Human iPSC-derived neurons with reliable synapses and large presynaptic action potentials.

Understanding the function of the human brain requires determining basic properties of synaptic transmission in human neurons. One of the most fundamental parameters controlling neurotransmitter release is the presynaptic action potential, but its amplitude and duration remain controversial. Presynaptic action potentials have so far been measured with high temporal resolution only in a limited number of vertebrate but not in human neurons. To uncover properties of human presynaptic action potentials, we exploited recently developed tools to generate human glutamatergic neurons by transient expression of Neurogenin 2 (Ngn2) in pluripotent stem cells. During maturation for 3 to 9 weeks of culturing in different established media, the proportion of cells with multiple axon initial segments decreased, while the amount of axonal tau protein and neuronal excitability increased. Super-resolution microscopy revealed the alignment of the pre- and postsynaptic proteins, Bassoon and Homer. Synaptic transmission was surprisingly reliable at frequencies of 20, 50, and 100 Hz. The synchronicity of synaptic transmission during high-frequency transmission increased during 9 weeks of neuronal maturation. To analyze the mechanisms of synchronous high-frequency glutamate release, we developed direct presynaptic patch-clamp recordings from human neurons. The presynaptic action potentials had large overshoots to ∼25 mV and short durations of ∼0.5 ms. Our findings show that Ngn2-induced neurons represent an elegant model system allowing for functional, structural, and molecular analyses of glutamatergic synaptic transmission with high spatio-temporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.Significance statement Presynaptic physiology remains poorly understood despite its relevance to neurological and psychiatric diseases. Studying presynaptic functions in human iPSC-derived neurons offers the important advantage of characterizing molecular mechanisms of neurotransmitter release in neurons derived from diseased patients. As a first step towards this goal, we established direct presynaptic whole-cell patch-clamp recordings from human glutamatergic neurons induced by transient Neurogenin 2 overexpression. We furthermore analyzed the structure of the synapses with super-resolution light microscopy and the synaptic short-term plasticity during high-frequency transmission. Our findings show that synchronous high-frequency transmission is mediated by rapid and large presynaptic action potentials in human neurons, similar to small conventional nerve terminals of rodent neurons.

KATP channel mutation disrupts hippocampal network activity and nocturnal gamma shifts.

ATP-sensitive potassium (KATP) channels couple cell metabolism to cellular electrical activity. Humans affected by severe activating mutations in KATP channels suffer from developmental delay, epilepsy, and neonatal diabetes (DEND syndrome). While the aetiology of diabetes in DEND syndrome is well understood, the pathophysiology of the neurological symptoms remains unclear. We hypothesised that impaired activity of parvalbumin-positive interneurons (PV-INs) may result in seizures and cognitive problems. We found, by performing electrophysiological experiments, that expressing the DEND mutation Kir6.2-V59M selectively in mouse PV-INs reduced intrinsic gamma frequency preference and short-term depression as well as disturbed cognition-associated gamma oscillations and hippocampal sharp waves. Furthermore, the risk of seizures was increased and the day-night shift in gamma activity disrupted. Blocking KATP channels with tolbutamide partially rescued the network oscillations. The non-reversible part may, to some extent, result from observed altered PV-IN dendritic branching and PV-IN arrangement within CA1. In summary, PV-INs play a key role in DEND syndrome, and this provides a framework for establishing treatment options.

Fully-primed slowly-recovering vesicles mediate presynaptic LTP at neocortical neurons.

Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

Cav2.2 Channels Sustain Vesicle Recruitment at a Mature Glutamatergic Synapse.

