ex-vivo brain slices preparation from mouse brain tissue for studying hippocampal-cortical and olfactory bulb circuits

Multimodal Large-Scale Neural Recordings and Analysis on HD-MEA 

Neuronal ensembles, often termed cell assemblies, are pivotal in neural coding, facilitating intricate computations for processing multiscale neural information1,2,3. These ensembles underpin the formation of expansive neuronal networks and their nuanced microcircuits4. Such networks and their oscillatory patterns drive advanced brain functions, including perception and cognition. While extensive research has explored specific neuronal types and synaptic pathways, a deeper understanding of how neurons collaboratively form cell assemblies and influence spatiotemporal information processing across circuits and networks remains elusive5.
Acute, ex-vivo brain slices are pivotal electrophysiological tools for studying intact neural circuits, offering a controlled setting to probe oscillatory activity patterns of neural function, synaptic transmission, and connectivity, with implications in pharmacological testing and disease modeling6,7,8. This study protocol highlights two key brain circuits - the hippocampal-cortical (HC) involved in learning and memory processes9,10, and the olfactory bulb (OB) responsible for odor discrimination11,12,13. In these two regions, new functional neurons are continuously generated by adult neurogenesis throughout life in mammalian brains14. Both circuits demonstrate multidimensional dynamic neural activity patterns and inherent plasticity that participate in rewiring the existing neural network and facilitate alternative information processing strategies when required15,16.
Acute, ex-vivo brain slice models are indispensable for delving into brain functionality and understanding disease mechanisms at the microcircuit level. However, in-vitro cell cultures derived from human induced pluripotent stem cells (iPSCs) neuronal networks offer a promising avenue of translational research, seamlessly connecting findings from animal experiments to potential human clinical treatment17,18. These human-centric in-vitro assays serve as a reliable platform for assessing pharmacological toxicity, enabling precise drug screening, and furthering research into innovative cell-based therapeutic strategies19,20. Recognizing the pivotal role of the iPSC neuronal model, we have dedicated the third module of this protocol study to thoroughly investigate the functional characteristics of its derived networks and to fine-tune the associated cell culture protocols.
These electrogenic neural modules have been commonly studied using techniques like calcium (Ca2+ imaging), patch-clamp recordings, and low-density microelectrode arrays (LD-MEA). While Ca2+ imaging offers single-cell activity mapping, it is a cell-labeling-based method hindered by its low temporal resolution and challenges in long-term recordings. LD-MEAs lack spatial precision, while patch-clamp, being an invasive single-site technique and laborious, often yields a low success rate21,22,23. To address these challenges and effectively probe network-wide activity, large-scale simultaneous neural recordings have emerged as a pivotal approach for understanding the computational principles of neural dynamics underlying brain complexity and their implications in health and disease24,25.
In this JoVE protocol, we demonstrate a large-scale neural recording method based on the high-density MEA (HD-MEA) for capturing spatiotemporal neuronal activity across various brain modalities, including hippocampal and olfactory bulb circuits from ex-vivo mouse brain acute slices (Figures 1A-C) and in-vitro human iPSC-derived neuronal networks (Figures 1D-E), previously reported by our group and other colleagues26,27,28,29,30,31,32,33,34,35. The HD-MEA, built on complementary-metal-oxide-semiconductor (CMOS) technology, boasts on-chip circuitry and amplification, allowing sub-millisecond recordings across a 7mm2 array size36. This non-invasive approach captures multi-site, label-free extracellular firing patterns from thousands of neuronal ensembles simultaneously using 4096 microelectrodes at a high spatiotemporal resolution, revealing the intricate dynamics of local field potentials (LFPs) and multiunit spiking activity (MUA)26,29.
Given the vastness of the data generated by this methodology, a sophisticated analytical framework is essential, yet poses challenges37. We have developed computational tools that encompass automatic event detection, classification, graph theory, machine learning, and other advanced techniques (Figure 1F)26,29,38,39. Integrating the HD-MEA with these analytical tools, a holistic approach is devised to probe the intricate dynamics from individual cell assemblies to broader neural networks across diverse neural modalities. This combined approach deepens our grasp of the computational dynamics in normal brain functions and offers insights into anomalies present in pathological conditions28. Moreover, insights from this approach can propel advancements in brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Ultimately, this method holds promise in uncovering the core mechanisms behind neural network disruptions, potentially identifying biomarkers, and guiding the creation of precise diagnostic tools and targeted treatments for neurological conditions.

Author Spotlight: Advancing Large-Scale Neural Dynamics Through HD-MEA Technology

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Our research explores the frontiers of neural technology by integrating high-density CMOS-based microelectrode array for decoding neural communication and large networks. We aim to answer how neural information across scales is encoded in unique detail, enhancing our understanding of brain function and dysfunction in health and disease. Navigating the complex area of neuronal ensemble research, we confront challenges like achieving precise signal resolution amidst brain activity, and ensuring the biocompatibility of our CMOS-based microelectrode arrays.These hurdles are pivotal in accurately capturing and interpreting the rich tapestry of neural interaction using multimodal recordings. Our research addresses a critical gap in neuroscience, the lack of a comprehensive method to recode and analyze the dynamics of larger scale neuronal ensemble with high spatial and temporal resolution. This gap hinders our understanding of complex brain networks and function in health and disease.Our protocol enables multimodal, label-free, high-resolution recordings across the hippocampus, olfactory bulb, and human IPSC-derived neurons, providing a versatile tool for diverse experiments. This unique approach facilitates unparalleled insight into neuronal dynamics, bridging the research gap between various brain regions and model systems, significantly advancing our understanding of neural function and disorder. Future endeavors in our lab will deeply investigate neural computations and dynamics from genes to networks, aiming to bridge molecular and functional signatures in health and disease.Through advanced bioelectronics and neural technology, we'll focus on neuroplasticity, olfactory coding, developing AI, and memory-enhancing strategies for novel therapeutics and brain machine interfaces.

