The authors would like to thank V. Jayaraman, D.S. Kim, L.L. Looger and K. Svoboda from the Genetically- Encoded Neuronal Indicator and Effector (GENIE) Project at Janelia Research Campus of the Howard Hughes Medical Institute for their generous donation of AAV1-GCaMP6f to University of Pennsylvania Vector Core. They would also like to thank the A. Olson and Neuroscience Microscopy Core at Stanford University supported by NIH NS069375 grant for their confocal microscopy services.

<ol>
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The authors have read the journal's policy and have the following competing interests: SG, SO and VC are paid employees at Inscopix.

Understanding neural circuit activity during awake behavior is a vital level of neuroscientific investigation needed to effectively dissect brain function in health and disease. The cortex is a particularly important region to study in the context of awake behavior, as it plays an important role in many vital sensory, cognitive and executive functions28,29.
The cortical column is thought to be the basic functional unit in the cortex and population-level activity of cortical cells is known to differ based on their physical location within the column. For example, excitatory neurons in layers 2/3 in the somatosensory cortex project primarily to other neocortical regions and modulate other cortical networks30, while cells in deeper layers project primarily to subcortical regions like the thalamus31. Recording the activity of hundreds of pre-specified cortical cells simultaneously and reliably over time across different lamina in freely behaving subjects would greatly advance our understanding of cortical information flow, allowing for finer functional dissection of cortical columns informed by real-time behavioral information and task-relevant time-scales.
Collecting this level of neural circuit data is made possible with the use of an efficient and streamlined miniaturized microscopy platform to conduct large-scale Ca2+ imaging in freely behaving subjects (or head-fixed subjects as desired). Used with genetically encoded calcium indicators to enable cell-type specific targeting, and imaging a multi-layer field of view provided by a chronically implanted prism probe, this protocol explored one case among many possible applications: observing laminar differences in somatosensory cortical processing when mice physically engaged with a novel object (Figure 5). This is the first procedural illustration of this kind of cell-type specific, in vivo approach to study multiple cortical layers in awake, freely behaving animals, and broadens the spectrum of experimental methods available to understand laminar structures in the active brain.
The periscopic field of view enabled by the prism probe in this technique can feasibly be applied to other brain structures when preservation of the tissue directly dorsal to a region of interest is desired; for example, CA3 imaging could be achieved without disruption of hippocampal function.
Prism probe based approach for imaging Ca2+ activity requires the physical insertion and permanent implantation of a microprism into the cortex, which equates to the creation of a cortical lesion where the lens probe is inserted. This may result in disruptions to the local neural circuitry, including the severing of apical dendrites and processes. This procedure will also cause an initial activation of glial cells in the region, though this is expected to be localized to the tissue about 150 &#181;m from the prism face, and to subside after the brain has healed22. It is very important to consider if this technique will affect the normal circuit anatomy and/or behavior of the animals when planning experiments. Behavioral control groups should always be conducted to ensure that there are no significant alterations in baseline behaviors that could produce confounding experimental results.
Using this miniaturized, mobile Ca2+ imaging technique with neuropharmacological manipulation, various cognitive, social, motor or intrinsic behavioral paradigms, and combining it with other physiological metrics can deepen and enrich studies focused on understanding the functional roles of neural circuits in behavior and signal processing32. Suppression or activation of certain pathways modulated by drugs can influence associated behaviors, which can be easily studied using this technology33. Branching out into different cell types by modifying the targeting of the calcium indicator is another powerful and useful application, and enables many creative combinations of experimental tools to address diverse neural circuit questions.

