Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging.

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.

Multiline Orthogonal Scanning Temporal Focusing (mosTF) Microscopy for Scattering Reduction in High-speed in vivo Brain Imaging.

Temporal focusing two-photon microscopy enables high resolution imaging of fine structures in vivo over a large volume. A limitation of temporal focusing is that signal-to-background ratio and resolution degrade rapidly with increasing imaging depth. This degradation originates from the scattered emission photons are widely distributed resulting in a strong background. We have developed Multiline Orthogonal Scanning Temporal Focusing (mosTF) microscopy that overcomes this problem. mosTF captures a sequence of images at each scan location of the excitation line, followed by a reconstruction algorithm reassigns scattered photons back to the correct scan position. We demonstrate mosTF by acquiring mice neuronal images in vivo. Our results show remarkably improvements with mosTF for in vivo brain imaging while maintaining its speed advantage.

De-scattering Deep Neural Network Enables Fast Imaging of Spines through Scattering Media by Temporal Focusing Microscopy.

Today the gold standard for in vivo imaging through scattering tissue is point-scanning two-photon microscopy (PSTPM). Especially in neuroscience, PSTPM is widely used for deep-tissue imaging in the brain. However, due to sequential scanning, PSTPM is slow. Temporal focusing microscopy (TFM), on the other hand, focuses femtosecond pulsed laser light temporally while keeping wide-field illumination, and is consequently much faster. However, due to the use of a camera detector, TFM suffers from the scattering of emission photons. As a result, TFM produces images of poor quality, obscuring fluorescent signals from small structures such as dendritic spines. In this work, we present a de-scattering deep neural network (DeScatterNet) to improve the quality of TFM images. Using a 3D convolutional neural network (CNN) we build a map from TFM to PSTPM modalities, to enable fast TFM imaging while maintaining high image quality through scattering media. We demonstrate this approach for in vivo imaging of dendritic spines on pyramidal neurons in the mouse visual cortex. We quantitatively show that our trained network rapidly outputs images that recover biologically relevant features previously buried in the scattered fluorescence in the TFM images. In vivo imaging that combines TFM and the proposed neural network is one to two orders of magnitude faster than PSTPM but retains the high quality necessary to analyze small fluorescent structures. The proposed approach could also be beneficial for improving the performance of many speed-demanding deep-tissue imaging applications, such as in vivo voltage imaging.

Mapping thalamic innervation to individual L2/3 pyramidal neurons and modeling their 'readout' of visual input.

The thalamus is the main gateway for sensory information from the periphery to the mammalian cerebral cortex. A major conundrum has been the discrepancy between the thalamus's central role as the primary feedforward projection system into the neocortex and the sparseness of thalamocortical synapses. Here we use new methods, combining genetic tools and scalable tissue expansion microscopy for whole-cell synaptic mapping, revealing the number, density and size of thalamic versus cortical excitatory synapses onto individual layer 2/3 (L2/3) pyramidal cells (PCs) of the mouse primary visual cortex. We find that thalamic inputs are not only sparse, but remarkably heterogeneous in number and density across individual dendrites and neurons. Most surprising, despite their sparseness, thalamic synapses onto L2/3 PCs are smaller than their cortical counterparts. Incorporating these findings into fine-scale, anatomically faithful biophysical models of L2/3 PCs reveals how individual neurons with sparse and weak thalamocortical synapses, embedded in small heterogeneous neuronal ensembles, may reliably 'read out' visually driven thalamic input.

Epitope-preserving magnified analysis of proteome (eMAP).

Synthetic tissue-hydrogel methods have enabled superresolution investigation of biological systems using diffraction-limited microscopy. However, chemical modification by fixatives can cause loss of antigenicity, limiting molecular interrogation of the tissue gel. Here, we present epitope-preserving magnified analysis of proteome (eMAP) that uses purely physical tissue-gel hybridization to minimize the loss of antigenicity while allowing permanent anchoring of biomolecules. We achieved success rates of 96% and 94% with synaptic antibodies for mouse and marmoset brains, respectively. Maximal preservation of antigenicity allows imaging of nanoscopic architectures in 1000-fold expanded tissues without additional signal amplification. eMAP-processed tissue gel can endure repeated staining and destaining without epitope loss or structural damage, enabling highly multiplexed proteomic analysis. We demonstrated the utility of eMAP as a nanoscopic proteomic interrogation tool by investigating molecular heterogeneity in inhibitory synapses in the mouse brain neocortex and characterizing the spatial distributions of synaptic proteins within synapses in mouse and marmoset brains.

Genetic variants in the bipolar disorder risk locus SYNE1 that affect CPG2 expression and protein function.

