IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

BACKGROUND: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. METHODS: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. RESULTS: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. CONCLUSION: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

A 21-bp deletion in the complement regulator CD55 promotor region is associated with multifocal motor neuropathy and its disease course.

BACKGROUND AND AIMS: To further substantiate the role of antibody-mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor polymorphisms of genes encoding the membrane-bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course. METHODS: We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters. RESULTS: The genotype frequencies of rs28371582, a 21-bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 [1.27-4.58]), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council [MRC] points/year; 95% confidence interval [CI]: 0.040-0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre-diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083-1.80, p .032). INTERPRETATION: MMN susceptibility is associated with a 21-bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long-term disease outcome. Taken together, these results point out the relevance of the pre-C5 level of the complement cascade in the inflammatory processes underlying MMN.

Offline orbitofrontal cortex reactivation depends on recency of place-reward changes and coheres with hippocampal replay.

The orbitofrontal cortex, one of the key neocortical areas in valuation and emotion, is critical for cognitive flexibility but its role in the consolidation of recently acquired information remains unclear. Here, we demonstrate orbitofrontal offline replay in the context of a place-reward association task on a maze with varying goal locations. When switches in place-reward coupling were applied, replay was enhanced relative to sessions with stable contingencies. Moreover, replay strength was positively correlated with the subsequent overnight change in behavioral performance. Interrogating relationships between orbitofrontal and hippocampal activity, we found that orbitofrontal and hippocampal replay could occur independently but became coordinated during a type of cortical state with strong spiking activity. These findings reveal a structured form of offline orbitofrontal ensemble activity that is correlated with cognitive flexibility required to adapt to changing task contingencies, and becomes associated with hippocampal replay only during a specific state of high cortical excitability.

OXA-48-Producing Uropathogenic Escherichia coli Sequence Type 127, the Netherlands, 2015-2022.

During 2015-2022, a genetic cluster of OXA-48-producing uropathogenic Escherichia coli sequence type 127 spread throughout the Netherlands. The 20 isolates we investigated originated mainly from urine, belonged to Clermont phylotype B2, and carried 18 genes encoding putative uropathogenicity factors. The isolates were susceptible to first-choice antimicrobial drugs for urinary tract infections.

Multifocal motor neuropathy is not associated with altered innate immune responses to endotoxin.

OBJECTIVE: Antibody- and complement-mediated peripheral nerve inflammation are central in the pathogenesis of MMN. Here, we studied innate immune responses to endotoxin in patients with MMN and controls to further our understanding of MMN risk factors and disease modifiers. METHODS: We stimulated whole blood of 52 patients with MMN and 24 controls with endotoxin and collected plasma. With a multiplex assay, we determined levels of the immunoregulating proteins IL-1RA, IL-1ß, IL-6, IL-10, IL-21, TNF-α, IL-8 and CD40L in unstimulated and LPS-stimulated plasma. We compared baseline and stimulated protein levels between patients and controls and correlated concentrations to clinical parameters. RESULTS: Protein level changes after stimulation were comparable between groups (p > 0.05). IL-1RA, IL-1ß, IL-6 and IL-21 baseline concentrations showed a positive correlation with monthly IVIg dosage (all corrected p-values < 0.016). Patients with anti-GM1 IgM antibodies showed a more pronounced IL-21 increase after stimulation (p 0.048). CONCLUSIONS: Altered endotoxin-induced innate immune responses are unlikely to be a susceptibility factor for MMN.

Spike-based coupling between single neurons and populations across rat sensory cortices, perirhinal cortex, and hippocampus.

