GluN2A- and GluN2B-containing pre-synaptic N-methyl-d-aspartate receptors differentially regulate action potential-evoked Ca2+ influx via modulation of SK channels.

N-methyl-d-aspartate receptors (NMDARs) play a pivotal role in synaptic plasticity. While the functional role of post-synaptic NMDARs is well established, pre-synaptic NMDAR (pre-NMDAR) function is largely unexplored. Different pre-NMDAR subunit populations are documented at synapses, suggesting that subunit composition influences neuronal transmission. Here, we used electrophysiological recordings at Schaffer collateral-CA1 synapses partnered with Ca2+ imaging and glutamate uncaging at boutons of CA3 pyramidal neurones to reveal two populations of pre-NMDARs that contain either the GluN2A or GluN2B subunit. Activation of the GluN2B population decreases action potential-evoked Ca2+ influx via modulation of small-conductance Ca2+-activated K+ channels, while activation of the GluN2A population does the opposite. Critically, the level of functional expression of the subunits is subject to homeostatic regulation, bidirectionally affecting short-term facilitation, thus providing a capacity for a fine adjustment of information transfer. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Repeated imaging through a multimode optical fiber using adaptive optics.

Multimode optical fibers (MMF) have shown considerable potential for minimally invasive diffraction-limited fluorescence imaging of deep brain regions owing to their small size. They also look to be suitable for imaging across long time periods, with repeated measurements performed within the same brain region, which is useful to assess the role of synapses in normal brain function and neurological disease. However, the approach is not without challenge. Prior to imaging, light propagation through a MMF must be characterized in a calibration procedure. Manual repositioning, as required for repeated imaging, renders this calibration invalid. In this study, we provide a two-step solution to the problem consisting of (1) a custom headplate enabling precise reinsertion of the MMF implant achieving low-quality focusing and (2) sensorless adaptive optics to correct translational shifts in the MMF position enabling generation of high-quality imaging foci. We show that this approach achieves fluorescence imaging after repeated removal and reinsertion of a MMF.

Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning.

Rapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications.

Long-term depression links amyloid-ß to the pathological hyperphosphorylation of tau.

In Alzheimer's disease, soluble oligomers of the amyloid-ß peptide (Aßo) trigger a cascade of events that includes abnormal hyperphosphorylation of the protein tau, which is essential for pathogenesis. However, the mechanistic link between these two key pathological proteins remains unclear. Using hippocampal slices, we show here that an Aßo-mediated increase in glutamate release probability causes enhancement of synaptically evoked N-methyl-d-aspartate subtype glutamate receptor (NMDAR)-dependent long-term depression (LTD). We also find that elevated glutamate release probability is required for Aßo-induced pathological hyperphosphorylation of tau, which is likewise NMDAR dependent. Finally, we show that chronic, repeated chemical or optogenetic induction of NMDAR-dependent LTD alone is sufficient to cause tau hyperphosphorylation without Aßo. Together, these results support a possible causal chain in which Aßo increases glutamate release probability, thus leading to enhanced LTD induction, which in turn drives hyperphosphorylation of tau. Our data identify a mechanistic pathway linking the two critical pathogenic proteins of AD.

Volumetric two-photon fluorescence imaging of live neurons using a multimode optical fiber.

Multimode optical fibers (MMFs), combined with wavefront control methods, have achieved minimally invasive in vivo imaging of neurons in deep-brain regions with diffraction-limited spatial resolution. Here, we report a method for volumetric two-photon fluorescence imaging with a MMF-based system requiring a single transmission matrix measurement. Central to this method is the use of a laser source able to generate both continuous wave light and femtosecond pulses. The chromatic dispersion of pulses generated an axially elongated excitation focus, which we used to demonstrate volumetric imaging of neurons and their dendrites in live rat brain slices through a 60 µm core MMF.

A Novel Optical Quantal Analysis of Miniature Events Reveals Enhanced Frequency Following Amyloid ß Exposure.

