CellBoost: A pipeline for machine assisted annotation in Neuroanatomy.

One of the important yet labor intensive tasks in neuroanatomy is the identification of select populations of cells. Current high-throughput techniques enable marking cells with histochemical fluorescent molecules as well as through the genetic expression of fluorescent proteins. Modern scanning microscopes allow high resolution multi-channel imaging of the mechanically or optically sectioned brain with thousands of marked cells per square millimeter. Manual identification of all marked cells is prohibitively time consuming. At the same time, simple segmentation algorithms suffer from high error rates and sensitivity to variation in fluorescent intensity and spatial distribution. We present a methodology that combines human judgement and machine learning that serves to significantly reduce the labor of the anatomist while improving the consistency of the annotation. As a demonstration, we analyzed murine brains with marked premotor neurons in the brainstem. We compared the error rate of our method to the disagreement rate among human anatomists. This comparison shows that our method can reduce the time to annotate by as much as ten-fold without significantly increasing the rate of errors. We show that our method achieves significant reduction in labor while achieving an accuracy that is similar to the level of agreement between different anatomists.

A lone spike in blood glucose can enhance the thrombo-inflammatory response in cortical venules.

How transient hyperglycemia contributes to cerebro-vascular disease has been a challenge to study under controlled physiological conditions. We use amplified, ultrashort laser-pulses to physically disrupt brain-venule endothelium at targeted locations. This vessel disruption is performed in conjunction with transient hyperglycemia from a single injection of metabolically active D-glucose into healthy mice. The observed real-time responses to laser-induced disruption include rapid serum extravasation, platelet aggregation, and neutrophil recruitment. Thrombo-inflammation is pharmacologically ameliorated by a platelet inhibitor, by a scavenger of reactive oxygen species, and by a nitric oxide donor. As a control, vessel thrombo-inflammation is significantly reduced in mice injected with metabolically inert L-glucose. Venules in mice with diabetes show a similar response to laser-induced disruption and damage is reduced by restoration of normo-glycemia. Our approach provides a controlled method to probe synergies between transient metabolic and physical vascular perturbations and can reveal new aspects of brain pathophysiology.

Construction and use of an adaptive optics two-photon microscope with direct wavefront sensing.

Two-photon microscopy, combined with the appropriate optical labelling, enables the measurement and tracking of submicrometer structures within brain cells, as well as the spatiotemporal mapping of spikes in individual neurons and of neurotransmitter release in individual synapses. Yet, the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths of more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300-400 µm of the neocortex. To obviate this limitation, we shape the focal volume, generated by the excitation beam, by modulating the incident wavefront via guidestar-assisted adaptive optics. Here, we describe the construction, calibration and operation of a two-photon microscope that incorporates adaptive optics to restore diffraction-limited resolution at depths close to 900 µm in the mouse cortex. Our setup detects a guidestar formed by the excitation of a red-shifted dye in blood serum, used to directly measure the wavefront. We incorporate predominantly commercially available optical, optomechanical, mechanical and electronic components, and supply computer-aided design models of other customized components. The resulting adaptive optics two-photon microscope is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in the mouse neocortex by imaging the morphology of somatostatin-expressing neurons that lie 700 µm beneath the pia, calcium dynamics of layer 5b projection neurons and thalamocortical glutamate transmission to L4 neurons. The protocol requires ~30 d to complete and is suitable for users with graduate-level expertise in optics.

Elephant trunks: Strength and dexterity from mini-fascicles.

Muscular hydrostats, such as the elephant trunk, can perform precise motor actions. A new study has revealed that the elephant trunk contains a dense network of tiny muscle fascicles, suggesting that muscle miniaturization may be a key toward understanding how soft organs achieve both strength and dexterity.

Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

Low- and high-level coordination of orofacial motor actions.