Cortical computations require coordination of neuronal activity within and across multiple areas. We characterized spiking relationships within and between areas by quantifying coupling of single neurons to population firing patterns. Single-neuron population coupling (SNPC) was investigated using ensemble recordings from hippocampal CA1 region and somatosensory, visual, and perirhinal cortices. Within-area coupling was heterogeneous across structures, with area CA1 showing higher levels than neocortical regions. In contrast to known anatomical connectivity, between-area coupling showed strong firing coherence of sensory neocortices with CA1, but less with perirhinal cortex. Cells in sensory neocortices and CA1 showed positive correlations between within- and between-area coupling; these were weaker for perirhinal cortex. All four areas harbored broadcasting cells, connecting to multiple external areas, which was uncorrelated to within-area coupling strength. When examining correlations between SNPC and spatial coding, we found that, if such correlations were significant, they were negative. This result was consistent with an overall preservation of SNPC across different brain states, suggesting a strong dependence on intrinsic network connectivity. Overall, SNPC offers an important window on cell-to-population synchronization in multi-area networks. Instead of pointing to specific information-coding functions, our results indicate a primary function of SNPC in dynamically organizing communication in systems composed of multiple, interconnected areas.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

BACKGROUND AND OBJECTIVES: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN. METHODS: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed. RESULTS: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs. DISCUSSION: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

Multidrug-resistant organisms in patients from Ukraine in the Netherlands, March to August 2022.

Since March 2022, there has been an emergence of multidrug-resistant organisms (MDRO) in the Netherlands in patients originating from Ukraine (58 patients, 75 isolates). For about half of these patients, recent hospitalisation in Ukraine was reported. Genomic surveillance revealed that the majority of the MDRO represent globally spread epidemic lineages and that 60% contain New Delhi metallo-ß-lactamase (NDM) genes. Professionals should be aware of an increase in such MDRO associated with migration and medical evacuation of people from Ukraine.

SMN1 Duplications Are Associated With Progressive Muscular Atrophy, but Not With Multifocal Motor Neuropathy and Primary Lateral Sclerosis.

OBJECTIVE: To assess the association between copy number (CN) variation in the survival motor neuron (SMN) locus and multifocal motor neuropathy (MMN), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS) susceptibility and to determine the association of SMN1 and SMN2 CN with MMN, PMA, and PLS disease course. METHODS: In this monocenter study, we used multiplex ligation-dependent probe amplification to determine SMN1 and SMN2 CN in Dutch patients with MMN, PMA, and PLS and controls. We stratified clinical parameters for SMN1 and SMN2 CN. We analyzed SMN1 and SMN2 exons 1-6, intron 6, and exon 8 CN to study the genetic architecture of SMN1 duplications. RESULTS: SMN1 and SMN2 CN were determined in 132 patients with MMN, 150 patients with PMA, 104 patients with PLS, and 956 control subjects. MMN and PLS were not associated with CN variation in SMN1 or SMN2. By contrast, patients with PMA more often than controls carried SMN1 duplications (≥3 SMN1 copies, 12.0% vs 5.0%, odds ratio 2.69 (1.43-4.91), p 0.0020). SMN1 and SMN2 CN status was not associated with MMN, PLS, or PMA disease course. In case of SMN1 exon 7 duplications, exons 1-6, exon 8, and introns 6 and 7 were also duplicated, suggesting full SMN1 duplications. CONCLUSIONS: SMN1 duplications are associated with PMA, but not with PLS and MMN. SMN1 duplications in PMA are balanced duplications. The results of this study highlight the primary effect of altered SMN CN on lower motor neurons.

Reconciling the object and spatial processing views of the perirhinal cortex through task-relevant unitization.

The perirhinal cortex is situated on the border between sensory association cortex and the hippocampal formation. It serves an important function as a transition area between the sensory neocortex and the medial temporal lobe. While the perirhinal cortex has traditionally been associated with object coding and the "what" pathway of the temporal lobe, current evidence suggests a broader function of the perirhinal cortex in solving feature ambiguity and processing complex stimuli. Besides fulfilling functions in object coding, recent neurophysiological findings in freely moving rodents indicate that the perirhinal cortex also contributes to spatial and contextual processing beyond individual sensory modalities. Here, we address how these two opposing views on perirhinal cortex-the object-centered and spatial-contextual processing hypotheses-may be reconciled. The perirhinal cortex is consistently recruited when different features can be merged perceptually or conceptually into a single entity. Features that are unitized in these entities include object information from multiple sensory domains, reward associations, semantic features and spatial/contextual associations. We propose that the same perirhinal network circuits can be flexibly deployed for multiple cognitive functions, such that the perirhinal cortex performs similar unitization operations on different types of information, depending on behavioral demands and ranging from the object-related domain to spatial, contextual and semantic information.

