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.

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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><tbody><tr><td>Neurostar Motorized&lt;br/&gt; 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&quot;/1.6 mm diameter) - 1/4&quot; Jack</td><td>FHC</td><td>40-90-5D-02</td><td>Surgery</td></tr><tr><td>Heating Pad 5 X 12.5 cm&amp;nbsp;</td><td>FHC</td><td>40-90-2-07</td><td>Surgery</td></tr><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 &amp;mu;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></tr><tr><td>Omnidrill35, 115 - 230 V</td><td>World Precision Instruments</td><td>503598</td><td>Surgery</td></tr><tr><td>Burrs for Micro Drill</td><td>Fine Science Tools</td><td>19007-05</td><td>Surgery</td></tr><tr><td>nVista</td><td>Inscopix</td><td>100-001048</td><td>Imaging</td></tr><tr><td>AAV1.CaMKII.GCaMP6f.WPRE.SV40</td><td>Penn Vector Core</td><td>AV-1-PV3435</td><td>Surgery</td></tr><tr><td>Ketoprofen</td><td>Victor Medical</td><td>5487</td><td>Surgery</td></tr><tr><td>Carprofen</td><td>Victor Medical</td><td>1699008&amp;nbsp;</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 12 cm x 7 mm)</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><tr><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 1.0 mm-Dia. ~4.3 mm 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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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="http://cloudflare.jove.com/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

Research

JoVE Journal

Behavior

31.0K Views.  Inscopix Inc..  Hier presenteren we een procedure voor het uitvoeren van grootschalig Ca 2+ beeldvorming met cellulaire resolutie over meerdere corticale lagen in vrij bewegende muizen. Honderden actieve cellen kunnen tegelijkertijd worden waargenomen met behulp van een miniatuur kop microscoop gekoppeld met een geïmplanteerde prisma sonde. 

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

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 ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Neuroscience

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Two-Photon and Wide-Field Calcium Imaging in the Superior Colliculus

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Author Spotlight: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

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 ...

Recording Neuronal Activity During Odor Plume Navigation with Miniscope Imaging

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

Author Spotlight: Understanding Processing of Olfactory and Spatial Information by Brain with Real-Time Behavioral Analysis

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to ...

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

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