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Visual Abstract, While BDNF is receiving considerable attention for its role in synaptic plasticity and in nervous system dysfunction, identifying brain circuits involving BDNF-expressing neurons has been challenging. BDNF levels are very low in most brain areas, except for the large mossy fiber terminals in the hippocampus where BDNF accumulates at readily detectable levels. This report describes the generation of a mouse line allowing the detection of single brain cells synthesizing BDNF. A bicistronic construct encoding BDNF tagged with a P2A sequence preceding GFP allows the translation of BDNF and GFP as separate proteins. Following its validation with transfected cells, this construct was used to replace the endogenous Bdnf gene. Viable and fertile homozygote animals were generated, with the GFP signal marking neuronal cell bodies translating the Bdnf mRNA. Importantly, the distribution of immunoreactive BDNF remained unchanged, as exemplified by its accumulation in mossy fiber terminals in the transgenic animals. GFP-labeled neurons could be readily visualized in distinct layers in the cerebral cortex where BDNF has been difficult to detect with currently available reagents. In the hippocampal formation, quantification of the GFP signal revealed that

Visual Abstract, Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell types. We have targeted the recently discovered microglia-specific Tmem119 gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous Tmem119 in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11b+CD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the Tmem119-EGFP and Tmem119-CreERT2 lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice.

Designer receptors exclusively activated by designer drugs (DREADD)-based chemogenetic tools are extensively used to manipulate neuronal activity in a cell type-specific manner. Whole-cell patch-clamp recordings indicate membrane depolarization, coupled with increased neuronal firing rate, following administration of the DREADD ligand, clozapine-N-oxide (CNO) to activate the Gq-coupled DREADD, hM3Dq. Although hM3Dq has been used to enhance neuronal firing in order to manipulate diverse behaviors, often within 30 min to 1 h after CNO administration, the physiological effects on excitatory neurotransmission remain poorly understood. We investigated the influence of CNO-mediated hM3Dq DREADD activation on distinct aspects of hippocampal excitatory neurotransmission at the Schaffer collateral-CA1 synapse in hippocampal slices derived from mice expressing hM3Dq in Ca2+/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons. Our results indicate a clear dose-dependent effect on field EPSP (fEPSP) slope, with no change noted at the lower dose of CNO (1 µM) and a significant, long-term decline in fEPSP slope observed at higher doses (5-20 µM). Further, we noted a robust θ burst stimulus (TBS) induced long-term potentiation (LTP) in the presence of the lower CNO (1 µM) dose, which was significantly attenuated at the higher CNO (20 µM) dose. Whole-cell patch-clamp recording revealed both complex dose-dependent regulation of excitability, and spontaneous and evoked activity of CA1 pyramidal neurons in response to hM3Dq activation across CNO concentrations. Our data indicate that CNO-mediated activation of the hM3Dq DREADD results in dose-dependent regulation of excitatory hippocampal neurotransmission and highlight the importance of careful interpretation of behavioral experiments involving chemogenetic manipulation. ispartof: ENEURO vol:6 issue:6 ispartof: location:United States status: published

The study of brain circuits depends on a clear understanding of the role played by different neuronal populations. Therefore, the unambiguous identification of different cell types is essential for the correct interpretation of experimental data. Here, we emphasize to the broader neuroscience community the importance of recognizing the persistent presence of Cajal–Retzius cells in the molecular layers of the postnatal hippocampus, and then we suggest a variety of criteria for distinguishing Cajal–Retzius cells from other neurons of the hippocampal molecular layers, such as GABAergic interneurons and semilunar granule cells. The toolbox of criteria that we have investigated (in male and female mice) can be useful both for anatomical and functional experiments, and relies on the quantitative study of neuronal somatic/nuclear morphology, location and developmental profile, expression of specific molecular markers (GAD67, reelin, COUP-TFII, calretinin, and p73), single cell anatomy, and electrophysiological properties. We conclude that Cajal–Retzius cells are small, non-GABAergic neurons that are tightly associated with the hippocampal fissure (HF), and that, within this area of interest, selectively express the proteins p73 and calretinin. We highlight the dangers of using markers such as reelin or COUP-TFII to identify Cajal–Retzius cells or GABAergic interneurons because of their poor specificity. Lastly, we examine neurons of the postnatal hippocampal molecular layers and show cell type-specific differences in their dendritic/axonal morphologies and density distributions, as well as in their membrane properties and spontaneous synaptic inputs. These parameters can be used to distinguish biocytin-filled and/or electrophysiologically recorded neurons and should be considered to avoid interpretational mistakes.

