A deep learning algorithm for 3D cell detection in whole mouse brain image datasets.

Understanding the function of the nervous system necessitates mapping the spatial distributions of its constituent cells defined by function, anatomy or gene expression. Recently, developments in tissue preparation and microscopy allow cellular populations to be imaged throughout the entire rodent brain. However, mapping these neurons manually is prone to bias and is often impractically time consuming. Here we present an open-source algorithm for fully automated 3D detection of neuronal somata in mouse whole-brain microscopy images using standard desktop computer hardware. We demonstrate the applicability and power of our approach by mapping the brain-wide locations of large populations of cells labeled with cytoplasmic fluorescent proteins expressed via retrograde trans-synaptic viral infection.

Synchronous Infra-Slow Oscillations Organize Ensembles of Accessory Olfactory Bulb Projection Neurons into Distinct Microcircuits.

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 ensembles in 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 STATEMENT Information 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 both in vitro and in vivo Infra-slow synchronous oscillatory activity within AOB microcircuits thus adds a new dimension to chemosensory coding along the accessory olfactory pathway.

Interdependent Conductances Drive Infraslow Intrinsic Rhythmogenesis in a Subset of Accessory Olfactory Bulb Projection Neurons.

The accessory olfactory system controls social and sexual behavior. However, key aspects of sensory signaling along the accessory olfactory pathway remain largely unknown. Here, we investigate patterns of spontaneous neuronal activity in mouse accessory olfactory bulb mitral cells, the direct neural link between vomeronasal sensory input and limbic output. Both in vitro and in vivo, we identify a subpopulation of mitral cells that exhibit slow stereotypical rhythmic discharge. In intrinsically rhythmogenic neurons, these periodic activity patterns are maintained in absence of fast synaptic drive. The physiological mechanism underlying mitral cell autorhythmicity involves cyclic activation of three interdependent ionic conductances: subthreshold persistent Na(+) current, R-type Ca(2+) current, and Ca(2+)-activated big conductance K(+) current. Together, the interplay of these distinct conductances triggers infraslow intrinsic oscillations with remarkable periodicity, a default output state likely to affect sensory processing in limbic circuits. SIGNIFICANCE STATEMENT: We show for the first time that some rodent accessory olfactory bulb mitral cells-the direct link between vomeronasal sensory input and limbic output-are intrinsically rhythmogenic. Driven by ≥ 3 distinct interdependent ionic conductances, infraslow intrinsic oscillations show remarkable periodicity both in vitro and in vivo. As a novel default state, infraslow autorhythmicity is likely to affect limbic processing of pheromonal information.

Extracellular pH regulates excitability of vomeronasal sensory neurons.

The mouse vomeronasal organ (VNO) plays a critical role in semiochemical detection and social communication. Vomeronasal stimuli are typically secreted in various body fluids. Following direct contact with urine deposits or other secretions, a peristaltic vascular pump mediates fluid entry into the recipient's VNO. Therefore, while vomeronasal sensory neurons (VSNs) sample various stimulatory semiochemicals dissolved in the intraluminal mucus, they might also be affected by the general physicochemical properties of the "solvent." Here, we report cycle stage-correlated variations in urinary pH among female mice. Estrus-specific pH decline is observed exclusively in urine samples from sexually experienced females. Moreover, patch-clamp recordings in acute VNO slices reveal that mouse VSNs reliably detect extracellular acidosis. Acid-evoked responses share the biophysical and pharmacological hallmarks of the hyperpolarization-activated current Ih. Mechanistically, VSN acid sensitivity depends on a pH-induced shift in the voltage-dependence of Ih activation that causes the opening of HCN channels at rest, thereby increasing VSN excitability. Together, our results identify extracellular acidification as a potent activator of vomeronasal Ih and suggest HCN channel-dependent vomeronasal gain control of social chemosignaling. Our data thus reveal a potential mechanistic basis for stimulus pH detection in rodent chemosensory communication.

Accurate determination of marker location within whole-brain microscopy images.

High-resolution whole-brain microscopy provides a means for post hoc determination of the location of implanted devices and labelled cell populations that are necessary to interpret in vivo experiments designed to understand brain function. Here we have developed two plugins (brainreg and brainreg-segment) for the Python-based image viewer napari, to accurately map any object in a common coordinate space. We analysed the position of dye-labelled electrode tracks and two-photon imaged cell populations expressing fluorescent proteins. The precise location of probes and cells were physiologically interrogated and revealed accurate segmentation with near-cellular resolution.