific diets showed significant improvement in the times spent active and at rest after 10 days (p &lt; 0.01 and p &lt; 0.05, respectively). These dogs also showed an overall significant improvement in clinical and behavioral symptoms. A specific sensor, along with its related hardware, was demonstrated to successfully monitor behavioral changes relating to movement in dogs.

We report a simple approach to evaluate the effectiveness of a specific diet in positively modulating the daily activity and clinical and behavioral symptoms of dogs with evident behavioral disturbances.

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

A Within-subjects Experimental Protocol to Assess the Effects of Social Input on Infant EEG

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants socially interact. Electroencephalography (EEG) power is an advantageous technique to assess infant functional neural activation. However, many studies record infant EEG only during one baseline condition. This protocol describes a paradigm that is designed to comprehensively assess infant EEG activity in both social and nonsocial contexts as well as tease apart how different types of social inputs differentially relate to infant EEG. The within-subjects paradigm includes four controlled conditions. In the nonsocial condition, infants view objects on computer screens. The joint attention condition involves an experimenter directing the infant's attention to pictures. The joint attention condition includes three types of social input: language, face-to-face interaction, and the presence of joint attention. Differences in infant EEG between the nonsocial and joint attention conditions could be due to any of these three types of input. Therefore, two additional conditions (one with language input while the experimenter is hidden behind a screen and one with face-to-face interaction) were included to assess the driving contextual factors in patterns of infant neural activation. Representative results demonstrate that infant EEG power varied by condition, both overall and differentially by brain region, supporting the functional nature of infant EEG power. This technique is advantageous in that it includes conditions that are clearly social or nonsocial and allows for examination of how specific types of social input relate to EEG power. This paradigm can be used to assess how individual differences in age, affect, socioeconomic status, and parent-infant interaction quality relate to the development of the social brain. Based on the demonstrated functional nature of infant EEG power, future studies should consider the role of EEG recording context and design conditions that are clearly social or nonsocial.

This novel protocol is designed to assess the neural bases of social interaction in infants. The paradigm is designed to tease apart how various social inputs such as language, joint attention, and face-to-face interaction relate to infant neural activation. Infant EEG power is recorded during both social and nonsocial conditions.

Despite the importance of social interactions for infant brain development, little research has assessed functional neural activation while infants ...