Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid–producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

Single-cell transcriptomic studies have identified a conserved set of neocortical cell types from small postmortem cohorts. We extended these efforts by assessing cell type variation across 75 adult individuals undergoing epilepsy and tumor surgeries. Nearly all nuclei map to one of 125 robust cell types identified in the middle temporal gyrus. However, we found interindividual variance in abundances and gene expression signatures, particularly in deep-layer glutamatergic neurons and microglia. A minority of donor variance is explainable by age, sex, ancestry, disease state, and cell state. Genomic variation was associated with expression of 150 to 250 genes for most cell types. This characterization of cellular variation provides a baseline for cell typing in health and disease.

Neocortical layer 1 (L1) is a site of convergence between pyramidal-neuron dendrites and feedback axons where local inhibitory signaling can profoundly shape cortical processing. Evolutionary expansion of human neocortex is marked by distinctive pyramidal neurons with extensive L1 branching, but whether L1 interneurons are similarly diverse is underexplored. Using Patch-seq recordings from human neurosurgical tissue, we identified four transcriptomic subclasses with mouse L1 homologs, along with distinct subtypes and types unmatched in mouse L1. Subclass and subtype comparisons showed stronger transcriptomic differences in human L1 and were correlated with strong morphoelectric variability along dimensions distinct from mouse L1 variability. Accompanied by greater layer thickness and other cytoarchitecture changes, these findings suggest that L1 has diverged in evolution, reflecting the demands of regulating the expanded human neocortical circuit.

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.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

The neocortex is disproportionately expanded in human compared with mouse 1 , 2 , both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth 3 . Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer’s disease 4 , 5 . Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.

Primary and metastatic brain tumors can overlap in traditional imaging features detected on preoperative conventional magnetic resonance imaging (MRI). The research objective was to determine whether morphological vascular characteristics present in routine preoperative imaging using traditional MRI sequences are predictive of primary versus metastatic brain tumors; secondarily to determine association of conventional and vascular-related imaging parameters with intraoperative blood loss, pathological invasion, and World Health Organization (WHO) tumor grade. A retrospective review analyzed 100 consecutive intracranial tumor surgeries, 50 WHO grade II-IV gliomas and 50 intracranial metastases. Two blinded expert readers independently evaluated preoperative MRIs, obtained via standard morphological imaging sequences, for adjacent or intra-tumoral arterial aneurysm, peritumoral venous ectasia, prominence, or engorgement ("aberrant peritumoral vessels"), and prominent intra-tumoral flow voids. Multivariate analysis was performed to develop models predictive of glioma and glioblastoma (GBM). Aberrant peritumoral vessels and prominent intra-tumoral flow voids were statistically significant predictors of glioma in univariate analyses (p = 0.048, p = 0.001, respectively) and when combined in multivariate analysis (OR = 5.23, p = 0.001), particularly for GBM (OR = 9.08, p < 0.001). Multivariate modeling identified prominent intra-tumoral flow voids and FLAIR invasion as the strongest combined predictors of gliomas and GBM. Aberrant peritumoral vessels and larger tumor volume predicted higher intraoperative blood loss in all analyses. No vascular-related parameters predicted pathological invasion on multivariate analysis. Aberrant peritumoral vessels and prominent intra-tumoral flow voids were predictive of gliomas, specifically GBM. These vascular characteristics, evaluated on routine clinical preoperative MRI imaging, may aid in distinguishinggliomafrom brainmetastases andmay predict intraoperative blood loss.