The cortex is an essential player in many complex mental and behavioral functions, from attention, sensory perception and top-down cognitive control1,2,3 to motivational, reward, and addiction pathways4,5. Understanding the computational processes that underlie its function is an important goal to drive a better clinical understanding of many mental and behavioral disorders.
Many current theories of psychiatric disease center around the idea that cortical neural circuit dysfunction or discoordination may underlie cognitive and behavioral abnormalities that are the hallmarks of conditions such as schizophrenia6, autism7 or obsessive-compulsive disorder8. Thus, obtaining population level neural activity data from cortical circuits within the proper context of simultaneous behavioral information is of great importance, and ideally can be targeted to specific cell types for finer neural circuit dissection.
Miniaturized microscopes in conjunction with implantable gradient refractive index (GRIN) microlenses enable optical access to neuronal ensembles under freely moving conditions from a diversity of possible brain regions9,10,11,12,13, including the cortex14,15,16. Using a mobile microscopy system coupled with genetically encoded calcium indicators allows for consistent imaging of the same cellular population encompassing hundreds of neurons over days to weeks in many brain regions9, and can be genetically targeted to specific cell types using viral vector or transgenic techniques.
As the cortex is known to support different functions and connect to different brain regions depending on the location of the cells within the cortical lamina17,18,19, we are interested in obtaining simultaneous multi-layer neural activity in awake behaving subjects. Here we demonstrate how to image hundreds of fluorescently labeled neurons in freely behaving mice over days, using the miniaturized fluorescence microscope20 paired with an implanted prism probe, which offers a multi-layer view of the cortex (Figure 1).
The prism probe used here is composed of two separate GRIN lenses: a prism and a cylindrical relay lens (Figure 1). The light from the microscope excites the fluorescently labeled cells located along the imaging face of the prism probe, after being reflected off the hypotenuse of the prism portion of the probe. The emitted light from the cells also reflects off the hypotenuse of the prism, is collected through the objective of the microscope and reaches the sensor in the microscope. The prism probe used in this procedure is adapted for easy use with standard stereotaxic equipment.
The miniaturized fluorescence microscope20 detects action potential-evoked Ca2+ transients in neuronal populations with single cell resolution, after those cells have been specifically labeled with Ca2+-sensitive genetically encoded fluorescent indicators. In this protocol, we inject Ca2+ indicator encoded in a viral vector (AAV1.CaMKII.GCaMP6f.WPRE.SV40), implant a prism probe, install the microscope, then obtain multiple days of somatosensory (S1 hind limb) neural activity data from an animal exposed to novel object surfaces during free exploration (Figure 2).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurostar Motorized 
			Ultra Precise Small Animal Stereotaxic Instrument</td>
			<td>Kopf</td>
			<td>Model 963SD</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Labomed</td>
			<td>Prima DNT</td>
			<td>Surgery and Imaging</td>
		</tr>
		<tr>
			<td>Mini Rectal Thermistor Probe (.062&#34;/1.6mm diameter) - 1/4&#34; Jack</td>
			<td>FHC</td>
			<td>40-90-5D-02</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Heating Pad 5 X 12.5cm&#160;</td>
			<td>FHC</td>
			<td>40-90-2-07</td>
			<td>Surgery</td>
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		<tr>
			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Microsyringe Pump</td>
			<td>World Precision Instruments</td>
			<td>UMP3 model; serial 155788 F110</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>NanoFil 10&#956;L Syringe</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>35 G Beveled Tip Nanofil NDL 2PK</td>
			<td>World Precision Instruments</td>
			<td>NF35BV-2</td>
			<td>Surgery</td>
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		<tr>
			<td>Omnidrill35, 115-230V</td>
			<td>World Precision Instruments</td>
			<td>503598</td>
			<td>Surgery</td>
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		<tr>
			<td>Burrs for Micro Drill</td>
			<td>Fine Science Tools</td>
			<td>19007-05</td>
			<td>Surgery</td>
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		<tr>
			<td>nVista</td>
			<td>Inscopix</td>
			<td>100-001048</td>
			<td>Imaging</td>
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			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr>
			<td>AAV1.CaMKII.GCaMP6f.WPRE.SV40</td>
			<td>Penn Vector Core</td>
			<td>AV-1-PV3435</td>
			<td>Surgery</td>
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			<td>Ketoprofen</td>
			<td>Victor Medical</td>
			<td>5487</td>
			<td>Surgery</td>
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		<tr>
			<td>Carprofen</td>
			<td>Victor Medical</td>
			<td>1699008&#160;</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Victor Medical</td>
			<td>1001054</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Gelfoam (Patterson Veterinary Supply Inc Gelfoam Sponge 12cmx7mm)</td>
			<td>Pfizer (Fisher Scientific)</td>
			<td>NC9841478</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Dumont #5/45 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td>Surgery</td>
		</tr>
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			<td>Dissecting knives</td>
			<td>Fine Science Tools</td>
			<td>10055-12</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>ProView Implant Kit</td>
			<td>Inscopix</td>
			<td>100-000756</td>
			<td>Surgery and Imaging</td>
		</tr>
		<tr>
			<td>ProView Prism Probe 1.0mm-Dia. ~4.3mm Length</td>
			<td>Inscopix</td>
			<td>100-000592</td>
			<td>Surgery and Imaging</td>
		</tr>
		<tr>
			<td>Kwik-Sil adhesive pack of 2</td>
			<td>World Precision Instruments</td>
			<td>KWIK-SIL</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Kwik-Cast Sealant</td>
			<td>World Precision Instruments</td>
			<td>KWIK-CAST</td>
			<td>Surgery and Imaging</td>
		</tr>
		<tr>
			<td>Miniature Optical Mounting Post</td>
			<td>Newport</td>
			<td>M-TSP-3</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Microscope Baseplate</td>
			<td>Inscopix</td>
			<td>BPL-2</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Microscope Baseplate Cover</td>
			<td>Inscopix</td>
			<td>BPC-2</td>
			<td>Imaging</td>
		</tr>
	</tbody>
</table>

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.

