Mouse Neocortex Research Articles

A disector-based framework for the automatic optical fractionator

Stereology-based methods provide the current state-of-the-art approaches for accurate quantification of numbers and other morphometric parameters of biological objects in stained tissue sections. The advent of artificial intelligence (AI)-based deep learning (DL) offers the possibility of improving throughput by automating the collection of stereology data. We have recently shown that DL can effectively achieve comparable accuracy to manual stereology but with higher repeatability, improved throughput, and less variation due to human factors by quantifying the total number of immunostained cells at their maximal profile of focus in extended depth of field (EDF) images. In the first of two novel contributions in this work, we propose a semi-automatic approach using a handcrafted Adaptive Segmentation Algorithm (ASA) to automatically generate ground truth on EDF images for training our deep learning (DL) models to automatically count cells using unbiased stereology methods. This update increases the amount of training data, thereby improving the accuracy and efficiency of automatic cell counting methods, without a requirement for extra expert time. The second contribution of this work is a Multi-channel Input and Multi-channel Output (MIMO) method using a U-Net deep learning architecture for automatic cell counting in a stack of z-axis images (also known as disector stacks). This DL-based digital automation of the ordinary optical fractionator ensures accurate counts through spatial separation of stained cells in the z-plane, thereby avoiding false negatives from overlapping cells in EDF images without the shortcomings of 3D and recurrent DL models. The contribution overcomes the issue of under-counting errors with EDF images due to overlapping cells in the z-plane (masking). We demonstrate the practical applications of these advances with automatic disector-based estimates of the total number of NeuN-immunostained neurons in a mouse neocortex. In summary, this work provides the first demonstration of automatic estimation of a total cell number in tissue sections using a combination of deep learning and the disector-based optical fractionator method.

Dysregulated Protein Phosphorylation in a Mouse Model of FTLD-Tau.

The neocortex of P301S mice, used as a model of fronto-temporal lobar degeneration linked to tau mutation (FTLD-tau), and wild-type mice, both aged 9 months, were analyzed with conventional label-free phosphoproteomics and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 328 corresponding to 524 phosphorylation sites. The majority of dysregulated phosphoproteins, most of them hyperphosphorylated, were proteins of the membranes, synapses, membrane trafficking, membrane vesicles linked to endo- and exocytosis, cytoplasmic vesicles, and cytoskeleton. Another group was composed of kinases. In contrast, proteins linked to DNA, RNA metabolism, RNA splicing, and protein synthesis were hypophosphorylated. Other pathways modulating energy metabolism, cell signaling, Golgi apparatus, carbohydrates, and lipids are also targets of dysregulated protein phosphorylation in P301S mice. The present results, together with accompanying immunohistochemical and Western-blotting studies, show widespread abnormal phosphorylation of proteins, in addition to protein tau, in P301S mice. These observations point to dysregulated protein phosphorylation as a relevant contributory pathogenic component of tauopathies.

Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer's disease transgenic mice.

Increased astrocytic Ca2+ signaling has been shown in Alzheimer's disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signaling in such mice. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signaling in 15-month-old plaque-bearing mice. Using the genetically encoded fluorescent norepinephrine sensor GRABNE, we demonstrate a reduced norepinephrine signaling during spontaneous running and startle responses in the transgenic mice, providing a possible mechanistic underpinning of the observed reduced astrocytic Ca2+ responses. Our data points to a dysfunction in the norepinephrine-astrocyte Ca2+ activity axis, which may account for some of the cognitive deficits observed in Alzheimer's disease.

Critical Role of Neuronal Vps35 in Blood Vessel Branching and Maturation in Developing Mouse Brain.