Non-evoked miniature release of neurotransmitters is increasingly recognized as playing an important role in neural function and is implicated in synaptic plasticity, metaplasticity, and homeostasis. Spontaneous miniature release events (minis) are usually measured electrophysiologically by recording the miniature postsynaptic currents (mEPSCs) that they evoke. However, this indirect technique can be confounded by changes within the postsynaptic neuron. Here, using the fluorescent probe SynaptopHluorin 2×, we have developed an optical method for the measurement of minis that enables direct assessment of release events. We use the technique to reveal that the frequency of minis following incubation of hippocampal neurons with Amyloid ß oligomers (Aßo) is increased. Electrophysiological mEPSC recordings obtained under the same conditions report a decrease in frequency, with the discrepancy likely due to Aßo-induced changes in quantal size. Optical quantal analysis of minis may therefore have a role in the study of minis in both normal physiology and disease, as it can circumvent potential confounds caused by postsynaptic changes.

Deconvolution for multimode fiber imaging: modeling of spatially variant PSF.

Focusing light through a step-index multimode optical fiber (MMF) using wavefront control enables minimally-invasive endoscopy of biological tissue. The point spread function (PSF) of such an imaging system is spatially variant, and this variation limits compensation for blurring using most deconvolution algorithms as they require a uniform PSF. However, modeling the spatially variant PSF into a series of spatially invariant PSFs re-opens the possibility of deconvolution. To achieve this we developed svmPSF: an open-source Java-based framework compatible with ImageJ. The approach takes a series of point response measurements across the field-of-view (FOV) and applies principal component analysis to the measurements' co-variance matrix to generate a PSF model. By combining the svmPSF output with a modified Richardson-Lucy deconvolution algorithm, we were able to deblur and regularize fluorescence images of beads and live neurons acquired with a MMF, and thus effectively increasing the FOV.

Compact and contactless reflectance confocal microscope for neurosurgery.

Visual guidance at the cellular level during neurosurgical procedures is essential for complete tumour resection. We present a compact reflectance confocal microscope with a 20 mm working distance that provided <1.2 µm spatial resolution over a 600 µm × 600 µm field of view in the near-infrared region. A physical footprint of 200 mm × 550 mm was achieved using only standard off-the-shelf components. Theoretical performance of the optical design was first evaluated via commercial Zemax software. Then three specimens from rodents: fixed brain, frozen calvaria and live hippocampal slices, were used to experimentally assess system capability and robustness. Results show great potential for the proposed system to be translated into use as a next generation label-free and contactless neurosurgical microscope.

A two-compartment model of synaptic computation and plasticity.

The synapse is typically viewed as a single compartment, which acts as a linear gain controller on incoming input. Traditional plasticity rules enable this gain control to be dynamically optimized by Hebbian activity. Whilst this view nicely captures postsynaptic function, it neglects the non-linear dynamics of presynaptic function. Here we present a two-compartment model of the synapse in which the presynaptic terminal first acts to filter presynaptic input before the postsynaptic terminal, acting as a gain controller, amplifies or depresses transmission. We argue that both compartments are equipped with distinct plasticity rules to enable them to optimally adapt synaptic transmission to the statistics of pre- and postsynaptic activity. Specifically, we focus on how presynaptic plasticity enables presynaptic filtering to be optimally tuned to only transmit information relevant for postsynaptic firing. We end by discussing the advantages of having a presynaptic filter and propose future work to explore presynaptic function and plasticity in vivo.

Focusing light in biological tissue through a multimode optical fiber: refractive index matching.

Controlling light propagation through a step-index multimode optical fiber (MMF) has several important applications, including biological imaging. However, little consideration has been given to the coupling of fiber and tissue optics. In this Letter, we characterized the effects of tissue-induced light distortions, in particular those arising from a mismatch in the refractive index of the pre-imaging calibration and biological media. By performing the calibration in a medium matching the refractive index of the brain, optimal focusing ability was achieved, as well as a gain in focus uniformity within the field-of-view. These changes in illumination resulted in a 30% improvement in spatial resolution and intensity in fluorescence images of beads and live brain tissue. Beyond refractive index matching, our results demonstrate that sample-induced aberrations can severely deteriorate images from MMF-based systems.

Intracellular Ca2+ Release and Synaptic Plasticity: A Tale of Many Stores.

Ca2+ is an essential trigger for most forms of synaptic plasticity. Ca2+ signaling occurs not only by Ca2+ entry via plasma membrane channels but also via Ca2+ signals generated by intracellular organelles. These organelles, by dynamically regulating the spatial and temporal extent of Ca2+ elevations within neurons, play a pivotal role in determining the downstream consequences of neural signaling on synaptic function. Here, we review the role of three major intracellular stores: the endoplasmic reticulum, mitochondria, and acidic Ca2+ stores, such as lysosomes, in neuronal Ca2+ signaling and plasticity. We provide a comprehensive account of how Ca2+ release from these stores regulates short- and long-term plasticity at the pre- and postsynaptic terminals of central synapses.

