Changes in the Excitability of Neocortical Neurons in a Mouse Model of Amyotrophic Lateral Sclerosis Are Not Specific to Corticospinal Neurons and Are Modulated by Advancing Disease.

Cell type-specific changes in neuronal excitability have been proposed to contribute to the selective degeneration of corticospinal neurons in amyotrophic lateral sclerosis (ALS) and to neocortical hyperexcitability, a prominent feature of both inherited and sporadic variants of the disease, but the mechanisms underlying selective loss of specific cell types in ALS are not known. We analyzed the physiological properties of distinct classes of cortical neurons in the motor cortex of hSOD1G93A mice of both sexes and found that they all exhibit increases in intrinsic excitability that depend on disease stage. Targeted recordings and in vivo calcium imaging further revealed that neurons adapt their functional properties to normalize cortical excitability as the disease progresses. Although different neuron classes all exhibited increases in intrinsic excitability, transcriptional profiling indicated that the molecular mechanisms underlying these changes are cell type specific. The increases in excitability in both excitatory and inhibitory cortical neurons show that selective dysfunction of neuronal cell types cannot account for the specific vulnerability of corticospinal motor neurons in ALS. Furthermore, the stage-dependent alterations in neuronal function highlight the ability of cortical circuits to adapt as disease progresses. These findings show that both disease stage and cell type must be considered when developing therapeutic strategies for treating ALS.SIGNIFICANCE STATEMENT It is not known why certain classes of neurons preferentially die in different neurodegenerative diseases. It has been proposed that the enhanced excitability of affected neurons is a major contributor to their selective loss. We show using a mouse model of amyotrophic lateral sclerosis (ALS), a disease in which corticospinal neurons exhibit selective vulnerability, that changes in excitability are not restricted to this neuronal class and that excitability does not increase monotonically with disease progression. Moreover, although all neuronal cell types tested exhibited abnormal functional properties, analysis of their gene expression demonstrated cell type-specific responses to the ALS-causing mutation. These findings suggest that therapies for ALS may need to be tailored for different cell types and stages of disease.

Cellular plasticity induced by anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis antibodies.

OBJECTIVE: Autoimmune-mediated anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood. METHODS: The effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses. RESULTS: We show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase. INTERPRETATION: These results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder.

Dynamics of mature myelin.

Myelin, which is produced by oligodendrocytes, insulates axons to facilitate rapid and efficient action potential propagation in the central nervous system. Traditionally viewed as a stable structure, myelin is now known to undergo dynamic modulation throughout life. This Review examines these dynamics, focusing on two key aspects: (1) the turnover of myelin, involving not only the renewal of constituents but the continuous wholesale replacement of myelin membranes; and (2) the structural remodeling of pre-existing, mature myelin, a newly discovered form of neural plasticity that can be stimulated by external factors, including neuronal activity, behavioral experience and injury. We explore the mechanisms regulating these dynamics and speculate that myelin remodeling could be driven by an asymmetry in myelin turnover or reactivation of pathways involved in myelin formation. Finally, we outline how myelin remodeling could have profound impacts on neural function, serving as an integral component of behavioral adaptation.

Features, Fates, and Functions of Oligodendrocyte Precursor Cells.

Oligodendrocyte precursor cells (OPCs) are a central nervous system resident population of glia with a distinct molecular identity and an ever-increasing list of functions. OPCs generate oligodendrocytes throughout development and across the life span in most regions of the brain and spinal cord. This process involves a complex coordination of molecular checkpoints and biophysical cues from the environment that initiate the differentiation and integration of new oligodendrocytes that synthesize myelin sheaths on axons. Outside of their progenitor role, OPCs have been proposed to play other functions including the modulation of axonal and synaptic development and the participation in bidirectional signaling with neurons and other glia. Here, we review OPC identity and known functions and discuss recent findings implying other roles for these glial cells in brain physiology and pathology.

Paired vagus nerve stimulation drives precise remyelination and motor recovery after myelin loss.

Myelin loss in the central nervous system can cause permanent motor or cognitive deficits in patients with multiple sclerosis (MS). While current immunotherapy treatments decrease the frequency of demyelinating episodes, they do not promote myelin repair or functional recovery. Vagus nerve stimulation (VNS) is a neuromodulation therapy which enhances neuroplasticity and the recovery of motor function after stroke, but its effects on myelin repair are not known. To determine if VNS influences myelin repair, we applied VNS following a demyelinating injury and measured longitudinal myelin dynamics and functional recovery. We found that VNS promotes remyelination by increasing the generation of myelinating oligodendrocytes. Pairing VNS with a skilled reach task leads to the regeneration of myelin sheaths on previously myelinated axon segments, enhancing the restoration of the original pattern of myelination. Moreover, the magnitude of sheath pattern restoration correlates with long-term motor functional improvement. Together, these results suggest that recovery of the myelin sheath pattern is a key factor in the restoration of motor function following myelin loss and identify paired VNS as a potential remyelination therapy to treat demyelinating diseases.

Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis.

The generation of new myelin-forming oligodendrocytes in the adult central nervous system is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions, suggesting that local cues drive regional differences in myelination and the capacity for regeneration. However, the layer- and region-specific regulation of oligodendrocyte populations is unclear due to the inability to monitor deep brain structures in vivo. Here we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of the entire cortical column and subcortical white matter in adult mice. We find that cortical oligodendrocyte populations expand at a higher rate in the adult brain than those of the white matter. Following demyelination, oligodendrocyte replacement is enhanced in the white matter, while the deep cortical layers show deficits in regenerative oligodendrogenesis and the restoration of transcriptional heterogeneity. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

Pathogenic myelin-specific antibodies in multiple sclerosis target conformational proteolipid protein 1-anchored membrane domains.

B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.

Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis.

The generation of new myelin-forming oligodendrocytes in the adult CNS is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions suggesting that local cues drive regional differences in myelination and the capacity for regeneration. Yet, the determination of regional variability in oligodendrocyte cell behavior is limited by the inability to monitor the dynamics of oligodendrocytes and their transcriptional subpopulations in white matter of the living brain. Here, we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of an entire cortical column and underlying subcortical white matter without cellular damage or reactivity. Using this approach, we found that the white matter generated substantially more new oligodendrocytes per volume compared to the gray matter, yet the rate of population growth was proportionally higher in the gray matter. Following demyelination, the white matter had an enhanced population growth that resulted in higher oligodendrocyte replacement compared to the gray matter. Finally, deep cortical layers had pronounced deficits in regenerative oligodendrogenesis and restoration of the MOL5/6-positive oligodendrocyte subpopulation following demyelinating injury. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy.

Significance: Three-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to studying multicellular interactions and dynamics in the intact brain. Aim: We characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain. Approach: We used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225- to 1360-nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP. Results: We find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have a high signal-to-background ratio in the 1300- to 1360-nm range, consistent with enhanced 3P cross-sections. Conclusions: These results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy.

Motor learning drives dynamic patterns of intermittent myelination on learning-activated axons.

Myelin plasticity occurs when newly formed and pre-existing oligodendrocytes remodel existing patterns of myelination. Myelin remodeling occurs in response to changes in neuronal activity and is required for learning and memory. However, the link between behavior-induced neuronal activity and circuit-specific changes in myelination remains unclear. Using longitudinal in vivo two-photon imaging and targeted labeling of learning-activated neurons in mice, we explore how the pattern of intermittent myelination is altered on individual cortical axons during learning of a dexterous reach task. We show that behavior-induced myelin plasticity is targeted to learning-activated axons and occurs in a staged response across cortical layers in the mouse primary motor cortex. During learning, myelin sheaths retract, which results in lengthening of nodes of Ranvier. Following motor learning, addition of newly formed myelin sheaths increases the number of continuous stretches of myelination. Computational modeling suggests that motor learning-induced myelin plasticity initially slows and subsequently increases axonal conduction speed. Finally, we show that both the magnitude and timing of nodal and myelin dynamics correlate with improvement of behavioral performance during motor learning. Thus, learning-induced and circuit-specific myelination changes may contribute to information encoding in neural circuits during motor learning.

Miniature structured illumination microscope for in vivo 3D imaging of brain structures with optical sectioning.

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 µm. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 260 µm is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 µm in the brain.

Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis.

We recently described a severe, potentially lethal, but treatment-responsive encephalitis that associates with autoantibodies to the NMDA receptor (NMDAR) and results in behavioral symptoms similar to those obtained with models of genetic or pharmacologic attenuation of NMDAR function. Here, we demonstrate that patients' NMDAR antibodies cause a selective and reversible decrease in NMDAR surface density and synaptic localization that correlates with patients' antibody titers. The mechanism of this decrease is selective antibody-mediated capping and internalization of surface NMDARs, as Fab fragments prepared from patients' antibodies did not decrease surface receptor density, but subsequent cross-linking with anti-Fab antibodies recapitulated the decrease caused by intact patient NMDAR antibodies. Moreover, whole-cell patch-clamp recordings of miniature EPSCs in cultured rat hippocampal neurons showed that patients' antibodies specifically decreased synaptic NMDAR-mediated currents, without affecting AMPA receptor-mediated currents. In contrast to these profound effects on NMDARs, patients' antibodies did not alter the localization or expression of other glutamate receptors or synaptic proteins, number of synapses, dendritic spines, dendritic complexity, or cell survival. In addition, NMDAR density was dramatically reduced in the hippocampus of female Lewis rats infused with patients' antibodies, similar to the decrease observed in the hippocampus of autopsied patients. These studies establish the cellular mechanisms through which antibodies of patients with anti-NMDAR encephalitis cause a specific, titer-dependent, and reversible loss of NMDARs. The loss of this subtype of glutamate receptors eliminates NMDAR-mediated synaptic function, resulting in the learning, memory, and other behavioral deficits observed in patients with anti-NMDAR encephalitis.

Astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis.

Astrocytes modulate the formation and function of glutamatergic synapses in the CNS, but whether astrocytes modulate GABAergic synaptogenesis is unknown. We demonstrate that media conditioned by astrocytes, but not other cells, enhanced GABAergic but not glutamatergic axon length and branching, and increased the number and density of presynaptically active GABAergic synapses in dissociated hippocampal cultures. Candidate mechanisms and factors, such as activity, neurotrophins, and cholesterol were excluded as mediating these effects. While thrombospondins secreted by astrocytes are necessary and sufficient to increase hippocampal glutamatergic synaptogenesis, they do not mediate astrocyte effects on GABAergic synaptogenesis. We show that the factors in astrocyte conditioned media that selectively affect GABAergic neurons are proteins. Taken together, our results show that astrocytes increase glutamatergic and GABAergic synaptogenesis via different mechanisms and release one or more proteins with the novel functions of increasing GABAergic axon length, branching and synaptogenesis.

Premyelinating Oligodendrocytes: Mechanisms Underlying Cell Survival and Integration.

In the central nervous system, oligodendrocytes produce myelin sheaths that enwrap neuronal axons to provide trophic support and increase conduction velocity. New oligodendrocytes are produced throughout life through a process referred to as oligodendrogenesis. Oligodendrogenesis consists of three canonical stages: the oligodendrocyte precursor cell (OPC), the premyelinating oligodendrocyte (preOL), and the mature oligodendrocyte (OL). However, the generation of oligodendrocytes is inherently an inefficient process. Following precursor differentiation, a majority of premyelinating oligodendrocytes are lost, likely due to apoptosis. If premyelinating oligodendrocytes progress through this survival checkpoint, they generate new myelinating oligodendrocytes in a process we have termed integration. In this review, we will explore the intrinsic and extrinsic signaling pathways that influence preOL survival and integration by examining the intrinsic apoptotic pathways, metabolic demands, and the interactions between neurons, astrocytes, microglia, and premyelinating oligodendrocytes. Additionally, we will discuss similarities between the maturation of newly generated neurons and premyelinating oligodendrocytes. Finally, we will consider how increasing survival and integration of preOLs has the potential to increase remyelination in multiple sclerosis. Deepening our understanding of premyelinating oligodendrocyte biology may open the door for new treatments for demyelinating disease and will help paint a clearer picture of how new oligodendrocytes are produced throughout life to facilitate brain function.

Mechanisms underlying autoimmune synaptic encephalitis leading to disorders of memory, behavior and cognition: insights from molecular, cellular and synaptic studies.

Recently, several novel, potentially lethal and treatment-responsive syndromes that affect hippocampal and cortical function have been shown to be associated with auto-antibodies against synaptic antigens, notably glutamate or GABA-B receptors. Patients with these auto-antibodies, sometimes associated with teratomas and other neoplasms, present with psychiatric symptoms, seizures, memory deficits and decreased levels of consciousness. These symptoms often improve dramatically after immunotherapy or tumor resection. Here we review studies of the cellular and synaptic effects of these antibodies in hippocampal neurons in vitro and preliminary work in rodent models. Our work suggests that patient antibodies lead to rapid and reversible removal of neurotransmitter receptors from synaptic sites, leading to changes in synaptic and circuit function that in turn are likely to lead to behavioral deficits. We also discuss several of the many questions raised by these and related disorders. Determining the mechanisms underlying these novel anti-neurotransmitter receptor encephalopathies will provide insights into the cellular and synaptic bases of the memory and cognitive deficits that are hallmarks of these disorders, and potentially suggest avenues for therapeutic intervention.

AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location.