Vps35 (vacuolar protein sorting 35), a key component of retromer, plays a crucial role in selective retrieval of transmembrane proteins from endosomes to trans-Golgi networks. Dysfunctional Vps35/retromer is a risk factor for the development of neurodegenerative diseases. Vps35 is highly expressed in developing pyramidal neurons, both in the mouse neocortex and hippocampus, Although embryonic neuronal Vps35’s function in promoting neuronal terminal differentiation and survival is evident, it remains unclear whether and how neuronal Vps35 communicates with other types of brain cells, such as blood vessels (BVs), which are essential for supplying nutrients to neurons. Dysfunctional BVs contribute to the pathogenesis of various neurodegenerative disorders. Here, we provide evidence for embryonic neuronal Vps35 as critical for BV branching and maturation in the developing mouse brain. Selectively knocking out (KO) Vps35 in mouse embryonic, not postnatal, neurons results in reductions in BV branching and density, arteriole diameter, and BV-associated pericytes and microglia but an increase in BV-associated reactive astrocytes. Deletion of microglia by PLX3397 enhances these BV deficits in mutant mice. These results reveal the function of neuronal Vps35 in neurovascular coupling in the developing mouse brain and implicate BV-associated microglia as underlying this event.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near‐Infrared Light‐Triggered Release and Optical Sensing**

AbstractNeuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell‐based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin‐14 (SST) can be rapidly released under near‐infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 μm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 μm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

A quantitative in vivo imaging platform for tracking pathological tau depositions and resultant neuronal death in a mouse model.

Depositions of tau fibrils are implicated in diverse neurodegenerative disorders, including Alzheimer's disease, and precise assessments of tau pathologies and their impacts on neuronal survival are crucial for pursuing the neurodegenerative tau pathogenesis with and without potential therapies. We aimed to establish an in vivo imaging system to quantify tau accumulations with positron emission tomography (PET) and brain atrophy with volumetric MRI in rTg4510 transgenic mice modeling neurodegenerative tauopathies. A total of 91 rTg4510 and non-transgenic control mice underwent PET with a tau radiotracer, 18F-PM-PBB3, and MRI at various ages (1.8-12.3 months). Using the cerebellum as reference, the radiotracer binding in target regions was estimated as standardized uptake value ratio (SUVR) and distribution volume ratio (DVR). Histopathological staining of brain sections derived from scanned animals was also conducted to investigate the imaging-neuropathology correlations. 18F-PM-PBB3 SUVR at 40-60 min in the neocortex, hippocampus, and striatum of rTg4510 mice agreed with DVR, became significantly different from control values around 4-5 months of age, and progressively and negatively correlated with age and local volumes, respectively. Neocortical SUVR also correlated with the abundance of tau inclusions labeled with PM-PBB3 fluorescence, Gallyas-Braak silver impregnation, and anti-phospho-tau antibodies in postmortem assays. The in vivo and ex vivo 18F-PM-PBB3 binding was blocked by non-radioactive PM-PBB3. 18F-PM-PBB3 yielded a 1.6-fold greater dynamic range for tau imaging than its ancestor, 11C-PBB3. Our imaging platform has enabled the quantification of tau depositions and consequent neuronal loss and is potentially applicable to the evaluation of candidate anti-tau and neuroprotective drugs.

Network States Classification based on Local Field Potential Recordings in the Awake Mouse Neocortex.

Recent studies using intracellular recordings in awake behaving mice revealed that cortical network states, defined based on membrane potential features, modulate sensory responses and perceptual outcomes. Single-cell intracellular recordings are difficult and have low yield compared to extracellular recordings of population signals, such as local field potentials (LFPs). However, it is currently unclear how to identify these behaviorally-relevant network states from the LFP. We used simultaneous LFP and intracellular recordings in the somatosensory cortex of awake mice to design a network state classification from the LFP, the Network State Index (NSI). We used the NSI to analyze the relationship between single-cell (intracellular) and population (LFP) signals over different network states of wakefulness. We found that graded levels of population signal faithfully predicted the levels of single-cell depolarization in nonrhythmic regimes whereas, in δ ([2–4 Hz]) oscillatory regimes, the graded levels of rhythmicity in the LFP mapped into a stereotypical oscillatory pattern of membrane potential. Finally, we showed that the variability of network states, beyond the occurrence of slow oscillatory activity, critically shaped the average correlations between single-cell and population signals. Application of the LFP-based NSI to mouse visual cortex data showed that this index increased with pupil size and during locomotion and had a U-shaped dependence on population firing rates. NSI-based characterization provides a ready-to-use tool to understand from LFP recordings how the modulation of local network dynamics shapes the flexibility of sensory processing during behavior.

