Uncovering kinesin dynamics in neurites with MINFLUX.

Neurons grow neurites of several tens of micrometers in length, necessitating active transport from the cell body by motor proteins. By tracking fluorophores as minimally invasive labels, MINFLUX is able to quantify the motion of those proteins with nanometer/millisecond resolution. Here we study the substeps of a truncated kinesin-1 mutant in primary rat hippocampal neurons, which have so far been mainly observed on polymerized microtubules deposited onto glass coverslips. A gentle fixation protocol largely maintains the structure and surface modifications of the microtubules in the cell. By analyzing the time between the substeps, we identify the ATP-binding state of kinesin-1 and observe the associated rotation of the kinesin-1 head in neurites. We also observed kinesin-1 switching microtubules mid-walk, highlighting the potential of MINFLUX to study the details of active cellular transport.

Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy.

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15µm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

Real-Time FRET-Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons.

Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.

Magnetic voluntary head-fixation in transgenic rats enables lifespan imaging of hippocampal neurons.

The precise neural mechanisms within the brain that contribute to the remarkable lifetime persistence of memory are not fully understood. Two-photon calcium imaging allows the activity of individual cells to be followed across long periods, but conventional approaches require head-fixation, which limits the type of behavior that can be studied. We present a magnetic voluntary head-fixation system that provides stable optical access to the brain during complex behavior. Compared to previous systems that used mechanical restraint, there are no moving parts and animals can engage and disengage entirely at will. This system is failsafe, easy for animals to use and reliable enough to allow long-term experiments to be routinely performed. Animals completed hundreds of trials per session of an odor discrimination task that required 2-4 s fixations. Together with a reflectance fluorescence collection scheme that increases two-photon signal and a transgenic Thy1-GCaMP6f rat line, we are able to reliably image the cellular activity in the hippocampus during behavior over long periods (median 6 months), allowing us track the same neurons over a large fraction of animals' lives (up to 19 months).

Three-dimensional liquid metal-based neuro-interfaces for human hippocampal organoids.

Human hippocampal organoids (hHOs) derived from human induced pluripotent stem cells (hiPSCs) have emerged as promising models for investigating neurodegenerative disorders, such as schizophrenia and Alzheimer's disease. However, obtaining the electrical information of these free-floating organoids in a noninvasive manner remains a challenge using commercial multi-electrode arrays (MEAs). The three-dimensional (3D) MEAs developed recently acquired only a few neural signals due to limited channel numbers. Here, we report a hippocampal cyborg organoid (cyb-organoid) platform coupling a liquid metal-polymer conductor (MPC)-based mesh neuro-interface with hHOs. The mesh MPC (mMPC) integrates 128-channel multielectrode arrays distributed on a small surface area (~2*2 mm). Stretchability (up to 500%) and flexibility of the mMPC enable its attachment to hHOs. Furthermore, we show that under Wnt3a and SHH activator induction, hHOs produce HOPX+ and PAX6+ progenitors and ZBTB20+PROX1+ dentate gyrus (DG) granule neurons. The transcriptomic signatures of hHOs reveal high similarity to the developing human hippocampus. We successfully detect neural activities from hHOs via the mMPC from this cyb-organoid. Compared with traditional planar devices, our non-invasive coupling offers an adaptor for recording neural signals from 3D models.

Location analysis of presynaptically active and silent synapses in single-cultured hippocampal neurons.

A morphologically present but non-functioning synapse is termed a silent synapse. Silent synapses are categorized into "postsynaptically silent synapses," where AMPA receptors are either absent or non-functional, and "presynaptically silent synapses," where neurotransmitters cannot be released from nerve terminals. The presence of presynaptically silent synapses remains enigmatic, and their physiological significance is highly intriguing. In this study, we examined the distribution and developmental changes of presynaptically active and silent synapses in individual neurons. Our findings show a gradual increase in the number of excitatory synapses, along with a corresponding decrease in the percentage of presynaptically silent synapses during neuronal development. To pinpoint the distribution of presynaptically active and silent synapses, i.e., their positional information, we employed Sholl analysis. Our results indicate that the distribution of presynaptically silent synapses within a single neuron does not exhibit a distinct pattern during synapse development in different distance from the cell body. However, irrespective of neuronal development, the proportion of presynaptically silent synapses tends to rise as the projection site moves farther from the cell body, suggesting that synapses near the cell body may exhibit higher synaptic transmission efficiency. This study represents the first observation of changes in the distribution of presynaptically active and silent synapses within a single neuron.