The composition of voltage-gated Ca2+ channel (Cav) subtypes that gate action potential (AP)-evoked release changes during the development of mammalian CNS synapses. Cav2.2 and Cav2.3 lose their function in gating-evoked release during postnatal synapse maturation. In mature boutons, Cav2.1 currents provide the almost exclusive trigger for evoked release, and Cav2.3 currents are required for the induction of presynaptic long-term potentiation. However, the functional significance of Cav2.2 remained elusive in mature boutons, although they remain present at active zones and continue contributing significantly to presynaptic Ca2+ influx. Here, we addressed the functional significance of Cav2.2 and Cav2.3 at mature parallel-fiber (PF) to Purkinje neuron synapses of mice of either sex. These synapses are known to exhibit the corresponding developmental Cav subtype changes in gating release. We addressed two hypotheses, namely that Cav2.2 and Cav2.3 are involved in triggering spontaneous glutamate release and that they are engaged in vesicle recruitment during repetitive evoked release. We found that spontaneous miniature release is Ca2+ dependent. However, experiments with Cav subtype-specific blockers excluded the spontaneous opening of Cavs as the Ca2+ source for spontaneous glutamate release. Thus, neither Cav2.2 nor Cav2.3 controls spontaneous release from PF boutons. Furthermore, vesicle recruitment during brief bursts of APs was also independent of Ca2+ influx through Cav2.2 and Cav2.3. However, Cav2.2, but not Cav2.3, currents significantly boosted vesicle recruitment during sustained high-frequency synaptic transmission. Thus, in mature PF boutons Cav2.2 channels are specifically required to sustain synaptic transmission during prolonged neuronal activity.SIGNIFICANCE STATEMENT At young CNS synapses, action potential-evoked release is gated via three subtypes of voltage-gated Ca2+ channels: Cav2.1, Cav2.2, and Cav2.3. During postnatal maturation, Cav2.2 and Cav2.3 lose their function in gating evoked release, such that at mature synapses Cav2.1 provides the almost exclusive source for triggering evoked release. Cav2.3 currents are required for the induction of presynaptic long-term potentiation. However, the function of the still abundant Cav2.2 in mature boutons remained largely elusive. Here, we studied mature cerebellar parallel-fiber synapses and found that Cav2.2 does not control spontaneous release. However, Ca2+ influx through Cav2.2 significantly boosted vesicle recruitment during trains of action potentials. Thus, Cav2.2 in mature parallel-fiber boutons participate in sustaining synaptic transmission during prolonged activity.

Direct whole-cell patch-clamp recordings from small boutons in rodent primary neocortical neuron cultures.

Direct electrical recordings from conventional boutons in the mammalian central nervous system have proven challenging due to their small size. Here, we provide a protocol for direct whole-cell patch-clamp recordings from small presynaptic boutons of primary dissociated cultured neurons of the rodent neocortex. We describe steps to prepare primary neocortical cultures and recording pipettes, followed by identifying boutons and establishing a whole-cell bouton recording. We then provide details on precise pipette capacitance compensation required for high-resolution current-clamp recordings from boutons. For further details on the use and execution of this protocol, please refer to Ritzau-Jost et al.1.

Transient astrocytic accumulation of fluorescein during spreading depolarizations.

