Sex Differences in Dopamine Release in Nucleus Accumbens and Dorsal Striatum Determined by Chronic Fast Scan Cyclic Voltammetry: Effects of social housing and repeated stimulation.

We investigated sex differences in dopamine (DA) release in the nucleus accumbens (NAc) and dorsolateral striatum (DLS) using a chronic 16-channel carbon fiber electrode and fast-scan cyclic voltammetry (FSCV). Electrical stimulation (ES; 60  Hz) induced DA release was recorded in the NAc of single or pair-housed male and female rats. When core (NAcC) and shell (NAcS) were recorded simultaneously, there was greater ES DA release in NAcC of pair-housed females compared with single females and males. Housing did not affect ES NAc DA release in males. In contrast, there was significantly more ES DA release from the DLS of female rats than male rats. This was true prior to and after treatment with methamphetamine. Furthermore, in castrated (CAST) males and ovariectomized (OVX) females, there were no sex differences in ES DA release from the DLS, demonstrating the hormone dependence of this sex difference. However, in the DLS of both intact and gonadectomized rats, DA reuptake was slower in females than in males. Finally, DA release following ES of the medial forebrain bundle at 60  Hz was studied over four weeks. ES DA release increased over time for both CAST males and OVX females, demonstrating sensitization. Using this novel 16-channel chronic FSCV electrode, we found sex differences in the effects of social housing in the NAcS, sex differences in DA release from intact rats in DLS, sex differences in DA reuptake in DLS of intake and gonadectomized rats, and we report sensitization of ES-induced DA release in DLS in vivo.Significance Statement Dopamine release is not uniform or fixed. In the nucleus accumbens, pair housing, compared with individual housing, is shown to differentially affect dopamine responsiveness to stimulation in a sex-dependent and region-specific way. There are also sex differences in stimulated dopamine release in the dorsolateral striatum of intact rats, which are not seen in gonadectomized rats, indicating the hormone dependence of this sex difference. However, reuptake of dopamine was slower in females than in males, independent of gonadal hormones. Importantly, the electrical stimulation-induced dopamine release in the dorsolateral striatum of gonadectomized rats demonstrated sensitization of dopamine release in vivo within animals for the first time. Thus, stimulated dopamine release exhibits sex-specific neuroplasticity that is modified in females by the housing conditions.

Monolithic 3D phase profile formation in glass for spatial and temporal control of optical waves.

Optical manufacturing technologies play a central role in modern science and engineering. Progress on both subtractive and additive fabrications is transforming the implementation of optical technologies. Despite the recent advances, modern fabrication still faces challenges in the accuracy, dimension, durability, intensity, and wavelength range. Here we present a direct monolithic 3D phase profile formation in glass and demonstrate its versatile applications for high-accuracy spatial and temporal control of optical waves in the extreme wavelength and intensity domains, direct fabrication of microlenses, and in situ aberration correction for refractive components. These advances and flexibilities will provide a new dimension for high-performance optical design and manufacture and enable novel applications in a broad range of disciplines.

Cellular-scale silicon probes for high-density, precisely localized neurophysiology.

Neural implants with large numbers of electrodes have become an important tool for examining brain functions. However, these devices typically displace a large intracranial volume compared with the neurons they record. This large size limits the density of implants, provokes tissue reactions that degrade chronic performance, and impedes the ability to accurately visualize recording sites within intact circuits. Here we report next-generation silicon-based neural probes at a cellular scale (5 × 10 µm cross section), with ultra-high-density packing (as little as 66 µm between shanks) and 64 or 256 closely spaced recording sites per probe. We show that these probes can be inserted into superficial or deep brain structures and record large spikes in freely behaving rats for many weeks. Finally, we demonstrate a slice-in-place approach for the precise registration of recording sites relative to nearby neurons and anatomical features, including striatal µ-opioid receptor patches. This scalable technology provides a valuable tool for examining information processing within neural circuits and potentially for human brain-machine interfaces.NEW & NOTEWORTHY Devices with many electrodes penetrating into the brain are an important tool for investigating neural information processing, but they are typically large compared with neurons. This results in substantial damage and makes it harder to reconstruct recording locations within brain circuits. This paper presents high-channel-count silicon probes with much smaller features and a method for slicing through probe, brain, and skull all together. This allows probe tips to be directly observed relative to immunohistochemical markers.