High-resolution mapping identifies HLA class II associations with multifocal motor neuropathy.

OBJECTIVE: To gain further insight in the immunopathology underlying multifocal motor neuropathy (MMN) by exploring the association between MMN and the human leukocyte antigen (HLA) class II DRB1, DQB1, and DQA loci in depth and by correlating associated haplotypes to detailed clinical and anti-ganglioside antibody data. METHODS: We performed high-resolution HLA-class II typing for the DRB1, DQB1, and DQA1 loci in 126 well-characterized MMN patients and assessed disease associations with haplotypes. We used a cohort of 1305 random individuals as a reference for haplotype distribution in the Dutch population. RESULTS: The DRB1*15:01-DQB1*06:02 haplotype (OR 1.6 [95% CI 1.1-2.2], p < 0.05) and the DRB1*12:01-DQB1*03:01 haplotype (OR 2.7 [95% CI 1.2-5.5], p < 0.05) were more frequent in patients with MMN than in controls. These haplotypes were not associated with disease course, response to treatment or anti-ganglioside antibodies. CONCLUSIONS: MMN is associated with the DRB1*15:01-DQB1*06:02 and DRB1*12:01-DQB1*03:01 haplotypes. These HLA molecules or gene variants in their immediate vicinity may promote the specific inflammatory processes underlying MMN.

Clinical outcomes in multifocal motor neuropathy: A combined cross-sectional and follow-up study.

OBJECTIVE: To assess the clinical course of multifocal motor neuropathy (MMN) in a large cohort of patients and to identify predictive factors of a progressive disease course. METHODS: Between May 2015 and February 2016, we collected clinical data from 100 patients with MMN, of whom 60 had participated in a nationwide cross-sectional cohort study in 2007. We documented clinical characteristics using standardized questionnaires and performed a standardized neurologic examination. We used multiple linear regression analysis to identify factors that correlated with worse outcome. RESULTS: We found that age at diagnosis (45.2 vs 48.6 years, p < 0.02) was significantly increased between 2007 and 2015-2016, whereas diagnostic delay decreased by 15 months. Seven out of 10 outcome measures deteriorated over time (all p < 0.01). Patients who had a lower Medical Research Council (MRC) sumscore and absence of 1 or more reflexes at the baseline visit showed a greater functional loss at follow-up (p = 0.007 and p = 0.016). CONCLUSIONS: Our study shows that MMN is a progressive disease. Although 87% of patients received maintenance treatment, muscle strength, reflexes, vibration sense, and the Self-Evaluation Scale score significantly deteriorated over time. Lower MRC sumscore and absence of reflexes predicted a more progressive disease course. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that lower MRC sumscore and the absence of reflexes predict a more progressive disease course in patients with MMN.

Shared Learnings on the New EMA First-in-Human and Early Clinical Trial Guideline: Proceedings From a DIAlogue Session at DIA Europe 2018.

The EU is a member of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH), and therefore adopts the ICH Guidelines, including the ICH M3 Guideline on Nonclinical Safety Studies. Following the 2016 incident in France with BIA 10-2474, and in light of the substantial evolvement of how early clinical development has been undertaken during the last 10 years, for example, conducting integrated (FIH) studies that include multiple parts (eg, single ascending doses, multiple ascending doses, food effect), EMA decided to update the existing 2007 FIH guideline. The key revisions to the 2007 guideline, now titled "Guideline on Strategies to Identify and Mitigate Risks for First-in-Human and Early Clinical Trials With Investigational Medicinal Products," include additional information. The revision reinforces the importance and impact of pharmacologic data, which supports the intended efficacy of the compound, risk assessment, and protocol design. The updates, effective February 2018, are intended to provide additional guidance and clarity for Sponsors developing FIH and early phase clinical research programs, and ultimately support subject safety. At the 2018 DIA Europe Annual Meeting in Basel, Switzerland, European regulators, industry representatives and academics convened a DIAlogue Session on April 17 to discuss how the revised 2017 guideline is being applied, and to establish recommendations for its application. Using two case studies as examples, the session participants discussed the nonclinical and clinical considerations for applying the newly revised recommendations, and interacted with a panel including regulators and industry representatives. The proceedings from this session reflect practical considerations for the implementation of the revised guideline.