Visual Abstract, The cytoarchitecture of a neuron is very important in defining morphology and ultrastructure. Although there is a wealth of information on the molecular components that make and regulate these ultrastructures, there is a dearth of understanding of how these changes occur or how they affect neurons in health and disease. Recent advances in nanoscale imaging which resolve cellular structures at the scale of tens of nanometers below the limit of diffraction enable us to understand these structures in fine detail. However, automated analysis of these images is still in its infancy. Towards this goal, attempts have been made to automate the detection and analysis of the cytoskeletal organization of microtubules. To date, evaluation of the nanoscale organization of filamentous actin (F-actin) in neuronal compartments remains challenging. Here, we present an objective paradigm for analysis which adopts supervised learning of nanoscale images of F-actin network in excitatory synapses, obtained by single molecule based super-resolution light microscopy. We have used the proposed analysis to understand the heterogeneity in the organization of F-actin in dendritic spines of primary neuronal cultures from rodents. Our results were validated using ultrastructural data obtained from platinum replica electron microscopy (PREM). The automated analysis approach was used to differentiate the heterogeneity in the nanoscale organization of F-actin in primary neuronal cultures from wild-type (WT) and a transgenic mouse model of Alzheimer’s disease (APPSwe/PS1ΔE9).

Despite the profound influence on coding capacity of sensory neurons, the measurements of noise correlations have been inconsistent. This is, possibly, because nonstationarity, i.e., drifting baselines, engendered the spurious long-term correlations even if no actual short-term correlation existed. Although attempts to separate them have been made previously, they weread hocfor specific cases or computationally too demanding. Here we proposed an information-geometric method to unbiasedly estimate pure short-term noise correlations irrespective of the background brain activities without demanding computational resources. First, the benchmark simulations demonstrated that the proposed estimator is more accurate and computationally efficient than the conventional correlograms and the residual correlations with Kalman filters or moving averages of length three or more, while the best moving average of length two coincided with the propose method regarding correlation estimates. Next, we analyzed the cat V1 neural responses to demonstrate that the statistical test accompanying the proposed method combined with the existing nonstationarity test enabled us to dissociate short-term and long-term noise correlations. When we excluded the spurious noise correlations of purely long-term nature, only a small fraction of neuron pairs showed significant short-term correlations, possibly reconciling the previous inconsistent observations on existence of significant noise correlations. The decoding accuracy was slightly improved by the short-term correlations. Although the long-term correlations deteriorated the generalizability, the generalizability was recovered by the decoder with trend removal, suggesting that brains could overcome nonstationarity. Thus, the proposed method enables us to elucidate the impacts of short-term and long-term noise correlations in a dissociated manner.

Microglia, the resident immune cells in the CNS, play multiple roles during development. In the embryonic cerebral wall, microglia modulate the functions of neural stem/progenitor cells through their distribution in regions undergoing cell proliferation and/or differentiation. Previous studies using CX3CR1-GFP transgenic mice demonstrated that microglia extensively survey these regions. To simultaneously visualize microglia and neural-lineage cells that interact with each other, we applied thein uteroelectroporation (IUE) technique, which has been widely used for gene-transfer in neurodevelopmental studies, to CX3CR1-GFP mice (males and females). However, we unexpectedly faced a technical problem: although microglia are normally distributed homogeneously throughout the mid-embryonic cortical wall with only limited luminal entry, the intraventricular presence of exogenously derived plasmid DNAs induced microglia to accumulate along the apical surface of the cortex and aggregate in the choroid plexus. This effect was independent of capillary needle puncture of the brain wall or application of electrical pulses. The microglial response occurred at plasmid DNA concentrations lower than those routinely used for IUE, and was mediated by activation of Toll-like receptor 9 (TLR9), an innate immune sensor that recognizes unmethylated cytosine-phosphate guanosine motifs abundant in microbial DNA. Administration of plasmid DNA together with oligonucleotide 2088, the antagonist of TLR9, partially restored the dispersed intramural localization of microglia and significantly decreased luminal accumulation of these cells. Thus, via TLR9, intraventricular plasmid DNA administration causes aberrant distribution of embryonic microglia, suggesting that the behavior of microglia in brain primordia subjected to IUE should be carefully interpreted.

