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.

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

Temporal focusing two-photon microscopy enables high resolution imaging of fine structures 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.

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

Today the gold standard for 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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Non-descanned multifocal multiphoton microscopy with a multianode photomultiplier tube.

Multifocal multiphoton microscopy (MMM) improves imaging speed over a point scanning approach by parallelizing the excitation process. Early versions of MMM relied on imaging detectors to record emission signals from multiple foci simultaneously. For many turbid biological specimens, the scattering of emission photons results in blurred images and degrades the signal-to-noise ratio (SNR).

Spectral-resolved multifocal multiphoton microscopy with multianode photomultiplier tubes.

Multiphoton excitation fluorescence microscopy is the preferred method for in vivo deep tissue imaging. Many biological applications demand both high imaging speed and the ability to resolve multiple fluorophores. One of the successful methods to improve imaging speed in a highly turbid specimen is multifocal multiphoton microscopy (MMM) based on use of multi-anode photomultiplier tubes (MAPMT).

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT).

Aberrant development and plasticity of excitatory visual cortical networks in the absence of cpg15.

During development, experience plays a crucial role in sculpting neuronal connections. Patterned neural activity guides formation of functional neural circuits through the selective stabilization of some synapses and the pruning of others. Activity-regulated factors are fundamental to this process, but their roles in synapse stabilization and maturation is still poorly understood.

Regulation of glutamate receptor internalization by the spine cytoskeleton is mediated by its PKA-dependent association with CPG2.

A key neuronal mechanism for adjusting excitatory synaptic strength is clathrin-mediated endocytosis of postsynaptic glutamate receptors (GluRs). The actin cytoskeleton is critical for clathrin-mediated endocytosis, yet we lack a mechanistic understanding of its interaction with the endocytic process and how it may be regulated. Here we show that F-actin in dendritic spines physically binds the synaptic nuclear envelope 1 gene product candidate plasticity gene 2 (CPG2) in a PKA-dependent manner, and that this association is required for synaptic GluR internalization.