Iterative expansion microscopy.

We recently developed a method called expansion microscopy, in which preserved biological specimens are physically magnified by embedding them in a densely crosslinked polyelectrolyte gel, anchoring key labels or biomolecules to the gel, mechanically homogenizing the specimen, and then swelling the gel-specimen composite by ∼4.5× in linear dimension. Here we describe iterative expansion microscopy (iExM), in which a sample is expanded ∼20×. After preliminary expansion a second swellable polymer mesh is formed in the space newly opened up by the first expansion, and the sample is expanded again. iExM expands biological specimens ∼4.5 × 4.5, or ∼20×, and enables ∼25-nm-resolution imaging of cells and tissues on conventional microscopes. We used iExM to visualize synaptic proteins, as well as the detailed architecture of dendritic spines, in mouse brain circuitry.

Multispectral tracing in densely labeled mouse brain with nTracer.

SUMMARY: This note describes nTracer, an ImageJ plug-in for user-guided, semi-automated tracing of multispectral fluorescent tissue samples. This approach allows for rapid and accurate reconstruction of whole cell morphology of large neuronal populations in densely labeled brains. AVAILABILITY AND IMPLEMENTATION: nTracer was written as a plug-in for the open source image processing software ImageJ. The software, instructional documentation, tutorial videos, sample image and sample tracing results are available at https://www.cai-lab.org/ntracer-tutorial. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Long-range remote focusing by image-plane aberration correction.

Laser scanning plays an important role in a broad range of applications. Toward 3D aberration-free scanning, a remote focusing technique has been developed for high-speed imaging applications. However, the implementation of remote focusing often suffers from a limited axial scan range as a result of unknown aberration. Through simple analysis, we show that the sample-to-image path length conservation is crucially important to the remote focusing performance. To enhance the axial scan range, we propose and demonstrate an image-plane aberration correction method. Using a static correction, we can effectively improve the focus quality over a large defocusing range. Experimentally, we achieved ∼three times greater defocusing range than that of conventional methods. This technique can broadly benefit the implementations of high-speed large-volume 3D imaging.

Improved tools for the Brainbow toolbox.

In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.

Improved tools for the Brainbow toolbox.

In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.

Pulse-shaping based two-photon FRET stoichiometry.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

Tubulin modifications and their cellular functions.

All microtubules are built from a basic alpha/beta-tubulin building block, yet subpopulations of microtubules can be differentially marked by a number of post-translational modifications. These modifications, conserved throughout evolution, are thought to act individually or in combination to control specific microtubule-based functions, analogous to how histone modifications regulate chromatin functions. Here we review recent studies demonstrating that tubulin modifications influence microtubule-associated proteins such as severing proteins, plus-end tracking proteins, and molecular motors. In this way, tubulin modifications play an important role in regulating microtubule properties, such as stability and structure, as well as microtubule-based functions, such as ciliary beating, cell division, and intracellular trafficking.

Sex Differences in Dopamine Release in Nucleus Accumbens and Dorsal Striatum Determined by Chronic Fast Scan Cyclic Voltammetry: Effects of social housing and repeated stimulation.

We investigated sex differences in dopamine (DA) release in the nucleus accumbens (NAc) and dorsolateral striatum (DLS) using a chronic 16-channel carbon fiber electrode and fast-scan cyclic voltammetry (FSCV). Electrical stimulation (ES; 60Hz) induced DA release was recorded in the NAc of single or pair-housed male and female rats. When core (NAcC) and shell (NAcS) were recorded simultaneously, there was greater ES DA release in NAcC of pair-housed females compared with single females and males. Housing did not affect ES NAc DA release in males. In contrast, there was significantly more ES DA release from the DLS of female rats than male rats. This was true prior to and after treatment with methamphetamine. Furthermore, in castrated (CAST) males and ovariectomized (OVX) females, there were no sex differences in ES DA release from the DLS, demonstrating the hormone dependence of this sex difference. However, in the DLS of both intact and gonadectomized rats, DA reuptake was slower in females than in males. Finally, DA release following ES of the medial forebrain bundle at 60Hz was studied over four weeks. ES DA release increased over time for both CAST males and OVX females, demonstrating sensitization. Using this novel 16-channel chronic FSCV electrode, we found sex differences in the effects of social housing in the NAcS, sex differences in DA release from intact rats in DLS, sex differences in DA reuptake in DLS of intake and gonadectomized rats, and we report sensitization of ES-induced DA release in DLS in vivo.