Biallelic variants in POLR3GL cause endosteal hyperostosis and oligodontia.

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A, POLR3B, and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report, we describe three individuals with biallelic variants in POLR3GL, a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual, we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis, oligodontia, short stature, and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A, POLR3B, POLR1C, and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected.

Multiplexing of Information about Self and Others in Hippocampal Ensembles.

In addition to coding a subject's location in space, the hippocampus has been suggested to code social information, including the spatial position of conspecifics. "Social place cells" have been reported for tasks in which an observer mimics the behavior of a demonstrator. We examine whether rat hippocampal neurons may encode the behavior of a minirobot, but without requiring the animal to mimic it. Rather than finding social place cells, we observe that robot behavioral patterns modulate place fields coding animal position. This modulation may be confounded by correlations between robot movement and changes in the animal's position. Although rat position indeed significantly predicts robot behavior, we find that hippocampal ensembles code additional information about robot movement patterns. Fast-spiking interneurons are particularly informative about robot position and global behavior. In conclusion, when the animal's own behavior is conditional on external agents, the hippocampus multiplexes information about self and others.

Functional determinants of enhanced and depressed interareal information flow in nonrapid eye movement sleep between neuronal ensembles in rat cortex and hippocampus.

Compared with wakefulness, neuronal activity during nonrapid eye movement (NREM) sleep is characterized by a decreased ability to integrate information, but also by the reemergence of task-related information patterns. To investigate the mechanisms underlying these seemingly opposing phenomena, we measured directed information flow by computing transfer entropy between neuronal spiking activity in three cortical regions and the hippocampus of rats across brain states. State-dependent information flow was jointly determined by the anatomical distance between neurons and by their functional specialization. We distinguished two regimes, operating at short and long time scales, respectively. From wakefulness to NREM sleep, transfer entropy at short time scales increased for interareal connections between neurons showing behavioral task correlates. Conversely, transfer entropy at long time scales became stronger between nontask modulated neurons and weaker between task-modulated neurons. These results may explain how, during NREM sleep, a global interareal disconnection is compatible with highly specific task-related information transfer.

Perirhinal firing patterns are sustained across large spatial segments of the task environment.

Spatial navigation and memory depend on the neural coding of an organism's location. Fine-grained coding of location is thought to depend on the hippocampus. Likewise, animals benefit from knowledge parsing their environment into larger spatial segments, which are relevant for task performance. Here we investigate how such knowledge may be coded, and whether this occurs in structures in the temporal lobe, supplying cortical inputs to the hippocampus. We found that neurons in the perirhinal cortex of rats generate sustained firing patterns that discriminate large segments of the task environment. This contrasted to transient firing in hippocampus and sensory neocortex. These spatially extended patterns were not explained by task variables or temporally discrete sensory stimuli. Previously it has been suggested that the perirhinal cortex is part of a pathway processing object, but not spatial information. Our results indicate a greater complexity of neural coding than captured by this dichotomy.

Brachial plexus magnetic resonance imaging differentiates between inflammatory neuropathies and does not predict disease course.

