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 ...

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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. 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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>

The authors have nothing to disclose.

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).
<p class="jove_content" fo:keep...

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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8.2K visualizações.  Katholieke Universiteit Leuven. 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 st...