BACKGROUND:Noxious stimuli are conveyed to and integrated in the dorsal horn of the spinal cord before being transmitted to supraspinal centres, where pain perception is generated. Descending pathways from the brainstem dynamically modulate this integration, either facilitating or inhibiting nociceptive information based on physiological, emotional, genetic and environmental factors. Serotonergic neurones in the nucleus raphe magnus (NRM), activating different spinal 5-hydroxytryptamine (5-HT) receptors, exert bidirectional control, both facilitatory and inhibitory, but the underlying mechanisms of this control remain unclear. METHODS:Serotonergic modulation by the NRM of nociception was investigated in adult mice using imaging, behavioural, pharmacological, electrophysiological, chemogenetic and optogenetic approaches. RESULTS:The action of serotonergic neurones in the NRM on spinal nociceptive transmission depends on their activation pattern, which targets different spinal 5-HT receptors likely associated with different spinal microcircuits. Serotonergic neurones of the NRM exert a tonic analgesic effect mediated by 5-HT2c receptor. Low increases in 5-HT activity led to increased analgesia through spinal inhibitory interneurones expressing 5-HT2c and 5-HT2A receptors. Prolonged stimulation of serotonergic neurones led to hyperalgesia mediated by 5-HT3 receptors. Comparison of 5-HT receptors in spinal tissue from mice and humans shows that 5-HT2c receptors have high expression level, comparable between both species. CONCLUSIONS:These results propose a bidirectional model of action by serotonin neurones of nociceptive transmission depending on their level of activity and show that 5-HT2c receptors mediate the serotonin-induced analgesia.

BACKGROUND:Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects hippocampal excitatory synaptic transmission. RESULTS:Here we provide in vitro evidence that LPA elicits intracellular calcium concentration ([Ca2+]i) transients by LPA2 receptor activation in primary cultured hippocampal mouse neurons. Downstream and via Gi-coupling, this led to phospholipase C (PLC) activation, inositol (1,4,5) trisphosphate (IP3)-induced Ca2+ release (IICR) and voltage gated Ca2+ channel activation. In addition, we found that LPA elevated [Ca2+]i, not only in the soma but also in presynaptic terminals. This altered the frequency of spontaneous vesicle release specifically in excitatory synapses. However, against our expectations, LPA reduced the frequency of miniature excitatory postsynaptic currents. This was due to a depletion of releasable vesicles resulting from a slowed recycling. SynaptopHluorin based measurements indicated a transient augmentation of release followed by prolonged persistence of vesicles at the membrane. Concordant to our previous findings on ex vivo brain slices, LPA increased spontaneous glutamatergic vesicle release in Banker style astrocytic co-cultures. Our results indicate that pro-excitatory LPA effects critically depend on stable vesicle pools. CONCLUSIONS:Taken together, our data further support membrane derived phospholipids as active modulators of excitatory synaptic transmission.

Vertebrate axons can be wrapped by myelin produced by oligodendrocytes. This cellular interaction ensures fast and accurate propagation of action potentials, but the physiology of the myelin sheath is almost completely unknown. To investigate the physiology of the myelin sheath, we implemented an imaging strategy that allowed optical measurements of myelin membrane voltage, with the aim to identify physiological changes of the myelin membrane during neuronal firing. We expressed the genetically encoded voltage indicator ASAP3 in mouse oligodendrocytes in vivo and subsequently investigated myelin physiology by optically measuring myelin membrane voltage. We found that myelin depolarizes during neuronal activity, which is blocked by inhibiting neuronal action potentials. Pharmacological and knock-out experiments of Kir4.1 showed that potassium uptake channels mediate action potential induced depolarization. Blocking myelin dependent potassium uptake and direct application of high potassium to identified axons induced axonal initiated and antidromic propagating action potentials. Our study shows that myelin is not an electrically passive insulator, but exhibits ion dynamics and its physiological response is fine tuned to neuronal activity. By facilitating potassium removal during action potentials, myelin supports high precision axonal firing. Genetically encoded sensors are thus a useful tool to study physiological properties of myelin, inaccessible by classical techniques. ### Competing Interest Statement The authors have declared no competing interest.