OBJECTIVE: The main objective of this study was to evaluate the correlation between the distribution of brachial plexus magnetic resonance imaging (MRI) abnormalities and clinical weakness, and to evaluate the value of brachial plexus MRI in predicting disease course and response to treatment in multifocal motor neuropathy (MMN), Lewis-Sumner syndrome (LSS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: Sixty-seven patients with an inflammatory neuropathy diagnosed at our tertiary referral center for neuromuscular diseases had undergone bilateral T2-weighted short tau inversion recovery (STIR) MRI of the brachial plexus. We obtained clinical follow-up data and scored all MRIs for abnormalities and the symmetry of their distribution. RESULTS: Brachial plexus MRI abnormalities were detected in 45% of the patients. An abnormal MRI did not predict disease course in terms of patterns of weakness, sensory disturbances or response to treatment. Within the spectrum of radiological abnormalities, asymmetrical clinical syndromes, MMN and LSS were significantly associated with asymmetrical radiological abnormalities, whereas symmetrical abnormalities predominated in CIDP (p < .001, phi 0.791). CONCLUSION: T2 STIR brachial plexus MRI abnormalities correspond with the distribution of neurological deficits in inflammatory neuropathies, but do not correlate with specific clinical characteristics, response to treatment or disease course.

Comparative study of peripheral nerve Mri and ultrasound in multifocal motor neuropathy and amyotrophic lateral sclerosis.

INTRODUCTION: Differentiating multifocal motor neuropathy (MMN) from amyotrophic lateral sclerosis (ALS) is important, as MMN is a difficult, but treatable disorder. METHODS: We studied peripheral nerve imaging techniques in differentiating MMN from ALS by measuring the cross-sectional area (CSA) of the median and ulnar nerves in the forearms using high resolution ultrasound (HRUS) and MRI. RESULTS: HRUS CSA values of the median nerve in the forearm (P = 0.002) and the ulnar nerve distal to the sulcus (P = 0.009) were significantly enlarged in patients with MMN. There was a positive correlation between CSA as measured with HRUS and MRI (Spearman rho 0.60; P < 0.001). CONCLUSIONS: Peripheral nerve imaging is a potentially powerful technique to distinguish MMN from ALS. Muscle Nerve, 2016 Muscle Nerve 54: 1133-1135, 2016.

Spike-Based Functional Connectivity in Cerebral Cortex and Hippocampus: Loss of Global Connectivity Is Coupled to Preservation of Local Connectivity During Non-REM Sleep.

UNLABELLED: Behavioral states are commonly considered global phenomena with homogeneous neural determinants. However, recent studies indicate that behavioral states modulate spiking activity with neuron-level specificity as a function of brain area, neuronal subtype, and preceding history. Although functional connectivity also strongly depends on behavioral state at a mesoscopic level and is globally weaker in non-REM (NREM) sleep and anesthesia than wakefulness, it is unknown how neuronal communication is modulated at the cellular level. We hypothesize that, as for neuronal activity, the influence of behavioral states on neuronal coupling strongly depends on type, location, and preceding history of involved neurons. Here, we applied nonlinear, information-theoretical measures of functional connectivity to ensemble recordings with single-cell resolution to quantify neuronal communication in the neocortex and hippocampus of rats during wakefulness and sleep. Although functional connectivity (measured in terms of coordination between firing rate fluctuations) was globally stronger in wakefulness than in NREM sleep (with distinct traits for cortical and hippocampal areas), the drop observed during NREM sleep was mainly determined by a loss of inter-areal connectivity between excitatory neurons. Conversely, local (intra-area) connectivity and long-range (inter-areal) coupling between interneurons were preserved during NREM sleep. Furthermore, neuronal networks that were either modulated or not by a behavioral task remained segregated during quiet wakefulness and NREM sleep. These results show that the drop in functional connectivity during wake-sleep transitions globally holds true at the cellular level, but confine this change mainly to long-range coupling between excitatory neurons. SIGNIFICANCE STATEMENT: Studies performed at a mesoscopic level of analysis have shown that communication between cortical areas is disrupted in non-REM sleep and anesthesia. However, the neuronal determinants of this phenomenon are not known. Here, we applied nonlinear, information-theoretical measures of functional coupling to multi-area tetrode recordings from freely moving rats to investigate whether and how brain state modulates coordination between individual neurons. We found that the previously observed drop in functional connectivity during non-REM (NREM) sleep can be explained by a decrease in coupling between excitatory neurons located in distinct brain areas. Conversely, intra-area communication and coupling between interneurons are preserved. Our results provide significant new insights into the neuron-level mechanisms responsible for the loss of consciousness occurring in NREM sleep.