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
<ol>
	<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 the same recording session in order to test the neuron extensively and obtain a better understanding of the features driving the neural response (Figure 2-3).&#160;
	<ol>
		<li>Train the animal in Visually-Guided Grasping (VGG; grasping &#8216;in the light&#8217;) to evaluate the visuomotor components of the response. Note: independently from the task chosen, gradually restrict fluid intake at least three days before the start of the training phase.&#160;
		<ol>
			<li>Restrain the monkey&#8217;s head for the whole duration of the experimental session.</li>
			<li>In the first sessions, hold the hand contralateral to the recording chamber at the resting position and help the animal to reach and grasp the object, giving manual reward after each attempt.</li>
			<li>Place back the monkey&#8217;s hand on the resting position at the end of each trial.</li>
			<li>Every few trials, release the hand of the monkey, and wait a few seconds to observe if the animal initiates the movement spontaneously.</li>
			<li>Apply manual reward whenever the monkey reaches towards the object.</li>
			<li>When the reaching phase is acquired correctly, help the animal to lift (or pull) the object and reward manually.</li>
			<li>As in 1.1.1.4 and 1.1.1.5, release the monkey&#8217;s hand, and wait a few seconds to observe if the animal initiates the movement spontaneously. Give reward whenever the movement is performed correctly.</li>
			<li>Correct the reaching, hand position, and wrist orientation as many times as necessary during the procedure.</li>
			<li>Repeat the steps above until the animal performs the sequence automatically.</li>
			<li>Load the automatic task. The animal gets rewarded automatically when it performs the reach and grasp movements for a predetermined time.</li>
			<li>Gradually increase the holding time of the object.</li>
			<li>Introduce the laser that projects the fixation point at the base of the object. Add then the eye tracker to monitor the eye position around the object-to-be-grasped.&#160;</li>
		</ol>
		</li>
		<li>Train the animal in Memory-Guided Grasping (MGG) to investigate the motor component of the response, not affected by the visual component of the stimulus.
		<ol>
			<li>Restrain the monkey&#8217;s head.</li>
			<li>Follow the same steps described for the VGG making sure that the animal maintains fixation on the laser during the task within an electronically defined window. For this version of the task, the light goes off at the end of the fixation period.</li>
		</ol>
		</li>
		<li>Train the monkey in Passive Fixation to address visual responsiveness and shape selectivity.&#160;
		<ol>
			<li>Restrain the monkey&#8217;s head.</li>
			<li>Present the visual stimuli to the monkey using either a CRT (Passive fixation of 3D stimuli) or an LCD monitor (Passive Fixation of 2D stimuli).</li>
			<li>Present a fixation spot at the center of the screen, superimposed on the visual stimuli.</li>
			<li>Reward the animal after each presentation of the stimulus and increase gradually the fixation period until reaching the standards of the task.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform Surgery, using sterile tools, drapes and gowns.
	<ol>
		<li>Anesthetize the animal with ketamine (15 mg/kg, intramuscularly) and medetomidine hydrochloride (0.01-0.04 mL/kg intramuscularly) and confirm the anesthesia regularly by checking the animal&#8217;s response to stimuli, heart rate, respiration rate and blood pressure.&#160;</li>
		<li>Maintain general anesthesia (propofol 10 mg/kg/h intravenously) and administer oxygen with a tracheal tube. Use a lanolim-based ointment to prevent eye dryness while under anesthesia.</li>
		<li>Provide analgesia using 0.5 cc of Buprenorphine (0.3mg/ml intravenously). In case of increase of heart rate during the surgery, an extra dosage can be administered.&#160;</li>
		<li>Implant an MRI compatible head post with ceramic screws and dental acrylic. Perform all survival surgeries under strict aseptic conditions. For an adequate maintenance of the sterile field, use disposable sterile gloves, masks and sterile instruments.&#160;</li>
		<li>Guided by anatomical magnetic resonance imaging (MRI; Horsley-Clark coordinates), make a craniotomy above the area of interest and implant the recording chamber on the monkey&#8217;s skull. Use a standard recording chamber for single unit extracellular recordings or a multielectrode microdrive, for the simultaneous recording of multiple neurons.</li>
		<li>After the surgery, discontinue the intravenous administration of propofol until spontaneous breathing resumes. Do not leave the animal unattended until it has regained consciousness and introduce the animal in the social group only after complete recovery.</li>
		<li>Provide post-operative analgesia as recommended by the institutional veterinarian; use for example Meloxicam (5mg/ml intramuscularly).</li>