Exosomes define a local and systemic communication network in healthy pancreas and pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a lethal disease, requires a grasp of its biology for effective therapies. Exosomes, implicated in cancer, are poorly understood in living systems. Here we use the genetically engineered mouse model (ExoBow) to map the spatiotemporal distribution of exosomes from healthy and PDAC pancreas in vivo to determine their biological significance. We show that, within the PDAC microenvironment, cancer cells establish preferential communication routes through exosomes with cancer associated fibroblasts and endothelial cells. The latter being a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios enhances angiogenesis, underscoring their contribution to vascularization and to cancer. Inter-organ communication is significantly increased in PDAC with specific organs as most frequent targets of exosomes communication occurring in health with the thymus, bone-marrow, brain, and intestines, and in PDAC with the kidneys, lungs and thymus. In sum, we find that exosomes mediate an organized intra- and inter- pancreas communication network with modulatory effects in vivo.

Two-photon imaging of multiple fluorescent proteins by phase-shaping and linear unmixing with a single broadband laser.

Imaging multiple fluorescent proteins (FPs) by two-photon microscopy has numerous applications for studying biological processes in thick and live samples. Here we demonstrate a setup utilizing a single broadband laser and a phase-only pulse-shaper to achieve imaging of three FPs (mAmetrine, TagRFPt, and mKate2) in live mammalian cells. Phase-shaping to achieve selective excitation of the FPs in combination with post-imaging linear unmixing enables clean separation of the fluorescence signal of each FP. This setup also benefits from low overall cost and simple optical alignment, enabling easy adaptation in a regular biomedical research laboratory.

A Comparison of Lossless Compression Methods in Microscopy Data Storage Applications.

Modern high-throughput microscopy methods such as light-sheet imaging and electron microscopy are capable of producing petabytes of data inside of a single experiment. Storage of these large images, however, is challenging because of the difficulty of moving, storing, and analyzing such vast amounts of data, which is often collected at very high data rates (>1GBps). In this report, we provide a comparison of the performance of several compression algorithms using a collection of published and unpublished datasets including confocal, fMOST, and pathology images. We also use simulated data to demonstrate the efficiency of each algorithm as image content or entropy increases. As a result of this work, we recommend the use of the BLOSC algorithm combined with ZSTD for various microscopy applications, as it produces the best compression ratio over a collection of conditions.

CELL INSTANCE SEGMENTATION VIA MULTI-SCALE NON-LOCAL CORRELATION.

For cell instance segmentation on Electron Microscopy (EM) images, state-of-the-art methods either conduct pixel-wise classification or follow a detection and segmentation manner. However, both approaches suffer from the enormous cell instances of EM images where cells are tightly close to each other and show inconsistent morphological properties and/or homogeneous appearances. This fact can easily lead to over-segmentation and under-segmentation problems for model prediction, i.e., falsely splitting and merging adjacent instances. In this paper, we propose a novel approach incorporating non-local correlation in the embedding space to make pixel features distinct or similar to their neighbors and thus address the over- and under-segmentation problems. We perform experiments on five different EM datasets where our proposed method yields better results than several strong baselines. More importantly, by using non-local correlation, we observe fewer false separations within one cell and fewer false fusions between cells.

Hacking brain development to test models of sensory coding.