Subcellular spatial resolution achieved for deep-brain imaging in vivo using a minimally invasive multimode fiber.

Achieving intravital optical imaging with diffraction-limited spatial resolution of deep-brain structures represents an important step toward the goal of understanding the mammalian central nervous system1-4. Advances in wavefront-shaping methods and computational power have recently allowed for a novel approach to high-resolution imaging, utilizing deterministic light propagation through optically complex media and, of particular importance for this work, multimode optical fibers (MMFs)5-7. We report a compact and highly optimized approach for minimally invasive in vivo brain imaging applications. The volume of tissue lesion was reduced by more than 100-fold, while preserving diffraction-limited imaging performance utilizing wavefront control of light propagation through a single 50-µm-core MMF. Here, we demonstrated high-resolution fluorescence imaging of subcellular neuronal structures, dendrites and synaptic specializations, in deep-brain regions of living mice, as well as monitored stimulus-driven functional Ca2+ responses. These results represent a major breakthrough in the compromise between high-resolution imaging and tissue damage, heralding new possibilities for deep-brain imaging in vivo.

Neuronal Receptors Display Cytoskeleton-Independent Directed Motion on the Plasma Membrane.

Directed transport of transmembrane proteins is generally believed to occur via intracellular transport vesicles. However, using single-particle tracking in rat hippocampal neurons with a pH-sensitive quantum dot probe that specifically reports surface movement of receptors, we have identified a subpopulation of neuronal EphB2 receptors that exhibit directed motion between synapses within the plasma membrane itself. This receptor movement occurs independently of the cytoskeleton but is dependent on cholesterol and is regulated by neuronal activity.

Hippocampal mGluR1-dependent long-term potentiation requires NAADP-mediated acidic store Ca2+ signaling.

Acidic organelles, such as endosomes and lysosomes, store Ca2+ that is released in response to intracellular increases in the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). In neurons, NAADP and Ca2+ signaling contribute to synaptic plasticity, a process of activity-dependent long-term potentiation (LTP) [or, alternatively, long-term depression (LTD)] of synaptic strength and neuronal transmission that is critical for neuronal function and memory formation. We explored the function of and mechanisms regulating acidic Ca2+ store signaling in murine hippocampal neurons. We found that metabotropic glutamate receptor 1 (mGluR1) was coupled to NAADP signaling that elicited Ca2+ release from acidic stores. In turn, this released Ca2+-mediated mGluR1-dependent LTP by transiently inhibiting SK-type K+ channels, possibly through the activation of protein phosphatase 2A. Genetically removing two-pore channels (TPCs), which are endolysosomal-specific ion channels, switched the polarity of plasticity from LTP to LTD, indicating the importance of specific receptor store coupling and providing mechanistic insight into how mGluR1 can produce both synaptic potentiation and synaptic depression.

Inhibition of lysosomal Ca2+ signalling disrupts dendritic spine structure and impairs wound healing in neurons.

A growing body of evidence suggests that lysosomes, which have traditionally been regarded as degradative organelles, can function as Ca2+ stores, regulated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). We previously demonstrated that in hippocampal pyramidal neurons, activity-dependent Ca2+ release from these stores triggers fusion of the lysosome with the plasma membrane. We found that the physiological role of this Ca2+-dependent fusion was to maintain the long-term structural enlargement of dendritic spines induced by synaptic activity. Here, we examined the pathophysiological consequences of lysosomal dysfunction in hippocampal pyramidal neurons by chronically inhibiting lysosomal Ca2+ signalling using the NAADP antagonist, NED-19. We found that within just 20 hours, inhibition of lysosomal function led to a profound intracellular accumulation of lysosomal membrane. This was accompanied by a significant change in dendritic spine structure, which included a lengthening of dendritic spines, an increase in the number of filipodia, and an overall decrease in spine number. Inhibition of lysosomal function also inhibited wound healing in neurons by preventing lysosomal fusion with the plasma membrane. Neurons were therefore more susceptible to injury. Our findings suggest that dysfunction in lysosomal Ca2+ signalling and lysosomal fusion with the plasma membrane may contribute to the loss of dendritic spines and neurons seen in neurological disorders, such as Niemann-Pick disease type C1, in which lysosomal function is impaired.