Exertional heat stroke-induced changes in gut microbiota cause cognitive impairment in mice.

BACKGROUND: The incidence of exertional heat stroke (EHS) escalates during periods of elevated temperatures, potentially leading to persistent cognitive impairment postrecovery. Currently, effective prophylactic or therapeutic measures against EHS are nonexistent. METHODS: The selection of days 14 and 23 postinduction for detailed examination was guided by TEM of neuronal cells and HE staining of intestinal villi and the hippocampal regions. Fecal specimens from the ileum and cecum at these designated times were analyzed for changes in gut microbiota and metabolic products. Bioinformatic analyses facilitated the identification of pivotal microbial species and metabolites. The influence of supplementing these identified microorganisms on behavioral outcomes and the expression of functional proteins within the hippocampus was subsequently assessed. RESULTS: TEM analyses of neurons, coupled with HE staining of intestinal villi and the hippocampal region, indicated substantial recovery in intestinal morphology and neuronal injury on Day 14, indicating this time point for subsequent microbial and metabolomic analyses. Notably, a reduction in the Lactobacillaceae family, particularly Lactobacillus murinus, was observed. Functional annotation of 16S rDNA sequences suggested diminished lipid metabolism and glycan biosynthesis and metabolism in EHS models. Mice receiving this intervention (EHS + probiotics group) exhibited markedly reduced cognitive impairment and increased expression of BDNF/TrKB pathway molecules in the hippocampus during behavioral assessment on Day 28. CONCLUSION: Probiotic supplementation, specifically with Lactobacillus spp., appears to mitigate EHS-induced cognitive impairment, potentially through the modulation of the BDNF/TrKB signaling pathway within the hippocampus, illustrating the therapeutic potential of targeting the gut-brain axis.

Control of working memory by phase-amplitude coupling of human hippocampal neurons.

Retaining information in working memory is a demanding process that relies on cognitive control to protect memoranda-specific persistent activity from interference1,2. However, how cognitive control regulates working memory storage is unclear. Here we show that interactions of frontal control and hippocampal persistent activity are coordinated by theta-gamma phase-amplitude coupling (TG-PAC). We recorded single neurons in the human medial temporal and frontal lobe while patients maintained multiple items in their working memory. In the hippocampus, TG-PAC was indicative of working memory load and quality. We identified cells that selectively spiked during nonlinear interactions of theta phase and gamma amplitude. The spike timing of these PAC neurons was coordinated with frontal theta activity when cognitive control demand was high. By introducing noise correlations with persistently active neurons in the hippocampus, PAC neurons shaped the geometry of the population code. This led to higher-fidelity representations of working memory content that were associated with improved behaviour. Our results support a multicomponent architecture of working memory1,2, with frontal control managing maintenance of working memory content in storage-related areas3-5. Within this framework, hippocampal TG-PAC integrates cognitive control and working memory storage across brain areas, thereby suggesting a potential mechanism for top-down control over sensory-driven processes.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

[Screening of place cell and analysis of its influencing factors for pigeons].

Place cell with location tuning characteristics play an important role in brain spatial cognition and navigation, but there is relatively little research on place cell screening and its influencing factors. Taking pigeons as model animals, the screening process of pigeon place cell was given by using the spike signal in pigeon hippocampus under free activity. The effects of grid number and filter kernel size on the place field of place cells during the screening process were analyzed. The results from the real and simulation data showed that the proposed place cell screening method presented in this study could effectively screen out place cell, and the research found that the size of place field was basically inversely proportional to the number of grids divided, and was basically proportional to the size of Gaussian filter kernel in the overall trend. This result will not only help to determine the appropriate parameters in the place cell screening process, but also promote the research on the neural mechanism of spatial cognition and navigation of birds such as pigeons.

Functional sensory circuits built from neurons of two species.

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair.

Centripetal integration of past events in hippocampal astrocytes regulated by locus coeruleus.