Metabolic lactate production coordinates vasculature development and progenitor behavior in the developing mouse neocortex.

Proper neural progenitor behavior in conjunction with orderly vasculature formation is fundamental to the development of the neocortex. However, the mechanisms coordinating neural progenitor behavior and vessel growth remain largely elusive. Here we show that robust metabolic production of lactate by radial glial progenitors (RGPs) co-regulates vascular development and RGP division behavior in the developing mouse neocortex. RGPs undergo a highly organized lineage progression program to produce diverse neural progeny. Systematic single-cell metabolic state analysis revealed that RGPs and their progeny exhibit distinct metabolic features associated with specific cell types and lineage progression statuses. Symmetrically dividing, proliferative RGPs preferentially express a cohort of genes that support glucose uptake and anaerobic glycolysis. Consequently, they consume glucose in anaerobic metabolism and produce a high level of lactate, which promotes vessel growth. Moreover, lactate production enhances RGP proliferation by maintaining mitochondrial length. Together, these results suggest that specific metabolic states and metabolites coordinately regulate vasculature formation and progenitor behavior in neocortical development.

Prenatal reduction of E14.5 embryonically fate-mapped pyramidal neurons in a mouse model of autism.

Although several observations suggest that the constitutive biological, genetic or physiological changes leading to autism spectrum disorders (ASD) start in utero, their early impact on the number and density of neurons in the brain remains unknown. Using genetic fate mapping associated with the immunollabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) clearing method we identified and counted a selective population of neocortical and hippocampal pyramidal neurons in the in utero valproate (VPA) mouse model of autism. We report that 1 day before birth, the number of pyramidal neurons born at E14.5 in the neocortex and hippocampus of VPA mice is smaller than in age-matched controls. VPA also induced a reduction of the neocortical-but not hippocampal-volume 1 day before birth. Interestingly, VPA mice present an increase in both neocortical and hippocampal volumes 2days after birth compared with controls. These results suggest that the VPA-exposed hippocampus and neocortex differ substantially from controls during the highly complex perinatal period, and specially 1 day before birth, reflecting the early pathogenesis of ASD.

DIALOGUE maps multicellular programs in tissue from single-cell or spatial transcriptomics data.

Deciphering the functional interactions of cells in tissues remains a major challenge. Here we describe DIALOGUE, a method to systematically uncover multicellular programs (MCPs)-combinations of coordinated cellular programs in different cell types that form higher-order functional units at the tissue level-from either spatial data or single-cell data obtained without spatial information. Tested on spatial datasets from the mouse hypothalamus, cerebellum, visual cortex and neocortex, DIALOGUE identified MCPs associated with animal behavior and recovered spatial properties when tested on unseen data while outperforming other methods and metrics. In spatial data from human lung cancer, DIALOGUE identified MCPs marking immune activation and tissue remodeling. Applied to single-cell RNA sequencing data across individuals or regions, DIALOGUE uncovered MCPs marking Alzheimer's disease, ulcerative colitis and resistance to cancer immunotherapy. These programs were predictive of disease outcome and predisposition in independent cohorts and included risk genes from genome-wide association studies. DIALOGUE enables the analysis of multicellular regulation in health and disease.

Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Cell Type-Specific Transcriptional Control of Gsk3β in the Developing Mammalian Neocortex.

Temporal control of neurogenesis is central for the development and evolution of species-specific brain architectures. The balance between progenitor expansion and neuronal differentiation is tightly coordinated by cell-intrinsic and cell-extrinsic cues. Wnt signaling plays pivotal roles in the proliferation and differentiation of neural progenitors in a temporal manner. However, regulatory mechanisms that adjust intracellular signaling amplitudes according to cell fate progression remain to be elucidated. Here, we report the transcriptional controls of Gsk3β, a critical regulator of Wnt signaling, in the developing mouse neocortex. Gsk3β expression was higher in ventricular neural progenitors, while it gradually declined in differentiated neurons. We identified active cis-regulatory module (CRM) of Gsk3β that responded to cell type-specific transcription factors, such as Sox2, Sox9, and Neurogenin2. Furthermore, we found extensive conservation of the CRM among mammals but not in non-mammalian amniotes. Our data suggest that a mammalian-specific CRM drives the cell type-specific activity of Gsk3β to fine tune Wnt signaling, which contributes to the tight control of neurogenesis during neocortical development.