Recent developments in CRISPR-based gene editing have provided new avenues to interrogate gene function. However, application of these tools in the central nervous system has been delayed due to difficulties in transgene expression in post-mitotic neurons. Here, we present a highly efficient, neuron-optimized dual lentiviral CRISPR-based transcriptional activation (CRISPRa) system to drive gene expression in primary neuronal cultures and the adult brain of rodent model systems. We demonstrate robust, modular, and tunable induction of endogenous target genes as well as multiplexed gene regulation necessary for investigation of complex transcriptional programs. CRISPRa targeting unique promoters in the complex multi-transcript gene Brain-derived neurotrophic factor (Bdnf) revealed both transcript- and genome-level selectivity of this approach, in addition to highlighting downstream transcriptional and physiological consequences of Bdnf regulation. Finally, we illustrate that CRISPRa is highly efficient in vivo, resulting in increased protein levels of a target gene in diverse brain structures. Taken together, these results demonstrate that CRISPRa is an efficient and selective method to study gene expression programs in brain health and disease.

Visual Abstract, Frontal eye field (FEF) in macaque monkeys contributes to visual attention, visual–motor transformations and production of eye movements. Traditionally, neurons in FEF have been classified by the magnitude of increased discharge rates following visual stimulus presentation, during a waiting period, and associated with eye movement production. However, considerable heterogeneity remains within the traditional visual, visuomovement, and movement categories. Cluster analysis is a data-driven method of identifying self-segregating groups within a dataset. Because many cluster analysis techniques exist and outcomes vary with analysis assumptions, consensus clustering aggregates over multiple analyses, identifying robust groups. To describe more comprehensively the neuronal composition of FEF, we applied a consensus clustering technique for unsupervised categorization of patterns of spike rate modulation measured during a memory-guided saccade task. We report 10 functional categories, expanding on the traditional 3 categories. Categories were distinguished by latency, magnitude, and sign of visual response; the presence of sustained activity; and the dynamics, magnitude and sign of saccade-related modulation. Consensus clustering can include other metrics and can be applied to datasets from other brain regions to provide better information guiding microcircuit models of cortical function.

Visual Abstract, Speech is an ecologically essential signal, whose processing crucially involves the subcortical nuclei of the auditory brainstem, but there are few experimental options for studying these early responses in human listeners under natural conditions. While encoding of continuous natural speech has been successfully probed in the cortex with neurophysiological tools such as electroencephalography (EEG) and magnetoencephalography, the rapidity of subcortical response components combined with unfavorable signal-to-noise ratios signal-to-noise ratio has prevented application of those methods to the brainstem. Instead, experiments have used thousands of repetitions of simple stimuli such as clicks, tone-bursts, or brief spoken syllables, with deviations from those paradigms leading to ambiguity in the neural origins of measured responses. In this study we developed and tested a new way to measure the auditory brainstem response (ABR) to ongoing, naturally uttered speech, using EEG to record from human listeners. We found a high degree of morphological similarity between the speech-derived ABRs and the standard click-evoked ABR, in particular, a preserved Wave V, the most prominent voltage peak in the standard click-evoked ABR. Because this method yields distinct peaks that recapitulate the canonical ABR, at latencies too short to originate from the cortex, the responses measured can be unambiguously determined to be subcortical in origin. The use of naturally uttered speech to measure the ABR allows the design of engaging behavioral tasks, facilitating new investigations of the potential effects of cognitive processes like language and attention on brainstem processing.

Visual Abstract, Establishing the molecular diversity of cell types is crucial for the study of the nervous system. We compiled a cross-laboratory database of mouse brain cell type-specific transcriptomes from 36 major cell types from across the mammalian brain using rigorously curated published data from pooled cell type microarray and single-cell RNA-sequencing (RNA-seq) studies. We used these data to identify cell type-specific marker genes, discovering a substantial number of novel markers, many of which we validated using computational and experimental approaches. We further demonstrate that summarized expression of marker gene sets (MGSs) in bulk tissue data can be used to estimate the relative cell type abundance across samples. To facilitate use of this expanding resource, we provide a user-friendly web interface at www.neuroexpresso.org.