Homeostatic Presynaptic Plasticity Is Specifically Regulated by P/Q-type Ca2+ Channels at Mammalian Hippocampal Synapses.

Voltage-dependent Ca2+ channels (VGCC) represent the principal source of Ca2+ ions driving evoked neurotransmitter release at presynaptic boutons. In mammals, presynaptic Ca2+ influx is mediated mainly via P/Q-type and N-type VGCC, which differ in their properties. Changes in their relative contributions tune neurotransmission both during development and in Hebbian plasticity. However, whether this represents a functional motif also present in other forms of activity-dependent regulation is unknown. Here, we study the role of VGCC in homeostatic plasticity (HSP) in mammalian hippocampal neurons using optical techniques. We find that changes in evoked Ca2+ currents specifically through P/Q-type, but not N-type, VGCC mediate bidirectional homeostatic regulation of both neurotransmitter release efficacy and the size of the major synaptic vesicle pools. Selective dependence of HSP on P/Q-type VGCC in mammalian terminals has important implications for phenotypes associated with P/Q-type channelopathies, including migraine and epilepsy.

Synaptic Transmission Optimization Predicts Expression Loci of Long-Term Plasticity.

Long-term modifications of neuronal connections are critical for reliable memory storage in the brain. However, their locus of expression-pre- or postsynaptic-is highly variable. Here we introduce a theoretical framework in which long-term plasticity performs an optimization of the postsynaptic response statistics toward a given mean with minimal variance. Consequently, the state of the synapse at the time of plasticity induction determines the ratio of pre- and postsynaptic modifications. Our theory explains the experimentally observed expression loci of the hippocampal and neocortical synaptic potentiation studies we examined. Moreover, the theory predicts presynaptic expression of long-term depression, consistent with experimental observations. At inhibitory synapses, the theory suggests a statistically efficient excitatory-inhibitory balance in which changes in inhibitory postsynaptic response statistics specifically target the mean excitation. Our results provide a unifying theory for understanding the expression mechanisms and functions of long-term synaptic transmission plasticity.

Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines.

Lysosomes have traditionally been viewed as degradative organelles, although a growing body of evidence suggests that they can function as Ca2+ stores. Here we examined the function of these stores in hippocampal pyramidal neurons. We found that back-propagating action potentials (bpAPs) could elicit Ca2+ release from lysosomes in the dendrites. This Ca2+ release triggered the fusion of lysosomes with the plasma membrane, resulting in the release of Cathepsin B. Cathepsin B increased the activity of matrix metalloproteinase 9 (MMP-9), an enzyme involved in extracellular matrix (ECM) remodelling and synaptic plasticity. Inhibition of either lysosomal Ca2+ signaling or Cathepsin B release prevented the maintenance of dendritic spine growth induced by Hebbian activity. This impairment could be rescued by exogenous application of active MMP-9. Our findings suggest that activity-dependent exocytosis of Cathepsin B from lysosomes regulates the long-term structural plasticity of dendritic spines by triggering MMP-9 activation and ECM remodelling.

Rapid regulation of endoplasmic reticulum dynamics in dendritic spines by NMDA receptor activation.

Endoplasmic reticulum (ER) is motile within dendritic spines, but the mechanisms underlying its regulation are poorly understood. To address this issue, we have simultaneously imaged morphology and ER content of dendritic spines in cultured dissociated mouse hippocampal neurons. Over a 10 min period, spines were highly dynamic, with spines both increasing and decreasing in volume. ER was present in approximately 50% of spines and was also highly dynamic, with a net increase over this period of time. Inhibition of the endogenous activation of NMDA receptors resulted in a reduction in ER growth. Conversely, augmentation of the synaptic activation of NMDA receptors, by elimination of striatal-enriched protein tyrosine phosphatase (STEP), resulted in enhanced ER growth. Therefore, NMDA receptors rapidly regulate spine ER dynamics.

Wavelength-orthogonal photolysis of neurotransmitters in vitro.

Irradiation of a mixture of 4-methoxyphenacyl-caged (S)-glutamate and 4,5-dimethoxy-2-nitrobenzyl-caged γ-amino butyric acid (GABA) on neurons, at ~260 nm, evokes selective photorelease of (S)-glutamate (Glu) whereas photolysis at 405 nm causes selective photorelease of GABA.