Name: investigating object representations in the macaque dorsal visual stream using single-unit recordings
Uploaded: 2018-08-01T15:00:00
Description: Watch this Scientific Journal Video about Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings at JoVE.com

We thank Inez Puttemans, Marc De Paep, Sara De Pril, Wouter Depuydt, Astrid Hermans, Piet Kayenbergh, Gerrit Meulemans, Christophe Ulens, and Stijn Verstraeten for technical and administrative assistance.

All technical procedures were performed in accordance with the National Institute of Health&#39;s Guide for the Care and Use of Laboratory Animals and EU Directive 2010/63/EU and were approved by the Ethical Committee of KU Leuven.
1. General Methods for Extracellular Recordings in Awake Behaving Monkeys
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	<li>Train the animals to perform the visual and motor tasks required to address your specific research question. Ensure that the animal is able to flexibly switch between tasks during ...

<ol>
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C., Pani, P., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coding+of+shape+features+in+the+macaque+anterior+intraparietal+area.">Coding of shape features in the macaque anterior intraparietal area.</a> J. Neurosci. 34 (11), 4006-4021 (2014).</li><li>Sakata, H., Taira, M., Kusonoki, M., Murata, A., Tanaka, Y., Tsutsui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+coding+of+3D+features+of+objects+for+hand+action+in+the+parietal+cortex+of+the+monkey.">Neural coding of 3D features of objects for hand action in the parietal cortex of the monkey.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353 (1373), 1363-1373 (1998).</li><li>Taira, M., Mine, S., Georgopoulos, A. 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Neurosci. 8 (38), 69-86 (2015).</li><li>Theys, T., Pani, P., van Loon, J., Goffin, J., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectivity+for+three-dimensional+contours+and+surfaces+in+the+anterior+intraparietal+area.">Selectivity for three-dimensional contours and surfaces in the anterior intraparietal area.</a> J. Neurophysiol. 107 (3), 995-1008 (2012).</li><li>Goffin, J., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+shape+coding+in+grasping+circuits:+a+comparison+between+the+anterior+intraparietal+area+and+ventral+premotor+area+F5a.">Three-dimensional shape coding in grasping circuits: a comparison between the anterior intraparietal area and ventral premotor area F5a.</a> J. Cogn. Neurosci. 25 (3), 352-364 (2013).</li><li>Raos, V., Umilt&#225;, M. A., Murata, A., Fogassi, L., Gallese, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+grasping-related+neurons+in+the+ventral+premotor+area+F5+of+the+macaque+monkey.">Functional properties of grasping-related neurons in the ventral premotor area F5 of the macaque monkey.</a> J. Neurophysiol. 95 (2), 709-729 (2006).</li><li>Umilta, M. A., Brochier, T., Spinks, R. L., Lemon, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recording+of+macaque+premotor+and+primary+motor+cortex+neuronal+populations+reveals+different+functional+contributions+to+visuomotor+grasp.">Simultaneous recording of macaque premotor and primary motor cortex neuronal populations reveals different functional contributions to visuomotor grasp.</a> J. Neurophysiol. 98 (1), 488-501 (2007).</li><li>Denys, K., 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=The+processing+of+visual+shape+in+the+cerebral+cortex+of+human+and+nonhuman+primates:+a+functional+magnetic+resonance+imaging+study.">The processing of visual shape in the cerebral cortex of human and nonhuman primates: a functional magnetic resonance imaging study.</a> J. Neurosci. 24 (10), 2551-2565 (2004).</li><li>Theys, T., Pani, P., van Loon, J., Goffin, J., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectivity+for+three-dimensional+shape+and+grasping-related+activity+in+the+macaque+ventral+premotor+cortex.">Selectivity for three-dimensional shape and grasping-related activity in the macaque ventral premotor cortex.</a> J.Neurosci. 32 (35), 12038-12050 (2012).</li><li>Nelissen, K., Luppino, G., Vanduffel, W., Rizzolatti, G., Orban, 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=Observing+others:+multiple+action+representation+in+the+frontal+lobe.">Observing others: multiple action representation in the frontal lobe.</a> Science. 310 (5746), 332-336 (2005).</li><li>Janssen, P., Srivastava, S., Ombelet, S., Orban, 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=Coding+of+shape+and+position+in+macaque+lateral+intraparietal+area.">Coding of shape and position in macaque lateral intraparietal area.</a> J. Neurosci. 28 (26), 6679-6690 (2008).</li><li>Romero, M. C., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptive+field+properties+of+neurons+in+the+macaque+anterior+intraparietal+area.">Receptive field properties of neurons in the macaque anterior intraparietal area.</a> J. Neurophysiol. 115 (3), 1542-1555 (2016).</li><li>Decramer, T., Premereur, E., Theys, T., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-electrode+recordings+in+the+macaque+frontal+cortex+reveal+common+processing+of+eye-,+arm-+and+hand+movements.+Program+No.+495.15/GG14.">Multi-electrode recordings in the macaque frontal cortex reveal common processing of eye-, arm- and hand movements. Program No. 495.15/GG14.</a> Neuroscience Meeting Planner. , (2017).</li><li>Pani, P., Theys, T., Romero, M. C., Janssen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grasping+execution+and+grasping+observation+activity+of+single+neurons+in+macaque+anterior+intraparietal+area.">Grasping execution and grasping observation activity of single neurons in macaque anterior intraparietal area.</a> J. Cogn. Neurosci. 26 (10), 2342-2355 (2014).</li><li>Turriziani, P., Smirni, D., Oliveri, M., Semenza, C., Cipolotti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+prefrontal+cortex+in+familiarity+and+recollection+processes+during+verbal+and+non-verbal+recognition+memory.">The role of the prefrontal cortex in familiarity and recollection processes during verbal and non-verbal recognition memory.</a> Neuroimage. 52 (1), 469-480 (2008).</li><li>Tsao, D. Y., Schweers, N., Moeller, S., Freiwald, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patches+of+faces-selective+cortex+in+the+macaque+frontal+lobe.">Patches of faces-selective cortex in the macaque frontal lobe.</a> Nat. Neurosci. 11 (8), 877-879 (2008).</li></ol>