One of the principal functions of the brain is to control movement and rapidly adapt behavior to a changing external environment. Over the last decades our ability to monitor activity in the brain, manipulate it while also manipulating the environment the animal moves through, has been tackled with increasing sophistication. However, our ability to track the movement of the animal in real time has not kept pace. Here, we use a dynamic vision sensor (DVS) based event-driven neuromorphic camera system to implement real-time, low-latency tracking of a single whisker that mice can move at ∼25 Hz. The customized DVS system described here converts whisker motion into a series of events that can be used to estimate the position of the whisker and to trigger a position-based output interactively within 2 ms. This neuromorphic chip-based closed-loop system provides feedback rapidly and flexibly. With this system, it becomes possible to use the movement of whiskers or in principal, movement of any part of the body to reward, punish, in a rapidly reconfigurable way. These methods can be used to manipulate behavior, and the neural circuits that help animals adapt to changing values of a sequence of motor actions.

Genetically modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here, we validate these lines usingin situhybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways, respectively. We used whole-genome sequencing to confirm the lack of off-target effects and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

Visual Abstract, Physical activity is a critical behavioral variable in many research studies and is, therefore, important to quantify. However, existing methods for measuring physical activity have limitations which include high expense, specialized caging or equipment, and high computational overhead. To address these limitations, we present an open-source, cost-effective, device for measuring rodent activity. Our device is battery powered and designed to be placed in vivarium home cages to enable high-throughput, long-term operation with minimal investigator intervention. The primary aim of this study was to assess the feasibility of using passive infrared (PIR) sensors and microcontroller-based dataloggers in a rodent home cages to collect physical activity records. To this end, we developed an open-source PIR based data-logging device called the rodent activity detector (RAD). We publish the design files and code so others can readily build the RAD in their own labs. To demonstrate its utility, we used the RAD to collect physical activity data from 40 individually housed mice for up to 10 weeks. This dataset demonstrates the ability of the RAD to (1) operate in a high-throughput installation, (2) detect high-fat diet (HFD)-induced changes in physical activity, and (3) quantify circadian rhythms in individual animals. We further validated the data output of the RAD with simultaneous video tracking of mice in multiple caging configurations, to determine the features of physical activity that it detects. The RAD is easy to build, economical, and fits in vivarium caging. The scalability of such devices will enable high-throughput studies of physical activity in research studies.

In the parkinsonian state, the motor cortex and basal ganglia (BG) undergo dynamic remodeling of movement representation. One such change is the loss of the normal contralateral lateralized activity pattern. The increase in the number of movement-related neurons responding to ipsilateral or bilateral limb movements may cause motor problems, including impaired balance, reduced bimanual coordination, and abnormal mirror movements. However, it remains unknown how individual types of motor cortical neurons organize this reconstruction. To explore the effect of dopamine depletion on lateralized activity in the parkinsonian state, we used a partial hemiparkinsonian model [6-hydroxydopamine (6-OHDA) lesion] in Long–Evans rats performing unilateral movements in a right–left pedal task, while recording from primary (M1) and secondary motor cortex (M2). The lesion decreased contralateral preferred activity in both M1 and M2. In addition, this change differed among identified intratelencephalic (IT) and pyramidal tract (PT) cortical projection neurons, depending on the cortical area. We detected a decrease in lateralized activity only in PT neurons in M1, whereas in M2, this change was observed in IT neurons, with no change in the PT population. Our results suggest a differential effect of dopamine depletion in the lateralized activity of the motor cortex, and suggest possible compensatory changes in the contralateral hemisphere.

Microglia play a pivotal role in maintaining homeostasis of the CNS. There is growing interest in understanding how microglia influence normal brain function and disease progression. Several microglia-specific Cx3cr1-Cre lines have been developed and have become indispensable tools in many investigations of microglial function. However, some recent studies have reported that these lines may have significant leakage into neurons. Other studies have reported that Cx3cr1 is expressed in non-microglial cells, including neurons and astrocytes, in vitro or in vivo either during brain development or upon neurological insult. All these reports raise serious concerns about the trustworthiness of these Cre-lines and whether the conclusions drawn from previous studies are valid. Here, we found that a floxed fluorescent reporter mouse line which has been frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines. We further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia. Finally, we clarified that these two mouse lines maintain microglia-specific expression even following excitatory injury. Together, our findings confirm that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia. Moreover, to ensure the quality of data generated and the soundness of conclusions drawn from such data, it should be compulsory to thoroughly examine reporter lines for spontaneous leakiness when labeling cells to study CNS function and diseases.

