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.

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 spatiotemporal resolution in human neurons. Furthermore, our data predict that glutamatergic transmission is mediated by large and rapid presynaptic action potentials in the human brain.

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.

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.

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.

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.

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.

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.

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.

Developmental tightening of cerebellar cortical synaptic influx-release coupling.

Tight coupling between Ca(2+) channels and the sensor for vesicular transmitter release at the presynaptic active zone (AZ) is crucial for high-fidelity synaptic transmission. It has been hypothesized that a switch from a loosely coupled to a tightly coupled transmission mode is a common step in the maturation of CNS synapses. However, this hypothesis has never been tested at cortical synapses. We addressed this hypothesis at a representative small cortical synapse: the synapse connecting mouse cerebellar cortical parallel fibers to Purkinje neurons. We found that the slow Ca(2+) chelator EGTA affected release significantly stronger at immature than at mature synapses, while the fast chelator BAPTA was similarly effective in both groups. Analysis of paired-pulse ratios and quantification of release probability (pr) with multiple-probability fluctuation analysis revealed increased facilitation at immature synapses accompanied by reduced pr. Cav2.1 Ca(2+) channel immunoreactivity, assessed by quantitative high-resolution immuno-electron microscopy, was scattered over immature boutons but confined to putative AZs at mature boutons. Presynaptic Ca(2+) signals were quantified with two-photon microscopy and found to be similar between maturation stages. Models adjusted to fit EGTA dose-response curves as well as differential effects of the Ca(2+) channel blocker Cd(2+) indicate looser and less homogenous coupling at immature terminals compared with mature ones. These results demonstrate functionally relevant developmental tightening of influx-release coupling at a single AZ cortical synapse and corroborate developmental tightening of coupling as a prevalent phenomenon in the mammalian brain.

Munc13-3 superprimes synaptic vesicles at granule cell-to-basket cell synapses in the mouse cerebellum.

Munc13-3 is a presynaptic protein implicated in vesicle priming that is strongly expressed in cerebellar granule cells (GCs). Mice deficient of Munc13-3 (Munc13-3(-/-)) show an increased paired-pulse ratio (PPR), which led to the hypothesis that Munc13-3 increases the release probability (pr) of vesicles. In the present study, we analyzed unitary synaptic connections between GCs and basket cells in acute cerebellar slices from wild-type and Munc13-3(-/-) mice. Unitary EPSCs recorded from Munc13-3(-/-) GCs showed normal kinetics and synaptic latency but a significantly increased PPR and fraction of synaptic failures. A quantal analysis revealed that neither the charge of single quanta nor the binominal parameter N were affected by loss of Munc13-3 but that pr was almost halved in Munc13-3(-/-). Neither presynaptic Ca(2+) influx was affected by deletion of Munc13-3 nor replenishment of the readily releasable vesicle pool. However, a high concentration of EGTA led to a reduction in EPSCs that was significantly stronger in Munc13-3(-/-). We conclude that Munc13-3 is responsible for an additional step of molecular and/or positional "superpriming" that substantially increases the efficacy of Ca(2+)-triggered release.

KV 10.1 opposes activity-dependent increase in Ca²âº influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse.

KEY POINTS: Voltage-gated KV 10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV 10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV 10.1 regulates Ca(2+) influx and neurotransmitter release during repetitive high-frequency activity. Our results describe the physiological role of mammalian KV 10.1 for the first time and help understand the fine-tuning of synaptic transmission. The voltage-gated potassium channel KV 10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV 10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV 10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca(2+) imaging in KV 10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca(2+) influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre-Purkinje cell synapse at high firing rates. We propose that KV 10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation.

STIM1, STIM2, and Orai1 regulate store-operated calcium entry and purinergic activation of microglia.

Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store-operated Ca(2+) entry (SOCE), the prevalent Ca(2+) influx mechanism in non-excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca(2+) sensors and Orai Ca(2+) channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down-regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca(2+) entry induced by cyclopiazonic acid, ATP, the P2Y6 receptor agonist UDP, or the P2Y12 receptor agonist 2-methylthio-ADP (2-MeSADP) was clearly affected in microglia from Stim1(-/-) , Stim2(-/-) , and Orai1(-/-) mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide-induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia.

A method for long-term live imaging of tissue macrophages in adipose tissue explants.

