The unique pH and temperature responsiveness of PNIPAM-based microgels make them a promising target for novel biomedical applications such as cellular drug delivery systems. However, we lack a comprehensive understanding of how the physicochemical properties of microgels relate to their interaction with cells. Here, we show that HEK293T cells take up PNIPAMbased microgels on a second-to-minute time scale. Uptake rates are determined by microgel size and cross-linker content. Using fluorescence confocal live-cell microscopy, we observe microgel uptake in real time and describe cellular uptake kinetics. Experiments reveal that small and less cross-linked microgels show faster uptake kinetics than microgels of larger size or higher cross-linker content. Only microgels that are larger than 800 nm in diameter and have cross-linking contents of 10−15 mol % do not show translocation into cells. Together, these results provide insight into microgel−cell interactions and generate quantitative information on the deterministic role of microgel architecturei.e., size and rigidityfor uptake by a prototypical human cell line.

The accessory olfactory system controls social and sexual behavior. In the mouse accessory olfactory bulb, the first central stage of information processing along the accessory olfactory pathway, projection neurons (mitral cells) display infra-slow oscillatory discharge with remarkable periodicity. The physiological mechanisms that underlie this default output state, however, remain controversial. Moreover, whether such rhythmic infra-slow activity patterns exist in awake behaving mice and whether such activity reflects the functional organization of the accessory olfactory bulb circuitry remain unclear. Here, we hypothesize that mitral cell ensembles form synchronized microcircuits that subdivide the accessory olfactory bulb into segregated functional clusters. We use a miniature microscope to image the Ca2+dynamics within the apical dendritic compartments of large mitral cell ensemblesin vivo. We show that infra-slow periodic patterns of concerted neural activity, indeed, reflect the idle state of accessory olfactory bulb output in awake male and female mice. Ca2+activity profiles are distinct and glomerulus-specific. Confocal time-lapse imaging in acute slices reveals that groups of mitral cells assemble into microcircuits that exhibit correlated Ca2+signals. Moreover, electrophysiological profiling of synaptic connectivity indicates functional coupling between mitral cells. Our results suggest that both intrinsically rhythmogenic neurons and neurons entrained by fast synaptic drive are key elements in organizing the accessory olfactory bulb into functional microcircuits, each characterized by a distinct default pattern of infra-slow rhythmicity.SIGNIFICANCE STATEMENTInformation processing in the accessory olfactory bulb (AOB) plays a central role in conspecific chemosensory communication. Surprisingly, many basic physiological principles that underlie neuronal signaling in the AOB remain elusive. Here, we show that AOB projection neurons (mitral cells) form parallel synchronized ensembles bothin vitroandin vivo. Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway.

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ. Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca 2+ level, and increased firing. The Ca 2+ -activated chloride channels TMEM16A and TMEM16B are co-expressed within microvilli of vomeronasal neurons, but their physiological role remains elusive. Here, we investigate the contribution of each of these channels to vomeronasal neuron firing activity by comparing wild-type and knockout mice. Performing loose-patch recordings from neurons in acute vomeronasal organ slices, we show that spontaneous activity is modified by Tmem16a knockout, indicating that TMEM16A, but not TMEM16B, is active under basal conditions. Upon exposure to diluted urine, a rich source of mouse pheromones, we observe significant changes in activity. Vomeronasal sensory neurons from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals compared to WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs. Significance StatementVomeronasal sensory neurons express two Ca 2+ -activated chloride channels TMEM16A and TMEM16B, however their physiological role is still unclear. Using a loss of function approach, we found that TMEM16A modulates the pattern of VSN spontaneous spike activity, while TMEM16A and TMEM16B reduced the instant frequency of pheromone-evoked activity. These new findings call for a reconsideration of the patterns of the peripheral coding of sensory stimuli.

A new triangle-shaped microfluidic channel system for defined cell trapping is presented. Different variants of the same basic geometry were produced to reveal the best fitting parameter combinations regarding efficiency and sensitivity. Variants with differences in the trap gap width and the inter-trap distance were analyzed in detail by Computational Fluid Dynamics simulations and in experiments with artificial beads of different sizes (30, 60, 80 m). Simulation analysis of flow dynamics and pressure profiles revealed strongly reduced pressure conditions and balanced flow rates inside the microfluidic channels compared to commonly used systems with meandering channels. Quantitative experiments with beads showed very good trapping results in all channel types with slight variations due to geometrical differences. Highest efficiency in terms of fast trap filling and low particle loss was shown with channel types having a larger trap gap width (20m) and/or a larger inter-trap distance (400 m). Here, experimental success was achieved in almost 85% to 100% of all cases. Particle loss appeared significantly more often with large beads than with small beads. A significantly reduced trapping efficiency of about 50% was determined by using narrow trap gaps and a small inter-trap distance in combination with large 80m beads. The combination of the same parameters with small and medium beads led to an only slight decrease in trapping efficiency (80%). All channel types were tested qualitatively with invertebrate neurons from the pond snail . The systems were appropriate to trap those sensitive neurons and to keep their viability in the trapping area at the same time.