This work was supported by NSERC &amp; AHFMR. The authors thank Mariam Alaverdashvili and Amanda Mauthe-Kaddoura for comments on the manuscript and Hiroe Yamazaki for help on the surgery preparation.

1. Surgery Preparation
<ol>
	<li>Anesthetize the rat with the appropriate type and dose of anesthetics. Here we use urethane (1.5 g/kg, Sigma-Aldrich Co., St. Luis, MO) - prepared as 20% solution by diluting 20 g of urethane in 100 ml of phosphate buffered saline (PBS) i.e. the animal will receive 7.5 ml/kg of urethane solution intraperitonealy (IP). Because of its dose-response curve, the total dose of urethane is divided in to four applications, each separated by approximately 30 minutes. Before the first two applications of urethane, animal is anesthetized with isoflurane administered at a concentration of 3-4% (oxygen at 2 liters per minute) and maintained at 2% (oxygen at up to 2 liters per minute) to prevent an animal from getting stressed by injection. During surgery, an additional administration of isofluorane (2% concentration for 2 minutes) can be used if needed.</li>
	<li>Shave the fur along the incision site (dorsal side of the head) to ensure that the fur will not contaminate the wound and that a sufficient area can be disinfected around the incision site.</li>
	<li>Prepare surgical site antiseptically using organic iodine or chlorhexidine soap and antiseptics (ethanol).</li>
	<li>Prepare silicon probes (NeuroNexus Technologies, Ann Arbor, MI). Probes can be disinfected using 70% ethanol followed by rinsing with distilled water. If needed, probes can be painted with a fluorescent marker prepared antiseptically (e.g. Dye I, Invitrogen Co.), to later reveal the position of the probe in histological analysis. To do this we use a small and very soft paint brush and delicately touch the back of the silicon probe with the dye. This procedure should always be done over a microscope to make sure the probe is not being damaged or bent. Always check to make sure that the dye is not blocking any of the channels. Any residual dye left on the probe can be later removed with alcohol, followed by rinsing with water.</li>
	<li>Silicon electrodes care: Because of their small size, silicon probes are delicate and extremely susceptible to breaking even when using it with considerable care. Thus, we recommend monitoring potential hazards at all times, such as: (i) Keep the probes in an appropriately designated box; (ii) Prevent crashing or dropping probes when handling them; (iii) Make sure that the stereotaxic manipulators are well fixed to prevent any accidents; (iv) When recording, surrounding tissue and/or blood can attach to the probes. Therefore, it is important to be also careful when removing the probes after recording. Using a constant flow of PBS or saline solution is strongly recommended; (v) Always clean the probes after recording. To clean the probes, it is strongly recommended to submerge them in a liquid enzymatic cleaner (Boston, Bausch &amp; Lomb) for a few minutes. Always rinse the probes with distilled water and return them to their dedicated box. In our experience, adequate care of the silicon probes ensures good quality recordings for more than 10 experiments. If a channel 'gets noisy' during use, it should be excluded from subsequent analyses. We have found that cross-talk between channels is minimal; therefore a noisy channel usually has very little effect on a signal in other channels.</li>
</ol>
2. Surgery
<ol>
	<li>Apply an eye-ointment on eye surface before any surgery manipulations.</li>
	<li>Administer Dexamethasone 5 mg/ml subcutaneously to reduce brain inflammation.</li>
	<li>Fix the animal in stereotaxic frame throughout surgery.</li>
	<li>Administer Lidocaine HCl 2% with Epinephrine (2-4 mg/kg diluted to 0.5 % solution) subcutaneously at the incision site before the incision is made to reduce the potential risk of the wound bleeding and for local anesthesia.</li>
	<li>Make a 2 cm incision through the scalp midline and displace the skin laterally to expose the skull surface.</li>
	<li>Clear the dorsal scalp from fascia by blunt dissection and control the bleeding by microcautery or by using bone wax.</li>
	<li>Make two drilling holes to attach small screws in the occipital bone of the skull for animal grounding and reference. The reference screw must be in contact with dura. In order to reduce electrical noise during the recordings, it is recommended to keep the screws wet at all times. To do this, a small acrylic barrier surrounding the screws can be built or the screws can be covered with agar.</li>
	<li>Make a 1-2 mm diameter craniotomy in the coordinates corresponding to the cortical and/or subcortical area(s) of interest 5. Use compressed air to remove bone dust as needed during craniotomy (drilling hole). Prevent skull warm up from drilling by applying drops of PBS. Make a second craniotomy if needed to insert the second probe. In this video, we are presenting a recording from two cortical areas simultaneously, therefore two craniotomies are made.</li>
	<li>Build a small barrier of acrylic resin (e.g.: Lang Dental Manufacturing, Co., Inc., Wheeting, IL) surrounding the two craniotomies in order to keep PBS on top of exposed dura, preventing it from drying. Apply PBS constantly during the whole experiment.</li>
	<li>Open the dura matter of both craniotomies. Two needles (30 &frac12; gauge) bended on the tips (forming a hook), may be used for this manipulation.</li>
	<li>Attach the connector of the silicon probes to the stereotaxic manipulatiors. Connectors are attached to a pole in order to have the shanks of the probes perfectly parallel to the stereotaxic manipulator. This is crucial to maintain stable recordings and avoid probe damage.</li>
	<li>Connect the reference screw to the silicon probe reference pin. To reduce electrical noise the ground screws have to be connected to: i) the ground of the probe plug; ii) the chassis ground from the recording device; and iii) the stereotaxic frame.</li>
	<li>Place the tip of the silicon probes just in contact with the pia.</li>
	<li>Slowly introduce the silicon probes and make sure that the shanks of the silicon probes (Fig. 1 &amp; 2) can be inserted into the brain without resistance or bending.</li>
	<li>Carefully lower the probes until reaching desired recording area. If needed, add drops of PBS on the top of craniotomies to avoid drying of the tissue.</li>
	<li>Because urethane anesthesia should only be used for terminal procedures, the animal is euthanized by an i.p. injection of at least 200mg/kg of sodium pentobarbital diluted to a concentration of no more than 60 mg/ml. This is followed by decapitation of the animal.</li>
</ol>
3. Representative Results:
Representative recordings of local field potentials and spiking activity are illustrated in Figure 3A. After performing spike sorting (discriminating spikes originating from different neurons; Fig 3B,C; 6, 7, 8) silicon probe recordings provide the ability to investigate neuronal population activity involved in, among other, the following processes: memory and decision making 9, plasticity 10, stimulus coding 11, spontaneous activity 12 and effects of various drugs 13.
<img alt="Figure 1" src="/files/ftp_upload/3282/3282fig1.jpg" /> 
Figure 1. A. Example of a silicon probe with a single shank is superimposed on a montage of reconstructed neurons (courtesy of S. Sakata 14). B. Example of a probe with a tetrode configuration at each of the 8 shanks. C. Schematic of a rat skull. The green boxes represent the extent of the craniotomy over the right and left somatosensory cortex.
<img alt="Figure 2" src="/files/ftp_upload/3282/3282fig2.jpg" /> 
Figure 2. Example of experiment with silicon probes inserted in the prefrontal cortex and hippocampus. Drops of PBS cover the craniotomy to protect the exposed brain from drying. Two screws located over the cerebellum will be connected to ground and reference, respectively.
<img alt="Figure 3" src="/files/ftp_upload/3282/3282fig3.jpg" /> 
Figure 3. A. Image shows 500ms of a representative electrophysiological recording of Local Field Potential (LFP) from one tetrode (note: negative voltage is plotted up on the ordinate). Insert shows - a waveform of a spike. B. Two-dimensional views of unit clusters in feature space from 1 tetrode. Each cluster represents spikes from a single unit (neuron). C. Average spike waveforms from representative units (color coded) at each of the four recording sites from a single tetrode. D. Waveform energy (related to amplitude) of recorded spikes over time. Note the relatively constant energy values for each unit; this indicates a stable recording over the duration of the displayed period.

