SN and AGZ were supported by the PON 01-01297 VIRTUALAB funds.

Ethical Statement
To study how sensory stimuli are represented by neuronal activity there is no alternative to the use of animals and the use of an in vivo approach. All the animals have been treated along the Italian and European Laws on animal treatment in Scientific Research (Italian Bioethical Committee, Law Decree on the Treatment of Animals in Research, 27 Jan 1992, No. 116). The National Research Council, where the experiments have been performed, adheres to the International Committee on Laboratory Animal Science (ICLAS) on behalf of the United Nations Educational, Scientific and Cultural Organizations (UNESCO), the Council for International Organizations of Medical Sciences (CIOMS) and the International Union of Biological Sciences (IUBS). As such, no protocol-specific approval was required. The approval of the Ministry of Health is classified as &#34;Biella 1, 3/2011&#34; into the files of the Ethical Committee of the University of Milan.
1. Preparation of the Experimental Animals
<ol>
	<li>Select male rats approximately 300-400 g in size.</li>
	<li>Anesthetize the rats for the experimental preparation.
		<ol>
			<li>Inject intraperitoneally a barbiturate solution (pentobarbital, 50 mg/kg for induction, 10 mg/kg for maintenance).</li>
			<li>Check the anesthesia level by low threshold and high threshold mechanical stimuli over a paw ensuring that no retraction reflex occurs.</li>
			<li>Prepare the cranial skin by shaving the cranial vault accurately and then make an incision to expose the cranial surface.</li>
			<li>Cannulate the trachea, by a tracheal cannula (inner diameter of 2 mm and outer diameter of 2.5 mm) fixing it by surgical ligation around the trachea itself.</li>
			<li>Laterally to the tracheal segment expose the jugular vein and cannulate it inserting a cannula (PE10) connected to a syringe fixing it by surgical ligation around the vessel.</li>
		</ol>
	</li>
	<li>Mount the rat onto a stereotaxic apparatus.
		<ol>
			<li>Secure rat body and place in a supine position.</li>
			<li>Fix the head by ear bars and block the snout. Put one or two drops of local anaesthetic (lidocaine) in the rat&#39;s ear before placing ear bars.</li>
		</ol>
	</li>
	<li>Regulate the temperature of the stereotaxic pad by an electronically controlled heating pad, maintaining the temperature at 37.5 &#176;C.</li>
	<li>Connect the tracheal tube to the respiratory anesthesia device.
		<ol>
			<li>Set the respiratory anesthesia device at 1 stroke/s delivering Isoflurane (2.5% 0.4-0.8 L/min) and O2 (0.15-0.2 L/min).</li>
		</ol>
	</li>
	<li>Clean carefully the cranial vault by a povidone-iodine topical antiseptic.</li>
	<li>Take a sterile scalpel and cut longitudinally on the midline from the basis of the snout to the angle of the occipital bone.
		<ol>
			<li>Divaricate the wound borders by a retractor and fix them by two small cocker forceps applied to the wound borders.</li>
			<li>Identify the periosteum by pointing a light over a skull vault observing the translucent surface. Scratch carefully the bone vault removing the periosteum and exposing the bone surface.</li>
			<li>Provide hemostasis with a cotton tip or surgical sponge over the bone surface if focal hemorrhages appear over the bone.</li>
		</ol>
	</li>
	<li>With a fine tip pen, identify the stereotactic point bregma at the cross point of the mediosagittal and coronal suturas.
		<ol>
			<li>Under surgical microscope control, identify the stereotactic area (S1HL) overlaying the somatosensory cortex contralateral to the posterior paw used for the experiment (Bregma, AP -0.5 to 1.2 mm, LL -2.3 to 2.5 mm).</li>
		</ol>
	</li>
	<li>With a fine tip pen draw the square perimeter delimiting the hole to be drilled.
		<ol>
			<li>Drill a 9 mm2 hole on the previously identified blue square removing the bone tile.</li>
			<li>Clean and absorb potential bone bleedings.</li>
			<li>Carefully remove the dura mater and cover the cortical surface with a surgical sponge soaked in artificial cerebrospinal fluid maintained at 37.5 &#176;C.</li>
		</ol>
	</li>
	<li>Fix the electrode matrix to a holder connected to an electronic micromanipulator.
		<ol>
			<li>Connect the head of the matrix to the preamplifier by a microconnector.</li>
			<li>Drive manually the electrode matrix (under surgical microscope control) up to the cortical surface (without touching it).</li>
			<li>Switch on the amplifiers with auditory signal.</li>
			<li>Drive, by the electronic control, the electrode matrix to touch the cortical surface until the contact is reached, signaled by a clear noise sound change.</li>
			<li>Pull down the electrode matrix by electronically controlled steps (2 &#956;m/step) until a depth of 350-400 &#956;m is reached (cortical layer IV).</li>
			<li>Check the responsiveness of neurons by light touches on the contralateral posterior paw.</li>
			<li>Adjust the depth by a few further steps until a clear spiking response is observed.</li>