Bipolar disorder (BD) is a common mood disorder characterized by recurrent episodes of mania and depression. Both genetic and environmental factors have been implicated in BD etiology, but the biological underpinnings remain elusive. Recently, genome-wide association studies (GWAS) of neuropsychiatric disorders have identified a risk locus for BD containing the SYNE1 gene, a large gene encoding multiple proteins. The BD association signal spans, almost exclusively, the part of SYNE1 encoding CPG2, a brain-specific protein localized to excitatory postsynaptic sites, where it regulates glutamate receptor internalization. Here we show that CPG2 protein levels are significantly decreased in postmortem brain tissue from BD patients, as compared to control subjects, as well as schizophrenia and depression patients. We identify genetic variants within the postmortem brains that map to the CPG2 promoter region, and show that they negatively affect gene expression. We also identify missense single nucleotide polymorphisms (SNPs) in CPG2 coding regions that affect CPG2 expression, localization, and synaptic function. Our findings link genetic variation in the CPG2 region of SYNE1 with a mechanism for glutamatergic synapse dysfunction that could underlie susceptibility to BD in some individuals. Few GWAS hits in human genetics for neuropsychiatric disorders to date have afforded such mechanistic clues. Further, the potential for genetic distinction of susceptibility to BD from other neuropsychiatric disorders with overlapping clinical traits holds promise for improved diagnostics and treatment of this devastating illness.

Neuron class-specific responses govern adaptive myelin remodeling in the neocortex.

Myelin plasticity is critical for neurological function, including learning and memory. However, it is unknown whether this plasticity reflects uniform changes across all neuronal subtypes, or whether myelin dynamics vary between neuronal classes to enable fine-tuning of adaptive circuit responses. We performed in vivo two-photon imaging of myelin sheaths along single axons of excitatory callosal neurons and inhibitory parvalbumin-expressing interneurons in adult mouse visual cortex. We found that both neuron types show homeostatic myelin remodeling under normal vision. However, monocular deprivation results in adaptive myelin remodeling only in parvalbumin-expressing interneurons. An initial increase in elongation of myelin segments is followed by contraction of a separate cohort of segments. This data indicates that distinct classes of neurons individualize remodeling of their myelination profiles to diversify circuit tuning in response to sensory experience.

CPG15/Neuritin Mimics Experience in Selecting Excitatory Synapses for Stabilization by Facilitating PSD95 Recruitment.

A key feature of brain plasticity is the experience-dependent selection of optimal connections, implemented by a set of activity-regulated genes that dynamically adjust synapse strength and number. The activity-regulated gene cpg15/neuritin has been previously implicated in stabilization and maturation of excitatory synapses. Here, we combine two-photon microscopy with genetic and sensory manipulations to dissect excitatory synapse formation in vivo and examine the role of activity and CPG15 in dendritic spine formation, PSD95 recruitment, and synapse stabilization. We find that neither visual experience nor CPG15 is required for spine formation. However, PSD95 recruitment to nascent spines and their subsequent stabilization requires both. Further, cell-autonomous CPG15 expression is sufficient to replace experience in facilitating PSD95 recruitment and spine stabilization. CPG15 directly interacts with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on immature dendritic spines, suggesting a signaling mode for this small extracellular molecule acting as an experience-dependent "selector" for spine stabilization and synapse maturation.

Scanless volumetric imaging by selective access multifocal multiphoton microscopy.

Simultaneous, high-resolution imaging across a large number of synaptic and dendritic sites is critical for understanding how neurons receive and integrate signals. Yet, functional imaging that targets a large number of submicrometer-sized synaptic and dendritic locations poses significant technical challenges. We demonstrate a new parallelized approach to address such questions, increasing the signal-to-noise ratio by an order of magnitude compared to previous approaches. This selective access multifocal multiphoton microscopy uses a spatial light modulator to generate multifocal excitation in three dimensions (3D) and a Gaussian-Laguerre phase plate to simultaneously detect fluorescence from these spots throughout the volume. We test the performance of this system by simultaneously recording Ca2+ dynamics from cultured neurons at 98-118 locations distributed throughout a 3D volume. This is the first demonstration of 3D imaging in a "single shot" and permits synchronized monitoring of signal propagation across multiple different dendrites.

Glutamate Receptors: Not Just for Excitation.

In this issue of Neuron, Fossati et al. (2019) report a new constellation of players regulating inhibitory synaptogenesis. They show that GluD1, through a non-canonical ionotropic-independent mechanism, controls GABAergic synapse formation via trans-synaptic interactions mediated by extracellular cerebellin-4. They identify ARHGEF12 and PPP1R12A as GluD1 intracellular interactors and downstream effectors.

Scattering reduction by structured light illumination in line-scanning temporal focusing microscopy.