The dentate gyrus (DG) of the hippocampus is a mosaic of dentate granule neurons (DGNs) accumulated throughout life. While many studies focused on the morpho-functional properties of adult-born DGNs, much less is known about DGNs generated during development, and in particular those born during embryogenesis. One of the main reasons for this gap is the lack of methods available to specifically label and manipulate embryonically-born DGNs. Here, we have assessed the relevance of the PenkCre mouse line as a genetic model to target this embryonically-born population. In young animals, PenkCre expression allows to tag neurons in the DG with positional, morphological and electrophysiological properties characteristic of DGNs born during the embryonic period. In addition, PenkCre+ cells in the DG are distributed in both blades along the entire septo-temporal axis. This model thus offers new possibilities to explore the functions of this underexplored population of embryonically-born DGNs.

Background Oligodendrocyte lineage cells interact with the vasculature in the gray matter. Physical and functional interactions between blood vessels and oligodendrocyte precursor cells play an essential role in both the developing and adult brain. Oligodendrocyte precursor cells have been shown to migrate along the vasculature and subsequently detach from it during their differentiation to oligodendrocytes. However, the association of mature oligodendrocytes with blood vessels has been noted since the discovery of this glial cell type almost a century ago, but this interaction remains poorly explored. Results Here, we systematically investigated the extent of mature oligodendrocyte interaction with the vasculature in mouse brain. We found that ~ 17% of oligodendrocytes were in contact with blood vessels in the neocortex, the hippocampal CA1 region and the cerebellar cortex. Contacts were made mainly with capillaries and sparsely with larger arterioles or venules. By combining light and serial electron microscopy, we demonstrated that oligodendrocytes are in direct contact with the vascular basement membrane, raising the possibility of direct signaling pathways and metabolite exchange with endothelial cells. During experimental remyelination in the adult, oligodendrocytes were regenerated and associated with blood vessels in the same proportion compared to control cortex, suggesting a homeostatic regulation of the vasculature-associated oligodendrocyte population. Conclusions Based on their frequent and close association with blood vessels, we propose that vasculature-associated oligodendrocytes should be considered as an integral part of the brain vasculature microenvironment. This particular location could underlie specific functions of vasculature-associated oligodendrocytes, while contributing to the vulnerability of mature oligodendrocytes in neurological diseases.

Core planar cell polarity (PCP) genes, which are involved in various neurodevelopmental disorders such as neural tube closure, epilepsy, and autism spectrum disorder, have poorly defined molecular signatures in neurons, mostly synapse-centric. Here, we show that the core PCP protein Prickle-like protein 2 (Prickle2) controls neuronal polarity and is a previously unidentified member of the axonal initial segment (AIS) proteome. We found that Prickle2 is present and colocalizes with AnkG480, the AIS master organizer, in the earliest stages of axonal specification and AIS formation. Furthermore, by binding to and regulating AnkG480, Prickle2 modulates its ability to bundle microtubules, a crucial mechanism for establishing neuronal polarity and AIS formation. Prickle2 depletion alters cytoskeleton organization, and Prickle2 levels determine both axon number and AIS maturation. Last, early Prickle2 depletion produces impaired action potential firing.