The protocol outlined here describes an effective and efficient way to perform longitudinal multi-layer Ca2+ imaging from hundreds of cortical neurons in freely behaving mice using prism probes (Figure 1). Previous approaches towards multi-layer cortical imaging have primarily been restricted to head fixed animals 22,23,24,25,26,27. In order to acquire this level of data in a freely behaving context, a miniaturized microscope platform was used for behavioral flexibility; a genetically encoded calcium indicator (GCaMP6f) was used to target a specific cell population (CAMKII+ cells in the cortex); and a prism probe was chosen to provide a chronic, multi-layer field of view.
We demonstrated the workflow for preparing the animal for imaging. A viral vector encoding an appropriate calcium indicator was injected into the cortex (Figure 2, Step 1), before chronically implanting a prism probe to enable optical access to the labeled cells (Figure 2, Step 2). A baseplate that serves as a secure, temporary dock for positioning of the microscope during imaging sessions was then installed over the head of the animal (Figure 2, Step 3), enabling the visualization of cortical activity across multiple cell layers in an awake behaving experimental setup (Figure 2, Step 4).
To ensure that the desired cellular population was being targeted, a post- mortem coronal brain section from a representative mouse is shown in Figure 3 with the prism probe tract and field of view marked relative to the GCaMP6f labeled neurons in Layers 2/3 and 5 of the somatosensory cortex.
During awake behaving imaging with the system, the activity of the somatosensory cortical neurons was recorded when the mouse was exposed to three different environments- Open Field (Day 1), Object Familiarization (Day 2-4) and Novel Object (Day 5) (Figure 4). On Day 1 the mouse was placed in a behavioral arena devoid of any objects. On Day 2-4 the mouse was placed in the arena with the same two texturally different objects (a gel pad and a wood block). On Day 5, one of the objects was replaced with a novel object. The animal was imaged across 5 days for 20 min each day.
Following cell extraction using the Ca2+ image data analysis software, spatial filters corresponding to cell locations were overlaid on the mean fluorescent intensity projection of the microscope recording data (Figure 5). A white dashed line separates layers 2/3 and 5 cells. Corresponding Ca2+ traces from 5 cells from each of the layers show the firing pattern of the cells in two different behavioral contexts- Object Familiarization and Novel Object Exposure. Layer 2/3 cells were more active compared to layer 5 cells on the day when the mouse was exposed to a novel object. This is also evident from the raster plots that show the thresholded firing activity of all imaged cells on days 1, 4 and 5.
<img alt="Figure 1" src="/files/ftp_upload/55579/55579fig1.jpg" /> 
Figure 1: In Vivo Ca2+ Imaging Across Multiple Cortical Layers in Freely Moving Mice. (A) Prism probe specifications and depiction of imaging plane. The reflective coating on the inside of the hypotenuse allows for imaging 90&#176; from the insertion plane of the prism probe. The lens cuff integrates with the lens holder, which streamlines the implantation procedure and allows for potential viewing of ambient tissue fluorescence during implantation (B) (i). Illustration of placement of prism probe craniotomy and knife incision relative to the virus injection site, and (ii) illustration of location of prism probe flat side relative to the knife incision and virus injection site. (C) Illustration of in-vivo Ca2+ imaging setup showing the light path for a small area within the full field of view through prism probe implanted in mouse cortex. (D) Example field of view during prism probe installation. Miniature microscope is attached to the lens holder, which holds the prism probe allowing for checking the virus expression during prism probe installation. (E) Integration of microscope with prism probe for multi-layer cortical imaging of GCaMP6f labeled S1 cells. F Example field of view during baseplate installation. Clear blood vessel pattern is visible at the time of baseplate install with some cells in raw image. More cells are clearly visible when DF/F is turned on in the acquisition software window. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55579/55579fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55579/55579fig2.jpg" /> 
Figure 2: Schematic Showing the Timeline of Workflow Events for Prism Probe Implantation and Microscope Installation. Number of weeks is represented on the X-axis and the workflow steps of the procedures along the Y axis. (A) Graphic illustrating viral injection (AAV1.CaMKII.GCaMP6f.WPRE.SV40) along the same dorso-ventral axis, to label multiple layers of the mouse somatosensory cortex. (B) 2 weeks post virus injections, a prism probe is implanted at an axis that is parallel to the virus injection sites. (C) Approximately one week after the prism probe implantation, the animal is checked for expression with the microscope and a baseplate is mounted on the head if a population of cells is visible. (D) The animal is then ready for chronic imaging during relevant behavioral tasks (Mouse clip art modified after permission from- UW-Madison Biochemistry MediaLab). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55579/55579fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55579/55579fig3.jpg" /> 
Figure 3: Postmortem Histological Validation of Prism Probe Location and GCaMP Expression. (A) Coronal section from a representative mouse brain showing the prism probe tract and with its imaging side facing the GCaMP6f expressing cells (AAV1.CaMKII.GCaMP6f expressed in neurons in layers 2/3 and 5). (B) Same coronal brain section following staining for DAPI. Scale bar = 250 &#181;m (C) Zoomed in view of GCaMP6f expressing cells in somatosensory cortex. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55579/55579fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55579/55579fig4.jpg" /> 
Figure 4: Mouse Activity During Habituation, Familiarization, and Novel Object Exposure Testing was Video-tracked Using Video Software. (A) On Day 1, the animal was placed in a behavioral arena devoid of any objects (Open Field). (B) On days 2-4, the same two texturally different objects (gel pad and wood block) were placed in the arena (Object Familiarization). (C) On Day 5, one of the objects was replaced with a novel object (wood block with sand paper) (Novel Object Exposure). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55579/55579fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55579/55579fig5.jpg" /> 
Figure 5: Calcium Dynamics from Superficial and Deep Layers of Somatosensory Cortex of a Representative Mouse Imaged with the Microscope. (A) Merged image of neuronal spatial filters (green blobs) and mean fluorescence intensity projection of the microscope recording through prism probe field of view. Border between supragranular and infragranular layers indicated by a white dashed line. Scale bar = 100 &#181;m. (B) Calcium traces from five example superficial and deep layer cells (filled blue and red cells in panel A), indicating units of standard deviation of fluorescence following principal and independent component analysis. Horizontal scale bar 50 s and vertical scale bar 10 S.D. (C) Raster plot of cells from superficial (layers 2/3) and deep layers (layer 5) shown over Open field, Object Familiarization and Novel object exploration. Scale bar = 100 s. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55579/55579fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy at JoVE.com