Spreading depolarizations (SDs) occur frequently in acute cerebral injuries. They are characterized by a breakdown of transmembrane ion gradients resulting in a reduced extracellular sodium ([Na+]o) and increased extracellular potassium concentration ([K+]o). Elevated [K+]o induces astrocytic swelling, another feature of SD; however, the solutes that drive astrocytic swelling remain incompletely understood. We incidentally found astrocytic accumulation of fluorescein (Fluo) - a low molecular weight anionic dye - during SDs induced by elevated [K+]o. Herein, we aimed to explore the properties of astrocytic Fluo accumulation during SDs, electrical stimulation, [K+]o and glutamate elevation and elucidate underlying mechanisms and its relation to swelling. Experiments were performed in acute neocortical slices from adult male C57Bl6 mice and transgenic mice expressing tdTomato in parvalbumin (PV)-positive neurons. We labeled astrocytes with sulforhodamine-101 (SR-101), measured Fluo kinetics using 2-photon laser scanning microscopy and recorded local field potentials (LFP) to detect SDs. Elevations of [K+]o lead to an increase of the astrocytic Fluo intensity in parallel with astrocytic swelling. Pharmacological inhibitors of sodium­potassium ATPase (Na/K-ATPase), secondary-active transporters and channels were used to address the underlying mechanisms. Fluo accumulation as well as swelling were only prevented by inhibition of the sodium­potassium ATPase. Application of glutamate or hypoosmolar solution induced astrocytic swelling independent of Fluo accumulation and glutamate opposed Fluo accumulation when co-administered with high [K+]o. Astrocytes accumulated Fluo and swelled during electrical stimulation and even more during SDs. Taken together, Fluo imaging can be used as a tool to visualize yet unidentified anion fluxes during [K+]o- but not glutamate- or hypoosmolarity induced astrocytic swelling. Fluo imaging may thereby help to elucidate mechanisms of astrocytic swelling and associated fluid movements between brain compartments during physiological and pathological conditions, e.g. SDs.

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity.

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD+ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD+ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K+ , typical correlates of neuronal activity, induced a more reduced NADH/NAD+ redox ratio. While application of glutamate decreased [ATP], K+ as well as the combination of glutamate and K+ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K+ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg2+ caused a shift of the NADH/NAD+ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.

Calcium dependence of neurotransmitter release at a high fidelity synapse.

The Ca2+-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 µM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.

Active zone compaction correlates with presynaptic homeostatic potentiation.

Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.

Adipocyte death triggers a pro-inflammatory response and induces metabolic activation of resident macrophages.

A chronic low-grade inflammation within adipose tissue (AT) seems to be the link between obesity and some of its associated diseases. One hallmark of this AT inflammation is the accumulation of AT macrophages (ATMs) around dead or dying adipocytes, forming so-called crown-like structures (CLS). To investigate the dynamics of CLS and their direct impact on the activation state of ATMs, we established a laser injury model to deplete individual adipocytes in living AT from double reporter mice (GFP-labeled ATMs and tdTomato-labeled adipocytes). Hence, we were able to detect early ATM-adipocyte interactions by live imaging and to determine a precise timeline for CLS formation after adipocyte death. Further, our data indicate metabolic activation and increased lipid metabolism in ATMs upon forming CLS. Most importantly, adipocyte death, even in lean animals under homeostatic conditions, leads to a locally confined inflammation, which is in sharp contrast to other tissues. We identified cell size as cause for the described pro-inflammatory response, as the size of adipocytes is above a critical threshold size for efferocytosis, a process for anti-inflammatory removal of dead cells during tissue homeostasis. Finally, experiments on parabiotic mice verified that adipocyte death leads to a pro-inflammatory response of resident ATMs in vivo, without significant recruitment of blood monocytes. Our data indicate that adipocyte death triggers a unique degradation process and locally induces a metabolically activated ATM phenotype that is globally observed with obesity.

Large, Stable Spikes Exhibit Differential Broadening in Excitatory and Inhibitory Neocortical Boutons.

Presynaptic action potential spikes control neurotransmitter release and thus interneuronal communication. However, the properties and the dynamics of presynaptic spikes in the neocortex remain enigmatic because boutons in the neocortex are small and direct patch-clamp recordings have not been performed. Here, we report direct recordings from boutons of neocortical pyramidal neurons and interneurons. Our data reveal rapid and large presynaptic action potentials in layer 5 neurons and fast-spiking interneurons reliably propagating into axon collaterals. For in-depth analyses, we establish boutons of mature cultured neurons as models for excitatory neocortical boutons, demonstrating that the presynaptic spike amplitude is unaffected by potassium channels, homeostatic long-term plasticity, and high-frequency firing. In contrast to the stable amplitude, presynaptic spikes profoundly broaden during high-frequency firing in layer 5 pyramidal neurons, but not in fast-spiking interneurons. Thus, our data demonstrate large presynaptic spikes and fundamental differences between excitatory and inhibitory boutons in the neocortex.

