Maarten H. P. Kole

Myelin membranes are tightly connected to the axon, forming an electrical route for rapid saltatory conduction of action potentials from node to node. Beneath the myelin, adjacent to the node, lie voltage-gated potassium (K ⁺ ) channels, whose fundamental role in conduction remains poorly understood. Constrained by biological data, we developed a finite element model of a node of Ranvier encompassing an adaxonal membrane separating oligodendrocyte cytoplasm from the periaxonal space, and solved the full coupled set of physical equations governing electrodiffusion and voltage profiles at the nanoscale. Remarkably, we find that nodal action potentials invert at the inner adaxonal myelin membrane. The hyperpolarization-driven opening of myelinic Kir4.1 channels produces an instantaneous, powerful K ⁺ prebuffering, clamping the periaxonal K ⁺ concentration and enabling high-frequency action potential generation. Together, these findings indicate that the periaxon maintains an electrical feedback loop with the node, and suggest a key evolutionary advantage of juxtaparanodal K ⁺ channel positioning.

Divisive normalization describes how neuronal responses are scaled by the activity of a broader pool of inputs and accounts for several nonlinear response properties in primary visual cortex (V1). It accurately describes contrast gain control, as well as the response to overlapping gratings. Nevertheless, how this computation is performed by the brain is unclear. One mechanism suggested to underlie divisive normalization in V1 is short-term depression (STD) at thalamocortical synapses. Here, we used a Vglut2-Cre dependent heterozygous deletion of Stxbp1 in mice as an in vivo model for increased STD. This enabled us to test whether STD is responsible for divisive normalization in V1. As expected, responses were shorter in the mutant mice compared to wild-type siblings. Although we did find other differences in visual responses, notably in direction selectivity and response linearity, neither the contrast response function, nor the amount of cross orientation suppression differed between the two groups. We conclude that STD is likely not involved in divisive normalization of visual responses in mouse V1. Instead, short-term plasticity shapes the temporal integration through which selective cortical responses are constructed.

Summary: Neuronal development and function rely on precise sorting of membrane proteins to distinct neuronal domains, yet the underlying mechanisms remain incompletely understood. Here, we investigated the trafficking of two highly homologous NLGNs (NLGN1 and 2) which distinctly localize and function at excitatory and inhibitory synapses. By using spatiotemporal proteomics, genetic engineering and microscopy in CNS cultured neurons and organotypic slices, we dissected the itinerary of biosynthetic NLGNs, identifying co-cargoes and sorting routes to dendrites and the AIS. At the AIS, which contains only inhibitory synapses, both NLGN1 and NLGN2 are locally exocytosed. While NLGN2 is stably recruited to axo-axonic synapses by its extracellular domain and modulated by activity, NLGN1 is retrieved by endocytic mechanisms. We propose that biosynthetic NLGNs are co-sorted to the AIS PM where they detect pre-synaptic type. These findings may help to elucidate the role of AIS-localized NLGNs in shaping AIS structural and functional plasticity. Highlights: Spatiotemporal proteomics elucidates interactome of biosynthetic NLGN1 in neurons NLGN1 and NLGN2 are targeted to the AIS by kinesin-1 NLGN2, but not NLGN1, is retained at axo-axonic synapses and modulated by activity NLGN stability at the AIS plasma membrane is determined by the ectodomain Graphical

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1–7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

Mitochondria are diverse and multifaceted intracellular organelles regulating oxidative energy supply, lipid metabolism and calcium (Ca ²⁺ ) signalling. In neurons the spatial sequestration of cytoplasmic Ca ²⁺ by mitochondria plays a critical role in determining activity‐dependent spine plasticity, shaping the presynaptic transmitter release characteristics and contributing to sustained action potential firing. Here, we tested the hypothesis that mitochondria at the axon initial segment (AIS) affect the microdomain cytoplasmic Ca ²⁺ transients, thereby regulating Ca ²⁺ ‐dependent voltage‐gated ion channels at the plasma membrane and initiation of action potentials. Using 3D electron microscopy reconstructions and virally injecting genetically encoded fluorescence indicators we visualized the ultrastructure and distribution of mitochondria selectively in thick‐tufted layer 5 pyramidal neurons. We found that most mitochondria were stably clustered to the proximal AIS, while few were observed at distal sites. Simultaneous two‐photon imaging of action potential‐dependent cytoplasmic and mitochondrial Ca ²⁺ , combined with electrophysiological recordings showed that AIS mitochondria exhibit powerful activity‐dependent cytosolic Ca ²⁺ uptake. However, while intracellular application of the mitochondrial Ca ²⁺ uniporter inhibitor Ru360 fully blocked mitochondrial Ca ²⁺ import and increased the slow afterhyperpolarization duration, it did not affect action potential input–output function, action potential dynamics nor the ability to produce high‐frequency burst output. Together, the results indicate that AIS mitochondria are dispensable for temporal and rate encoding, suggesting that mt‐Ca ²⁺ buffering at the AIS may be involved in non‐electrical roles. image Key points: Mitochondrial Ca ²⁺ buffering controls multiple Ca ²⁺ ‐dependent intracellular processes and their subcellular location of the organelles defines local physiological properties in neurons. Recent studies implicate mitochondrial Ca ²⁺ uptake in the slow afterhyperpolarization and maintenance of action potential firing. Using electron microscopy and virally delivered genetically encoded tools we examined mitochondria in the layer 5 pyramidal neuron axon initial segment (AIS), the site where action potentials initiate, and found that cytoplasmic Ca ²⁺ influx is powerfully buffered by proximally clustered mitochondria. Electrophysiological recordings during the block of the mitochondrial calcium uniporter reveal a role in the slow afterhyperpolarization, while AIS action potential initiation and action potential waveforms are independent from mitochondria. These findings indicate AIS mitochondria under physiological conditions exert non‐electrical roles.

