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Detailed characterization of interneuron types in primary visual cortex (V1) has greatly contributed to understanding visual perception, yet the role of chandelier cells (ChCs) in visual processing remains poorly characterized. Using viral tracing we found that V1 ChCs predominantly receive monosynaptic input from local layer 5 pyramidal cells and higher-order cortical regions. Two-photon calcium imaging and convolutional neural network modeling revealed that ChCs are visually responsive but weakly selective for stimulus content. In mice running in a virtual tunnel, ChCs respond strongly to events known to elicit arousal, including locomotion and visuomotor mismatch. Repeated exposure of the mice to the virtual tunnel was accompanied by reduced visual responses of ChCs and structural plasticity of ChC boutons and axon initial segment length. Finally, ChCs only weakly inhibited pyramidal cells. These findings suggest that ChCs provide an arousal-related signal to layer 2/3 pyramidal cells that may modulate their activity and/or gate plasticity of their axon initial segments during behaviorally relevant events.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have unexpectedly fast input-output properties: Rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human pyramidal neurons can explain their fast input-output properties. Human Na+ and K+ currents exhibited more depolarized voltage dependence, slower inactivation, and faster recovery from inactivation compared with their mouse counterparts. Computational modeling showed that despite lower Na+ channel densities in human neurons, the biophysical properties of Na+ channels resulted in higher channel availability and contributed to fast AP kinetics stability. Last, human Na+ channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+ and K+ channels enable fast input-output properties of large human pyramidal neurons.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, but the molecular mechanisms regulating their plasticity remain largely unknown. Here, we developed genetic tools to label endogenous sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons in slices and primary cultures. We find that N-methyl-d-aspartate receptor activation causes both long-term synaptic depression and rapid internalization of AIS sodium channels within minutes. The clathrin-mediated endocytosis of sodium channels at the distal AIS increases the threshold for action potential generation. These data reveal a fundamental mechanism for rapid activity-dependent AIS reorganization and suggests that plasticity of intrinsic excitability shares conserved features with synaptic plasticity.

Objective Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon–myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. Methods Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon–myelin unit in MS NAWM (n = 8) and control white matter (WM) in the optic nerve. Results In the MS NAWM, there were more activated and phagocytic microglia cells (HLA+P2RY12− and Iba1+CD68+) and more T cells (CD3+) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. Interpretation These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon–myelin unit in MS NAWM may contribute to disease progression. ANN NEUROL 2023;93:856–870

Objective: Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon–myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. Methods: Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon–myelin unit in MS NAWM (n = 8) and control white matter (WM) in the optic nerve. Results: In the MS NAWM, there were more activated and phagocytic microglia cells (HLA +P2RY12 − and Iba1 +CD68 +) and more T cells (CD3 +) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. Interpretation: These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon–myelin unit in MS NAWM may contribute to disease progression. ANN NEUROL 2023;93:856–870.

OBJECTIVE: Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon-myelin unit in MS NAWM and determined how this correlates with low grade inflammation.METHODS: Human brain tissue obtained with short post-mortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohistochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon-myelin unit in MS NAWM (n=8) and control white matter (WM) in the optic nerve.RESULTS: In the MS NAWM, there were more activated and phagocytic microglia cells (HLA+ P2RY12- and Iba1+ CD68+ ) and more T cells (CD3+ ) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve.INTERPRETATION: These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These sub-clinical alterations to the axon-myelin unit in MS NAWM may be contributing to disease progression. This article is protected by copyright. All rights reserved.

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer 5 pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

OBJECTIVE: In metachromatic leukodystrophy, a lysosomal storage disorder due to decreased arylsulfatase A activity, hematopoietic stem cell transplantation may stop brain demyelination and allow remyelination, thereby halting white matter degeneration. This is the first study to define the effects and therapeutic mechanisms of hematopoietic stem cell transplantation on brain tissue of transplanted metachromatic leukodystrophy patients.METHODS: Autopsy brain tissue was obtained from eight (two transplanted and six nontransplanted) metachromatic leukodystrophy patients, and two age-matched controls. We examined the presence of donor cells by immunohistochemistry and microscopy. In addition, we assessed myelin content, oligodendrocyte numbers, and macrophage phenotypes. An unpaired t-test, linear regression or the nonparametric Mann-Whitney U-test was performed to evaluate differences between the transplanted, nontransplanted, and control group.RESULTS: In brain tissue of transplanted patients, we found metabolically competent donor macrophages expressing arylsulfatase A distributed throughout the entire white matter. Compared to nontransplanted patients, these macrophages preferentially expressed markers of alternatively activated, anti-inflammatory cells that may support oligodendrocyte survival and differentiation. Additionally, transplanted patients showed higher numbers of oligodendrocytes and evidence for remyelination. Contrary to the current hypothesis on therapeutic mechanism of hematopoietic cell transplantation in metachromatic leukodystrophy, we detected no enzymatic cross-correction to resident astrocytes and oligodendrocytes.INTERPRETATION: In conclusion, donor macrophages are able to digest accumulated sulfatides and may play a neuroprotective role for resident oligodendrocytes, thereby enabling remyelination, albeit without evidence of cross-correction of oligo- and astroglia. These results emphasize the importance of immunomodul

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.

Ensheathment of axons by myelin is a highly complex and multi-cellular process. Cytosolic calcium (Ca2+) changes in the myelin sheath have been implicated in myelin synthesis, but the source of this Ca2+ and the role of neuronal activity is not well understood. Using one-photon Ca2+ imaging, we investigated myelin sheath formation in the mouse somatosensory cortex and found a high rate of spontaneous microdomain Ca2+ transients and large-amplitude Ca2+ waves propagating along the internode. The frequency of Ca2+ transients and waves rapidly declines with maturation and reactivates during remyelination. Unexpectedly, myelin microdomain Ca2+ transients occur independent of neuronal action potential generation or network activity but are nearly completely abolished when the mitochondrial permeability transition pores are blocked. These findings are supported by the discovery of mitochondria organelles in non-compacted myelin. Together, the results suggest that myelin microdomain Ca2+ signals are cell-autonomously driven by high activity of mitochondria during myelin remodeling.

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking.METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole cell patch-clamp recordings and K+-sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC.RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+dynamics and neuronal network activity are abnormal in MLC mice.INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. This article is protected by copyright. All rights reserved.