OBJECTIVE: To report the clinical and immunological features of a novel autoantigen related to limbic encephalitis (LE) and the effect of patients' antibodies on neuronal cultures. METHODS: We conducted clinical analyses of 10 patients with LE. Immunoprecipitation and mass spectrometry were used to identify the antigens. Human embryonic kidney 293 cells expressing the antigens were used in immunocytochemistry and enzyme-linked immunoabsorption assay. The effect of patients' antibodies on cultures of live rat hippocampal neurons was determined with confocal microscopy. RESULTS: Median age was 60 (38-87) years; 9 were women. Seven had tumors of the lung, breast, or thymus. Nine patients responded to immunotherapy or oncological therapy, but neurological relapses, without tumor recurrence, were frequent and influenced the long-term outcome. One untreated patient died of LE. All patients had antibodies against neuronal cell surface antigens that by immunoprecipitation were found to be the glutamate receptor 1 (GluR1) and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Human embryonic kidney 293 cells expressing GluR1/2 reacted with all patients' sera or cerebrospinal fluid, providing a diagnostic test for the disorder. Application of antibodies to cultures of neurons significantly decreased the number of GluR2-containing AMPAR clusters at synapses with a smaller decrease in overall AMPAR cluster density; these effects were reversed after antibody removal. INTERPRETATION: Antibodies to GluR1/2 associate with LE that is often paraneoplastic, treatment responsive, and has a tendency to relapse. Our findings support an antibody-mediated pathogenesis in which patients' antibodies alter the synaptic localization and number of AMPARs.

Neuron-oligodendroglia interactions: Activity-dependent regulation of cellular signaling.

Oligodendrocyte lineage cells (oligodendroglia) and neurons engage in bidirectional communication throughout life to support healthy brain function. Recent work shows that changes in neuronal activity can modulate proliferation, differentiation, and myelination to support the formation and function of neural circuits. While oligodendroglia express a diverse collection of receptors for growth factors, signaling molecules, neurotransmitters and neuromodulators, our knowledge of the intracellular signaling pathways that are regulated by neuronal activity remains largely incomplete. Many of the pathways that modulate oligodendroglia behavior are driven by changes in intracellular calcium signaling, which may differentially affect cytoskeletal dynamics, gene expression, maturation, integration, and axonal support. Additionally, activity-dependent neuron-oligodendroglia communication plays an integral role in the recovery from demyelinating injuries. In this review, we summarize the modalities of communication between neurons and oligodendroglia and explore possible roles of activity-dependent calcium signaling in mediating cellular behavior and myelination.

Motor learning promotes remyelination via new and surviving oligodendrocytes.

Oligodendrocyte loss in neurological disease leaves axons vulnerable to damage and degeneration, and activity-dependent myelination may represent an endogenous mechanism to improve remyelination following injury. Here we report that, while learning a forelimb reach task transiently suppresses oligodendrogenesis, it subsequently increases oligodendrocyte precursor cell differentiation, oligodendrocyte generation and myelin sheath remodeling in the forelimb motor cortex. Immediately following demyelination, neurons exhibit hyperexcitability, learning is impaired and behavioral intervention provides no benefit to remyelination. However, partial remyelination restores neuronal and behavioral function, allowing learning to enhance oligodendrogenesis, remyelination of denuded axons and the ability of surviving oligodendrocytes to generate new myelin sheaths. Previously considered controversial, we show that sheath generation by mature oligodendrocytes is not only possible but also increases myelin pattern preservation following demyelination, thus presenting a new target for therapeutic interventions. Together, our findings demonstrate that precisely timed motor learning improves recovery from demyelinating injury via enhanced remyelination from new and surviving oligodendrocytes.

Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome.

The roles of astrocytes in the CNS have been expanding beyond the long held view of providing passive, supportive functions. Recent evidence has identified roles in neuronal development, extracellular matrix maintenance, and response to inflammatory challenges. Therefore, insights into astrocyte secretion are critically important for understanding physiological responses and pathological mechanisms in CNS diseases. Primary astrocyte cultures were treated with inflammatory cytokines for either a short (1 day) or sustained (7 days) exposure. Increased interleukin-6 secretion, nitric oxide production, cyclooxygenase-2 activation, and nerve growth factor (NGF) secretion confirmed the astrocytic response to cytokine treatment. MS/MS analysis, computational prediction algorithms, and functional classification were used to compare the astrocyte protein secretome from control and cytokine-exposed cultures. In total, 169 secreted proteins were identified, including both classically and nonconventionally secreted proteins that comprised components of the extracellular matrix and enzymes involved in processing of glycoproteins and glycosaminoglycans. Twelve proteins were detected exclusively in the secretome from cytokine-treated astrocytes, including matrix metalloproteinase-3 (MMP-3) and members of the chemokine ligand family. This compilation of secreted proteins provides a framework for identifying factors that influence the biochemical environment of the nervous system, regulate development, construct extracellular matrices, and coordinate the nervous system response to inflammation.