Introduction Group I metabotropic glutamate receptors (mGluRs) play a critical role in regulating neuronal excitability, synaptic strength, and cortical network activity. Although their physiological functions and involvement in neurological disorders are well established, direct experimental evidence for their role in human cortical neurons remains limited.Methods We investigated the effects of group I mGluR activation on excitatory synaptic transmission in the human supragranular cortex using paired whole-cell patch-clamp recordings from synaptically connected pyramidal cells and interneurons in acute slices of human neocortex resected during neurosurgery.Results Activation of mGluRs with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) altered excitatory synaptic efficacy in an interneuron subtype-dependent manner. Specifically, we observed acute enhancement of excitatory postsynaptic current (EPSC) amplitudes in 54% of fast-spiking interneurons and in 15% of non-fast-spiking interneuron types. Applying the same experimental protocol in slices from Wistar rats resulted in a similar increase in synaptic strength in fast-spiking interneurons. However, paired-pulse ratio analysis showed species-dependent differences, which may reflect distinct contributions of pre- and postsynaptic factors to the observed modulation.Discussion Together, these results demonstrate that acute modulation of pyramidal cell-fast-spiking interneuron synapses via group I mGluRs is conserved between human and rodent neocortex, while pointing to species-specific underlying mechanisms. Moreover, mGluR-mediated modulation exhibits cell-type specificity in human cortical circuits. Collectively, these findings provide direct functional evidence for group I mGluR-dependent synaptic regulation in the human cortex and highlight important species- and cell-type-specific differences that should be considered when extrapolating rodent data to human cortical physiology and disease mechanisms.

IntroductionIn pyramidal neurons, backpropagating action potentials (bAPs) activate voltage-gated calcium channels (VGCCs), producing compartment-specific dendritic Ca2+ transients. While extensively characterized in rodent models, little is known about the spatial properties and channel-specific contributions of bAP-induced Ca2+ signals in human cortical neurons.MethodsWe used simultaneous whole-cell patch-clamp recordings and two-photon Ca2+ imaging in acute human cortical slices to characterize bAP-evoked Ca2+ transients along the apical dendrites of layer 2/3 pyramidal neurons.ResultsWe found that Ca2+ signal amplitudes followed a non-linear spatial profile, increasing proximally and peaking between 50-100 µm from the soma before declining in more distal regions. Oblique dendrites exhibited significantly higher Ca2+ amplitudes compared to the primary apical branches. Morphological parameters, such as dendritic diameter, spine density, and branching, were correlated with the spatial profile of Ca2+ transients to the peak of the calcium signal profile. Pharmacological blockade of VGCCs revealed that major channel subtypes (L-, N-, R-, and T-type) contribute to dendritic Ca2+ influx, with distinct spatial effects. In particular, N-type channel blockade produced the largest attenuation in the medial dendritic segments, while T-type channel inhibition affected all regions.DiscussionThese findings highlight spatial heterogeneity and channel-specific contributions to dendritic Ca2+ signaling in human neocortical neurons and underscore the influence of dendritic morphology on signal propagation.

The hydroxyquinoline derivative Q134R is a promising drug candidate for the treatment of Alzheimer's disease with cytoprotective and cognition-enhancing properties. Radioligand binding assays showed that Q134R reduced [³⁵S]TBPS binding, consistent with modulation of the picrotoxin site or conformational states that regulate TBPS accessibility. Electrophysiological recordings in mouse brain slices revealed that Q134R significantly increased tonic inhibitory currents in cortical neurogliaform and dentate gyrus granule cells, both known to express delta subunit-containing GABAA receptors. This effect was abolished in mice deficient in the GABAA delta subunit confirming the delta subunit dependency of Q134R's action. Furthermore, in a scopolamine-induced amnesia model, Q134R treatment significantly improved spatial memory performance in wild-type mice, but not in mice lacking in the delta subunit. These results suggest that Q134R enhances tonic inhibition through delta subunit-containing GABAA receptors, which may contribute to the modulation of memory processes and serve as a protective mechanism in early-stage neurodegenerations. These receptor-mediated effects likely contribute to its broader therapeutic efficacy and may complement its previously reported interactions with signaling pathways such as NFAT and HIF-1.