Multinucleated Giant Cells in Adipose Tissue Are Specialized in Adipocyte Degradation.

Obesity is associated with chronic low-grade inflammation of visceral adipose tissue (AT) characterized by an increasing number of AT macrophages (ATMs) and linked to type 2 diabetes. AT inflammation is histologically indicated by the formation of so-called crown-like structures, as ATMs accumulate around dying adipocytes, and the occurrence of multinucleated giant cells (MGCs). However, to date, the function of MGCs in obesity is unknown. Therefore, the aim of this study was to characterize MGCs in AT and unravel the function of these cells. We demonstrated that MGCs occurred in obese patients and after 24 weeks of a high-fat diet in mice, accompanying signs of AT inflammation and then representing ∼3% of ATMs in mice. Mechanistically, we found evidence that adipocyte death triggered MGC formation. Most importantly, MGCs in obese AT had a higher capacity to phagocytize oversized particles, such as adipocytes, as shown by live imaging of AT, 45-µm bead uptake ex vivo, and higher lipid content in vivo. Finally, we showed that interleukin-4 treatment was sufficient to increase the number of MGCs in AT, whereas other factors may be more important for endogenous MGC formation in vivo. Most importantly, our data suggest that MGCs are specialized for clearance of dead adipocytes in obesity.

Undisturbed climbing fiber pruning in the cerebellar cortex of CX3 CR1-deficient mice.

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the PC layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3 CR1) signaling during postnatal development using genetic ablation of the CX3 CR1 receptor and an in-depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX3 CR1 signaling is not mandatory for correct cerebellar circuit formation. MAIN POINTS: Ablation of CX3 CR1 results in a transient migration defect in cerebellar microglia. CX3 CR1 is not required for functional pruning of cerebellar climbing fibers. Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX3 CR1-deficient mice.

Neocortical High Probability Release Sites Are Formed by Distinct Ca2+ Channel-to-Release Sensor Topographies during Development.

Coupling distances between Ca2+ channels and release sensors regulate vesicular release probability (pv). Tight coupling is thought to provide a framework for high pv and loose coupling for high plasticity at low pv. At synapses investigated during development, coupling distances decrease, thereby increasing pv and transmission fidelity. We find that neocortical high-fidelity synapses deviate from these rules. Paired recordings from pyramidal neurons with "slow" and "fast" Ca2+ chelators combined with experimentally constrained simulations suggest that coupling tightens significantly during development. However, fluctuation analysis revealed that neither pv (∼0.63) nor the number of release sites (∼8) changes concomitantly. Moreover, the amplitude and time course of presynaptic Ca2+ transients are not different between age groups. These results are explained by high-pv release sites with Ca2+ microdomains in young synapses and nanodomains in mature synapses. Thus, at neocortical synapses, a developmental reorganization of the active zone leaves pv unaffected, emphasizing developmental and functional synaptic diversity.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Developmental Easing of Short-Term Depression in "Winner" Climbing Fibers.

The postnatal development of cerebellar climbing fiber (CF) to Purkinje neuron (PN) synapses is characterized by a substantial pruning during the first 3 weeks after birth, switching from multiple- to single-CF innervation. Previous studies suggested that CF maturation is governed by bidirectional changes of synaptic plasticity. The strengthening of surviving "winner" CFs, which translocate from the PN soma to the dendrite, is thought to be guided by long-term potentiation (LTP), while weakening of to-be-eliminated "loser" CFs, which remain on the soma, was proposed to be due to long-term depression (LTD). However, there are conflicting results from previous studies, whether or not strengthening of winner and weakening of loser CFs during postnatal development is accompanied by changes in short-term plasticity and, thus, whether pre- or postsynaptic forms of LTD and LTP are operational. We, therefore, analyzed the developmental profile of paired-pulse depression (PPD) in "weak" and "strong" CFs in 3-21-day old Igsf9-eGFP mice, which allow visual identification of GFP-labeled CFs. We found that in 3-8-day old mice strong CFs are marked by a stronger PPD compared to weak CFs. Surprisingly, PPD of strong CFs eases during maturation, while PPD in weak CFs remains unchanged. This easing of PPD is neither due to changes in presynaptic influx-release coupling nor to an increased saturation of postsynaptic receptors. Thus, our results imply that synaptic contacts of CFs show distinct features of PPD depending on their affiliation to winner or loser CFs and depending on their somatic or dendritic location.