		<li>Wait 6 weeks after the surgery before starting the experiment. This allows a better anchorage of the head post to the skull and guarantees that the animal has fully recovered from the intervention.</li>
	</ol>
	</li>
	<li>Localize the recording area using MRI (for single unit extracellular recordings) and computed tomography (CT; for multielectrode recordings).
	<ol>
		<li>Fill glass capillaries with a 2% copper sulfate solution and insert them into a recording grid.</li>
		<li>Perform structural MRI (slice thickness: 0.6 mm).&#160;</li>
	</ol>
	</li>
	<li>Monitoring of&#160; neural activity.
	<ol>
		<li>Use tungsten microelectrodes with an impedance of 0.8 &#8211; 1 M&#937;.</li>
		<li>Insert the electrode through the dura using a 23G stainless steel guide tube and a hydraulic microdrive.&#160;</li>
		<li>For spike discrimination, amplify and filter the neural activity between 300 and 5,000 Hz.&#160;</li>
		<li>For local field potential (LFP) recordings, amplify and filter the signal between 1 and 170 Hz.</li>
	</ol>
	</li>
	<li>Monitor the eye signal
	<ol>
		<li>Adjust an infrared camera in front of the animal&#8217;s eyes to obtain an adequate image of the pupil and of the corneal reflex.&#160;</li>
		<li>Use an infrared-based camera to sample the pupil position at 500 Hz.</li>
	</ol>
	</li>
</ol>
2. Investigating Object Selectivity in Dorsal Areas&#160;
<ol>
	<li>Perform Visually-Guided Grasping (VGG).
	<ol>
		<li>Choose the right grasping setup depending on the goal of research: carousel setup or robot setup (Figure 3).&#160;</li>
		<li>For the carousel setup, run the VGG task:
		<ol>
			<li>Let the monkey place the hand contralateral to the recorded hemisphere in the resting position in complete darkness to initiate the sequence.&#160;</li>
			<li>After a variable time (intertrial interval: 2,000-3,000 ms), apply a red laser (fixation point) at the base of the object (distance: 28 cm from the monkeys&#8217; eyes). If the animal maintains its gaze inside an electronically-defined fixation window (+/- 2.5&#176;) for 500 ms, illuminate the object from above with a light source.&#160;</li>
			<li>After a variable delay (300-1500 ms), program a dimming of the laser (visual GO cue) instructing the monkey to lift the hand from the resting position, and reach, grasp and hold the object for a variable interval (holding time: 300-900 ms).&#160;</li>
			<li>Whenever the animal performs the whole sequence correctly, reward it with a drop of juice.</li>
		</ol>
		</li>
		<li>Use a similar task sequence for the robot setup.
		<ol>
			<li>As for the carousel setup, let the monkey place the hand contralateral to the recorded hemisphere in the resting position in complete darkness to initiate the sequence.&#160;</li>
			<li>After a variable time (intertrial interval: 2,000-3,000 ms), illuminate the LED (fixation point) on the object (from within; distance: 28 cm from the monkeys&#8217; eyes). Again, if the animal maintains its gaze inside an electronically-defined fixation window (+/- 2.5&#176;) for 500 ms, illuminate the object from within with a white light source.</li>
			<li>After a variable delay (300-1500 ms), switch off the LED (visual GO cue), instructing the monkey to lift the hand from the resting position, and reach, grasp and hold the object for a variable interval (holding time: 300-900 ms).&#160;</li>
			<li>Whenever the animal performs the whole sequence correctly, reward it with a drop of juice.</li>
		</ol>
		</li>
		<li>During the task, quantify the performance of the monkey, paying special attention to the timing. Measure both the time elapsed between the go-signal and the onset of the hand movement (reaction time), and between the start of the movement and the lift of the object (grasping time).</li>
	</ol>
	</li>
	<li>Perform Memory-Guided Grasping (MGG; &#8216;Grasping in the dark&#8217;). Use the MGG task to determine if neurons are visuomotor or motor-dominant.&#160; 
	&#8203;Note: The sequence is similar to that described for the VGG, but the object is grasped in total darkness.
	<ol>
		<li>Identical to the VGG task, let the monkey place the hand contralateral to the recorded hemisphere in the resting position in complete darkness to initiate the sequence.&#160;</li>
		<li>After a variable time (intertrial interval: 2,000-3,000 ms), apply a red laser/LED (fixation point) to indicate the fixation point (at the base of the object for the carousel setup, at the center of the object for the robot setup; distance: 28 cm from the monkeys&#8217; eyes). If the animal maintains its gaze inside an electronically-defined fixation window (+/- 2.5&#176;) for 500 ms, illuminate the object.</li>
		<li>After a fixed time (400 ms), switch off the light.&#160;</li>
		<li>After a variable delay period (300-1500 ms) following&#160; light offset, dim/switch off the fixation point (GO CUE) to instruct the monkey to lift the hand and reach, grasp, and hold the object (holding time: 300-900 ms).&#160;</li>
