Name: long-term imaging of identified neural populations using microprisms in freely moving and head-fixed animals
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We thank Ms. Charu Reddy and Professor Matteo Carandini (Cortex Lab) for their advice on surgical protocol and sharing of transgenic mouse strain. We thank Dr Norbert Hogrefe (Inscopix) for his guidance and assistance through the development of the surgery. We thank Ms Andreea Aldea (Sun Lab) for her assistance with the surgical setup and data processing. This work was supported by the Moorfields Eye Charity.

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

<ol>
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The authors declare no competing financial interest or conflict of interest.

Here, we have shown the ability to observe and directly compare neurons in head-fixed and freely moving conditions in the same neural populations. While we demonstrated the application in the visual cortex, this protocol can be adapted to a multitude of other brain areas, both cortical areas and deep nuclei24,25,26,27,28, as well as other data acquisition and behavioral setups29,30.
It is important to make note of the critical steps within this protocol as they allow optimal data acquisition. Firstly, when performing z-stacks, it is crucial to maintain consistent luminance throughout. As described earlier, light will scatter the more tissue it has to penetrate; thus, by increasing the gain, the laser has more power to be able to penetrate the tissue further and excite our ROIs. However, it is not advised to start the z-stack at the gain required to excite ROIs in the deep tissue, as this will cause overexposure of fluorescent structures and phototoxicity and may bleach the tissue at the surface of the cortex. Therefore, selecting the Relative exponential gradient option allows the software to calculate a steady gradient of power needed to be able to be optimal at every step of the z-stack. Secondly, the next critical step to help with consistent registration of cell sets over time is to ensure to save the mean image of the movie taken on the first one-photon imaging session. This allows the user to compare any subsequent imaging sessions to the original image to ensure continuity in cell sets and data acquisition.
It is worth noting that lightweight miniaturized two-photon microscopes have been independently designed31,32, which allow high-resolution imaging in freely behaving animals. While it highlights an exciting advancement for high-resolution in vivo imaging, the restricted field-of-view (FOV) and the highly specialized design make it challenging for general end-users. The method we described integrates several well-established platforms readily available from commercial suppliers, making it an accessible choice. In theory, it can also be replaced with a customized, self-assembled microprism compatible with an open-source miniscope and a generic two-photon microscope33,34.
Nonetheless, this protocol faces challenges such as accurate microprism implantation since errors could lead to nonviable FOVs and, consequently, compromised data. Maintaining consistent FOVs across different modalities is also challenging due to the different objectives used for imaging acquisition. However, as the XY axes of the microprism remain fixed, the variation in FOV is along the Z axis; therefore, specific landmarks present in both imaging modes (such as blood vessels, artifacts in the cortical tissue, and identical neurons) are employed to synchronize the FOVs. Furthermore, it is crucial to apply registration to align and identify the same neurons under different modalities. Another challenge to be aware of is to minimize photobleaching of the GCaMP tissue during acquisition in both modalities. Therefore, it is important to reduce the amount of time spent recording, optimize the signal-to-noise ratio (by increasing the gain rather than the LED/laser power), and wait 24-48 h between imaging sessions.
Despite these inherent complexities, our protocol, when executed with surgical precision and suitable image processing techniques, provides a robust platform for comparing neural activities. It enables the comparison between head-fixed states in strictly controlled tasks and freely moving states that mimic more natural behaviors, thus expanding the potential applications in the field of neuroscience.