Obesity is frequently associated with a chronic low-grade inflammation within adipose tissue (AT). Although classical signs of inflammation are missing in AT inflammation, there is a significant increase in macrophages and, to a lesser extent, other immune cells, such as T cells, B cells, mast cells, and neutrophils. The spatial and temporal activation of these cells as well as their accumulation in the AT seem to be tightly linked to so-called crown-like structures (CLS). CLS are accumulations of adipose tissue macrophages (ATMs) around dead adipocytes and are thought to reflect a scavenger response. At present, data on the life cycle of CLS are missing. To better understand the cellular events underlying AT inflammation, we developed an approach that allows long-term imaging of ATMs, adipocytes, and CLS within live AT explants. We tested three putative reporter mouse lines for myeloid cells in regard to their suitability for live imaging. Thereby, we identified ATMs from CSF1R-eGFP mice to exhibit the most robust expression of eGFP. AT explants from these mice allowed stable live imaging for more than 7 days without significant phototoxicity. Long-term imaging thus revealed the accumulation of ATMs around dying adipocytes, migration of ATMs within AT, and also the degradation of the lipid remnants of perishing adipocytes. The observed behavior of ATMs in the context of AT inflammation is in line with previous studies but for the first time provides data on the specific behavior of individual ATMs and on the life cycle of CLS with unprecedented spatiotemporal resolution.

Local proliferation of macrophages in adipose tissue during obesity-induced inflammation.

AIMS/HYPOTHESIS: Obesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans. METHODS: We studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n = 239). The cell fate of dividing macrophages was assessed by live imaging of AT explants. RESULTS: We show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68(+) macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients. CONCLUSIONS/INTERPRETATION: Local proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.

Paired-pulse facilitation at recurrent Purkinje neuron synapses is independent of calbindin and parvalbumin during high-frequency activation.

Paired-pulse facilitation (PPF) is a dynamic enhancement of transmitter release considered crucial in CNS information processing. The mechanisms of PPF remain controversial and may differ between synapses. Endogenous Ca(2+) buffers such as parvalbumin (PV) and calbindin-D28k (CB) are regarded as important modulators of PPF, with PV acting as an anti-facilitating buffer while saturation of CB can promote PPF. We analysed transmitter release and PPF at intracortical, recurrent Purkinje neuron (PN) to PN synapses, which show PPF during high-frequency activation (200 Hz) and strongly express both PV and CB. We quantified presynaptic Ca(2+) dynamics and quantal release parameters in wild-type (WT), and CB and PV deficient mice. Lack of CB resulted in increased volume averaged presynaptic Ca(2+) amplitudes and in increased release probability, while loss of PV had no significant effect on these parameters. Unexpectedly, none of the buffers significantly influenced PPF, indicating that neither CB saturation nor residual free Ca(2+) ([Ca(2+)]res) was the main determinant of PPF. Experimentally constrained, numerical simulations of Ca(2+)-dependent release were used to estimate the contributions of [Ca(2+)]res, CB, PV, calmodulin (CaM), immobile buffer fractions and Ca(2+) remaining bound to the release sensor after the first of two action potentials ('active Ca(2+)') to PPF. This analysis indicates that PPF at PN-PN synapses does not result from either buffer saturation or [Ca(2+)]res but rather from slow Ca(2+) unbinding from the release sensor.

Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca²âº-dependent interactions via its EF-hand 5 domain.

Ca²âº-binding proteins (CaBPs) are important regulators of neuronal Ca²âº signalling, acting either as buffers that shape Ca²âº transients and Ca²âº diffusion and/or as Ca²âº sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca²âº transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca²âº-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca²âº buffer and point towards a Ca²âº sensor function of CR.

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.

Rapid active zone remodeling during synaptic plasticity.

How can synapses change the amount of neurotransmitter released during synaptic plasticity? Although release in general is intensely investigated, its determinants during plasticity are still poorly understood. As a model for plastic strengthening of synaptic release, we here use the well-established presynaptic homeostatic compensation during interference with postsynaptic glutamate receptors at the Drosophila neuromuscular junction. Combining short-term plasticity analysis, cumulative EPSC analysis, fluctuation analysis, and quantal short-term plasticity modeling, we found an increase in the number of release-ready vesicles during presynaptic strengthening. High-resolution light microscopy revealed an increase in the amount of the active zone protein Bruchpilot and an enlargement of the presynaptic cytomatrix structure. Furthermore, these functional and structural alterations of the active zone were not only observed after lifelong but already after minutes of presynaptic strengthening. Our results demonstrate that presynaptic plasticity can induce active zone remodeling, which regulates the number of release-ready vesicles within minutes.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.