High-throughput volumetric mapping of synaptic transmission.

Volumetric imaging of synaptic transmission in vivo requires high spatial and high temporal resolution. Shaping the wavefront of two-photon fluorescence excitation light, we developed Bessel-droplet foci for high-contrast and high-resolution volumetric imaging of synapses. Applying our method to imaging glutamate release, we demonstrated high-throughput mapping of excitatory inputs at >1,000 synapses per volume and >500 dendritic spines per neuron in vivo and unveiled previously unseen features of functional synaptic organization in the mouse primary visual cortex.

A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

A Distinct Population of L6 Neurons in Mouse V1 Mediate Cross-Callosal Communication.

Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.

The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.

Aging results in a decline in neural stem cells (NSCs), neurogenesis, and cognitive function, and evidence is emerging to demonstrate disrupted adult neurogenesis in the hippocampus of patients with several neurodegenerative disorders. Here, single-cell RNA sequencing of the dentate gyrus of young and old mice shows that the mitochondrial protein folding stress is prominent in activated NSCs/neural progenitors (NPCs) among the neurogenic niche, and it increases with aging accompanying dysregulated cell cycle and mitochondrial activity in activated NSCs/NPCs in the dentate gyrus. Increasing mitochondrial protein folding stress results in compromised NSC maintenance and reduced neurogenesis in the dentate gyrus, neural hyperactivity, and impaired cognitive function. Reducing mitochondrial protein folding stress in the dentate gyrus of old mice improves neurogenesis and cognitive function. These results establish the mitochondrial protein folding stress as a driver of NSC aging and suggest approaches to improve aging-associated cognitive decline.

maskNMF: A denoise-sparsen-detect approach for extracting neural signals from dense imaging data.

A number of calcium imaging methods have been developed to monitor the activity of large populations of neurons. One particularly promising approach, Bessel imaging, captures neural activity from a volume by projecting within the imaged volume onto a single imaging plane, therefore effectively mixing signals and increasing the number of neurons imaged per pixel. These signals must then be computationally demixed to recover the desired neural activity. Unfortunately, currently-available demixing methods can perform poorly in the regime of high imaging density (i.e., many neurons per pixel). In this work we introduce a new pipeline (maskNMF) for demixing dense calcium imaging data. The main idea is to first denoise and temporally sparsen the observed video; this enhances signal strength and reduces spatial overlap significantly. Next we detect neurons in the sparsened video using a neural network trained on a library of neural shapes. These shapes are derived from segmented electron microscopy images input into a Bessel imaging model; therefore no manual selection of "good" neural shapes from the functional data is required here. After cells are detected, we use a constrained non-negative matrix factorization approach to demix the activity, using the detected cells' shapes to initialize the factorization. We test the resulting pipeline on both simulated and real datasets and find that it is able to achieve accurate demixing on denser data than was previously feasible, therefore enabling faithful imaging of larger neural populations. The method also provides good results on more "standard" two-photon imaging data. Finally, because much of the pipeline operates on a significantly compressed version of the raw data and is highly parallelizable, the algorithm is fast, processing large datasets faster than real time.

Adaptive optics enables aberration-free single-objective remote focusing for two-photon fluorescence microscopy.

Two-photon fluorescence microscopy has been widely applied to three-dimensional (3D) imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. In this paper, using a liquid-crystal spatial light modulator to control the divergence of the excitation beam through a single objective, we systematically characterized the aberrations introduced by divergence control through microscope objectives of NA 0.45, 0.8, and 1.05. We used adaptive optics to correct the divergence-induced-aberrations and maintain diffraction-limited focal quality over up to 800-µm axial range. We further demonstrated aberration-free remote focusing for in vivo imaging of neurites and synapses in the mouse brain.

Rats use memory confidence to guide decisions.

Memory enables access to past experiences to guide future behavior. Humans can determine which memories to trust (high confidence) and which to doubt (low confidence). How memory retrieval, memory confidence, and memory-guided decisions are related, however, is not understood. In particular, how confidence in memories is used in decision making is unknown. We developed a spatial memory task in which rats were incentivized to gamble their time: betting more following a correct choice yielded greater reward. Rat behavior reflected memory confidence, with higher temporal bets following correct choices. We applied machine learning to identify a memory decision variable and built a generative model of memories evolving over time that accurately predicted both choices and confidence reports. Our results reveal in rats an ability thought to exist exclusively in primates and introduce a unified model of memory dynamics, retrieval, choice, and confidence.