Flow cytometry and fluorescence-activated sorting are powerful techniques that hold great promise for studying heterogeneous populations of submicron particles such as synaptosomes, but many technical challenges arise in these experiments. To date, most flow cytometry studies of synaptosomes have relied on particle detection using forward scatter (FSC) measurements and size estimation with polystyrene (PS) bead standards. However, these practices have serious limitations, and special care must be taken to overcome the poor sensitivity of conventional flow cytometers in the analysis of submicron particles. Technical artifacts can confound these experiments, especially the detection of multiple particles as a single event. Here, we compared analysis of P2 crude synaptosomal preparations from murine forebrain on multiple flow cytometers using both FSC-triggered and fluorescence-triggered detection. We implemented multicolor fluorescent dye-based assays to quantify coincident particle detection and aggregation, and we assessed the false colocalization of antigens in immunostaining analyses. Our results demonstrate that fluorescence triggering and proper dilution can control for coincident particle detection, but not particle aggregation. We confirmed previous studies showing that FSC-based size estimation with PS beads underestimates biological particle size, and we identified pervasive aggregation in the FSC range analyzed in most synaptosome flow cytometry studies. We found that analyzing P2 samples in sucrose/EDTA/tris (SET) buffer reduces aggregation compared to PBS, but does not completely eliminate the presence of aggregates, especially in immunostaining experiments. Our study highlights challenges and pitfalls in synaptosome flow cytometry and provides a methodological framework for future studies.

Genome editing techniques have facilitated significant advances in our understanding of fundamental biological processes, and the Cre-Lox system has been instrumental in these achievements. Driving Cre expression specifically in injured neurons has not been previously possible: we sought to address this limitation in mice using a Cre-ERT2 construct driven by a reliable indicator of axotomy, activating transcription factor 3 (ATF3). When crossed with reporter mice, a significant amount of recombination was achieved (without tamoxifen treatment) in peripherally-projecting sensory, sympathetic, and motoneurons after peripheral nerve crush in hemizygotes (65–80% by 16 d) and was absent in uninjured neurons. Importantly, injury-induced recombination did not occur in Schwann cells distal to the injury, and with a knock-out-validated antibody we verified an absence of ATF3 expression. Functional recovery following sciatic nerve crush in ATF3-deficient mice (both hemizygotes and homozygotes) was delayed, indicating previously unreported haploinsufficiency. In a proof-of-principle experiment, we crossed the ATF3-CreERT2 line with a floxed phosphatase and tensin homolog (PTEN) line and show significantly improved axonal regeneration, as well as more complete recovery of neuromuscular function. We also demonstrate the utility of the ATF3-CreERT2 hemizygous line by characterizing recombination after lateral spinal hemisection (C8/T1), which identified specific populations of ascending spinal cord neurons (including putative spinothalamic and spinocerebellar) and descending supraspinal neurons (rubrospinal, vestibulospinal, reticulospinal and hypothalamic). We anticipate these mice will be valuable in distinguishing axotomized from uninjured neurons of several different classes (e.g., via reporter expression), and in probing the function of any number of genes as they relate to neuronal injury and regeneration.

The β-band oscillation in the subthalamic nucleus (STN) is a therapeutic target for Parkinson’s disease. Previous studies demonstrated thatl-DOPA decreases the β-band (13–30 Hz) oscillations with improvement of motor symptoms. However, it has not been elucidated whether patients with Parkinson’s disease are able to control the β-band oscillation voluntarily. Here, we hypothesized that neurofeedback training to control the β-band power in the STN induces plastic changes in the STN of individuals with Parkinson’s disease. We recorded the signals from STN deep-brain stimulation electrodes during operations to replace implantable pulse generators in eight human patients (3 male) with bilateral electrodes. Four patients were induced to decrease the β-band power during the feedback training (down-training condition), whereas the other patients were induced to increase (up-training condition). All patients were blinded to their assigned condition. Adjacent contacts that showed the highest β-band power were selected for the feedback. During the 10 min training, patients were shown a circle whose diameter was controlled by the β-band power of the selected contacts. Powers in the β-band during 5 min resting sessions recorded before and after the feedback were compared. In the down-training condition, the β-band power of the selected contacts decreased significantly after feedback in all four patients (p< 0.05). In contrast, the β-band power significantly increased after feedback in two of four patients in the up-training condition. Overall, the patients could voluntarily control the β-band power in STN in the instructed direction (p< 0.05) through neurofeedback.