		<li>Whenever the animal performs the whole sequence correctly, give a drop of juice as a reward.&#160;</li>
	</ol>
	</li>
	<li>Perform Passive fixation. As for the VGG task, choose the most appropriate setup (carousel or robot setup) depending on the goal of the research.&#160; 
	&#8203;Note: Two different passive fixation tasks can be performed: passive fixation of real-world objects (using the objects-to-be-grasped in the carousel and robot setups) and passive fixation of 3D/2D images of objects.
	<ol>
		<li>Perform passive fixation of real-world objects.&#160;
		<ol>
			<li>Present the fixation point (red laser for the carousel setup projected at the base of the object and red LED in the robot setup).</li>
			<li>If the animal maintains its gaze inside an electronically-defined fixation window (+/- 2.5&#176;) for 500 ms, illuminate the object for 2,000 ms.&#160;</li>
			<li>If the animal maintains its gaze within the window for 1,000 ms, reward it with a drop of juice.&#160;</li>
		</ol>
		</li>
		<li>Perform passive fixation of 3D/2D images of objects.
		<ol>
			<li>Present all visual stimuli on a black background (luminance of 8 cd/m2) using a monitor (resolution of 1,280 &#215; 1,024 pixels) equipped with a fast-decay P46-phosphor and operated at 120 Hz (viewing distance: 86 cm).</li>
			<li>In the 3D tests, present the stimuli stereoscopically by alternating the left and right eye images on a display (CRT monitor), in combination with two ferroelectric liquid crystal shutters. Locate these shutters in front of the monkey&#8217;s eyes, operate at 60 Hz and synchronize to the vertical retrace of the monitor.&#160;</li>
			<li>Start the trial by presenting a small square in the center of the screen (fixation point; 0.2&#176; &#215; 0.2&#176;). If the eye position remains within an electronically defined 1&#176; square window (much smaller than for real-world objects) for at least 500 ms, present the visual stimulus on the screen, for a total time of 500 ms.&#160;</li>
			<li>When the monkey maintains a stable fixation until the stimulus offset, reward it with a drop of juice.&#160;</li>
			<li>For an adequate study of shape selectivity, run a comprehensive battery of tests with 2D images during passive fixation task, in the following sequence.</li>
			<li>Run a Search test. Test the visual selectivity of the cell using a wide set of images (surface images; Figure 4A), including the pictures of the object that is grasped in the VGG. For this and all subsequent visual tasks, compare the image evoking the strongest response (termed &#8216;preferred image&#8217;) to a second image to which the neuron is responding weakly (termed &#8216;nonpreferred image&#8217;). If the neuron under study responds also to the images of objects, search for specific stimulus components driving the cell&#8217;s responsiveness (Contour test, Receptive Field test and Reduction test).</li>
			<li>Run a Contour test. From the original surface images of real objects (2D or 3D images containing texture, shading and perspective), obtain progressively simplified versions of the same stimulus shape (silhouettes and outlines; Figure 4B). Collect at least 10 trials per condition in order to determine whether the neuron prefers the original surface, the silhouette or the outline from the original shape.</li>
			<li>Run a Receptive Field (RF) test. To map the RF of a neuron, present the images of objects at different positions on a display (in this experiment, 35 positions; stimulus size of 3&#176;), covering the central visual field19,20. To collect enough stimulus repetitions at all possible positions in a reasonable time, reduce the stimulus duration (flashed stimuli; stimulus duration: 300 ms, intertrial interval: 300 ms).</li>
			<li>Run a Reduction test. Run a Reduction test with contour fragments presented at the center of the RF to identify the Minimum Effective Shape Feature (MESF). Generate the set of stimuli in Photoshop by cropping the contour of each of the original contour shapes along the main axes (Figure 3B). Design the MESF as the smallest shape fragment evoking a response that is at least 70% of the intact outline response and not significantly smaller than that response8.&#160;</li>
			<li>For a better estimation of position dependency (the effect of stimulus position on fragment selectivity), run two different tests. Run a Reduction Test with the fragments located at the position occupied in the original outline shape. Run a Reduction Test with the fragments at the center of mass of the shape.&#160;</li>
			<li>At this stage, run a new RF mapping using the MESF.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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/&amp;dagger;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&#039;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 ...