Apparent calcium dependence of vesicle recruitment.

KEY POINTS: Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium-dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium-dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium-independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast-recruited vesicles but does not change the vesicle recruitment rates themselves. ABSTRACT: During sustained synaptic transmission, recruitment of new transmitter-filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca2+ concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca2+ buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity-dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca2+ buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin-Munc13 pathway, which has been proposed to mediate Ca2+ -dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca2+ -independent recruitment rates can explain the slowed recovery from depression upon increased Ca2+ buffering. Our data do not rule out the involvement of the calmodulin-Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca2+ -dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well-established two-pool models predict an apparent Ca2+ -dependence of vesicle recruitment. Thus, previous conclusions of Ca2+ -dependent vesicle recruitment based solely on increased intracellular Ca2+ buffering should be considered with caution.

Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors.

Spatial relationships between Cav channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Cav2.1 and Cav2.2 channels, alters the localization of Cav2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca2+-channel pharmacology in electrophysiological and two-photon Ca2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca2+ influx through Cav2.1 and Cav2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Cav2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Cav2.1 and Cav2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains.

The transgenic mouse line Igsf9-eGFP allows targeted stimulation of inferior olive efferents.

BACKGROUND: The inferior olive (IO) innervates the cerebellum forming synapses in the deep cerebellar nuclei (DCN) and the cerebellar cortex. Beside the well-known exception of synapses on Purkinje neurons, synapses between IO efferents and other neuronal targets have not been studied intensively, mostly due to the technical challenge of unequivocally identifying IO efferents in electrophysiological experiments. NEW METHOD: We describe the transgenic mouse line Igsf9-eGFP, which expresses GFP in IO neurons, as a suitable tool for studying IO efferents using live imaging, immunohistochemistry and electrophysiology. RESULTS: In the Igsf9-eGFP line, GFP-positive neurons are found in all IO subnuclei. Their efferents show the expected trajectories innervating the DCN and, as climbing fibers (CFs), the cerebellar cortex. In the DCN the dentate nucleus shows the strongest innervation, and, within the cerebellar cortex, CFs show a rostral-to-caudal gradient. GFP-positive CFs undergo a normal postnatal maturation, and evoke normal synaptic responses in Purkinje neurons and DCN neurons. COMPARISON WITH EXISTING METHODS: IO efferents have been labelled via anterograde labelling, viral transfection and in transgenic mouse lines. The latter approach does not require stereotactic injections. However, available mouse lines show only a sparse GFP expression, harbor GFP-positive axons of other cerebellar fibers, or have not been characterized in detail. CONCLUSIONS: The Igsf9-eGFP line is characterized by a moderate density of GFP-positive IO efferents, which can be visually targeted for extracellular stimulation with micrometer precision. The mouse line will allow studying fiber-specific responses in all neurons targeted by the IO.

Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research.

To characterize LysM-Cre mediated gene targeting in mice, we crossed LysM-Cre mice to two independent reporter-mouse lines (tdTomato or YFP). Surprisingly, we found that more than 90% of cells with LysM-Cre mediated recombination in the brain were neurons, rather than myeloid cells, such as microglia. Hence, by using the LysM-Cre mouse line for conditional knockout approaches, a significant neuronal recombination needs to be considered.