Watch this Scientific Journal Video about Ex-Vivo Brain Slices Preparation from Mouse Brain Tissue for Studying Hippocampal-Cortical and Olfactory Bulb Circuits at JoVE.com

Ex-Vivo Brain Slices Preparation from Mouse Brain Tissue for Studying Hippocampal-Cortical and Olfactory Bulb Circuits

ex-vivo-brain-slices-preparation-from-mouse-brain-tissue-for-studying

Research

JoVE Journal

Neuroscience

568 次观看.  German Center for Neurodegenerative Diseases (DZNE). 首先，设置脑切片恢复和维护工作区。准备脑切片准备工作区并设置振动切片机。接下来，设置大脑提取和准备工作区。然后将提取的小鼠大脑转移到大脑制备工作区中带有冷却碳酸切割溶液的 90 毫米塑料培养皿中。定向大脑以在琼脂糖模具中定位。将强力胶涂在琼脂糖模具的喙端。用刮刀将大脑放在模具中，背侧朝下进行水平切片。切片海马体和嗅球组织。并?...

视频: 从小鼠脑组织制备离体脑切片，用于研究海马-皮质和嗅球回路

recording and analyzing multimodal large-scale neuronal ensemble dynamics on cmos-integrated high-density microelectrode array

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological...

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ex-vivo circuit-wide recordings from hippocampal-cortical and olfactory bulb slices using high-density microelectrode array (hd-mea)

Ex-Vivo Circuit-Wide Recordings from Hippocampal-Cortical and Olfactory Bulb Slices Using High-Density Microelectrode Array (HD-MEA)

ex vivo optogenetic dissection of fear circuits in brain slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

horizontal hippocampal slices of the mouse brain

Horizontal Hippocampal Slices of the Mouse Brain

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological...

obtaining horizontal mouse hippocampal brain slices

This video demonstrates a detailed protocol for preparing horizontal hippocampal brain slices from mice, focusing on preserving the integrity of hippocampal fiber pathways. The process involves carefully bisecting and securing the brain, using a vibratome for slicing, and maintaining optimal tissue conditions with a carbogenated fluid, resulting in hippocampal brain slices ready for further...

Obtaining Horizontal Mouse Hippocampal Brain Slices

ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Use two additional filter paper straps to position the second hemisphere onto the glue as just demonstrated, and place the specimen plate into the slicing chamber. Then, quickly but carefully, cover the plate with ice-cold high-sucrose slice solution slush. To acquire sections of the brain tissue, position the vibratome blade in front of the medial side of the hemispheres, and lift the vibratome table so that the blade is on the same height as the ventral sides of the hemispheres, which are now...

combined mechanical and enzymatic dissociation of mouse brain hippocampal tissue

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining...

isolation of the brain secretome from ex vivo brain slice cultures

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

The brain secretome plays a pivotal role in the development and normal functioning of the central nervous system. In this article, we provide a detailed protocol for isolation of the brain secretome from ex vivo brain slice...

Isolation of the Brain Secretome from Ex Vivo Brain Slice Cultures

preparation of colliculo-thalamocortical slices from a mouse pup brain

The video demonstrates the preparation of colliculo-thalamocortical brain slices from mouse pup brains. An isolated brain is aligned on a marked slide and cut at an angle to align the auditory midbrain and forebrain in the same plane. The brain is then sliced using a vibratome, and the slices containing the major auditory midbrain and forebrain structures are transferred to a holding chamber for...

Preparation of Colliculo-Thalamocortical Slices from a Mouse Pup Brain

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular...

evaluation of synapse density in hippocampal rodent brain slices

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this...

Next, glue the backstop onto the stage with cyanoacrylate adhesive, and then glue the bump on the right side of the backstop such that they form an 80-degree angle. On a slide marked with two lines at 90 degrees and a diagonal line at 17 degrees from the top left to the bottom right, place the brain dorsal side up at the intersecting point of the two...

analysis of ex-vivo mouse brain slices and in-vitro human ipsc-derived neuronal networks

Analysis of Ex-Vivo Mouse Brain Slices and In-Vitro Human iPSC-Derived Neuronal Networks

ex utero electroporation and organotypic slice culture of mouse hippocampal tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

in vivo intracerebral stereotaxic injections for optogenetic stimulation of long-range inputs in mouse brain slices

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain...

ex vivo culture and imaging of oculomotor slices from transgenic mouse embryos

This video demonstrates the ex vivo culture and imaging of oculomotor slices from transgenic mouse embryos. The embryos are isolated and embedded in agarose. Using a vibratome, slices containing the oculomotor nuclei and eyes are obtained. The slices are maintained in culture and imaged using a fluorescence...

Ex Vivo Culture and Imaging of Oculomotor Slices from Transgenic Mouse Embryos

minimizing hypoxia in hippocampal slices from adult and aging mice

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and...

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...