High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics.

Understanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

An Actuated Neural Probe Architecture for Reducing Gliosis-Induced Recording Degradation.

Glial encapsulation of chronically implanted neural probes inhibits recording and stimulation, and this signal loss is a significant factor limiting the clinical viability of most neural implant topologies for decades-long implantation. We demonstrate a mechanical proof of concept for silicon shank-style neural probes intended to minimize gliosis near the recording sites. Compliant whiskers on the edges of the probe fold inward to minimize tissue damage during insertion. Once implanted to the target depth and retracted slightly, these whiskers splay outward. The splayed tips, on which recording sites could be patterned, extend beyond the typical 50-100 [Formula: see text] radius of a glial scar. The whiskers are micrometer-scale to minimize or avoid glial scarring. Electrically inactive devices with whiskers of varying widths and curvature were designed and monolithically fabricated from a 5- [Formula: see text] silicon-on-insulator (SOI) wafer, and their mechanical functionality was demonstrated in a 0.6% agar brain phantom. Deflection was plotted versus deflection speed, and those that were most compliant actuated successfully. This probe requires no preparation for use beyond what is typical for a shank-style silicon probe.

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays.

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas. The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days. This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

A microfabricated, 3D-sharpened silicon shuttle for insertion of flexible electrode arrays through dura mater into brain.

OBJECTIVE: Electrode arrays for chronic implantation in the brain are a critical technology in both neuroscience and medicine. Recently, flexible, thin-film polymer electrode arrays have shown promise in facilitating stable, single-unit recordings spanning months in rats. While array flexibility enhances integration with neural tissue, it also requires removal of the dura mater, the tough membrane surrounding the brain, and temporary bracing to penetrate the brain parenchyma. Durotomy increases brain swelling, vascular damage, and surgical time. Insertion using a bracing shuttle results in additional vascular damage and brain compression, which increase with device diameter; while a higher-diameter shuttle will have a higher critical load and more likely penetrate dura, it will damage more brain parenchyma and vasculature. One way to penetrate the intact dura and limit tissue compression without increasing shuttle diameter is to reduce the force required for insertion by sharpening the shuttle tip. APPROACH: We describe a novel design and fabrication process to create silicon insertion shuttles that are sharp in three dimensions and can penetrate rat dura, for faster, easier, and less damaging implantation of polymer arrays. Sharpened profiles are obtained by reflowing patterned photoresist, then transferring its sloped profile to silicon with dry etches. MAIN RESULTS: We demonstrate that sharpened shuttles can reliably implant polymer probes through dura to yield high quality single unit and local field potential recordings for at least 95 days. On insertion directly through dura, tissue compression is minimal. SIGNIFICANCE: This is the first demonstration of a rat dural-penetrating array for chronic recording. This device obviates the need for a durotomy, reducing surgical time and risk of damage to the blood-brain barrier. This is an improvement to state-of-the-art flexible polymer electrode arrays that facilitates their implantation, particularly in multi-site recording experiments. This sharpening process can also be integrated into silicon electrode array fabrication.

High-Density, Long-Lasting, and Multi-region Electrophysiological Recordings Using Polymer Electrode Arrays.

The brain is a massive neuronal network, organized into anatomically distributed sub-circuits, with functionally relevant activity occurring at timescales ranging from milliseconds to years. Current methods to monitor neural activity, however, lack the necessary conjunction of anatomical spatial coverage, temporal resolution, and long-term stability to measure this distributed activity. Here we introduce a large-scale, multi-site, extracellular recording platform that integrates polymer electrodes with a modular stacking headstage design supporting up to 1,024 recording channels in freely behaving rats. This system can support months-long recordings from hundreds of well-isolated units across multiple brain regions. Moreover, these recordings are stable enough to track large numbers of single units for over a week. This platform enables large-scale electrophysiological interrogation of the fast dynamics and long-timescale evolution of anatomically distributed circuits, and thereby provides a new tool for understanding brain activity.

Cell type-specific long-range connections of basal forebrain circuit.