The advent of in vivo two-photon fluorescent imaging1,2, combining the new technologies in optical systems and genetically modified fluorescence indicators, has emerged as a powerful technique in neuroscience to investigate the intricate structure, function, and plasticity in the living brain3,4. In particular, this imaging modality offers an unparalleled advantage over traditional electrophysiology by capturing both the morphology and dynamic activities of neurons, thereby facilitating long-term tracking of identified neurons5,6,7,8.
Despite its noteworthy strengths, the application of high-resolution fluorescence imaging often necessitates a static, head-fixed setup that constrains the animal&#39;s mobility9,10,11. Additionally, the use of a transparent glass surface for visualizing neurons restricts observations to one or more horizontal planes, limiting the exploration of the dynamics of vertical processes that extend across different cortical depths12.
Addressing these limitations, the present study outlines an innovative surgical procedure that integrates head plate fixation, microprism, and miniscope to create an imaging modality with multilayer and multimodal capabilities. The microprism allows the observation of the vertical processing along the cortical column13,14,15,16, which is critical in understanding how information is processed and transformed as it moves through different layers of the cortex and how the vertical processing is altered during plastic changes. Moreover, it permits imaging of the same neural populations in a head-fixed paradigm and in a freely moving setting, encompassing the versatile experimental settings17,18,19: for example, head-fixation is often required for well-controlled paradigms like sensory perception assessment and stable recordings under 2-photon paradigm, while freely moving offers a more natural, flexible environment for behavioral studies. Therefore, the ability to conduct a direct comparison in both modes is crucial to furthering our understanding of the microcircuits that enable flexible, functional responses.
In essence, the integration of head plate fixation, microprism, and miniscope in fluorescence imaging offers a promising platform for probing the intricacies of the brain&#39;s structure and functionality. Researchers can sample identified cell populations across various depths spanning all cortical layers, directly compare their responses in both well-controlled and natural paradigms, and monitor their long-term alterations over months20. This approach offers valuable insight into how these neural populations interact and change over time under different experimental conditions, providing a window into the dynamic nature of neural circuits.