Oligodendrocyte lineage cells interact with the vasculature in the gray matter. Physical and functional interactions between blood vessels and oligodendrocyte precursor cells play an essential role in both the developing and adult brain. Oligodendrocyte precursor cells have been shown to migrate along the vasculature and subsequently detach from it during their differentiation to oligodendrocytes. However, the association of mature oligodendrocytes with blood vessels has been noted since the discovery of this glial cell type almost a century ago and this interaction remains poorly explored. Here, we systematically investigated the extent of mature oligodendrocyte interaction with the vasculature in mouse. We found that ~17% of oligodendrocytes were in contact with blood vessels in cortex, hippocampus and cerebellum. Contacts were made mainly with capillaries and sparsely with larger arterioles or venules. By combining light and serial electron microscopy, we demonstrated that oligodendrocytes are in direct contact with the vascular basement membrane, raising the possibility of direct signaling pathways and metabolite exchange with endothelial cells. During experimental remyelination in the adult, oligodendrocytes were regenerated and associated with blood vessels in the same proportion compared to control cortex, suggesting a homeostatic regulation of the vasculature-associated oligodendrocyte population. Based on the frequent and close association with blood vessels, we propose that vasculature-associated oligodendrocytes should be considered as an integral part of the vasculature microenvironment. This particular location could underlie specific functions of vasculature-associated oligodendrocytes, while contributing to the vulnerability of mature oligodendrocytes in neurological diseases.

Myelinating oligodendrocytes enable fast propagation of action potentials along the ensheathed axons. In addition, oligodendrocytes play diverse non-canonical roles including axonal metabolic support and activity-dependent myelination. An open question remains whether myelination also contributes to information processing in addition to speeding up conduction velocity. Here, we analyze the role of myelin in auditory information processing using paradigms that are also good predictors of speech understanding in humans. We compare mice with different degrees of dysmyelination using acute multiunit recordings in the auditory cortex, in combination with behavioral readouts. We find complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. We observe partially discriminable but similar deficits in well myelinated mice in which glial cells cannot fully support axons metabolically. We suggest a model in which myelination contributes to sustained stimulus perception in temporally complex paradigms, with a role of metabolically active oligodendrocytes in cortical information processing.

Combining fluorescence and transmitted light sources for microscopy is an invaluable method in cellular neuroscience to probe the molecular and cellular mechanisms of cells. This approach enables the targeted recording from fluorescent reporter protein expressing neurons or glial cells in brain slices and fluorescence-assisted electrophysiological recordings from subcellular structures. However, the existing tools to mix multiple light sources in one-photon microscopy are limited. Here, we present the development of several microcontroller devices that provide temporal and intensity control of light emitting diodes (LEDs) for computer controlled microscopy illumination. We interfaced one microcontroller with μManager for rapid and dynamic overlay of transmitted and fluorescent images. Moreover, on the basis of this illumination system we implemented an electronic circuit to combine two pulsed LED light sources for fast (up to 1 kHz) ratiometric calcium (Ca2+) imaging. This microcontroller enabled the calibration of intracellular Ca2+ concentration and furthermore the combination of Ca2+ imaging with optogenetic activation. The devices are based on affordable components and open-source hardware and software. Integration into existing bright-field microscope systems will take ∼1 day. The microcontroller based LED imaging substantially advances conventional illumination methods by limiting light exposure and adding versatility and speed.

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.

Myelinating oligodendrocytes enable fast impulse propagation along axons as revealed through studies of homogeneously myelinated white matter tracts. However, gray matter myelination patterns are different, with sparsely myelinated sections leaving large portions of the axons naked. The consequences of this patchy myelination for oligodendrocyte function are not understood but suggest other roles in information processing beyond the regulation of axonal conduction velocity. Here, we analyzed the contribution of myelin to auditory information processing using paradigms that are good predictors of speech understanding in humans. We compared mice with different degrees of dysmyelination using acute cortical multiunit recordings in combination with behavioral readouts. We identified complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. Partially discriminable but overall similar deficits were observed in mice with oligodendrocytes that can myelinate but cannot fully support axons metabolically. Thus, myelination contributes to sustained stimulus perception in temporally complex paradigms, revealing a role of oligodendrocytes in the CNS beyond the increase of axonal conduction velocity.

