Browsing by Author "Wang, Fan"

Ever since the initial discovery of general anesthetics almost 170 years ago, how general anesthesia (GA) induces loss of consciousness remains a century-long mystery. In addition, whether diverse anesthetic drugs and sleep share a common neural pathway is hotly debated and largely unknown. Previous studies have established that many GA drugs inhibit neural activity through targeting GABA receptors. Here, by using Fos staining, ex vivo brain slice recording, and eventually in vivo multichannel extracellular electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of peptidergic neuroendocrine cells, which are surprisingly and persistently activated by multiple classes of GA drugs. Strikingly, chemogenetic or optogenetic stimulation of these anesthesia-activated neurons (AANs) strongly potentiated slow-wave sleep and prolonged GA, whereas conditional ablation through diphtheria toxin receptor strategy or inhibition of AANs with optogenetics led to reduced slow-wave oscillation in the brain, significant loss of slow-wave and rapid-eye movement sleep, and shortened durations under GA. Together, these findings identify a previously unknown common neural substrate underlying diverse GA drugs and natural sleep, and further illustrate a crucial role of the neuroendocrine system in regulating global brain states.

With the unprecedented growth of the E-Commerce market, robotic warehouse automation has attracted much interest and capital investment. Compared to a conventional labor-intensive approach, an automated robot warehouse brings potential benefits such as increased uptime, higher total throughput, and lower accident rates. To date, warehouse automation has mostly developed in inventory mobilization and object picking.

Recently, one area that has attracted a lot of research attention is automated packaging or packing, a process during which robots stow objects into small confined spaces, such as shipping boxes. Automatic item packing is complementary to item picking in warehouse settings. Packing items densely improves the storage capacity, decreases the delivery cost, and saves packing materials. However, it is a demanding manipulation task that has not been thoroughly explored by the research community.

This dissertation focuses on packing objects of arbitrary shapes and weights into a single shipping box with a robot manipulator. I seek to advance the state-of-the-art in robot packing with regards to optimizing container size for a set of objects, planning object placements for stability and feasibility, and increasing robustness of packing execution with a robot manipulator.

The three main innovations presented in this dissertation are:

1. The implementation of a constrained packing planner that outputs stable and collision-free placements of objects when packed with a robot manipulator. Experimental evaluation of the method is conducted with a realistic physical simulator on a dataset of scanned real-world items, demonstrating stable and high-quality packing plans compared with other 3D packing methods.

2. The proposal and implementation of a framework for evaluating the ability to pack a set of known items presented in an unknown order of arrival within a given container size. This allows packing algorithms to work in more realistic warehouse scenarios, as well as provides a means of optimizing container size to ensure successful packing under unknown item arrival order conditions.

3. The systematic evaluation of the proposed planner under real-world uncertainties such as vision, grasping, and modeling errors. To conduct this evaluation, I built a hardware and software packing testbed that is representative of the current state-of-the-art in sensing, perception, and planing. An evaluation of the testbed is then performed to study the error sources and to model their magnitude. Subsequently, robustness measures are proposed to improve the packing success rate under such errors.

Overall, empirical results demonstrate that a success rate of up to 98\% can be achieved by a physical robot despite real-world uncertainties, demonstrating that these contributions have the potential to realize robust, dense automatic object packing.

Human speech is indispensable for effective communication. However, central or peripheral deficits can lead to the permanent loss of this crucial ability, significantly impacting the quality of life. To help develop future therapies to restore vocal communication capability, we first need a precise understanding of the neural circuits underlying the generation of speech. All animals that can vocalize produce vocal sounds through precise neural control of coordinated actions of multiple laryngeal, orofacial, and respiratory muscles. Here, I utilize mouse vocalization as a model system to investigate the brainstem neural circuits and mechanisms controlling laryngeal muscles—the essential components for phonation. First, I mapped the entire laryngeal premotor circuitry in adult mice. Subsequently, I showed that viral-genetically identified excitatory laryngeal premotor neurons located in the Retroambiguus nucleus (RAmVOC) as both necessary and sufficient for driving vocal-cord closure and eliciting mouse ultrasonic vocalizations (USVs). The duration of RAmVOC activation determines the lengths of USV syllables and post-inspiration phases. I further discovered that RAmVOC-neurons receive inhibitory inputs from the respiratory rhythm generator, the preBötzinger complex, and inspiration needs can override RAmVOC-mediated vocal cord closure. Ablating inhibitory synapses in RAmVOC-neurons compromised this inspiration gating of laryngeal adduction, resulting in de-coupling of vocalization and respiration. My dissertation study revealed the hitherto unknown critical circuits for vocal-pattern generation and vocal-respiratory coupling in the brainstem. I further discuss the implications of my work for building neural prosthesis to restore speech in human patients.

The sensation of touch is constructed from multiple types of unique sensory receptors acting in parallel. How the structure of different mechanoreceptors in the hair and skin relates to the functional output of their accompanying neurons remains poorly understood. One challenge in studying this problem is being able to provide natural stimuli to awake, behaving animals while also being able to experimentally isolate these different sensory receptors. We describe how the gene NetrinG-1 is selectively expressed in two specific sensory receptors in the mouse whisker follicle: club-like and lanceolate endings. We then selectively recorded from primary sensory afferents with club-like and lanceolate endings in vivo by photo-identifying them and recorded their activities during behavior. We found that both ending types were rapidly adapting to touch. Most were extremely direction selective, and displayed rapid responses to contact. Their patterns of activity could be well described by modeling the forces at the base of the whisker follicle, and were best explained by firing in response to a combination of lateral, axial, and dynamic forces. This study for the first time definitively links two morphological subtypes of mechanosensory receptors with their electrophysiological responses.

Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis.

We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons.

To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway.

These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.