Line-scanning temporal focusing microscopy (LineTFM) is capable of imaging biological samples more than 10 times faster than two-photon laser point-scanning microscopy (TPLSM), while achieving nearly the same lateral and axial spatial resolution. However, the image contrast taken by LineTFM is lower than that by TPLSM because LineTFM is severely influenced by biological tissue scattering. To reject the scattered photons, we implemented LineTFM using both structured illumination and uniform illumination combined with the HiLo post-processing algorithm, called HiLL microscopy (HiLo-Line-scanning temporal focusing microscopy). HiLL microscopy significantly reduces tissue scattering and improves image contrast. We demonstrate HiLL microscopy with in vivo brain imaging. This approach could potentially find applications in monitoring fast dynamic events and in mapping high resolution structures over a large volume.

Interneuron Simplification and Loss of Structural Plasticity As Markers of Aging-Related Functional Decline.

Changes in excitatory neuron and synapse structure have been recognized as a potential physical source of age-related cognitive decline. Despite the importance of inhibition to brain plasticity, little is known regarding aging-associated changes to inhibitory neurons. Here we test for age-related cellular and circuit changes to inhibitory neurons of mouse visual cortex. We find no substantial difference in inhibitory neuron number, inhibitory neuronal subtypes, or synapse numbers within the cerebral cortex of aged mice compared with younger adults. However, when comparing cortical interneuron morphological parameters, we find differences in complexity, suggesting that arbors are simplified in aged mice. In vivo two-photon microscopy has previously shown that in contrast to pyramidal neurons, inhibitory interneurons retain a capacity for dendritic remodeling in the adult. We find that this capacity diminishes with age and is accompanied by a shift in dynamics from balanced branch additions and retractions to progressive prevalence of retractions, culminating in a dendritic arbor that is both simpler and more stable. Recording of visually evoked potentials shows that aging-related interneuron dendritic arbor simplification and reduced dynamics go hand in hand with loss of induced stimulus-selective response potentiation (SRP), a paradigm for adult visual cortical plasticity. Chronic treatment with the antidepressant fluoxetine reversed deficits in interneuron structural dynamics and restored SRP in aged animals. Our results support a structural basis for age-related impairments in sensory perception, and suggest that declines in inhibitory neuron structural plasticity during aging contribute to reduced functional plasticity.SIGNIFICANCE STATEMENT Structural alterations in neuronal morphology and synaptic connections have been proposed as a potential physical basis for age-related decline in cognitive function. Little is known regarding aging-associated changes to inhibitory neurons, despite the importance of inhibitory circuitry to adult cortical plasticity and the reorganization of cortical maps. Here we show that brain aging goes hand in hand with progressive structural simplification and reduced plasticity of inhibitory neurons, and a parallel decline in sensory map plasticity. Fluoxetine treatment can attenuate the concurrent age-related declines in interneuron structural and functional plasticity, suggesting it could provide an important therapeutic approach for mitigating sensory and cognitive deficits associated with aging.

Functional implications of inhibitory synapse placement on signal processing in pyramidal neuron dendrites.

A rich literature describes inhibitory innervation of pyramidal neurons in terms of the distinct inhibitory cell types that target the soma, axon initial segment, or dendritic arbor. Less attention has been devoted to how localization of inhibition to specific parts of the pyramidal dendritic arbor influences dendritic signal detection and integration. The effect of inhibitory inputs can vary based on their placement on dendritic spines versus shaft, their distance from the soma, and the branch order of the dendrite they inhabit. Inhibitory synapses are also structurally dynamic, and the implications of these dynamics depend on their dendritic location. Here we consider the heterogeneous roles of inhibitory synapses as defined by their strategic placement on the pyramidal cell dendritic arbor.

Spine Dynamics: Are They All the Same?

Since Cajal's first drawings of Golgi stained neurons, generations of researchers have been fascinated by the small protrusions, termed spines, studding many neuronal dendrites. Most excitatory synapses in the mammalian CNS are located on dendritic spines, making spines convenient proxies for excitatory synaptic presence. When in vivo imaging revealed that dendritic spines are dynamic structures, their addition and elimination were interpreted as excitatory synapse gain and loss, respectively. Spine imaging has since become a popular assay for excitatory circuit remodeling. In this review, we re-evaluate the validity of using spine dynamics as a straightforward reflection of circuit rewiring. Recent studies tracking both spines and synaptic markers in vivo reveal that 20% of spines lack PSD-95 and are short lived. Although they account for most spine dynamics, their remodeling is unlikely to impact long-term network structure. We discuss distinct roles that spine dynamics can play in circuit remodeling depending on synaptic content.

Filling the (SR)GAP in Excitatory/Inhibitory Balance.

In this issue of Neuron, Fossati et al. (2016) report that through its domain structure, SRGAP2A, a Rho-GTPase-activating protein, can co-regulate excitatory and inhibitory synapse development, offering a putative evolutionary genetic mechanism for preserving excitatory/inhibitory balance during speciation.

