Summary: How the topography of neural circuits relates to their function remains unclear. Although topographic maps exist for sensory and motor variables, they are rarely observed for cognitive variables. Using calcium imaging during virtual navigation, we investigated the relationship between the anatomical organization and functional properties of grid cells, which represent a cognitive code for location during navigation. We found a substantial degree of grid cell micro-organization in mouse medial entorhinal cortex: grid cells and modules all clustered anatomically. Within a module, the layout of grid cells was a noisy two-dimensional lattice, in which the anatomical distribution of grid cells largely matched their spatial tuning phases. This micro-arrangement of phases demonstrates the existence of a topographical map encoding a cognitive variable in rodents. It contributes to a foundation for evaluating circuit models of the grid cell network, and is consistent with continuous attractor models as the mechanism of grid formation.

High throughput single cell imaging is a critical enabling and driving technology in molecular and cellular biology, biotechnology, medicine and related areas. Imaging flow cytometry combines single-cell imaging capabilities of microscopy with the high-throughput capabilities of conventional flow cytometry. Recent advances in imaging flow cytometry are remarkably revolutionizing the single-cell analysis. This article describes recent imaging flow cytometry technologies and their challenges.

In vivo two-photon microscopy provides the foundation for an array of powerful techniques for optically measuring and perturbing neural circuits. However, challenging tissue properties and geometry have prevented high-resolution optical access to regions situated within deep fissures. These regions include the medial prefrontal and medial entorhinal cortex (mPFC and MEC), which are of broad scientific and clinical interest. Here, we present a method for in vivo, subcellular resolution optical access to the mPFC and MEC using microprisms inserted into the fissures. We chronically imaged the mPFC and MEC in mice running on a spherical treadmill, using two-photon laser-scanning microscopy and genetically encoded calcium indicators to measure network activity. In the MEC, we imaged grid cells, a widely studied cell type essential to memory and spatial information processing. These cells exhibited spatially modulated activity during navigation in a virtual reality environment. This method should be extendable to other brain regions situated within deep fissures, and opens up these regions for study at cellular resolution in behaving animals using a rapidly expanding palette of optical tools for perturbing and measuring network structure and function.two-photon imaging | medial prefrontal cortex | medial entorhinal cortex | grid cell O ptical tools provide powerful means to measure and perturb the structure and function of neural circuits (1). However, a key set of brain regions remains outside the reach of cellularresolution optical methods: those situated within deep fissures. In the rodent brain, these areas include the medial prefrontal and medial entorhinal cortex (mPFC and MEC), two of the most widely studied regions supporting cognitive functions. The mPFC is centrally important to planning, executive function, learning, and memory (2, 3). Understanding how the mPFC implements these functions at a mechanistic, circuit level remains a longstanding goal of the field. The entorhinal cortex forms the interface between the neocortex and hippocampus and is believed to play an essential role in episodic memory and spatial information processing. These functions are particularly associated with MEC grid cells, which fire on a hexagonal lattice as an animal moves through an environment (4). Grid cells have galvanized a large body of work investigating their computational role and the mechanisms underlying grid formation, which remain important, open questions (4, 5). Moreover, mPFC or MEC dysfunction is associated with addiction, depression, schizophrenia, Alzheimer's disease, and epilepsy.The study of the mPFC, MEC, and neighboring regions could be greatly advanced by in vivo, high-resolution optical tools. However, tissue properties and geometry present significant challenges to deploying these tools within deep fissures. For example, the depth of two-photon microscopy is limited by scattering and out-of-plane excitation (6). Access in the mammalian brain is therefore typically restricted to superficial regions (<500 ...

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABA A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25-syntaxin1A/B-VAMP2 complexes, whereas insertion of GABA A receptors relies on SNAP23-syntaxin1A/B-VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABA A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.AMPA receptor | GABA A receptor | constitutive exocytosis | TIRFM | SNARE I n the mammalian central nervous system, neurons receive excitatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neurotransmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA A receptors, which are predominantly heteropentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a "plasma membrane sorting model," different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a "vesicle sorting model," different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the e...

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Cell classification based on phenotypical, spatial, and genetic information greatly advances our understanding of the physiology and pathology of biological systems. Technologies derived from next generation sequencing and fluorescent activated cell sorting are cornerstones for cell‐ and genomic‐based assays supporting cell classification and mapping. However, there exists a deficiency in technology space to rapidly isolate cells based on high content image information. Fluorescence‐activated cell sorting can only resolve cell‐to‐cell variation in fluorescence and optical scattering. Utilizing microfluidics, photonics, computation microscopy, real‐time image processing and machine learning, we demonstrate an image‐guided cell sorting and classification system possessing the high throughput of flow cytometer and high information content of microscopy. We demonstrate the utility of this technology in cell sorting based on (1) nuclear localization of glucocorticoid receptors, (2) particle binding to the cell membrane, and (3) DNA damage induced γ‐H2AX foci. © 2019 International Society for Advancement of Cytometry

During spatial navigation, animals use self-motion to estimate positions through path integration. However, estimation errors accumulate over time and it is unclear how they are corrected. Here we report a new cell class (‘cue cell’) encoding visual cues that could be used to correct errors in path integration in mouse medial entorhinal cortex (MEC). During virtual navigation, individual cue cells exhibited firing fields only near visual cues and their population response formed sequences repeated at each cue. These cells consistently responded to cues across multiple environments. On a track with cues on left and right sides, most cue cells only responded to cues on one side. During navigation in a real arena, they showed spatially stable activity and accounted for 32% of unidentified, spatially stable MEC cells. These cue cell properties demonstrate that the MEC contains a code representing spatial landmarks, which could be important for error correction during path integration.

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