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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</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.
<p c...

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

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8.3K 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 ...

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

Behavior

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 ...

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 ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

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 ...

Development of an X-Y Electrode Stage for Precise Neural Recording in Marmosets

Electrophysiology of Laminar Cortical Activity in the Common Marmoset

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with linear arrays. The marmoset has recently grown in popularity due to its similar neural functional organization to other primates and its technical advantages for recording and imaging. However, neurophysiology in this model poses some unique challenges due to the small size and lack of gyri as anatomical landmarks. Using custom-built micro-drives, researchers can manipulate linear array placement to sub-millimeter precision and reliably record at the same retinotopically targeted location across recording days. This protocol describes the step-by-step construction of the micro-drive positioning system and the neurophysiological recording technique with silicon linear electrode arrays. With precise control of electrode placement across recording sessions, researchers can easily traverse the cortex to identify areas of interest based on their retinotopic organization and the tuning properties of the recorded neurons. Further, using this laminar array electrode system, it is possible to apply a current source density analysis (CSD) to determine the recording depth of individual neurons. This protocol also demonstrates examples of laminar recordings, including spike waveforms isolated in Kilosort, which span multiple channels on the arrays.

Author Spotlight: Optimizing Micro-Drive Systems for Precise Electrode Positioning in Marmoset

Custom-built micro-drives enable the sub-millimeter targeting of cortical recording sites with linear silicon arrays.

The marmoset monkey provides an ideal model for examining laminar cortical circuits due to its smooth cortical surface, which facilitates recordings with ...

Neuronal Activity Mapping in Developing Mouse Sensory Cortex

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

Author Spotlight: Deciphering Neural Circuit Formation from Two-Photon Microscopy and Single Neuron Imaging

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

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

Summary

Explore More Videos

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

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

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

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

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

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

Electrophysiology of Laminar Cortical Activity in the Common Marmoset

In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

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