An essential feature of neurons is their ability to centrally integrate information from their dendrites. The activity of astrocytes, in contrast, has been described as mostly uncoordinated across cellular compartments without clear central integration. Here we report conditional integration of calcium signals in astrocytic distal processes at their soma. In the hippocampus of adult mice of both sexes, we found that global astrocytic activity, as recorded with population calcium imaging, reflected past neuronal and behavioral events on a timescale of seconds. Salient past events, indicated by pupil dilations, facilitated the propagation of calcium signals from distal processes to the soma. Centripetal propagation to the soma was reproduced by optogenetic activation of the locus coeruleus, a key regulator of arousal, and reduced by pharmacological inhibition of α1-adrenergic receptors. Together, our results suggest that astrocytes are computational units of the brain that slowly and conditionally integrate calcium signals upon behaviorally relevant events.

Lifelong absence of microglia alters hippocampal glutamatergic networks but not synapse and spine density.

Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1rΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r+/+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1rΔFIRE/ΔFIRE mice. Notably, these changes in Csf1rΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.

Loose patch clamp membrane current measurements in cornus ammonis 1 neurons in murine hippocampal slices.

Hippocampal pyramidal neuronal activity has been previously studied using conventional patch clamp in isolated cells and brain slices. We here introduce the loose patch clamping study of voltage-activated currents from in situ pyramidal neurons in murine cornus ammonis 1 hippocampal coronal slices. Depolarizing pulses of 15-ms duration elicited early transient inward, followed by transient and prolonged outward currents in the readily identifiable junctional region between the stratum pyramidalis (SP) and oriens (SO) containing pyramidal cell somas and initial segments. These resembled pyramidal cell currents previously recorded using conventional patch clamp. Shortening the depolarizing pulses to >1-2 ms continued to evoke transient currents; hyperpolarizing pulses to varying voltages evoked decays whose time constants could be shortened to <1 ms, clarifying the speed of clamping in this experimental system. The inward and outward currents had distinct pharmacological characteristics and voltage-dependent inactivation and recovery from inactivation. Comparative recordings from the SP, known to contain pyramidal cell somas, demonstrated similar current properties. Recordings from the SO and stratum radiatum demonstrated smaller inward and outward current magnitudes and reduced transient outward currents, consistent with previous conventional patch clamp results from their different interneuron types. The loose patch clamp method is thus useful for in situ studies of neurons in hippocampal brain slices.

Effect of Oxygen-Glucose Deprivation of Microglia-Derived Exosomes on Hippocampal Neurons: A Study on miR-124 and Inflammatory Cytokines.

Stroke is a cerebrovascular disease that threatens human health. Developing safe and effective drugs and finding therapeutic targets has become an urgent scientific problem. The aim of this study was to investigate the effect of oxygen-glucose deprivation of the microglia-derived exosome on hippocampal neurons and its relationship to miR-124 in the exosome. We incubated hippocampal neurons with exosomes secreted by oxygen-glucose deprivation/ reoxygenation (OGD/R) microglia. The levels of glutamic acid (GLU) and gamma-aminobutyric acid (GABA) in the culture supernatant were detected by ELISA. CCK-8 was used to measure neuronal survival rates. The mRNA levels of TNF-α and IL-6 were detected by RT-qPCR to evaluate the effect of exosomes on neurons. RT-qPCR was then used to detect miR-124 in microglia and their secreted exosomes. Finally, potential targets of miR-124 were analyzed through database retrieval, gene detection with dual luciferase reporters, and western blotting experiments. The results showed that the contents of GLU, TNF-α and IL-6 mRNA increased in the supernatant of cultured hippocampal neurons, the content of GABA decreased, and the survival rate of neurons decreased. Oxygen-glucose deprivation increases miR-124 levels in microglia and their released exosomes. miR-124 acts as a target gene on cytokine signaling suppressor molecule 1(SOCS1), while miR-124 inhibitors reduce the expression of TNF-α and IL-6 mRNA in neurons. These results suggest that oxygen- and glucose-deprived microglia regulate inflammatory cytokines leading to reduced neuronal survival, which may be achieved by miR-124 using SOCS1 as a potential target.

JNK activity modulates postsynaptic scaffold protein SAP102 and kainate receptor dynamics in dendritic spines.

Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the nature of this functional interaction in dendritic spines requires further investigation. Here, using a combination of biochemical methods and live-cell imaging strategies, we show that the dynamics of the synaptic scaffold molecule SAP102 are negatively regulated by JNK inhibition, that SAP102 is a direct phosphorylation target of JNK3, and that SAP102 regulation by JNK is restricted to neurons that harbor mature synapses. We further demonstrate that SAP102 and JNK3 cooperate in the regulated trafficking of kainate receptors to the cell membrane. Specifically, we observe that SAP102, JNK3, and the kainate receptor subunit GluK2 exhibit overlapping expression at synaptic sites and that modulating JNK activity influences the surface expression of the kainate receptor subunit GluK2 in a neuronal context. We also show that SAP102 participates in this process in a JNK-dependent fashion. In summary, our data support a model in which JNK-mediated regulation of SAP102 influences the dynamic trafficking of kainate receptors to postsynaptic sites, and thus shed light on common pathophysiological mechanisms underlying the cognitive developmental defects associated with diverse mutations.

Subicular neurons encode concave and convex geometries.

Animals in the natural world constantly encounter geometrically complex landscapes. Successful navigation requires that they understand geometric features of these landscapes, including boundaries, landmarks, corners and curved areas, all of which collectively define the geometry of the environment1-12. Crucial to the reconstruction of the geometric layout of natural environments are concave and convex features, such as corners and protrusions. However, the neural substrates that could underlie the perception of concavity and convexity in the environment remain elusive. Here we show that the dorsal subiculum contains neurons that encode corners across environmental geometries in an allocentric reference frame. Using longitudinal calcium imaging in freely behaving mice, we find that corner cells tune their activity to reflect the geometric properties of corners, including corner angles, wall height and the degree of wall intersection. A separate population of subicular neurons encode convex corners of both larger environments and discrete objects. Both corner cells are non-overlapping with the population of subicular neurons that encode environmental boundaries. Furthermore, corner cells that encode concave or convex corners generalize their activity such that they respond, respectively, to concave or convex curvatures within an environment. Together, our findings suggest that the subiculum contains the geometric information needed to reconstruct the shape and layout of naturalistic spatial environments.

Barcoding of episodic memories in the hippocampus of a food-caching bird.

The hippocampus is critical for episodic memory. Although hippocampal activity represents place and other behaviorally relevant variables, it is unclear how it encodes numerous memories of specific events in life. To study episodic coding, we leveraged the specialized behavior of chickadees-food-caching birds that form memories at well-defined moments in time whenever they cache food for subsequent retrieval. Our recordings during caching revealed very sparse, transient barcode-like patterns of firing across hippocampal neurons. Each "barcode" uniquely represented a caching event and transiently reactivated during the retrieval of that specific cache. Barcodes co-occurred with the conventional activity of place cells but were uncorrelated even for nearby cache locations that had similar place codes. We propose that animals recall episodic memories by reactivating hippocampal barcodes. Similarly to computer hash codes, these patterns assign unique identifiers to different events and could be a mechanism for rapid formation and storage of many non-interfering memories.

Unpredictable chronic mild stress shows neuronal remodeling in multipolar projection neurons of hippocampal complex in postnatal chicks.

The hippocampal complex of birds is a narrow-curved strip of tissue that plays a crucial role in learning, memory, spatial navigation, and emotional and sexual behavior. This study was conducted to evaluate the effect of unpredictable chronic mild stress in multipolar neurons of 3-, 5-, 7-, and 9-week-old chick's hippocampal complex. This study revealed that chronic stress results in neuronal remodeling by causing alterations in dendritic field, axonal length, secondary branching, corrected spine number, and dendritic branching at 25, 50, 75, and 100 µm. Due to stress, the overall dendritic length was significantly retracted in 3-week-old chick, whereas no significant difference was observed in 5- and 7-week-old chick, but again it was significantly retracted in 9-week-old chick along with the axonal length. So, this study indicates that during initial days of stress exposure, the dendritic field shows retraction, but when the stress continues up to a certain level, the neurons undergo structural modifications so that chicks adapt and survive in stressful conditions. The repeated exposure to chronic stress for longer duration leads to the neuronal structural disruption by retraction in the dendritic length as well as axonal length. Another characteristic which leads to structural alterations is the dendritic spines which significantly decreased in all age groups of stressed chicks and eventually leads to less synaptic connections, disturbance in physiology, and neurology, which affects the learning, memory, and coping ability of an individual.