Orofacial motor actions are movements that, in rodents, involve whisking of the vibrissa, deflection of the nose, licking and lapping with the tongue, and consumption through chewing. These actions, along with bobbing and turning of the head, coordinate to subserve exploration while not conflicting with life-supporting actions such as breathing and swallowing. Orofacial and head movements are comprised of two additive components: a rhythm that can be entrained by the breathing oscillator and a broadband component that directs the actuator to the region of interest. We focus on coordinating the rhythmic component of actions into a behavior. We hypothesize that the precise timing of each constituent action is continually adjusted through the merging of low-level oscillator input with sensory-derived, high-level rhythmic feedback. Supporting evidence is discussed.

A brainstem circuit for the expression of defensive facial reactions in rat.

The brainstem houses neuronal circuits that control homeostasis of vital functions. These include the depth and rate of breathing1,2 and, critically, apnea, a transient cessation of breathing that prevents noxious vapors from entering further into the respiratory tract. Current thinking is that this reflex is mediated by two sensory pathways. One known pathway involves vagal and glossopharyngeal afferents that project to the nucleus of the solitary tract.3,4,5 Yet, apnea induced by electrical stimulation of the nasal epithelium or delivery of ammonia vapors to the nose persists after brainstem transection at the pontomedullary junction, indicating that the circuitry that mediates this reflex is intrinsic to the medulla.6 A second potential pathway, consistent with this observation, involves trigeminal afferents from the nasal cavity that project to the muralis subnucleus of the spinal trigeminal complex.7,8 Notably, the apneic reflex is not dependent on olfaction as it can be initiated even after disruption of olfactory pathways.9 We investigated how subnucleus muralis cells mediate apnea in rat. By means of electrophysiological recordings and lesions in anesthetized rats, we identified a pathway from chemosensors in the nostrils through the muralis subnucleus and onto both the preBötzinger and facial motor nuclei. We then monitored breathing and orofacial reactions upon ammonia delivery near the nostril of alert, head-restrained rats. The apneic reaction was associated with a grimace, characterized by vibrissa protraction, wrinkling of the nose, and squinting of the eyes. Our results show that a brainstem circuit can control facial expressions for nocifensive and potentially pain-inducing stimuli.

Interchangeable Role of Motor Cortex and Reafference for the Stable Execution of an Orofacial Action.

Animals interact with their environment through mechanically active, mobile sensors. The efficient use of these sensory organs implies the ability to track their position; otherwise, perceptual stability or prehension would be profoundly impeded. The nervous system may keep track of the position of a sensorimotor organ via two complementary feedback mechanisms-peripheral reafference (external, sensory feedback) and efference copy (internal feedback). Yet, the potential contributions of these mechanisms remain largely unexplored. By training male rats to place one of their vibrissae within a predetermined angular range without contact, a task that depends on knowledge of vibrissa position relative to their face, we found that peripheral reafference is not required. The presence of motor cortex is not required either, except in the absence of peripheral reafference to maintain motor stability. Finally, the red nucleus, which receives descending inputs from motor cortex and cerebellum and projects to facial motoneurons, is critically involved in the execution of the vibrissa positioning task. All told, our results point toward the existence of an internal model that requires either peripheral reafference or motor cortex to optimally drive voluntary motion.SIGNIFICANCE STATEMENT How does an animal know where a mechanically active, mobile sensor lies relative to its body? We address this basic question in sensorimotor integration using the motion of the vibrissae in rats. We show that rats can learn to reliably position their vibrissae in the absence of sensory feedback or in the absence of motor cortex. Yet, when both sensory feedback and motor cortex are absent, motor precision is degraded. This suggests the existence of an internal model able to operate in closed- and open-loop modes, requiring either motor cortex or sensory feedback to maintain motor stability.

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

A change in behavioral state switches the pattern of motor output that underlies rhythmic head and orofacial movements.