Animals can discriminate myriad sensory stimuli but can also generalize from learned experience. You can probably distinguish the favorite teas of your colleagues while still recognizing that all tea pales in comparison to coffee. Tradeoffs between detection, discrimination, and generalization are inherent at every layer of sensory processing. During development, specific quantitative parameters are wired into perceptual circuits and set the playing field on which plasticity mechanisms play out. A primary goal of systems neuroscience is to understand how material properties of a circuit define the logical operations-computations--that it makes, and what good these computations are for survival. A cardinal method in biology-and the mechanism of evolution--is to change a unit or variable within a system and ask how this affects organismal function. Here, we make use of our knowledge of developmental wiring mechanisms to modify hard-wired circuit parameters in the Drosophila melanogaster mushroom body and assess the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input number, but not cell number, tunes odor selectivity. Simple odor discrimination performance is maintained when Kenyon cell number is reduced and augmented by Kenyon cell expansion.

Post-explant profiling of subcellular-scale carbon fiber intracortical electrodes and surrounding neurons enables modeling of recorded electrophysiology.

Objective.Characterizing the relationship between neuron spiking and the signals that electrodes record is vital to defining the neural circuits driving brain function and informing clinical brain-machine interface design. However, high electrode biocompatibility and precisely localizing neurons around the electrodes are critical to defining this relationship.Approach.Here, we demonstrate consistent localization of the recording site tips of subcellular-scale (6.8µm diameter) carbon fiber electrodes and the positions of surrounding neurons. We implanted male rats with carbon fiber electrode arrays for 6 or 12+ weeks targeting layer V motor cortex. After explanting the arrays, we immunostained the implant site and localized putative recording site tips with subcellular-cellular resolution. We then 3D segmented neuron somata within a 50µm radius from implanted tips to measure neuron positions and health and compare to healthy cortex with symmetric stereotaxic coordinates.Main results.Immunostaining of astrocyte, microglia, and neuron markers confirmed that overall tissue health was indicative of high biocompatibility near the tips. While neurons near implanted carbon fibers were stretched, their number and distribution were similar to hypothetical fibers placed in healthy contralateral brain. Such similar neuron distributions suggest that these minimally invasive electrodes demonstrate the potential to sample naturalistic neural populations. This motivated the prediction of spikes produced by nearby neurons using a simple point source model fit using recorded electrophysiology and the mean positions of the nearest neurons observed in histology. Comparing spike amplitudes suggests that the radius at which single units can be distinguished from others is near the fourth closest neuron (30.7 ± 4.6µm,X-± S) in layer V motor cortex.Significance.Collectively, these data and simulations provide the first direct evidence that neuron placement in the immediate vicinity of the recording site influences how many spike clusters can be reliably identified by spike sorting.

Utah array characterization and histological analysis of a multi-year implant in non-human primate motor and sensory cortices.

Objective.The Utah array is widely used in both clinical studies and neuroscience. It has a strong track record of safety. However, it is also known that implanted electrodes promote the formation of scar tissue in the immediate vicinity of the electrodes, which may negatively impact the ability to record neural waveforms. This scarring response has been primarily studied in rodents, which may have a very different response than primate brain.Approach.Here, we present a rare nonhuman primate histological dataset (n= 1 rhesus macaque) obtained 848 and 590 d after implantation in two brain hemispheres. For 2 of 4 arrays that remained within the cortex, NeuN was used to stain for neuron somata at three different depths along the shanks. Images were filtered and denoised, with neurons then counted in the vicinity of the arrays as well as a nearby section of control tissue. Additionally, 3 of 4 arrays were imaged with a scanning electrode microscope to evaluate any materials damage that might be present.Main results.Overall, we found a 63% percent reduction in the number of neurons surrounding the electrode shanks compared to control areas. In terms of materials, the arrays remained largely intact with metal and Parylene C present, though tip breakage and cracks were observed on many electrodes.Significance.Overall, these results suggest that the tissue response in the nonhuman primate brain shows similar neuron loss to previous studies using rodents. Electrode improvements, for example using smaller or softer probes, may therefore substantially improve the tissue response and potentially improve the neuronal recording yield in primate cortex.

Input density tunes Kenyon cell sensory responses in the Drosophila mushroom body.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells.

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)-labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.