Myelin damage is a hallmark of several neurological disorders, but how it occurs remains to be fully understood. In this study, we found that early damage in zebrafish and rodent demyelination models is characterized by myelin swelling. We show, through live imaging, that myelin swelling does not always lead to myelin loss and that swellings can sometimes resolve, allowing sheaths to remodel. Increased neuronal activity during early demyelination exacerbates myelin damage, whereas reducing neuronal activity mitigates myelin swelling in both zebrafish and mice. In human multiple sclerosis tissue, myelin swelling is also dynamic and is prominent around active lesions. Our data indicate that myelin swelling is a conserved feature of demyelination and that damage to myelin sheaths can resolve, opening opportunities for targeting human disease.

Myelin is essential for the rapid conduction of action potentials (APs) but its role in long-range processing of disparate inputs remains unclear. Here, using a cell-type-specific approach we recorded optogenetically evoked pyramidal neuron responses via in vivo juxtacellular patch-clamp and Neuropixels probes, tracking spike transmission from layer 5 (L5) to the posteromedial thalamic nucleus (POm) in the mouse. Cuprizone-induced demyelination caused millisecond-scale delays, increased temporal jitter and impaired transmission of high-frequency AP bursts. Computational modeling of the saltatory propagation from L5 to POm revealed that myelin loss from neocortical internodes acts as a low-pass filter, impeding high-frequency spikes within the burst. Finally, pairing optogenetic stimulation with whisker input showed that intact myelination is crucial for coincidence detection in the thalamus. These findings suggest that the continuous myelin pattern of L5 axons not only speeds conduction but also enables precise temporal integration of sensory and cortical signals across long-range pathways.

Oligodendrocytes make myelin for the electrical insulation of axons and saltatory impulse conduction. Myelin lipids and proteins undergo a slow turnover, but exactly how the multilamellar and compacted membrane sheaths are remodeled without compromising myelin sheath integrity has remained puzzling, in particular at advanced age when myelin abnormalities increase. Earlier EM studies had suggested myelin membranes are shed and subsequently phagocytosed by microglia. However, the formation of multilamellar myelinoid bodies (MBs), leaving a well-ordered myelin sheath behind, is difficult to reconcile with simple shedding mechanisms. Here, we show by three-dimensional FIB-SEM reconstructions of optic nerves in mice and by two-photon live-imaging of myelinated cortical slices that MBs are initially connected to their parental sheaths by long tethers, which are stretched by trogocytosing microglia and astrocytes. We observe ruptured tethers attached to both MBs and sheaths, suggesting a novel mechanism of tension-driven tether scission. Importantly, the successive fusion of the corresponding innermost myelin membranes in an extended tether can preserve myelin sheath integrity. Thus, the remodeling by tether-mediated MB extraction emerges as a mechanism of physiological maintenance of myelin sheaths in the CNS.

Mitochondria are diverse and multifaceted intracellular organelles regulating oxidative energy supply, lipid metabolism and calcium (Ca ²⁺ ) signaling. In neurons the spatial sequestration of cytoplasmic Ca ²⁺ by mitochondria plays a critical role in determining activity-dependent spine plasticity, shaping the presynaptic transmitter release characteristics and contributing to sustained action potential firing. Here, we tested the hypothesis that mitochondria at the axon initial segment (AIS) affect the microdomain cytoplasmic Ca ²⁺ transients, thereby regulating Ca ²⁺ -dependent voltage-gated ion channels at the plasma membrane and initiation of action potentials. Using 3D electron microscopy (EM) reconstructions and virally injecting genetically encoded fluorescence indicators we visualized the ultrastructure and distribution of mitochondria selectively in thick-tufted layer 5 pyramidal neurons. We found that most mitochondria were stably clustered to the proximal AIS, while few were observed at distal sites. Simultaneous two-photon imaging of action potential-dependent cytoplasmic and mitochondrial Ca ²⁺ , combined with electrophysiological recordings showed the AIS mitochondria exhibit powerful activity-dependent cytosolic Ca ²⁺ uptake. However, while intracellular application of the mitochondrial Ca ²⁺ uniporter inhibitor Ru360 fully blocked mitochondrial Ca ²⁺ import, it did not affect action potential input-output function, action potential dynamics nor the ability to produce high-frequency burst output. Together, the results indicate that AIS mitochondria are dispensable for temporal and rate encoding, suggesting that mt-Ca ²⁺ buffering at the AIS may be involved in non-electrical roles, including AIS maintenance or axonal transport.