OBJECTIVE:To determine preoperative factors contributing to postoperative hemorrhage after stereotactic brain biopsy (STB), clinical implications of postoperative hemorrhage, and the role of postoperative imaging in clinical management. METHODS:Retrospective review of STB (2005-2018) across 2 institutions including patients aged >18 years undergoing first STB. Patients with prior craniotomy, open biopsy, or prior STB were excluded. Preoperative variables included age, sex, neurosurgeon seniority, STB method. Postoperative variables included pathology, postoperative hemorrhage on computed tomography, immediate and 30-day postoperative seizure, infection, postoperative hospital stay duration, and 30-day return to operating room (OR). Analysis used the Fisher exact tests for categorical variables. RESULTS:Overall, 410 patients were included. Average age was 56.5 (±16.5) years; 60% (n = 248) were men. The majority of biopsies were performed by senior neurosurgeons (66%, n = 270); frontal lobe (42%, n = 182) and glioblastoma (45%, n = 186) were the most common location and pathology. Postoperative hemorrhage occurred in 28% (114) of patients with 20% <0.05 cm3 and 8% >0.05 cm3. Postoperative hemorrhage of any size was associated with increased rate of postoperative deficit within both 24 hours and 30 days, postoperative seizure, and length of hospital stay when controlling for pathology. Hemorrhages >0.05 cm3 had a 16% higher rate of return to the OR for evacuation, due to clinical deterioration as opposed to radiographic progression. CONCLUSIONS:Postbiopsy hemorrhage was associated with higher risk of immediate and delayed postoperative deficit and seizure. Postoperative computed tomography should be used to determine whether STB patients can be discharged same day or admitted for observation; clinical evaluation should determine return to OR for evacuation.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The highly distinctive morpho-electric properties of these neurons have mainly been described in rodents, where ET neurons can be labeled by retrograde tracers or transgenic lines. Similar labeling strategies are not possible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically-defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology and morphology are assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were also apparent but were often smaller than differences between cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy, but also highlight marked phenotypic divergence possibly related to functional specialization of human neocortex.

The neocortex is disproportionately expanded in human compared to mouse, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers that selectively make connections within the cortex and other telencephalic structures. Single-cell transcriptomic analyses of human and mouse cortex show an increased diversity of glutamatergic neuron types in supragranular cortex in human and pronounced gradients as a function of cortical depth. To probe the functional and anatomical correlates of this transcriptomic diversity, we describe a robust Patch-seq platform using neurosurgically-resected human tissues. We characterize the morphological and physiological properties of five transcriptomically defined human glutamatergic supragranular neuron types. Three of these types have properties that are specialized compared to the more homogeneous properties of transcriptomically defined homologous mouse neuron types. The two remaining supragranular neuron types, located exclusively in deep layer 3, do not have clear mouse homologues in supragranular cortex but are transcriptionally most similar to deep layer mouse intratelencephalic-projecting neuron types. Furthermore, we reveal the transcriptomic types in deep layer 3 that express high levels of non-phosphorylated heavy chain neurofilament protein that label long-range neurons known to be selectively depleted in Alzheimer’s disease. Together, these results demonstrate the power of transcriptomic cell type classification, provide a mechanistic underpinning for increased complexity of cortical function in human cortical evolution, and implicate discrete transcriptomic cell types as selectively vulnerable in disease.

Synchronous gliomas of different histopathology are quite rare in non-syndromic, non-irradiated patients. Although "mixed" gliomas are not infrequent, and malignant gliomas often contain areas of disparate differentiation (e.g., glioblastoma with ependymal differentiation), it is unusual to find gliomas of different lineage presenting concurrently. We present a case of synchronous gliomas, one dysembryoplastic neuroepithelial tumor (DNET) and the other oligo-dendroglioma.

Gene expression studies suggest that differential ion channel expression contributes to differences in rodent versus human neuronal physiology. We tested whether h-channels more prominently contribute to the physiological properties of human compared to mouse supragranular pyramidal neurons. Single cell/nucleus RNA sequencing revealed ubiquitous HCN1-subunit expression in excitatory neurons in human, but not mouse supragranular layers. Using patch-clamp recordings, we found stronger h-channel-related membrane properties in supragranular pyramidal neurons in human temporal cortex, compared to mouse supragranular pyramidal neurons in temporal association area. The magnitude of these differences depended upon cortical depth and was largest in pyramidal neurons in deep L3. Additionally, pharmacologically blocking h-channels produced a larger change in membrane properties in human compared to mouse neurons. Finally, using biophysical modeling, we provided evidence that h-channels promote the transfer of theta frequencies from dendrite-to-soma in human L3 pyramidal neurons. Thus, h-channels contribute to between-species differences in a fundamental neuronal property.