A comprehensive approach to the study of the dorsal stream requires a careful selection of behavioral tasks and visual tests: visual and grasping paradigms can be employed either combined or separately depending on the specific properties of the region.
In this article, we provide the examples of the neural activity recorded in both AIP and F5p in response to a subset of visual and motor tasks, but very similar responses can be observed in other frontal areas such as area 45B and F5a.
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Human and non-human primates share the capacity of performing complex motor actions including object grasping. To successfully perform these tasks, our brain needs to complete the transformation of intrinsic object properties into motor commands. This transformation relies on a sophisticated network of dorsal cortical areas located in parietal and ventral premotor cortex1,2,3 (Figure 1). 
From lesion studies in monkeys and humans4,5, we know that the dorsal visual stream - originating in primary visual cortex and directed towards posterior parietal cortex - is involved in both spatial vision and the planning of motor actions. However, the majority of dorsal stream areas are not devoted to a unique type of processing. For instance, the anterior intraparietal area (AIP), one of the end stage areas in the dorsal visual stream, contains a variety of neurons that fire not only during grasping6,7,8, but also during the visual inspection of the object7,8,9,10.
Similar to AIP, neurons in area F5, located in the ventral premotor cortex (PMv), also respond during visual fixation and object grasping, which is likely to be important for the transformation of visual information into motor actions11. The anterior portion of this region (subsector F5a) contains neurons responding selectively to three-dimensional (3D, disparity-defined) images12,13, while the subsector located in the convexity (F5c) contains neurons characterized by mirror properties1,3, firing both when an animal performs or observes an action. Finally, the posterior F5 region (F5p) is a hand-related field, with a high proportion of visuomotor neurons responsive to both observation and grasping of 3D objects14,15. Next to F5, area 45B, located in the inferior ramus of the arcuate sulcus, may also be involved in both shape processing16,17 and grasping18.
Testing object selectivity in parietal and frontal cortex is challenging, because it is difficult to determine which features these neurons respond to and what the receptive fields of these neurons are. For example, if a neuron responds to a plate but not to a cone, which feature of these objects is driving this selectivity: the 2D contour, the 3D structure, the orientation in depth, or a combination of many different features? To determine the critical object features for neurons that respond during object fixation and grasping, it is necessary to employ various visual tests using images of objects and reduced versions of the same images. 
A sizeable fraction of the neurons in AIP and F5 not only responds to the visual presentation of an object, but also when the animal grasps this object in the dark (i.e., in the absence of visual information). Such neurons may not respond to an image of an object that cannot be grasped. Hence, visual and motor components of the response are intimately connected, which makes it difficult to investigate the neuronal object representation in these regions. Since visuomotor neurons can only be tested with real-world objects, we need a flexible system for presenting different objects at different positions in the visual field and at different orientations if we want to determine which features are important for these neurons. The latter can only be achieved by means of a robot capable of presenting different objects at different locations in visual space.
This article intends to provide an experimental guide for researchers interested in the study of parieto-frontal neurons. In the following sections, we will provide the general protocol used in our laboratory for the analysis of grasping and visual object responses in awake macaque monkeys (Macaca mulatta).