			<li>Paralyze the rat by intravenous Gallamine thriethiodide (20 mg/kg/hr). Use refracted doses throughout the experiment to maintain curarization levels (0.2 ml/1 hr).</li>
		</ol>
	</li>
</ol>
2. Signal Treatment
<ol>
	<li>Set the software bandpass filter to [0.1 6000] Hz.
		<ol>
			<li>Record the neuronal signals of the 8 channels microelectrode matrix sampled at 32 kHz.</li>
		</ol>
	</li>
	<li>After the acquisition ends, export the recorded signals into a binary format suitable for post-processing.
		<ol>
			<li>Perform the spike sorting procedure by means of the Wave_clus toolbox11.</li>
		</ol>
	</li>
</ol>
3. Manufacture and Configuration of the Stimulation Device
<ol>
	<li>Mount a cactus stalk (with blunted tip) orthogonally to the surface of a mid-range speaker gluing the stalk basis to the cap.</li>
	<li>Program a microcontroller to deliver voltage pulses to a driver circuit for the speaker.
		<ol>
			<li>Program the microcontroller to deliver two paired pulses of current each second (see Figure 1C).</li>
		</ol>
	</li>
	<li>Assemble the speaker and the microcontroller by means of a L293D h-bridge with basic passive components (see Figure 1A).</li>
	<li>Connect the microcontroller to a 12 V rechargeable battery.</li>
</ol>
4. Stimulation Protocol
<ol>
	<li>Glue the dorsal aspect of the hind-paw to a solid frame, exposing the volar surface and avoiding articular torsions.</li>
	<li>Place the tip of the stimulation device onto the desired limb/paw position.</li>
	<li>Switch on the stimulation device.</li>
</ol>
Offline steps:
5. Evaluation of Spike Responses
<ol>
	<li>For each recorded neuron, compute the Shannon Mutual Information (MI) of the stimulus evoked spiking activity (stimulus category is either 1, stimulus, or 0, no stimulus).
		<ol>
			<li>Estimate the conditional response probability p(r | s) where s represents the stimulus category and r represents the number of spikes emitted within a fixed time window.</li>
			<li>Correct the MI estimate by using the shuffling procedure described9.</li>
		</ol>
	</li>
</ol>
6. Evaluation of LFP Responses
<ol>
	<li>Filter the recorded signal in the frequency range [0.1 100] Hz in order to obtain the LFP signal</li>
	<li>Compute LFPSNR, a measure of LFP responsiveness, as explained in the reference10.</li>
</ol>
7. Model Estimation
<ol>
	<li>Generate a model of the form 
		<img alt="" fo:content-width="2in" height="24" src="/files/ftp_upload/50941/50941eq1.jpg" width="155" /> 
		where the x arguments represent three different LFP features (x1the LFP itself, x2 its derivative and x3 the Hilbert phase of its derivative) and F is expressed as follows 
		<img alt="" fo:content-width="2in" height="57" src="/files/ftp_upload/50941/50941eq2.jpg" width="187" /> 
		the g coefficients are weights of a linear combination and f is an operator that takes either the absolute value, the power or the original value of each xi.</li>
	<li>Use the NSGAII algorithm to estimate the model parameters and operators by using the following three objects: i) a local, trial-to-trial basis, measure of fit <img alt="" fo:content-width="2in" height="40" src="/files/ftp_upload/50941/50941eq3.jpg" width="149" />, where Nsp and Nr represent respectively the overall spikes count and the length of the response vector; ii)a global measure of fit based on the average response <img alt="" fo:content-width="3in" height="45" src="/files/ftp_upload/50941/50941eq4.jpg" width="205" /> where Nresp represents the length of the average response; iii) a measure of model complexity (see also)10 <img alt="" fo:content-width="2in" height="23" src="/files/ftp_upload/50941/50941eq5.jpg" width="161" />.</li>
</ol>
8. Histological Confirmation
<ol>
	<li>Sacrifice the rat.
		<ol>
			<li>At the end of experimental recordings, put the animal under deep gaseous anaesthesia (Isoflurane 2%, 4 L/min) and inject intravenously a barbiturate overdose (&#62;50 mg/kg, pentobarbital). </li>
			<li>Wait the heart arrest.</li>
			<li>Unmount the rat from the stereotaxic apparatus</li>
		</ol>
	</li>
	<li>Perfusion.
		<ol>
			<li>Place the rat laying over a grid onto a basin to collect the blood and fluids from the perfusion.</li>
			<li>Open the thorax by dissecting the sternum separating from the rib insertions.</li>
			<li>Block the sternal xyphoid process with a cocker forceps and overturning cranially the sternum and divaricate the ribs over the heart.</li>
			<li>Identify the left ventricle and the right atrium, place a nine gauge blunted tip needle (connected to the perfusion cannula) into the ventricle and open with surgical fine scissors the right atrium.</li>
			<li>Start the perfusion with cold (4 &#176;C) heparinized physiological solution (250 ml) followed by the perfusion of a 4% formalin solution (250 ml).</li>
		</ol>
	</li>
	<li>Extract the brain from the skull by opening the cranial vault with a suitable forceps and place the brain in a 10% formalin solution.</li>
	<li>After a week, prepare the histological slices by a microtome (10 &#956;m thickness).</li>
	<li>Stain the brain coronal and sagittal sections with cresyl violet solution.</li>
</ol>