BACKGROUND: Stereotactic radiosurgery (SRS) is a treatment modality that is frequently used as salvage therapy for small nodular recurrent high-grade gliomas (HGG). Due to the infiltrative nature of HGG, it is unclear if this highly focused technique provides a durable local control benefit. OBJECTIVE: To determine how demographic or clinical factors influence the pattern of failure following SRS for recurrent high-grade gliomas. METHODS: We retrospectively reviewed clinical, radiographic, and follow-up information for 47 consecutive patients receiving SRS for recurrent HGG at our institution between June 2006 and July 2016. All patients initially presented with an HGG (WHO grade III and IV). Following SRS for recurrence, all patients experienced treatment failure, and we evaluated patterns of local, regional, and distant failure in relation to the SRS 50% isodose line. RESULTS: Most patients with recurrent HGG developed "in-field" treatment failure following SRS (n = 40; 85%). Higher SRS doses were associated with longer time to failure (hazards ratio = 0.80 per 1 Gy increase; 95% confidence interval 0.67-0.96; P = .016). There was a statistically significant increase in distant versus in-field failure among older patients (P = .035). This effect was independent of bevacizumab use (odds ratio = 0.54, P = 1.0). CONCLUSION: Based on our experience, the majority of treatment failures after SRS for recurrent HGG were "in-field." Older patients, however, presented with more distant failures. Our results indicate that higher SRS doses delivered to a larger area as fractioned or unfractioned regimen may prolong time to failure, especially in the older population.

Generating a comprehensive description of cortical networks requires a large-scale, systematic approach. To that end, we have begun a pipeline project using multipatch electrophysiology, supplemented with two-photon optogenetics, to characterize connectivity and synaptic signaling between classes of neurons in adult mouse primary visual cortex (V1) and human cortex. We focus on producing results detailed enough for the generation of computational models and enabling comparison with future studies. Here, we report our examination of intralaminar connectivity within each of several classes of excitatory neurons. We find that connections are sparse but present among all excitatory cell classes and layers we sampled, and that most mouse synapses exhibited short-term depression with similar dynamics. Synaptic signaling between a subset of layer 2/3 neurons, however, exhibited facilitation. These results contribute to a body of evidence describing recurrent excitatory connectivity as a conserved feature of cortical microcircuits.

BACKGROUND: Extent of resection (EOR) correlates with glioblastoma outcomes. Resectability and EOR depend on anatomical, clinical, and surgeon factors. Resectability likely influences outcome in and of itself, but an accurate measurement of resectability remains elusive. An understanding of resectability and the factors that influence it may provide ameans to control a confounder in clinical trials and provide reference for decision making.OBJECTIVE: To provide proof of concept of the use of the collectivewisdom of experienced brain tumor surgeons in assessing glioblastoma resectability.METHODS: We surveyed 13 academic tumor neurosurgeons nationwide to assess the resectability of newly diagnosed glioblastoma. Participants reviewed 20 cases, including digital imaging and communications in medicine-formatted pre- and postoperative magnetic resonance images and clinical vignettes. The selected cases involved a variety of anatomical locations and a range of EOR. Participants were asked about surgical goal, eg, gross total resection, subtotal resection (STR), or biopsy, and rationale for their decision. We calculated a "resectability index" for each lesion by pooling responses fromall 13 surgeons.RESULTS: Neurosurgeons' individual surgical goals varied significantly (P =.015), but the resectability index calculated from the surgeons' pooled responses was strongly correlated with the percentage of contrast-enhancing residual tumor (R = 0.817, P <.001). The collective STR goal predicted intraoperative decision of intentional STR documented on operative notes (P <.01) and nonresectable residual (P <.01), but not resectable residual.CONCLUSION: In this pilot study, we demonstrate the feasibility of measuring the resectability of glioblastoma through crowdsourcing. This tool could be used to quantify resectability, a potential confounder in neuro-oncology clinical trials.