Visual Abstract, A multielectrode system that can address widely separated targets at multiple sites across multiple brain regions with independent implant angling is needed to investigate neural function and signaling in systems and circuits of small animals. Here, we present the systemDrive, a novel multisite, multiregion microdrive that is capable of moving microwire electrode bundles into targets along independent and nonparallel drive trajectories. Our design decouples the stereotaxic surgical placement of individual guide cannulas for each trajectory from the placement of a flexible drive structure. This separation enables placement of many microwire multitrodes along widely spaced and independent drive axes with user-set electrode trajectories and depths from a single microdrive body, and achieves stereotaxic precision with each. The system leverages tight tube–cannula tolerances and geometric constraints on flexible drive axes to ensure concentric alignment of electrode bundles within guide cannulas. Additionally, the headmount and microdrive both have an open-center design to allow for the placement of additional sensing modalities. This design is the first, in the context of small rodent chronic research, to provide the capability to finely position microwires through multiple widely distributed cell groups, each with stereotaxic precision, along arbitrary and nonparallel trajectories that are not restricted to emanate from a single source. We demonstrate the use of the systemDrive in male Long–Evans rats to observe simultaneous single-unit and multiunit activity from multiple widely separated sleep–wake regulatory brainstem cell groups, along with cortical and hippocampal activity, during free behavior over multiple many-day continuous recording periods.

Visual Abstract, Microglia are dynamic immune cells of the central nervous system, and their morphology is commonly used as a readout of cellular function. However, current morphological analysis techniques rely on either tracing of cells or two-dimensional projection analysis, which are time-consuming, subject to bias, and may ignore important three-dimensional (3D) information. Therefore, we have created 3DMorph, a MATLAB-based script that analyzes microglial morphology from 3D data. The program initially requires input of threshold levels, cell size expectations, and preferred methods of skeletonization. This makes 3DMorph easily scalable and adaptable to different imaging parameters or cell types. After these settings are defined, the program is completely automatic and can batch process files without user input. Output data includes cell volume, territorial volume, branch length, number of endpoints and branch points, and average distance between cells. We show that 3DMorph is accurate compared to manual tracing, with significantly decreased user input time. Importantly, 3DMorph is capable of processing in vivo microglial morphology, as well as other 3D branching cell types, from mouse cranial windows or acute hippocampal slices. Therefore, we present a novel, user-friendly, scalable, and semiautomatic method of analyzing cell morphology in 3 dimensions. This method should improve the accuracy of cell measurements, remove user bias between conditions, increase reproducibility between experimenters and labs, and reduce user input time. We provide this open source code on GitHub so that it is free and accessible to all investigators.

It is widely assumed that distributed neuronal networks are fundamental to the functioning of the brain. Consistent spike timing between neurons is thought to be one of the key principles for the formation of these networks. This can involve synchronous spiking or spiking with time delays, forming spike sequences when the order of spiking is consistent. Finding networks defined by their sequence of time-shifted spikes, denoted here as spike timing networks, is a tremendous challenge. As neurons can participate in multiple spike sequences at multiple between-spike time delays, the possible complexity of networks is prohibitively large. We present a novel approach that is capable of (1) extracting spike timing networks regardless of their sequence complexity, and (2) that describes their spiking sequences with high temporal precision. We achieve this by decomposing frequency-transformed neuronal spiking into separate networks, characterizing each network’s spike sequence by a time delay per neuron, forming a spike sequence timeline. These networks provide a detailed template for an investigation of the experimental relevance of their spike sequences. Using simulated spike timing networks, we show network extraction is robust to spiking noise, spike timing jitter, and partial occurrences of the involved spike sequences. Using rat multineuron recordings, we demonstrate the approach is capable of revealing real spike timing networks with sub-millisecond temporal precision. By uncovering spike timing networks, the prevalence, structure, and function of complex spike sequences can be investigated in greater detail, allowing us to gain a better understanding of their role in neuronal functioning.