<ol>
	<li>Pesaran, B. <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+Mysterious+Origins+of+Local+Field+Potentials.">Uncovering the Mysterious Origins of Local Field Potentials.</a> Neuron. 61 (1-2), (2009).</li><li>Delmas, P., Hao, J., Rodat-Despoix, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Mechanotrasduction+in+Mammalian+Sensory+Neurons.">Molecular Mechanisms of Mechanotrasduction in Mammalian Sensory Neurons.</a> Nat. Rev. Neurosci. 12, 139-153 (2011).</li><li>Rasch, M. J., Gretton, A., Murayama, Y., Maass, W., Logothetis, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+spike+trains+from+local+field+potentials.">Inferring spike trains from local field potentials.</a> J. Neurophys. 99 (3), 1461-1476 (2008).</li><li>Galindo-Leon, E. E., Liu, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+stimulus-locked+single+unit+spiking+from+cortical+local+field+potentials.">Predicting stimulus-locked single unit spiking from cortical local field potentials.</a> J. Comput. Neurosci. 29 (3), 581-597 (2010).</li><li>Theunissen, F. E., David, S. V., Singh, N. C., Hsu, A., Vinje, W. E., Gallant, 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=Estimating+spatio-temporal+receptive+fields+of+auditory+and+visual+neurons+from+their+responses+to+natural+stimuli.">Estimating spatio-temporal receptive fields of auditory and visual neurons from their responses to natural stimuli.</a> Network. 12 (3), 289 (2001).</li><li>Victor, J. D., Purpura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metric-space+analysis+of+spike+trains:+theory,+algorithms,+and+application.">Metric-space analysis of spike trains: theory, algorithms, and application.</a> Network. 8, 127-164 (1997).</li><li>Foffani, G., Chapin, J. K., Moxon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+Role+of+Large+Receptive+Fields+in+the+Primary+Somatosensory+Cortex.">Computational Role of Large Receptive Fields in the Primary Somatosensory Cortex.</a> J. Neurophysiol. 100 (1), 268-280 (2008).</li><li>Panzeri, S., Senatore, R., Montemurro, M. A., Petersen, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correcting+for+the+sampling+bias+problem+in+spike+train+information+measures.">Correcting for the sampling bias problem in spike train information measures.</a> J. Neurophysiol. 98, 1064-1072 (2007).</li><li>Storchi, R., Zippo, A. G., Caramenti, G. C., Valente, M., Biella, G. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Spike+Occurrence+and+Neuronal+Responsiveness+from+LFPs+in+Primary+Somatosensory+Cortex.">Predicting Spike Occurrence and Neuronal Responsiveness from LFPs in Primary Somatosensory Cortex.</a> PLoS ONE. 7 (5), (2012).</li><li>Quiroga, R. Q., Nadasdy, Z., Ben-Shaul, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsupervised+Spike+Detection+and+Sorting+with+Wavelets+and+Superparamagnetic+Clustering.">Unsupervised Spike Detection and Sorting with Wavelets and Superparamagnetic Clustering.</a> Neural Comput. 16, 1661-1687 (2004).</li><li>Deb, K., Agrawal, A., Pratab, A., Meyarivan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast+elitist+non-dominated+sorting+genetic+algorithm+for+multi-objective+optimization:+NSGA-II+IEEE.">A fast elitist non-dominated sorting genetic algorithm for multi-objective optimization: NSGA-II IEEE.</a> Trans. Evol. Comput. 6 (2), 181-197 (2000).</li></ol>

The authors declare no competing financial interests.