Human cortical interneurons differ from their rodent counterparts in intrinsic membrane properties, yet the mechanisms regulating excitability across physiologically relevant membrane potentials remain poorly defined. Here, we investigated inwardly rectifying potassium (Kir) channel control of subthreshold excitability in parvalbumin-expressing (Pvalb) interneurons from human and mouse neocortex. Using whole-cell recordings, dynamic clamp, patch sequencing, immunofluorescence, and computational modeling, we show that membrane hyperpolarization induces a proportional decrease in input resistance mediated by Kir channels in both species, despite higher baseline input resistance in human neurons. Transcriptomic and anatomical analyses revealed somatic membrane expression of four major Kir channel subtypes with moderate interspecies differences. Kir activation suppresses intrinsic excitability through combined voltage-dependent and shunting inhibition, an effect occurring during inhibitory postsynaptic potentials evoked by neurogliaform cells. Together, these findings show that homologous Pvalb neurons in humans have evolved toward a conserved, archetypal excitability phenotype, despite substantial differences in baseline excitability between species.

Human-specific cognitive abilities depend on information processing in the cerebral cortex, where the neurons are significantly larger and their processes longer and sparser compared to rodents. We found that, in synaptically connected layer 2/3 pyramidal cells (L2/3 PCs), the delay in signal propagation from soma to soma is similar in humans and rodents. To compensate for the longer processes of neurons, membrane potential changes in human axons and/or dendrites must propagate faster. Axonal and dendritic recordings show that the propagation speed of action potentials (APs) is similar in human and rat axons, but the forward propagation of excitatory postsynaptic potentials (EPSPs) and the backward propagation of APs are 26 and 47% faster in human dendrites, respectively. Experimentally-based detailed biophysical models have shown that the key factor responsible for the accelerated EPSP propagation in human cortical dendrites is the large conductance load imposed at the soma by the large basal dendritic tree. Additionally, larger dendritic diameters and differences in cable and ion channel properties in humans contribute to enhanced signal propagation. Our integrative experimental and modeling study provides new insights into the scaling rules that help maintain information processing speed albeit the large and sparse neurons in the human cortex.

The basic excitatory neurons of the cerebral cortex, the pyramidal cells, are the most important signal integrators for the local circuit. They have quite characteristic morphological and electrophysiological properties that are known to be largely constant with age in the young and adult cortex. However, the brain undergoes several dynamic changes throughout life, such as in the phases of early development and cognitive decline in the aging brain. We set out to search for intrinsic cellular changes in supragranular pyramidal cells across a broad age range: from birth to 85 y of age and we found differences in several biophysical properties between defined age groups. During the first year of life, subthreshold and suprathreshold electrophysiological properties changed in a way that shows that pyramidal cells become less excitable with maturation, but also become temporarily more precise. According to our findings, the morphological features of the three-dimensional reconstructions from different life stages showed consistent morphological properties and systematic dendritic spine analysis of an infantile and an old pyramidal cell showed clear significant differences in the distribution of spine shapes. Overall, the changes that occur during development and aging may have lasting effects on the properties of pyramidal cells in the cerebral cortex. Understanding these changes is important to unravel the complex mechanisms underlying brain development, cognition, and age-related neurodegenerative diseases.

Compared to rodents, inhibitory interneurons in the human neocortex exhibit high input excitability because of reduced passive ion leakage across their extracellular membrane. However, the regulation of intrinsic excitability by voltage-gated ion channels activated over a wide range of membrane potentials in human interneurons remains poorly understood. We performed whole-cell patch-clamp microelectrode recordings in mouse and human neocortical slices obtained from surgically resected non-pathological brain tissue finding that Kir channels control the electrical resistance of parvalbumin (Pvalb) neurons in an identical manner in the human and mouse. Molecular analyses revealed predominantly Kir3.1 and Kir3.2 channels in Pvalb neurons in both species. Using whole-cell recordings from synaptically connected neuron pairs and a computational model, we demonstrated that physiological Kir activation inhibits human Pvalb interneurons during postsynaptic potentials evoked by presynaptic neurogliaform cells. The similarity of Kir-mediated inhibition across species suggests that it is an archetypal property of Pvalb neurons.