investigating-object-representations-macaque-dorsal-visual-stream

Research

JoVE Journal

Neuroscience

8.2K Views.  Katholieke Universiteit Leuven. A detailed protocol to analyze object selectivity of parieto-frontal neurons involved in visuomotor transformations is presented.

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

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 ...

A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation of sensory information processing by attention. However, little is known about which neurotransmitter systems are involved in such modulations. A practical way to address this question is to combine single-cell recording with local and temporary neuropharmacological manipulation in a suitable animal model. Here we demonstrate a technique combining acute single-cell recordings with the injection of neuropharmacological agents in the direct vicinity of the recording electrode. The video shows the preparation of the pressure injection/recording system, including preparation of the substance to be injected. We show a rhesus monkey performing a visual attention task and the procedure of single-unit recording with block-wise pharmacological manipulations.

Here, we show the pressure injection of neuropharmacological substances during single-cell recording in an awake, behaving macaque monkey. This procedure allows pharmacological manipulation in the direct vicinity of a cortical recording site.

The top-down modulation of feed-forward cortical information processing is functionally important for many cognitive processes, including the modulation ...

Behavior

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

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 ...

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during active behavior. An important tool for quantifying behavior is eye tracking, which is an indispensable tool for studying visual attention. However, eye tracking is challenging to use concurrently with invasive electrophysiology and this approach has consequently been little used. Here, we present a proven experimental protocol to conduct single-neuron recordings with simultaneous eye tracking in humans. We describe how the systems are connected and the optimal settings to record neurons and eye movements. To illustrate the utility of this method, we summarize results that were made possible by this setup. This data shows how using eye tracking in a memory-guided visual search task allowed us to describe a new class of neurons called target neurons, whose response was reflective of top-down attention to the current search target. Lastly, we discuss the significance and solutions to potential problems of this setup. Together, our protocol and results suggest that single-neuron recordings with simultaneous eye tracking in humans are an effective method to study human brain function. It provides a key missing link between animal neurophysiology and human cognitive neuroscience.

We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search.

Intracranial recordings from patients with intractable epilepsy provide a unique opportunity to study the activity of individual human neurons during ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

A Cost-Effective, Versatile Probe Implant System for Electrophysiology in Mice

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. However, current methods for chronically implanting probes present steep trade-offs between cost, ease of use, size, adaptability, and long-term stability.
This protocol introduces a novel chronic probe implant system for mice called the DREAM (Dynamic, Recoverable, Economical, Adaptable, and Modular), designed to overcome the trade-offs associated with currently available options. The system provides a lightweight, modular and cost-effective solution with standardized hardware elements that can be combined and implanted in straightforward steps and explanted safely for recovery and multiple reuse of probes, significantly reducing experimental costs.
The DREAM implant system integrates three hardware modules: (1) a microdrive that can carry all standard silicon probes, allowing experimenters to adjust recording depth across a travel distance of up to 7 mm; (2) a three-dimensional (3D)-printable, open-source design for a wearable Faraday cage covered in copper mesh for electrical shielding, impact protection, and connector placement, and (3) a miniaturized head-fixation system for improved animal welfare and ease of use. The corresponding surgery protocol was optimized for speed (total duration: 2 h), probe safety, and animal welfare.
The implants had minimal impact on animals&#39; behavioral repertoire, were easily applicable in freely moving and head-fixed contexts, and delivered clearly identifiable spike waveforms and healthy neuronal responses for weeks of post-implant data collection. Infections and other surgery complications were extremely rare.
As such, the DREAM implant system is a versatile, cost-effective solution for chronic electrophysiology in mice, enhancing animal well-being, and enabling more ethologically sound experiments. Its design simplifies experimental procedures across various research needs, increasing accessibility of chronic electrophysiology in rodents to a wide range of research labs.

Here, we introduce a lightweight, cost-effective probe implant system for chronic electrophysiology in rodents optimized for ease of use, probe recovery, experimental versatility, and compatibility with behavior.

Chronic electrophysiological recordings in rodents have significantly improved our understanding of neuronal dynamics and their behavioral relevance. ...

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

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

A Pressure Injection System for Investigating the Neuropharmacology of Information Processing in Awake Behaving Macaque Monkey Cortex

Multi-layer Cortical Ca²⁺ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

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

Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

The DREAM Implant: A Lightweight, Modular, and Cost-Effective Implant System for Chronic Electrophysiology in Head-Fixed and Freely Behaving Mice