The breathing rhythm serves as a reference that paces orofacial motor actions and orchestrates active sensing. Past work has reported that pacing occurs solely at a fixed phase relative to sniffing. We re-evaluated this constraint as a function of exploratory behavior. Allocentric and egocentric rotations of the head and the electromyogenic activity of the motoneurons for head and orofacial movements were recorded in free-ranging rats as they searched for food. We found that a change in state from foraging to rearing is accompanied by a large phase shift in muscular activation relative to sniffing, and a concurrent change in the frequency of sniffing, so that pacing now occurs at one of the two phases. Further, head turning is biased such that an animal gathers a novel sample of its environment upon inhalation. In total, the coordination of active sensing has a previously unrealized computational complexity. This can emerge from hindbrain circuits with fixed architecture and credible synaptic time delays.

Guide to the construction and use of an adaptive optics two-photon microscope with direct wavefront sensing.

Two-photon microscopy, combined with appropriate optical labeling, has enabled the study of structure and function throughout nervous systems. This methodology enables, for example, the measurement and tracking of sub-micrometer structures within brain cells, the spatio-temporal mapping of spikes in individual neurons, and the spatio-temporal mapping of transmitter release in individual synapses. Yet the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300 to 400 µm of neocortex. To obviate this limitation, we measure the wavefront at the focus of the excitation beam and utilize adaptive optics that alters the incident wavefront to achieve an improved focal volume. We describe the constructions, calibration, and operation of a two-photon microscopy that incorporates adaptive optics to restore diffraction-limited resolution throughout the nearly 900 µm depth of mouse cortex. Our realization utilizes a guide star formed by excitation of red-shifted dye within the blood serum to directly measure the wavefront. We incorporate predominantly commercial optical, optomechanical, mechanical, and electronic components; computer aided design models of the exceptional custom components are supplied. The design is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in mouse neocortex by imaging the morphology of somatostatin-expressing neurons at 700 µm beneath the pia, calcium dynamics of layer 5b projection neurons, and glutamate transmission to L4 neurons.

Correction: Assessment of single-vessel cerebral blood velocity by phase contrast fMRI.

[This corrects the article DOI: 10.1371/journal.pbio.3000923.].

Theory of hierarchically organized neuronal oscillator dynamics that mediate rodent rhythmic whisking.

Rodents explore their environment through coordinated orofacial motor actions, including whisking. Whisking can free-run via an oscillator of inhibitory neurons in the medulla and can be paced by breathing. Yet, the mechanics of the whisking oscillator and its interaction with breathing remain to be understood. We formulate and solve a hierarchical model of the whisking circuit. The first whisk within a breathing cycle is generated by inhalation, which resets a vibrissa oscillator circuit, while subsequent whisks are derived from the oscillator circuit. Our model posits, consistent with experiment, that there are two subpopulations of oscillator neurons. Stronger connections between the subpopulations support rhythmicity, while connections within each subpopulation induce variable spike timing that enhances the dynamic range of rhythm generation. Calculated cycle-to-cycle changes in whisking are consistent with experiment. Our model provides a computational framework to support longstanding observations of concurrent autonomous and driven rhythmic motor actions that comprise behaviors.

David Kleinfeld.

Interview with David Kleinfeld, who leads the Neurophysics laboratory at UC San Diego.

The whisking oscillator circuit.

Central oscillators are primordial neural circuits that generate and control rhythmic movements1,2. Mechanistic understanding of these circuits requires genetic identification of the oscillator neurons and their synaptic connections to enable targeted electrophysiological recording and causal manipulation during behaviours. However, such targeting remains a challenge with mammalian systems. Here we delimit the oscillator circuit that drives rhythmic whisking-a motor action that is central to foraging and active sensing in rodents3,4. We found that the whisking oscillator consists of parvalbumin-expressing inhibitory neurons located in the vibrissa intermediate reticular nucleus (vIRtPV) in the brainstem. vIRtPV neurons receive descending excitatory inputs and form recurrent inhibitory connections among themselves. Silencing vIRtPV neurons eliminated rhythmic whisking and resulted in sustained vibrissae protraction. In vivo recording of opto-tagged vIRtPV neurons in awake mice showed that these cells spike tonically when animals are at rest, and transition to rhythmic bursting at the onset of whisking, suggesting that rhythm generation is probably the result of network dynamics, as opposed to intrinsic cellular properties. Notably, ablating inhibitory synaptic inputs to vIRtPV neurons quenched their rhythmic bursting, impaired the tonic-to-bursting transition and abolished regular whisking. Thus, the whisking oscillator is an all-inhibitory network and recurrent synaptic inhibition has a key role in its rhythmogenesis.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 µm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