<ol>
	<li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+recording+of+neuronal+ensembles.">Large-scale recording of neuronal ensembles.</a> Nat. Neurosci. 7, 446-451 (2004).</li><li>Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+neurotechnologies+for+chronic+neural+interfaces:+new+horizons+and+clinical+opportunities.">Advanced neurotechnologies for chronic neural interfaces: new horizons and clinical opportunities.</a> J. Neurosci. 28, 11830-11838 (2008).</li><li>Csicsvari, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+parallel+recording+of+unit+and+local+field+potentials+with+silicon-based+electrodes.">Massively parallel recording of unit and local field potentials with silicon-based electrodes.</a> J. Neurophysiol. 90, 1314-1323 (2003).</li><li>Bartho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neocortical+principal+cells+and+interneurons+by+network+interactions+and+extracellular+features.">Characterization of neocortical principal cells and interneurons by network interactions and extracellular features.</a> J. Neurophysiol. 92, 600-608 (2004).</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 stereotaxic coordinates. , (1998).</li><li>Lewicki, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+methods+for+spike+sorting:+the+detection+and+classification+of+neural+action+potentials.">A review of methods for spike sorting: the detection and classification of neural action potentials.</a> Network. 9, R53-R78 (1998).</li><li>Harris, K. D., Henze, D. A., Csicsvari, J., Hirase, H., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+tetrode+spike+separation+as+determined+by+simultaneous+intracellular+and+extracellular+measurements.">Accuracy of tetrode spike separation as determined by simultaneous intracellular and extracellular measurements.</a> J. Neurophysiol. 84, 401-414 (2000).</li><li>Luczak, A., Narayanan, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+representation--analyzing+single-unit+activity+in+extracellularly+recorded+neuronal+data+without+spike+sorting.">Spectral representation--analyzing single-unit activity in extracellularly recorded neuronal data without spike sorting.</a> J. Neurosci. Methods. 144, 53-61 (2005).</li><li>Pastalkova, E., Itskov, V., Amarasingham, A., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internally+generated+cell+assembly+sequences+in+the+rat+hippocampus.">Internally generated cell assembly sequences in the rat hippocampus.</a> Science. 321, 1322-1327 (2008).</li><li>Fujisawa, S., Amarasingham, A., Harrison, M. T., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior-dependent+short-term+assembly+dynamics+in+the+medial+prefrontal+cortex.">Behavior-dependent short-term assembly dynamics in the medial prefrontal cortex.</a> Nat Neurosci. 11, 823-833 (2008).</li><li>Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+neurons+work+together?+Lessons+from+auditory+cortex.">How do neurons work together? Lessons from auditory cortex.</a> Hear. Res. 271, 37-53 (2011).</li><li>Luczak, A., Bartho, P., Marguet, S. L., Buzsaki, G., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+structure+of+neocortical+spontaneous+activity+in+vivo.">Sequential structure of neocortical spontaneous activity in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 347-352 (2007).</li><li>Robbe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+reveal+importance+of+spike+timing+coordination+in+hippocampal+function.">Cannabinoids reveal importance of spike timing coordination in hippocampal function.</a> Nat. Neurosci. 9, 1526-1533 (2006).</li><li>Sakata, S., Harris, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+structure+of+spontaneous+and+sensory-evoked+population+activity+in+auditory+cortex.">Laminar structure of spontaneous and sensory-evoked population activity in auditory cortex.</a> Neuron. 64, 404-418 (2009).</li></ol>