<table><tbody><tr><td>0.9% Sodium Chloride solution for infusion (Vetivex 11) 250ml</td><td>Dechra</td><td>20091607</td><td>Saline for hydration and drug reconsitution</td></tr><tr><td>18004-1 Trephine 1.8mm diameter bur</td><td>FST</td><td>18004-18</td><td>Drill bit</td></tr><tr><td>1ml syringe</td><td>Terumo</td><td>MDSS01SE</td><td>1ml syringe</td></tr><tr><td>23G x 5/8 inch 6% LUER needle</td><td>Terumo</td><td>NN-2316R</td><td>23G needle</td></tr><tr><td>71000 Automated stereotaxic apparatus w/ built-in software</td><td>RWD</td><td>-</td><td>RWD</td></tr><tr><td>Absorbable Haemostatic Gelatin Sponge (10x10x10mm)</td><td>Surgispon</td><td>SSP-101010</td><td>gel-foam</td></tr><tr><td>Alcohol pads 70% isopropyl alcohol</td><td>Braun</td><td>9160612</td><td>Alcohol pads</td></tr><tr><td>Aluminium foil</td><td>Any retailer</td><td>-</td><td>Foil to cover eyes during surgery</td></tr><tr><td>Articifical Cerebrospinal Fluid&amp;nbsp;</td><td>Tocris Bioscience a Bio-Techne Brand</td><td>3525/25ML</td><td>ACSF</td></tr><tr><td>Automated microinjection pump</td><td>WPI</td><td>8091</td><td></td></tr><tr><td>Betadine solution (10% iodinated Povidone) 500ml</td><td>Videne/Ecolab</td><td>3030440</td><td>Betadine</td></tr><tr><td>Bruker Ultime 2Pplus (customised)</td><td>Bruker</td><td>-</td><td>Two-photon imaging system&amp;nbsp;</td></tr><tr><td>Cardiff Aldasorber</td><td>Vet-Tech</td><td>AN006</td><td>Anaesthesia absorber</td></tr><tr><td>CFI S Plan Fluor ELWD ADM 20XC</td><td>Nikon</td><td>MRH48230</td><td>20x objective lens</td></tr><tr><td>Compact Anaesthesia system - single gas - isoflurane K/F, with oxygen concentrator model: ZY-5AC and scavenging unit</td><td>Vet-Tech</td><td>AN001</td><td>Compact anaesthesia system&amp;nbsp;</td></tr><tr><td>Contec Prochlor&amp;nbsp;</td><td>Aston Pharma</td><td>AP2111L1</td><td>Disinfectant (hypochlorous acid)</td></tr><tr><td>Dexamethasone Sodium Phosphate Injection, USP, 4mg/ml, NDC: 0641-6145-25</td><td>Hikma</td><td>Covetrus:70789</td><td>Dexamethasone</td></tr><tr><td>Dissecting Knife, cutting edge 4mm, thickness 0.5mm, stainless steel</td><td>Fine Science Tools</td><td>10055-12</td><td>Knife for incisino of cortex</td></tr><tr><td>Dual-Sided, Non-Puncture Mouse &amp;amp; Neonatal Rat Ear Bars</td><td>Stoelting</td><td>51649</td><td>Ear bar</td></tr><tr><td>Dummy microscope</td><td>Inscopix</td><td>Dummy microscope</td><td>To help with implantation</td></tr><tr><td>Ethanol (100%)&amp;nbsp;</td><td>VWR</td><td>40-1712-25</td><td>Used to make 70% ethanol&amp;nbsp;</td></tr><tr><td>Fisherbrand&amp;nbsp;Nitrile Indigo Disposable Gloves PPE Cat III</td><td>FischerScientific</td><td>17182182</td><td>Gloves</td></tr><tr><td>Homeothermic Monitor 50-7222-F</td><td>Harvard Apparatus</td><td>50-7222-F</td><td>Homeothermic monitoring system/heating pad</td></tr><tr><td>Image processing software</td><td>ImageJ</td><td>-</td><td>Image processing software</td></tr><tr><td>Inscopix Data Processing Software (IDPS)</td><td>Inscopix</td><td>-</td><td>One-photon calcium imaging processing software</td></tr><tr><td>Insight Duals-232, S/N 2043</td><td>InSight</td><td>Insight Spectra X3</td><td>Two-photon imaging laser</td></tr><tr><td>IsoFlo 250ml 100% w/w inhalation</td><td>Zoetis</td><td>WM 42058/4195</td><td>Isoflurane</td></tr><tr><td>Kwik-Sil Low Toxicity Silicone Adhesive</td><td>World Precision Intruments (WPI)</td><td>KWIK-SIL</td><td>Silicone adhesive</td></tr><tr><td>MICROMOT mains adapter NG 2/S, w/ Drill unit 60/E</td><td>PROXXON</td><td>NO 28 515</td><td>Handheld drill</td></tr><tr><td>nVoke Integrated Imaging and Optogenetics System package</td><td>Inscopix</td><td>-</td><td>One-photon Imaging system and software</td></tr><tr><td>ProView Implant Kit</td><td>Inscopix</td><td>ProView Implant Kit</td><td>Dummy microscope, stereotaxic arm and attachment&amp;nbsp;</td></tr><tr><td>ProView Prism Probe</td><td>Inscopix</td><td>1050-002203</td><td>Microprism lens</td></tr><tr><td>Rimadyl (50mg/ml)</td><td>Zoetis</td><td>VM 42058/4123</td><td>Carprofen&amp;nbsp;</td></tr><tr><td>Stereotaxis Microscope on Articulated arm with table clamp</td><td>WPI</td><td>PZMTIII-AAC&amp;nbsp;</td><td>Microscope</td></tr><tr><td>Super-Bond Universal kit, SUN Medical</td><td>Prestige-Dental</td><td>K058E</td><td>Adhesive cement</td></tr><tr><td>Two-photon calcium image software</td><td>Suite2P</td><td>-</td><td>Two-photon calcium imaging processing software</td></tr><tr><td>Vapouriser</td><td>Vet-Tech</td><td>-</td><td>Isoflurane vapouriser</td></tr><tr><td>Xailin Lubricating Eye Ointment 5g</td><td>Xailin-Night</td><td>MLG/28/1551</td><td>Ophthalmic ointment&amp;nbsp;</td></tr></tbody></table>

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.