<table>
	<tbody>
		<tr>
			<td>Grasping robot</td>
			<td>GIBAS Universal Robots</td>
			<td>UR-6-85-5-A</td>
			<td>Robot arm equipped with a gripper</td>
		</tr>
		<tr>
			<td>Carousel motor</td>
			<td>Siboni</td>
			<td>RD066/&#8224;20 MV6, 35x23 F02</td>
			<td>Motor to be implemented in a custom-made vertical carousel. It allows the rotation of the carousel.</td>
		</tr>
		<tr>
			<td>Eye tracker</td>
			<td>SR Research</td>
			<td>EyeLink II</td>
			<td>Infrared camera system sampling at 500 Hz</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Wavetek Rockland</td>
			<td>852</td>
			<td>Electronic filters perform a variety of signal-processing functions with the purpose of removing a signal&#39;s unwanted frequency components.</td>
		</tr>
		<tr>
			<td>Preamplifier</td>
			<td>BAK ELECTRONICS, INC.</td>
			<td>A-1</td>
			<td>The Model A-1 allows to reduce input capacity and noise pickup and allows to test impedance for metal micro-electrodes</td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>FHC</td>
			<td>UEWLEESE*N4G</td>
			<td>Metal microelectrodes (* = Impedance, to be chosen by the researcher)</td>
		</tr>
		<tr>
			<td>CRT monitor</td>
			<td>Vision Research Graphics</td>
			<td>M21L-67S01</td>
			<td>The CRT monitor is equipped with a fast-decay P46-phosphor operating at 120 Hz</td>
		</tr>
		<tr>
			<td>Ferroelectric liquid crystal shutters</td>
			<td>Display Tech</td>
			<td>FLC Shutter Panel; LV2500P-OEM</td>
			<td>The shutters operate at 60 Hz in front of the monkeys and are synchronized to the vertical retrace of the monitor</td>
		</tr>
	</tbody>
</table>

investigating object representations in the macaque dorsal visual stream using single-unit recordings

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined curved surfaces, and images of real-world objects (with and without disparity) in a similar manner as described in the ventral visual stream. In addition, parieto-frontal areas are believed to convert visual object information into appropriate motor outputs, such as the pre-shaping of the hand during grasping. To better characterize object selectivity in the cortical network involved in visuomotor transformations, we provide a battery of tests intended to analyze the visual object selectivity of neurons in parieto-frontal regions.

Figure 5 plots the responses of an example neuron recorded from area F5p tested with four objects: two different shapes -a sphere and a plate- shown in two different sizes (6 and 3 cm). This particular neuron responded not only to the large sphere (optimal stimulus; upper left panel), but also to the large plate (lower left panel). In comparison, the response to the smaller objects was weaker (upper and lower right panels).
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Watch this Scientific Journal Video about Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings at JoVE.com

Investigating Object Representations in the Macaque Dorsal Visual Stream Using Single-unit Recordings

A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

Previous studies have shown that neurons in parieto-frontal areas of the macaque brain can be highly selective for real-world objects, disparity-defined ...