No conflicts of interest declared.

This paper demonstrates how to use the
silicon electrode arrays to record from large population of
neurons (&gt;100) in multiple cortical areas simultaneously. In
order to be successful in surgery and recording, the following
issues should be considered:

<ol>
<li>Introduction of the probes to a desired area: When
inserting the probes in brain tissue, it is possible to cause
considerable damage. This can result in a low quality of recorded units. To avoid this problem we recommend the following: (i) Introduce the probes at a certain degree angle (recommended to use a 10 degree angle). By doing this,
dendritic damage of the recorded neurons can be reduced; (ii)
After the probes are inserted in the brain tissue, they are
initially lowered at a faster rate (approximately 50 to 100
microns per 10-30 sec) until they get closer to the desired
area of recording. When the probes are about 200 microns from
the target area, the position is adjusted more slowly
(approximately 10 microns every 2 or 3 minutes).</li>

<li>Stabilization of recordings: When the probes are in the designated area and considerable activity is detected (distinctive spikes in the majority of the channels), it is recommended to wait approximately 30 minutes before starting a recording. This will allow brain tissue to mechanically stabilize after the insertion of probes and ensure a more stable recording.</li>

<li>Brain pulsation: Occasionally it is possible to see brain
pulsation that can significantly reduce the quality of a
recording. A small craniotomy (just enough space to fit the
probe) can reduce brain pulsation in a recording area. If
necessary, the cisterna magna can be punctured. This reduces
cerebrospinal fluid pressure and decreases swelling and
pulsation.</li>

<li>Silicon probe configuration: Silicon probes can have a
variety of shapes and recording site configurations. For
example, they can vary in the number of shanks, the length and
thickness of shanks, and in the arrangement of recording sites
(e.g. tetrode vs. linear configuration; see:
www.neuronexustech.com). The choice of probe used with a
specific configuration depends on the scientific question
needing to be answered. For example, if the objective is to
record populations of neurons in multiple locations in one
specific layer (as presented in these recordings), the best
choice is to use a probe with eight shanks and a tetrode
configuration. This allows for recordings of multiple single
units at each tetrode and sampling of eight locations across a
1.4mm span. In another instance, if someone would like to study
activity propagations across cortical layers, the best choice
would be to use a probe with one shank with regularly
distributed recording sites along that shank which allows
recording in multiple cortical layers simultaneously
14.</li>
</ol>