A vibrissa pathway that activates the limbic system.

Vibrissa sensory inputs play a central role in driving rodent behavior. These inputs transit through the sensory trigeminal nuclei, which give rise to the ascending lemniscal and paralemniscal pathways. While lemniscal projections are somatotopically mapped from brainstem to cortex, those of the paralemniscal pathway are more widely distributed. Yet the extent and topography of paralemniscal projections are unknown, along with the potential role of these projections in controlling behavior. Here, we used viral tracers to map paralemniscal projections. We find that this pathway broadcasts vibrissa-based sensory signals to brainstem regions that are involved in the regulation of autonomic functions and to forebrain regions that are involved in the expression of emotional reactions. We further provide evidence that GABAergic cells of the Kölliker-Fuse nucleus gate trigeminal sensory input in the paralemniscal pathway via a mechanism of presynaptic or extrasynaptic inhibition.

The Global Configuration of Visual Stimuli Alters Co-Fluctuations of Cross-Hemispheric Human Brain Activity.

We tested how a stimulus gestalt, defined by the neuronal interaction between local and global features of a stimulus, is represented within human primary visual cortex (V1). We used high-resolution fMRI, which serves as a surrogate of neuronal activation, to measure co-fluctuations within subregions of V1 as (male and female) subjects were presented with peripheral stimuli, each with different global configurations. We found stronger cross-hemisphere correlations when fine-scale V1 cortical subregions represented parts of the same object compared with different objects. This result was consistent with the vertical bias in global processing and, critically, was independent of the task and local discontinuities within objects. Thus, despite the relatively small receptive fields of neurons within V1, global stimulus configuration affects neuronal processing via correlated fluctuations between regions that represent different sectors of the visual field.SIGNIFICANCE STATEMENT We provide the first evidence for the impact of global stimulus configuration on cross-hemispheric fMRI fluctuations, measured in human primary visual cortex. Our results are consistent with changes in the level of γ-band synchrony, which has been shown to be affected by global stimulus configuration, being reflected in the level fMRI co-fluctuations. These data help narrow the gap between knowledge of global stimulus configuration encoding at the single-neuron level versus at the behavioral level.

Contribution of animal models toward understanding resting state functional connectivity.

Functional connectivity, which reflects the spatial and temporal organization of intrinsic activity throughout the brain, is one of the most studied measures in human neuroimaging research. The noninvasive acquisition of resting state functional magnetic resonance imaging (rs-fMRI) allows the characterization of features designated as functional networks, functional connectivity gradients, and time-varying activity patterns that provide insight into the intrinsic functional organization of the brain and potential alterations related to brain dysfunction. Functional connectivity, hence, captures dimensions of the brain's activity that have enormous potential for both clinical and preclinical research. However, the mechanisms underlying functional connectivity have yet to be fully characterized, hindering interpretation of rs-fMRI studies. As in other branches of neuroscience, the identification of the neurophysiological processes that contribute to functional connectivity largely depends on research conducted on laboratory animals, which provide a platform where specific, multi-dimensional investigations that involve invasive measurements can be carried out. These highly controlled experiments facilitate the interpretation of the temporal correlations observed across the brain. Indeed, information obtained from animal experimentation to date is the basis for our current understanding of the underlying basis for functional brain connectivity. This review presents a compendium of some of the most critical advances in the field based on the efforts made by the animal neuroimaging community.