Satellite oligodendrocytes (s-OLs) are closely apposed to the soma of neocortical layer 5 pyramidal neurons but their properties and functional roles remain unresolved. Here we show that s-OLs form compact myelin and action potentials of the host neuron evoke precisely timed Ba(2+)-sensitive K(+) inward rectifying (Kir) currents in the s-OL. Unexpectedly, the glial K(+) inward current does not require oligodendrocytic Kir4.1. Action potential-evoked Kir currents are in part mediated by gap-junction coupling with neighbouring OLs and astrocytes that form a syncytium around the pyramidal cell body. Computational modelling predicts that glial Kir constrains the perisomatic [K(+)]o increase most importantly during high-frequency action potentials. Consistent with these predictions neurons with s-OLs showed a reduced probability for action potential burst firing during [K(+)]o elevations. These data suggest that s-OLs are integrated into a glial syncytium for the millisecond rapid K(+) uptake limiting activity-dependent [K(+)]o increase in the perisomatic neuron domain.

Central nervous system (CNS) inflammation involves the generation of inducible cytokines such as interferons (IFNs) and alterations in brain activity, yet the interplay of both is not well understood. Here, we show that in vivo elevation of IFNs by viral brain infection reduced hyperpolarization-activated currents (Ih) in cortical pyramidal neurons. In rodent brain slices directly exposed to type I IFNs, the hyperpolarization-activated cyclic nucleotide (HCN)-gated channel subunit HCN1 was specifically affected. The effect required an intact type I receptor (IFNAR) signaling cascade. Consistent with Ih inhibition, IFNs hyperpolarized the resting membrane potential, shifted the resonance frequency, and increased the membrane impedance. In vivo application of IFN-β to the rat and to the mouse cerebral cortex reduced the power of higher frequencies in the cortical electroencephalographic activity only in the presence of HCN1. In summary, these findings identify HCN1 channels as a novel neural target for type I IFNs providing the possibility to tune neural responses during the complex event of a CNS inflammation.

Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of Kv7 potassium channels and voltage-gated sodium (Nav) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these Kv7 channels and the functional impact of colocalization with Nav channels remain poorly understood. Here, we quantitatively examined Kv7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. Kv7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ∼12 (proximal) to 150 pS μm−2 (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by Kv7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (∼15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic Kv7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal Kv7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains Kv7.2/7.3 channels were found to increase Nav channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, Kv7 clustering near axonal Nav channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.

Background: Recently, we and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines. However, direct information on PRG3 effects in neuronal development and, in particular, its putative spatio-temporal distribution and conditions of action, is sparse.Results: We demonstrate here that PRG3 induces filopodia formation in HEK293 cells depending on its N-glycosylation status. The PRG3 protein was strongly expressed during mouse brain development in vivo from embryonic day 16 to postnatal day 5 (E16 - P5). From P5 on, expression declined. Furthermore, in early, not yet polarized hippocampal cultured neurons, PRG3 was expressed along the neurite shaft. Knock-down of PRG3 in these neurons led to a decreased number of neurites. This phenotype is rescued by expression of an shRNA-resistant PRG3 construct in PRG3 knock-down neurons. After polarization, endogenous PRG3 expression shifted mainly to axons, specifically to the plasma membrane along the neurite shaft. These PRG3 pattern changes appeared temporally and spatially related to ongoing synaptogenesis. Therefore we tested (i) whether dendritic PRG3 re-enhancement influences synaptic currents and (ii) whether synaptic inputs contribute to the PRG3 shift. Our results rendered both scenarios unlikely: (i) PRG3 over-expression had no influence on miniature excitatory postsynaptic currents (mEPSC) and (ii) blocking of incoming signals did not alter PRG3 distribution dynamics. In addition, PRG3 levels did not interfere with intrinsic neuronal properties.Conclusion: Taken together, our data indicate that endogenous PRG3 promotes neurite shaft protrusion and therefore contributes to regulating filopodia formation in immature neurons. PRG3 expression in more mature neurons, however, is predominantly localized in the axon. Changes in PRG3 levels did not influence intrinsic or synaptic neuronal properties.