Inhibitory Synapses Are Repeatedly Assembled and Removed at Persistent Sites In Vivo.

Older concepts of a hard-wired adult brain have been overturned in recent years by in vivo imaging studies revealing synaptic remodeling, now thought to mediate rearrangements in microcircuit connectivity. Using three-color labeling and spectrally resolved two-photon microscopy, we monitor in parallel the daily structural dynamics (assembly or removal) of excitatory and inhibitory postsynaptic sites on the same neurons in mouse visual cortex in vivo. We find that dynamic inhibitory synapses often disappear and reappear again in the same location. The starkest contrast between excitatory and inhibitory synapse dynamics is on dually innervated spines, where inhibitory synapses frequently recur while excitatory synapses are stable. Monocular deprivation, a model of sensory input-dependent plasticity, shortens inhibitory synapse lifetimes and lengthens intervals to recurrence, resulting in a new dynamic state with reduced inhibitory synaptic presence. Reversible structural dynamics indicate a fundamentally new role for inhibitory synaptic remodeling--flexible, input-specific modulation of stable excitatory connections.

CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors.

Internalization of glutamate receptors at the postsynaptic membrane via clathrin-mediated endocytosis (CME) is a key mechanism for regulating synaptic strength. A role for the F-actin cytoskeleton in CME is well established, and recently, PKA-dependent association of candidate plasticity gene 2 (CPG2) with the spine-cytoskeleton has been shown to mediate synaptic glutamate receptor internalization. Yet, how the endocytic machinery is physically coupled to the actin cytoskeleton to facilitate glutamate receptor internalization has not been demonstrated. Moreover, there has been no distinction of endocytic-machinery components that are specific to activity-dependent versus constitutive glutamate receptor internalization. Here, we show that CPG2, through a direct physical interaction, recruits endophilin B2 (EndoB2) to F-actin, thus anchoring the endocytic machinery to the spine cytoskeleton and facilitating glutamate receptor internalization. Regulation of CPG2 binding to the actin cytoskeleton by protein kinase A directly impacts recruitment of EndoB2 and clathrin. Specific disruption of EndoB2 or the CPG2-EndoB2 interaction impairs activity-dependent, but not constitutive, internalization of both NMDA- and AMPA-type glutamate receptors. These results demonstrate that, through direct interactions with F-actin and EndoB2, CPG2 physically bridges the spine cytoskeleton and the endocytic machinery, and this tripartite association is critical specifically for activity-dependent CME of synaptic glutamate receptors.

Genomic mapping and cellular expression of human CPG2 transcripts in the SYNE1 gene.

Bipolar disorder (BD) is a prevalent and severe mood disorder characterized by recurrent episodes of mania and depression. Both genetic and environmental factors have been implicated in BD etiology, but the biological underpinnings remain elusive. Recent genome-wide association studies (GWAS) for identifying genes conferring risk for schizophrenia, BD, and major depression, identified an association between single-nucleotide polymorphisms (SNPs) in the SYNE1 gene and increased risk of BD. SYNE1 has also been identified as a risk locus for multiple other neurological or neuromuscular genetic disorders. The BD associated SNPs map within the gene region homologous to part of rat Syne1 encompassing the brain specific transcripts encoding CPG2, a postsynaptic neuronal protein localized to excitatory synapses and an important regulator of glutamate receptor internalization. Here, we use RNA-seq, ChIP-seq and RACE to map the human SYNE1 transcriptome, focusing on the CPG2 locus. We validate several CPG2 transcripts, including ones not previously annotated in public databases, and identify and clone a full-length CPG2 cDNA expressed in human neocortex, hippocampus and striatum. Using lenti-viral gene knock down/replacement and surface receptor internalization assays, we demonstrate that human CPG2 protein localizes to dendritic spines in rat hippocampal neurons and is functionally equivalent to rat CPG2 in regulating glutamate receptor internalization. This study provides a valuable gene-mapping framework for relating multiple genetic disease loci in SYNE1 with their transcripts, and for evaluating the effects of missense SNPs identified by patient genome sequencing on neuronal function.

Experience-Dependent Structural Plasticity in the Visual System.

During development, the environment exerts a profound influence on the wiring of brain circuits. Due to the limited resolution of studies in fixed tissue, this experience-dependent structural plasticity was once thought to be restricted to a specific developmental time window. The recent introduction of two-photon microscopy for in vivo imaging has opened the door to repeated monitoring of individual neurons and the study of structural plasticity mechanisms at a very fine scale. In this review, we focus on recent work showing that synaptic structural rearrangements are a key mechanism mediating neural circuit adaptation and behavioral plasticity in the adult brain. We examine this work in the context of classic studies in the visual systems of model organisms, which have laid much of the groundwork for our understanding of activity-dependent synaptic remodeling and its role in brain plasticity.