Midazolam at Low Nanomolar Concentrations Affects Long-term Potentiation and Synaptic Transmission Predominantly via the α1-γ-Aminobutyric Acid Type A Receptor Subunit in Mice.

Midazolam amplifies synaptic inhibition via different γ-aminobutyric acid type A (GABAA) receptor subtypes defined by the presence of α1-, α2-, α3-, or α5-subunits in the channel complex. Midazolam blocks long-term potentiation and produces postoperative amnesia. The aims of this study were to identify the GABAA receptor subtypes targeted by midazolam responsible for affecting CA1 long-term potentiation and synaptic inhibition in neocortical neurons. The effects of midazolam on hippocampal CA1 long-term potentiation were studied in acutely prepared brain slices of male and female mice. Positive allosteric modulation on GABAA receptor-mediated miniature inhibitory postsynaptic currents was investigated in organotypic slice cultures of the mouse neocortex. In both experiments, wild-type mice and GABAA receptor knock-in mouse lines were compared in which α1-, α5-, α1/2/3-, α1/3/5- and α2/3/5-GABAA receptor subtypes had been rendered benzodiazepine-insensitive. Midazolam (10 nM) completely blocked long-term potentiation (mean ± SD, midazolam, 98 ± 11%, n = 14/8 slices/mice vs. control 156 ± 19%, n = 20/12; P < 0.001). Experiments in slices of α1-, α5-, α1/2/3-, α1/3/5-, and α2/3/5-knock-in mice revealed a dominant role for the α1-GABAA receptor subtype in the long-term potentiation suppressing effect. In slices from wild-type mice, midazolam increased (mean ± SD) charge transfer of miniature synaptic events concentration-dependently (50 nM: 172 ± 71% [n = 10/6] vs. 500 nM: 236 ± 54% [n = 6/6]; P = 0.041). In α2/3/5-knock-in mice, charge transfer of miniature synaptic events did not further enhance when applying 500 nM midazolam (50 nM: 171 ± 62% [n = 8/6] vs. 500 nM: 175 ± 62% [n = 6/6]; P = 0.454), indicating two different binding affinities for midazolam to α2/3/5- and α1-subunits. These results demonstrate a predominant role of α1-GABAA receptors in the actions of midazolam at low nanomolar concentrations. At higher concentrations, midazolam also enhances other GABAA receptor subtypes. α1-GABAA receptors may already contribute at sedative doses to the phenomenon of postoperative amnesia that has been reported after midazolam administration.

Local connectivity and synaptic dynamics in mouse and human neocortex.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex.

In the neocortex, functionally distinct areas process specific types of information. Area identity is established by morphogens and transcriptional master regulators, but downstream mechanisms driving area-specific neuronal specification remain unclear. Here, we reveal a role for RNA-binding proteins in defining area-specific cytoarchitecture. Mice lacking Pum2 or overexpressing human TDP-43 show apparent 'motorization' of layers IV and V of primary somatosensory cortex (S1), characterized by dramatic expansion of cells co-expressing Sox5 and Bcl11b/Ctip2, a hallmark of subcerebral projection neurons, at the expense of cells expressing the layer IV neuronal marker Rorβ. Moreover, retrograde labeling experiments with cholera toxin B in Pum2; Emx1-Cre and TDP43A315T mice revealed a corresponding increase in subcerebral connectivity of these neurons in S1. Intriguingly, other key features of somatosensory area identity are largely preserved, suggesting that Pum2 and TDP-43 may function in a downstream program, rather than controlling area identity per se. Transfection of primary neurons and in utero electroporation (IUE) suggest cell-autonomous and post-mitotic modulation of Sox5, Bcl11b/Ctip2, and Rorβ levels. Mechanistically, we find that Pum2 and TDP-43 directly interact with and affect the translation of mRNAs encoding Sox5, Bcl11b/Ctip2, and Rorβ. In contrast, effects on the levels of these mRNAs were not detectable in qRT-PCR or single-molecule fluorescent in situ hybridization assays, and we also did not detect effects on their splicing or polyadenylation patterns. Our results support the notion that post-transcriptional regulatory programs involving translational regulation and mediated by Pum2 and TDP-43 contribute to elaboration of area-specific neuronal identity and connectivity in the neocortex.