This work firstly presented a new, simple and low-cost device enabling to deliver fast and spatially point-like sensory stimuli. Then a randomized paired pulse stimulation protocol and a set of computational analyses were validated. The overall aim was to establish a framework for the estimation of LFP-spike relations in electrophysiological recordings during tactile stimulation.
The device, the protocol and the analytical approach have jointly contributed to the result, namely the first demon...

In the context of signal processing the impulse response provides a fundamental characterization of the behavior of a dynamical system.
Although the ideal impulse stimulus is practically not achievable, it is possible to obtain a reasonable approximation of it by using an actuator element that generates high frequency displacements. This type of light tactile-vibratory stimulation is known to target both deep skin (e.g. fast responding, fast adapting Pacinian corpuscles)2 and superficial receptors (e.g. low-threshold slowly adapting Merkel discoid structures)2.
Current stimulation devices, mainly piezoelectric actuators, are charged with a number of drawbacks, not least resonances and small displacements. To overcome these flaws, an alternative implementation of impulse-like stimulation is proposed by using a blunted tip (a cactus smoothed tip in our case) vertically mounted on the membrane center of a mid-range speaker cone. This provides the advantage of larger displacements and broader frequency spectrum.
An effective application of such a device was the study of the relevant neurophysiological problem of the LFPs to spikes dependency. Because of the subtle temporal association between these electrical events a finely regulated device was needed for delivering peripheral stimuli. The stimuli had to be as fast and spatially selective as possible in order to reduce the &#34;background noise&#34; and sharpen the signals of interest. To this purpose, the stimulation device and the stimulus delivery protocol were jointly optimized for the task. In this paper, we describe the technique and present some representative results.
A stimulation protocol based on randomized paired-pulses has been designed and optimized in order to avoid habituation. This protocol offered the advantage of classical paired pulses and reduced the possibility of spurious locking between stimuli and spontaneous periodical bursts of neuronal activity.
By using this randomized paired pulse it was possible to obtain fast and reliable LFP and spike responses and to capture the special feature of these responses related to the dependence of both LFPs and spikes on the stimulation history. Indeed, from the raw LFP responses, a set of three LFP features (the LFP itself, the LFP first derivative and phase of the first derivative) strongly correlating with the average spike response, was also extracted.
Few methods have been proposed to fit models that predict spikes from LFPs3,4. In general a critical point of the model fitting process, common also to the prediction of spike event from the stimulus signal, is constituted by the appropriate choice of the objective function to maximize/minimize. While a range of objective functions has been proposed (e.g. correlation and coherence)5 none of these jointly captures the whole complexity of spike responses. Accordingly, a novel framework based on multi-objective optimization is introduced. We show that by using the proposed devised and this computational framework it is possible to estimate a set of predictive models based on strong LFP to spike relationships.

<table border="0" cellpadding="0" cellspacing="0" width="1204">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Microstepper</td>
			<td style="width:348px;">AB Transvertex (Stockholm, Sweden)</td>
			<td style="width:119px;"> </td>
			<td style="width:424px;">The microstepper used to pull down the electrode matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">32 channels Cheetah System</td>
			<td style="width:348px;">Neuralynx (MT, USA)</td>
			<td style="width:119px;"> </td>
			<td>The electrophysiological recording system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L293D h-bridge</td>
			<td style="width:348px;">RS Components (Cinisello Balsamo, Italy)</td>
			<td style="width:119px;"> </td>
			<td>The bridge used to connect the microcontroller to the speaker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">H21A3 Optical Interrupter Switch</td>
			<td style="width:348px;">Fairchild Semiconductor Corporation (San Jose, California)</td>
			<td style="width:119px;"> </td>
			<td>The phototransistor used to estabilish the tip displacement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Arduino Uno</td>
			<td style="width:348px;">Arduino (Duemilanove, Italy)</td>
			<td style="width:119px;"> </td>
			<td>The microcontroller used to deliver current pulse to the speaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Microelectrode Matrices GB1</td>
			<td style="width:348px;">FHC</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Isoflurane</td>
			<td style="width:348px;">Rhodia Organique Fine Ltd.</td>
			<td style="width:119px;"> </td>
			<td>The anaestetic used to prepare animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Stereotaxic apparatus</td>
			<td style="width:348px;">Narishighe (Tokyo, Japan)</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sprague-Dawley male rats</td>
			<td style="width:348px;">Charles River (Calco, LC, Italy)</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Gallamine thriethiodide</td>
			<td style="width:348px;">Sigma-Aldrich</td>
			<td style="width:119px;"> </td>
			<td>The compound used to curarize the animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Cresyl violet</td>
			<td style="width:348px;">Sigma-Aldrich</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Topical antiseptics (Betadine 10%)</td>
			<td style="width:348px;">Meda Pharma (Milanm Italy)</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Heparine</td>
			<td style="width:348px;">Sigma-Aldrich</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Formaldehyde</td>
			<td style="width:348px;">Carlo Erba Reagents (Pomigliano Milanese, Milan, Italy)</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple stimulatory device for evoking point-like tactile stimuli: a searchlight for lfp to spike transitions