This method can help answering key questions in visual and motor systems neuroscience. For example, how do neurons encode objects and and object parts? And how do neural representations change from visual to motor?The main advantage of this method is that neurons can be studied with real-world objects during reaching and grasping, and with images of objects and object parts presented on a display. Demonstrating this procedure will be Maria Romero, a postdoc, and Irene Caprara, a PhD student from my laboratory. Begin with the test subject situated in the testing chair in front of the object-grasping setup.Adjust an infrared camera in front of the subject's eyes to obtain an adequate image of the pupil and corneal reflex. To run the visually-guided grasping task using a carousel setup, first allow the macaque to place the hand contralateral to the recorded hemisphere in the resting position in complete darkness to initiate the sequence. After a variable intertrial interval of 2, 000 to 3, 000 milliseconds, apply a red laser as a fixation point at the base of the object.If the animal maintains its gaze inside an electronically-defined fixation window for 500 milliseconds, illuminate the object from above with a light source. Then, after a variable delay of 300 to 1, 500 milliseconds, program a dimming of the laser as the visual go cue. Instruct the macaque to lift the hand from the resting position and reach, grasp, and hold the object for a randomized variable interval of 300 to 900 milliseconds.Whenever the macaque performs the whole sequence correctly, reward it with a drop of juice. As for the carousel setup, let the macaque place the hand contralateral to the recorded hemisphere in the resting position in complete darkness to initiate the sequence. After 2, 000 to 3, 000 milliseconds, illuminate the LED on the object.Again, if the animal maintains its gaze inside an electronically-defined fixation window, illuminate the object with a white light source. After the delay, switch off the LED as the visual go cue. Again, the monkey lifts the hand from the resting position and reaches, grasps, and holds the object.Reward the monkey with a drop of juice after each correct sequence. Continue the training with additional objects. During the task, the software should measure the reaction time between the go signal and the onset of the hand movement.And the grasping time between the start of the movement and lifting of the object. For the 2D tasks, use a standard LCD monitor. Project all visual stimuli on a black background.For the 3D tests, locate two ferroelectric liquid crystal shutters in front of the monkey's eyes. For the 3D test, locate two ferroelectric liquid crystal shutters in front of the monkey's eyes. Present the stimuli stereoscopically by alternating the left and right eye images on a cathode ray tube monitor equipped with a fast-decay P46 phosphor, while operating the shutters at 60 hertz with synchronization to the vertical retrace of the monitor.Start the trial by presenting a small square in the center of the screen as the fixation point. If the eye position remains within an electronically-defined one-degree-square window for at least 500 milliseconds, present the visual stimulus on the screen for a total time of 500 milliseconds. When the monkey maintains a stable fixation until the stimulus offset, reward it with a drop of juice.To study shape selectivity, run multiple tests with 2D images during the passive fixation task beginning with a search test. Test the visual selectivity of the cell using a wide set of images, including pictures of the object that was grasped in the VGG. For this and all subsequent visual tasks, compare the image evoking the strongest response, termed preferred image, to a second image to which the neuron is responding weakly, termed nonpreferred image.Next, run a contour test. From the original surface images of real objects, obtain progressively simplified versions of the same stimulus shape. Collect at least 10 trials per condition.To determine whether the neuron prefers the original surface, the silhouette, or the outline from the original shape. Then run a receptive field test. Present the images of objects at different positions on a display, covering the central visual field.A total of 35 positions at three degrees are used here. Finally, run a reduction test with contour fragments presented at the center of the receptive field to identify the minimum effective shape feature. Determine the minimum effective shape feature as the smallest shape fragment evoking a response that is at least 70%of the intact outline response.This image shows the responses of an example neuron recorded from area F5p, tested with a sphere and a plate, shown in two different sizes. This particular neuron responded not only to the large sphere optimal stimulus, but also to the large plate. In comparison, the response to the smaller objects was weaker.The following images show results from an example neuron recorded in the anterior intraparietal area, and tested during both the visually-guided grasping task and passive fixation. This neuron was responsive during grasping. The neuron was also responsive to the visual presentation of 2D images of objects, including pictures of the objects used in the grasping task.The preferred was not the object to be grasped, but another 2D picture with which the animal had no previous grasping experience. An example of the responses obtained in the reduction test is shown here. This example neuron responded to the smallest fragments in the test.Once mastered, this technique can be done in less than 90 minutes, if it's performed correctly. After watching this video, you should have a good understanding of how to test neurons in the dorsal visual stream with objects and images of objects to determine the critical features that drive the responses.

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Neuroscience

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

A Visual Guide to Sorting Electrophysiological Recordings Using 'SpikeSorter'

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be user friendly with a graphical user interface, able to read data files in a variety of formats, and provide users with a flexible set of tools giving them the ability to detect and sort extracellular voltage waveforms from different units with some degree of reliability. Previously published spike sorting methods are now available in a software program, SpikeSorter, intended to provide electrophysiologists with a complete set of tools for sorting, starting from raw recorded data file and ending with the export of sorted spikes times. Procedures are automated to the extent this is currently possible. The article explains and illustrates the use of the program. A representative data file is opened, extracellular traces are filtered, events are detected and then clustered. A number of problems that commonly occur during sorting are illustrated, including the artefactual over-splitting of units due to the tendency of some units to fire spikes in pairs where the second spike is significantly smaller than the first, and over-splitting caused by slow variation in spike height over time encountered in some units. The accuracy of SpikeSorter's performance has been tested with surrogate ground truth data and found to be comparable to that of other algorithms in current development.

The article shows how to use the program SpikeSorter to detect and sort spikes in extracellular recordings made with multi-electrode arrays.

Few stand-alone software applications are available for sorting spikes from recordings made with multi-electrode arrays. Ideally, an application should be ...