Principles of GABAergic signaling in developing cortical network dynamics.

GABAergic signaling provides inhibitory stabilization and spatiotemporally coordinates the firing of recurrently connected excitatory neurons in mature cortical circuits. Inhibition thus enables self-generated neuronal activity patterns that underlie various aspects of sensation and cognition. In this review, we aim to provide a conceptual framework describing how and when GABA-releasing interneurons acquire their network functions during development. Focusing on the developing visual neocortex and hippocampus in mice and rats invivo, we hypothesize that at the onset of patterned activity, glutamatergic neurons are stable by themselves and inhibitory stabilization is not yet functional. We review important milestones in the development of GABAergic signaling and illustrate how the cell-type-specific strengthening of synaptic inhibition toward eye opening shapes cortical network dynamics and allows the developing cortex to progressively disengage from extra-cortical synaptic drive. We translate this framework to human cortical development and discuss clinical implications for the treatment of neonatal seizures.

Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler

How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, to systematically map the regulatory landscape of neural differentiation in the mouse neocortex, we profiled gene expression and chromatin accessibility in single cells and integrated these data with measurements of enhancer activity, DNA methylation and three-dimensional genome architecture in purified cell populations. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex.

Neuronal Yin Yang1 in the prefrontal cortex regulates transcriptional and behavioral responses to chronic stress in mice

Although the synaptic alterations associated with the stress-related mood disorder major depression has been well-documented, the underlying transcriptional mechanisms remain poorly understood. Here, we perform complementary bulk nuclei- and single-nucleus transcriptome profiling and map locus-specific chromatin interactions in mouse neocortex to identify the cell type-specific transcriptional changes associated with stress-induced behavioral maladaptation. We find that cortical excitatory neurons, layer 2/3 neurons in particular, are vulnerable to chronic stress and acquire signatures of gene transcription and chromatin structure associated with reduced neuronal activity and expression of Yin Yang 1 (YY1). Selective ablation of YY1 in cortical excitatory neurons enhances stress sensitivity in both male and female mice and alters the expression of stress-associated genes following an abbreviated stress exposure. These findings demonstrate how chronic stress impacts transcription in cortical excitatory neurons and identify YY1 as a regulator of stress-induced maladaptive behavior in mice.

Chronic co-implantation of ultraflexible neural electrodes and a cranial window.

.Significance: Electrophysiological recording and optical imaging are two prevalent neurotechnologies with complementary strengths, the combined application of which can significantly improve our capacity in deciphering neural circuits. Flexible electrode arrays can support longitudinal optical imaging in the same brain region, but their mechanical flexibility makes surgical preparation challenging. Here, we provide a step-by-step protocol by which an ultraflexible nanoelectronic thread is co-implanted with a cranial window in a single surgery to enable chronic, dual-modal measurements.Aim: The method uses -thick polymer neural electrodes which conform to the site of implantation. The mechanical flexibility of the probe allows bending without breaking and enables long-lasting electrophysiological recordings of single-unit activities and concurrent, high-resolution optical imaging through the cranial window.Approach: The protocol describes methods and procedures to co-implant an ultraflexible electrode array and a glass cranial window in the mouse neocortex. The implantation strategy includes temporary attachment of flexible electrodes to a retractable tungsten-microwire insertion shuttle, craniotomy, stereotaxic insertion of the electrode array, skull fixation of the cranial window and electrode, and installation of a head plate.Results: The resultant implant allows simultaneous interrogation of brain activity both electrophysiologically and optically for several months. Importantly, a variety of optical imaging modalities, including wide-field fluorescent imaging, two-photon microscopy, and functional optical imaging, can be readily applied to the specific brain region where ultraflexible electrodes record from.Conclusions: The protocol describes a method for co-implantation of ultraflexible neural electrodes and a cranial window for chronic, multimodal measurements of brain activity in mice. Device preparation and surgical implantation are described in detail to guide the adaptation of these methods for other flexible neural implants and cranial windows.