<table border="1">
			<tbody>
				<tr>
					<td>Name</td>
					<td>Company</td>
					<td>Catalogue Number</td>
				</tr>
				<tr>
					<td>Urethane</td>
					<td>Sigma-Aldrich</td>
					<td>&nbsp;</td>
				</tr>
				<tr>
					<td>Phosphate Buffer Saline (PBS)</td>
					<td>&nbsp;</td>
					<td>&nbsp;</td>
				</tr>
				<tr>
					<td>Isoflurane</td>
					<td>Benson</td>
					<td>02241315</td>
				</tr>
				<tr>
					<td>Silicon probes </td>
					<td>NeuroNexus Technologies</td>
					<td>A 4 X 2 - Tet - 5mm - 150 - 200 &#x2013; 312</td>
				</tr>
				<tr>
					<td>Dye I (DiIC18(3))</td>
					<td>Invitrogen Co.</td>
					<td>617830</td>
				</tr>
				<tr>
					<td>Dexamethasone 5</td>
					<td>Vetoquinol</td>
					<td>0A0640B</td>
				</tr>
				<tr>
					<td>Eye ointment</td>
					<td>Vetoquinol</td>
					<td>005165414</td>
				</tr>
				<tr>
					<td>Lidocaine HCl 2% with Epinephrine </td>
					<td>Bimeda-MTC</td>
					<td>00141984</td>
				</tr>
				<tr>
					<td>Stereotaxic Frame</td>
					<td>KOPF Instruments</td>
					<td>1430-B</td>
				</tr>
				<tr>
					<td>Electrode manipulator and holder</td>
					<td>KOPF Instruments</td>
					<td>960 </td>
				</tr>
				<tr>
					<td>Rat ear-bars</td>
					<td>KOPF Instruments</td>
					<td>1955</td>
				</tr>
				<tr>
					<td>Rat adaptor</td>
					<td>KOPF Instruments</td>
					<td>920</td>
				</tr>
				<tr>
					<td>Anaesthesia mask</td>
					<td>KOPF Instruments</td>
					<td>906</td>
				</tr>
				<tr>
					<td>Scalpel</td>
					<td>Roboz Surgical Instruments</td>
					<td>RS-9843</td>
				</tr>
				<tr>
					<td>Scalpel blades</td>
					<td>Paragon</td>
					<td>0086</td>
				</tr>
				<tr>
					<td>Micro dissecting scissors </td>
					<td>Roboz Surgical Instruments</td>
					<td>RS-5910</td>
				</tr>
				<tr>
					<td>Screws</td>
					<td>Gould Pasteners Limited</td>
					<td>14084701</td>
				</tr>
				<tr>
					<td>Hemostatic forceps</td>
					<td>Roboz Surgical Instruments</td>
					<td>RS-7130 RS-7211</td>
				</tr>
				<tr>
					<td>30&frac12; gauge needles</td>
					<td>Becton-Dickinson</td>
					<td>305115</td>
				</tr><tr>
					<td>Micromotor control</td>
					<td>Foredom</td>
					<td>HP4-917</td>
				</tr>
				<tr>
					<td>Dissecting forceps</td>
					<td>Roboz Surgical Instruments</td>
					<td>RS-5237</td>
				</tr>
				<tr>
					<td>Bone wax</td>
					<td>Lukens</td>
					<td>M263500</td>
				</tr>
				<tr>
					<td>Compressed air bottle</td>
					<td>Falcon safety products, Inc.</td>
					<td>50-10521C</td>
				</tr>
				<tr>
					<td>Enzymatic cleaner </td>
					<td>Boston, Bausch & Lomb</td>
					<td>BAUSCH497628</td>
				</tr>
				<tr>
					<td>Distilled water </td>
					<td>&nbsp;</td>
					<td>&nbsp;</td>
				</tr>
				<tr>
					<td>Dental acrylic powder</td>
					<td>Lang Dental</td>
					<td>1220</td>
				</tr>
				<tr>
					<td>Dental acrylic liquid</td>
					<td>Lang Dental</td>
					<td>1404</td>
				</tr>
				<tr>
					<td>Digital Lynx SX System</td>
					<td>Neuralynx Inc.</td>
					<td>4SX-Z400</td>
				</tr>
				<tr>
					<td>Cheeta Softwere</td>
					<td>Neuralynx Inc.</td>
					<td>&nbsp;</td>
				</tr>
				
				
				
				<tr>
					<td>Alligator cables </td>
					<td>&nbsp;</td>
					<td>&nbsp;</td>
				</tr>
			</tbody>
</table>

recording large-scale neuronal ensembles with silicon probes in the anesthetized rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Watch this Scientific Journal Video about Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat at JoVE.com

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

recording-large-scale-neuronal-ensembles-with-silicon-probes

Research

JoVE Journal

Neuroscience

24.8K vues.  University of Lethbridge. Enregistrements extracellulaires de l'activité neuronale en utilisant des sondes de silicium chez le rat anesthésié seront décrites. Cette technique permet d'obtenir des informations à travers de multiples zones du cerveau de plus de 100 neurones simultanément. Il fournit des informations avec une résolution cellule unique sur la dynamique des ensembles neuronaux dans de multiples circuits locaux.

Vidéo: Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.