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.

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 ...

multi-layer-cortical-ca2-imaging-freely-moving-mice-with-prism-probes

Nanyang Technological University

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JoVE Journal

Behavior

30.8K Views.  Inscopix Inc.. 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.

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

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

Quantitative Assessment of Cortical Auditory-tactile Processing in Children with Disabilities

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess children&#39;s cortical processing of light touch, speech sounds and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing children discomfort. To accomplish this we developed a dual channel, time and strength calibrated air puff stimulator that allows both tactile stimulation and sham control. We combined this with the use of event-related potential methodology to allow for high temporal resolution of signals from the primary and secondary somatosensory cortices as well as higher order processing. This methodology also allowed us to measure a multisensory response to auditory-tactile stimulation.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology to quantitatively assess infants and children&#39;s cortical processing of light touch, speech sounds, and the multisensory processing of the 2 stimuli, without requiring active subject participation or causing discomfort in vulnerable patients.

Objective and easy measurement of sensory processing is extremely difficult in nonverbal or vulnerable pediatric patients. We developed a new methodology ...

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with schizophrenia who are at familial high risk (FHR) may show similar cognitive and emotional changes. However, the neurological changes underlying the emergence of these symptoms remain unclear. This study sought to identify differences in frontal, striatal, and limbic regions in children and adolescents with FHR using functional magnetic resonance imaging. Groups of 21 children and adolescents at FHR and 21 healthy controls completed an emotional oddball task that relied on selective attention and the suppression of task-irrelevant emotional information. The standard oddball task was modified to include aversive and neutral distractors in order to examine potential group differences in both emotional and executive processing. This task was designed specifically to allow for children and adolescents to complete by keeping the difficulty and emotional image content age-appropriate. Furthermore, we demonstrate a technique for suitable fMRI registration for children and adolescent participants. This paradigm may also be applied in future studies to measure changes in neural activity in other populations with hypothesized developmental changes in executive and emotional processing.

This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR).&#160;FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence.

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Simultaneous Transcranial Alternating Current Stimulation and Functional Magnetic Resonance Imaging

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs frequency-specific stimulation of the human brain through current applied to the scalp with surface electrodes. Most current knowledge of the technique is based on behavioral studies; thus, combining the method with brain imaging holds potential to better understand the mechanisms of tACS. Because of electrical and susceptibility artifacts, combining tACS with brain imaging can be challenging, however, one brain imaging technique that is well suited to be applied simultaneously with tACS is functional magnetic resonance imaging (fMRI). In our lab, we have successfully combined tACS with simultaneous fMRI measurements to show that tACS effects are state, current, and frequency dependent, and that modulation of brain activity is not limited to the area directly below the electrodes. This article describes a safe and reliable setup for applying tACS simultaneously with visual task fMRI studies, which can lend to understanding oscillatory brain function as well as the effects of tACS on the brain.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations, though its effects are not completely understood. This article describes a safe and reliable setup for applying tACS simultaneously with functional magnetic resonance imaging, which can increase understanding oscillatory brain function and effects of tACS.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...