The method of conducting chronic multilayer in vivo calcium imaging of the same neuronal population over a period of several weeks, using both one- and two-photon imaging modalities, under freely moving and head-fixed conditions has been shown. Here, the ability to identify matching neuronal populations under one-photon imaging while the animal explored an open arena in the dark has been demonstrated (Figure 7A). Calcium traces were extracted from the identified neurons and z-scored for comparison (Figure 7B). Neurons showed comparable levels of fluorescence and firing rates in sessions that were 3 weeks apart.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65387/65387fig07v2.jpg" /> 
Figure 7: Calcium dynamics from the primary visual cortex can be stably registered across sessions spanning 3 weeks. (A) Spatial filters of identified neurons overlaid upon the maximum projection images from three separate recordings of the same field of view under one-photon freely moving calcium imaging, spanning a duration of 3 weeks. ROIs are labeled in the order they are presented in (B). Dark regions in the second (middle) and third (right) sessions are the results of image registration, using the first (left) session as reference (Scale bar represents 0.5 mm). (B) Z-scored calcium activities of the registered ROIs in the first (left), second (middle), and third (right) sessions shown in (A). The horizontal scale bar is 10 s, and the vertical scale bar is 10 sd. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65387/65387fig07largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the registration of a neuronal population across different imaging modalities was demonstrated. One-photon and two-photon imaging sessions were conducted on the same day. ROIs were identified from one-photon data and manually curated to generate a cell map (Figure 8A). Similarly, two-photon data were processed to generate a cell map whereby cells are automatically identified and manually selected for demonstration. Then, the mean projection image from the one-photon session was aligned to the maximum projection image from the two-photon z-stack with the MIRA platform (Figure 8B). This warped cell map was then exported, superimposed onto the two-photon maximum projection image, and aligned with the two-photon cell map (Figure 8C). This allowed for cell identification that showed activities of the same cells under both imaging modalities (Figure 8D), which can be used for quantitative analysis of the neuronal responses.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65387/65387fig08.jpg" /> 
Figure 8: Calcium dynamics from the primary visual cortex registered between different imaging modalities. (A) Spatial filters of identified neurons overlaid upon the maximum projection image from a one-photon, freely moving session. Labeled neurons are those that were successfully registered to two-photon recording data presented in (C). (B) The same contours shown in (A) were warped to match the two-photon session data shown in (C). (C) Spatial filters of the ROIs shown in (A) after alignment (white), and ROIs identified using the 2P processing software from a two-photon head-fixed recording session in the same day (green), overlaid upon the maximum projection image from the two-photon session. Overlapping ROIs are considered registered and are chosen for further analysis (Scale bars represent 0.5 mm). (D) Z-scored calcium activities of the registered ROIs in one-photon (left) and two-photon (right) recording sessions (horizontal scale bar is 10 s, and vertical scale bar is 10 sd). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65387/65387fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals at JoVE.com

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.

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 ...

long-term-imaging-identified-neural-populations-using-microprisms

Nanyang Technological University

Research

JoVE Journal

Neuroscience

902 Views.  University College London. Systeemneurowetenschappen hebben veel baat bij vooruitgang in beeldvormingstechnologie, vooral in combinatie met moleculaire en genetische benaderingen. Onderzoekers maken steeds vaker gebruik van hoge resolutie, chronische opname en celtype-specifieke targeting om de functie van de hersenen op fijnere schaal, langere tijd en meer positie te ontleden. Een opmerkelijke verschuiving in experimentele paradigma's is de verschuiving van de verdoofde opname naar wakkere of vrij bewegende toestanden...

Video: Auteur Spotlight: vergelijkende beeldvorming van neurale activiteit in wakkere en vrij bewegende toestanden

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

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

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

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

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

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

Behavior

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

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 ...

Biology

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

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 ...

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

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