Backpropagating action potentials play important role in synaptic plasticity, dendritic excitability and compartment specific intracellular Ca2+ dynamics. Signal propagation in human dendrites shows potentially species and dendritic region specific properties and we asked whether action potential backpropagation in human dendrites follows a uniform or a segment regulated pattern. We studied human cortical layer 2/3 pyramidal apical dendrites in acute brain slices with somatic whole-cell stimulation and simultaneous dendritic two-photon Ca2+ imaging. Single action potentials produced detectable Ca2+ influx in segments of apical dendrites up to 270 μm from the soma of human pyramidal neurons. Evoked Ca2+ signals showed a stereotyped spatial profile along dendrites: the amplitude of the Ca2+ transients increased with distance from the soma, reached the maximum level on the dendritic region 50-100 µm from the soma then decreased towards the distal dendritic regions of primary and higher order dendrites. Non-specific blockage of Ca2+ channels and blockade of voltage-gated Na+ channels significantly reduced and completely abolished Ca2+ transients, respectively. Various Ca2+ channel types contributed to the Ca2+ signals as shown by selective blockade with N-type, L-type, T-type, or R-type Ca2+ channel subtypes. These results suggest a booster region in primary dendrites of human pyramidal cells for backpropagation induced Ca2+ influx in the dendritic tree. Declaration of Interest Statement: None

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.

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.

Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.

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.

1 Wigner RCP, Institute for Solid State Physics and Optics, P.O. Box 49, H-1525 Budapest, Hungary 2 R&D Ultrafast Lasers Ltd, Konkoly-Thege str 29-33, H-1121 Budapest, Hungary 3 Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, Hungary 4 Nanyang Technological University, Environmental Life Sciences Engineering, Singapore 639798, Singapore 5 MTA-SZTE Research Group for Cortical Microcircuits, University of Szeged, Szeged, H-6726, Hungary

Summation of ionotropic receptor-mediated responses is critical in neuronal computation by shaping input-output characteristics of neurons. However, arithmetics of summation for metabotropic signals are not known. We characterized the combined ionotropic and metabotropic output of neocortical neurogliaform cells (NGFCs) using electrophysiological and anatomical methods in the rat cerebral cortex. These experiments revealed that GABA receptors are activated outside release sites and confirmed coactivation of putative NGFCs in superficial cortical layers in vivo. Triple recordings from presynaptic NGFCs converging to a postsynaptic neuron revealed sublinear summation of ionotropic GABAA responses and linear summation of metabotropic GABAB responses. Based on a model combining properties of volume transmission and distributions of all NGFC axon terminals, we predict that in 83% of cases one or two NGFCs can provide input to a point in the neuropil. We suggest that interactions of metabotropic GABAergic responses remain linear even if most superficial layer interneurons specialized to recruit GABAB receptors are simultaneously active.

Microglia are the main immune cells in the brain with emerging roles in brain homeostasis and neurological diseases, while mechanisms underlying microglia-neuron communication remain elusive. Here, we identify a novel site of interaction between neuronal cell bodies and microglial processes in mouse and human brain. Somatic microglia-neuron junctions possess specialized nanoarchitecture optimized for purinergic signaling. Activity of neuronal mitochondria is linked with microglial junction formation, which is rapidly induced in response to neuronal activation and blocked by inhibition of P2Y12-receptors (P2Y12R). Brain injury-induced changes at somatic junctions trigger P2Y12R-dependent microglial neuroprotection, regulating neuronal calcium load and functional connectivity. Collectively, our results suggest that microglial processes at these junctions are in ideal position to monitor and protect neuronal functions in both the healthy and injured brain. One-sentence summary Neuronal cell bodies possess specialized, pre-formed sites, through which microglia monitor their status and exert neuroprotection.

Inhibitory autapses are self-innervating synaptic connections in GABAergic interneurons in the brain. Autapses in neocortical layers have not been systematically investigated, and their function in different mammalian species and specific interneuron types is poorly known. We investigated GABAergic parvalbumin-expressing basket cells (pvBCs) in layer 2/3 (L2/3) in human neocortical tissue resected in deep-brain surgery, and in mice as control. Most pvBCs showed robust GABAAR-mediated self-innervation in both species, but autapses were rare in nonfast-spiking GABAergic interneurons. Light- and electron microscopy analyses revealed pvBC axons innervating their own soma and proximal dendrites. GABAergic self-inhibition conductance was similar in human and mouse pvBCs and comparable to that of synapses from pvBCs to other L2/3 neurons. Autaptic conductance prolonged somatic inhibition in pvBCs after a spike and inhibited repetitive firing. Perisomatic autaptic inhibition is common in both human and mouse pvBCs of supragranular neocortex, where they efficiently control discharge of the pvBCs.

A 20 MHz repetition rate, sub ps Ti:sapphire (Ti:S) laser system is proposed for real time, high chemical contrast dual vibration resonance frequency (DVRF) CARS imaging of the skin suitable for in vivo histology.

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.