Current neurophysiological research has the aim to develop methodologies to investigate the signal route from neuron to neuron, namely in the transitions from spikes to Local Field Potentials (LFPs) and from LFPs to spikes.
LFPs have a complex dependence on spike activity and their relation is still poorly understood1. The elucidation of these signal relations would be helpful both for clinical diagnostics (e.g. stimulation paradigms for Deep Brain Stimulation) and for a deeper comprehension of neural coding strategies in normal and pathological conditions (e.g. epilepsy, Parkinson disease, chronic pain). To this aim, one has to solve technical issues related to stimulation devices, stimulation paradigms and computational analyses. Therefore, a custom-made stimulation device was developed in order to deliver stimuli well regulated in space and time that does not incur in mechanical resonance. Subsequently, as an exemplification, a set of reliable LFP-spike relationships was extracted.
The performance of the device was investigated by extracellular recordings, jointly spikes and LFP responses to the applied stimuli, from the rat Primary Somatosensory cortex. Then, by means of a multi-objective optimization strategy, a predictive model for spike occurrence based on LFPs was estimated.
The application of this paradigm shows that the device is adequately suited to deliver high frequency tactile stimulation, outperforming common piezoelectric actuators. As a proof of the efficacy of the device, the following results were presented: 1) the timing and reliability of LFP responses well match the spike responses, 2) LFPs are sensitive to the stimulation history and capture not only the average response but also the trial-to-trial fluctuations in the spike activity and, finally, 3) by using the LFP signal it is possible to estimate a range of predictive models that capture different aspects of the spike activity.

Tip excursion features
To characterize the dynamical properties of the proposed stimulating device, a series of experiments were set up. A specific device which consists of a gallium arsenide infrared emitting diode coupled with a silicon phototransistor was used to assess the tip displacement, the displacement duration and the possible displacement delays. By means of this optical interrupter switch we placed the stimulator tip on the edge of the emitting diode hole (height = 1 mm) and both the ...

Watch this Scientific Journal Video about A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions at JoVE.com

A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions

To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions.

Current neurophysiological research has the aim to develop methodologies to investigate the signal route from neuron to neuron, namely in the transitions ...

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9.8K Views.  National Research Council. To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions.

Video: A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

A Simple Way to Measure Ethanol Sensitivity in Flies

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A Simple Flight Mill for the Study of Tethered Flight in Insects

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have shown that flight mills are valuable tools for the experimental study of insect flight behavior, allowing researchers to examine how factors such as age, host plants, or population source can influence an insects' propensity to disperse. Flight mills allow researchers to measure components of flight such as speed and distance flown. Lack of detailed information about how to build such a device can make their construction appear to be prohibitively complex. We present a simple and relatively inexpensive flight mill for the study of tethered flight in insects. Experimental insects can be tethered with non-toxic adhesives and revolve around an axis by means of a very low friction magnetic bearing. The mill is designed for the study of flight in controlled conditions as it can be used inside an incubator or environmental chamber. The strongest points are the very simple electronic circuitry, the design that allows sixteen insects to fly simultaneously allowing the collection and analysis of a large number of samples in a short time and the potential to use the device in a very limited workspace. This design is extremely flexible, and we have adjusted the mill to accommodate different species of insects of various sizes.

Flight in insects is influenced by a number of factors and the propensity to disperse is an important variable in understanding insect ecology and biological control strategies. We describe the construction and use of a simple, relatively inexpensive, and flexible flight mill for measuring parameters of tethered flight in insects.

Flight in insects can be long-range migratory flights, intermediate-range dispersal flights, or short-range host-seeking flights. Previous studies have ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...