The basal forebrain (BF) plays key roles in multiple brain functions, including sleep-wake regulation, attention, and learning/memory, but the long-range connections mediating these functions remain poorly characterized. Here we performed whole-brain mapping of both inputs and outputs of four BF cell types - cholinergic, glutamatergic, and parvalbumin-positive (PV+) and somatostatin-positive (SOM+) GABAergic neurons - in the mouse brain. Using rabies virus -mediated monosynaptic retrograde tracing to label the inputs and adeno-associated virus to trace axonal projections, we identified numerous brain areas connected to the BF. The inputs to different cell types were qualitatively similar, but the output projections showed marked differences. The connections to glutamatergic and SOM+ neurons were strongly reciprocal, while those to cholinergic and PV+ neurons were more unidirectional. These results reveal the long-range wiring diagram of the BF circuit with highly convergent inputs and divergent outputs and point to both functional commonality and specialization of different BF cell types.

Organization of long-range inputs and outputs of frontal cortex for top-down control.

Long-range projections from the frontal cortex are known to modulate sensory processing in multiple modalities. Although the mouse has become an increasingly important animal model for studying the circuit basis of behavior, the functional organization of its frontal cortical long-range connectivity remains poorly characterized. Here we used virus-assisted circuit mapping to identify the brain networks for top-down modulation of visual, somatosensory and auditory processing. The visual cortex is reciprocally connected to the anterior cingulate area, whereas the somatosensory and auditory cortices are connected to the primary and secondary motor cortices. Anterograde and retrograde tracing identified the cortical and subcortical structures belonging to each network. Furthermore, using new viral techniques to target subpopulations of frontal neurons projecting to the visual cortex versus the superior colliculus, we identified two distinct subnetworks within the visual network. These findings provide an anatomical foundation for understanding the brain mechanisms underlying top-down control of behavior.

Individual karyotypes at the origins of cervical carcinomas.

BACKGROUND: In 1952 Papanicolaou et al. first diagnosed and graded cervical carcinomas based on individual "abnormal DNA contents" and cellular phenotypes. Surprisingly current papilloma virus and mutation theories of carcinomas do not mention these individualities. The viral theory holds that randomly integrated, defective genomes of papilloma viruses, which are often untranscribed, cause cervical carcinomas with unknown cofactors 20-50 years after infection. Virus-free carcinomas are attributed to mutations of a few tumor-suppressor genes, especially the p53 gene. But the paradox of how a few mutations or latent defective viral DNAs would generate carcinomas with endless individual DNA contents, degrees of malignancies and cellular phenotypes is unsolved. Since speciation predicts individuality, we test here the theory that cancers are autonomous species with individual clonal karyotypes and phenotypes. This theory postulates that carcinogens induce aneuploidy. By unbalancing mitosis genes aneuploidy catalyzes chain reactions of karyotypic evolutions. Most such evolutions end with non-viable karyotypes but a few become new cancer karyotypes. Despite congenitally unbalanced mitosis genes cancer karyotypes are stabilized by clonal selections for cancer-specific autonomy. RESULTS: To test the prediction of the speciation theory that individual carcinomas have individual clonal karyotypes and phenotypes, we have analyzed here the phenotypes and karyotypes of nine cervical carcinomas. Seven of these contained papilloma virus sequences and two did not. We determined phenotypic individuality and clonality based on the morphology and sociology of carcinoma cells in vitro. Karyotypic individuality and clonality were determined by comparing all chromosomes of 20 karyotypes of carcinomas in three-dimensional arrays. Such arrays list chromosome numbers on the x-axis, chromosome copy numbers on the y-axis and the number of karyotypes arrayed on the z-axis. We found (1) individual clonal karyotypes and phenotypes in all nine carcinomas, but no virus-specific markers, (2) 1-to-1 variations between carcinoma-specific karyotypes and phenotypes, e.g. drug-resistance and cell morphology, (3) proportionality between the copy numbers of chromosomes and the copy numbers of hundreds of over- and under-expressed mRNAs, (4) evidence that tobacco-carcinogens induce cervical carcinomas via aneuploidy, consistent with the speciation theory. CONCLUSIONS: Since the individual clonal karyotypes of nine carcinomas correlated and co-varied 1-to-1 with complex individual transcriptomes and phenotypes, we have classical genetic and functional transcriptomic evidence to conclude that these karyotypes encode carcinomas - much like the clonal karyotypes that encode conventional species. These individual karyotypes explain the individual "DNA contents", the endless grades of malignancies and the complex individual transcriptomes and phenotypes of carcinomas.