Visual Abstract, Human neurons expressing mutations associated with neurodegenerative disease are becoming more widely available. Hence, developing assays capable of accurately detecting changes that occur early in the disease process and identifying therapeutics able to slow these changes should become ever more important. Using automated live-cell imaging, we studied human motor neurons in the process of dying following neurotrophic factor withdrawal. We tracked different neuronal features, including cell body size, neurite length, and number of nodes. In particular, measuring the number of nodes in individual neurons proved to be an accurate predictor of relative health. Importantly, intermediate phenotypes were defined and could be used to distinguish between agents that could fully restore neurons and neurites and those only capable of maintaining neuronal cell bodies. Application of live-cell imaging to disease modeling has the potential to uncover new classes of therapeutic molecules that intervene early in disease progression.

Visual Abstract, Filters are widely used for the modulation, typically attenuation, of amplitudes of different frequencies within neurophysiological signals. Filters, however, also induce changes in the phases of different frequencies whose amplitude is unmodulated. These phase shifts cause time lags in the filtered signals, leading to a disruption of the timing information between different frequencies within the same signal and between different signals. The emerging time lags can be either constant in the case of linear phase (LP) filters or vary as a function of the frequency in the more common case of non-LP (NLP) filters. Since filters are used ubiquitously online in the early stages of data acquisition, the vast majority of neurophysiological signals thus suffer from distortion of the timing information even prior to their sampling. This distortion is often exacerbated by further multiple offline filtering stages of the sampled signal. The distortion of timing information may cause misinterpretation of the results and lead to erroneous conclusions. Here we present a variety of typical examples of filter-induced phase distortions and discuss the evaluation and restoration of the timing information underlying the original signal.

The dynamic clamp should be a standard part of every cellular electrophysiologist’s toolbox. That it is not, even 25 years after its introduction, comes down to three issues: money, the disruption that adding dynamic clamp to an existing electrophysiology rig entails, and the technical prowess required of experimenters. These have been valid and limiting issues in the past, but no longer. Technological advances associated with the so-called maker movement render them moot. We demonstrate this by implementing a fast (∼100 kHz) dynamic clamp system using an inexpensive microcontroller (Teensy 3.6). The overall cost of the system is less than USD$100, and assembling it requires no prior electronics experience. Modifying it—for example, to add Hodgkin–Huxley-style conductances—requires no prior programming experience. The system works together with existing electrophysiology data acquisition systems (for Macintosh, Windows, and Linux); it does not attempt to supplant them. Moreover, the process of assembling, modifying, and using the system constitutes a useful pedagogical exercise for students and researchers with no background but an interest in electronics and programming. We demonstrate the system’s utility by implementing conductances as fast as a transient sodium conductance and as complex as the Ornstein–Uhlenbeck conductances of the “point conductance” model of synaptic background activity.

Visual Abstract, Methods for resolving the three-dimensional (3D) microstructure of the brain typically start by thinly slicing and staining the brain, followed by imaging numerous individual sections with visible light photons or electrons. In contrast, X-rays can be used to image thick samples, providing a rapid approach for producing large 3D brain maps without sectioning. Here we demonstrate the use of synchrotron X-ray microtomography (µCT) for producing mesoscale (∼1 µm 3 resolution) brain maps from millimeter-scale volumes of mouse brain. We introduce a pipeline for µCT-based brain mapping that develops and integrates methods for sample preparation, imaging, and automated segmentation of cells, blood vessels, and myelinated axons, in addition to statistical analyses of these brain structures. Our results demonstrate that X-ray tomography achieves rapid quantification of large brain volumes, complementing other brain mapping and connectomics efforts.

Lesion-symptom mapping is often employed to define brain structures that are crucial for human behavior. Even though poststroke deficits result from gray matter damage as well as secondary white matter loss, the impact of structural disconnection is overlooked by conventional lesion-symptom mapping because it does not measure loss of connectivity beyond the stroke lesion. This study describes how traditional lesion mapping can be combined with structural connectome lesion symptom mapping (CLSM) and connectome dynamics lesion symptom mapping (CDLSM) to relate residual white matter networks to behavior. Using data from a large cohort of stroke survivors with aphasia, we observed improved prediction of aphasia severity when traditional lesion symptom mapping was combined with CLSM and CDLSM. Moreover, only CLSM and CDLSM disclosed the importance of temporal-parietal junction connections in aphasia severity. In summary, connectome measures can uniquely reveal brain networks that are necessary for function, improving the traditional lesion symptom mapping approach.