General anesthesia and anesthetics, in addition to the well-known properties of unconsciousness, amnesia, analgesia, and akinesia, also have anxiolytic properties. Albeit successfully used clinically to treat patients with various anxiety disorders, the common underlying mechanisms that these drugs and compounds rely on to attenuate anxiety largely remain unclear, partially due to the difficulties in investigating neuropsychological disorders in rodent models, and in comprehensively studying both behavioral and physiological aspects of anxiety. Using transgenic mouse models and a variety of histochemistry, behavioral, and physiological recording methods, we aimed to decipher the brain regions and circuit mechanisms of anesthesia-induced anxiolysis. We discovered a unique population of neurons in the mouse ovBNST to be commonly activated by multiple anesthetics and anxiolytics, to innervate brain regions critical for the regulation of emotional and autonomic responses to stressors, and to express peptides and markers previously known to be associated with anxiolysis. We further showed that optogenetic activation of these neurons could attenuate anxiety-like behaviors, while the inhibitions of these neurons had opposite behavioral effects. Lastly, we showed that optogenetic activation of these neurons led to changes in heart rate, heart rate variability, and other bodily reactions involved in stress management. Our study indicates ovBNST to be a potential therapeutic target for anxiolysis.

General anesthesia (GA) can produce analgesia (loss of pain) independent of inducing loss of consciousness, but the underlying mechanisms remain unclear. We hypothesized that GA suppresses pain in part by activating supraspinal analgesic circuits. We discovered a distinct population of GABAergic neurons activated by GA in the mouse central amygdala (CeAGA neurons). In vivo calcium imaging revealed that different GA drugs activate a shared ensemble of CeAGA neurons. CeAGA neurons also possess basal activity that mostly reflect animals’ internal state rather than external stimuli. Optogenetic activation of CeAGA potently suppressed both pain-elicited reflexive and self-recuperating behaviors across sensory modalities, and abolished neuropathic pain-induced mechanical (hyper-)sensitivity. Conversely, inhibition of CeAGA activity exacerbated pain, produced strong aversion, and cancelled the analgesic effect of low-dose ketamine. CeAGA neurons have widespread inhibitory projections to numerous affective pain-processing centers. Our study points to CeAGA as a potential powerful therapeutic target for alleviating chronic pain.

The mouth, throat, and face contain numerous muscles that participate in a large variety of orofacial behaviors. The jaw and tongue can move independently, and thus require a high degree of coordination among the muscles that move them to prevent self-injury. However, different orofacial behaviors require distinct patterns of coordination between these muscles. The method through which motor control circuitry might coordinate this activity has yet to be determined. Electrophysiological, immunohistochemical, and retrograde tracing studies have attempted to identify populations of premotor neurons which directly send information to orofacial motoneurons in an effort to identify sources of coordination. Yet these studies have not provided a complete picture of the population of neurons which monosynaptically connect to jaw and tongue motoneurons. Additionally, while many of these studies have suggested that premotor neurons projecting to multiple motor pools may play a role in coordination of orofacial muscles, no clear functional roles for these neurons in the coordination of natural orofacial movements has been identified.

In this dissertation, I took advantage of the recently developed monosynaptic rabies virus to trace the premotor circuits for the jaw-closing masseter muscle and tongue-protruding genioglossus muscle in the neonatal mouse, uncovering novel premotor inputs in the brainstem. Furthermore, these studies identified a set of neurons which form boutons onto motor neurons in multiple motor pools, providing a premotor substrate for orofacial coordination. I then combined a retrogradely traveling lentivirus with a split-intein mediated split-Cre recombinase system to isolate and manipulate a population of neurons which project to both left and right jaw-closing motor nuclei. I found that these bilaterally projecting neurons also innervate multiple other orofacial motor nuclei, premotor regions, and midbrain regions implicated in motor control. I anatomically and physiologically characterized these neurons and used optogenetic and chemicogenetic approaches to assess their role in natural jaw-closing behavior, specifically with reference to bilateral masseter muscle electromyogram (EMG) activity. These studies identified a population of bilaterally projecting neurons in the supratrigeminal nucleus as essential for maintenance of an appropriate level of masseter activation during natural chewing behavior in the freely moving mouse. Moreover, these studies uncovered two distinct roles of supratrigeminal bilaterally projecting neurons in bilaterally synchronized activation of masseter muscles, and active balancing of bilateral masseter muscle tone against an excitatory input. Together, these studies identify neurons which project to multiple motor nuclei as a mechanism by which the brain coordinates orofacial muscles during natural behavior.

Information about diverse visual features is encoded in distributed visual cortical areas. Which area/areas are required for perception for a given visual feature and how the encoded visual information is computed to guide visual perception are the main questions of this dissertation. Here I take advantage of the mouse visual system to tackle these questions. I first identify mouse visual cortical areas (primary visual cortex (V1) and higher visual areas (HVAs)) that are required for perception of different visual features via a combination of optogenetics and mouse psychophysics. I find that V1, LM (lateromedial) and AL (anterolateral) are required for discriminating orientation and detecting contrast. However, PM (posteromedial) is not involved in the orientation discrimination task. Instead, suppressing PM increases contrast detection threshold and consistently increases false alarm rate in both contrast and speed increment detection tasks. The effects of PM suppression on false alarm rate remain intact even when the visual stimuli are presented outside of the affected visual field, suggesting a non-visual specific role. To understand the computation that transforms sensory encoding to decision choice, I next use visual adaptation as a tool to determine a decoder adopted by the mouse to discriminate orientation (target versus. distractor orientation) via a combination of in vivo calcium imaging and modeling approaches. This decoder sums the neuronal population response suboptimally with higher positive weights biased towards target preferring neurons without negatively weighting the distractor preferring neurons. This decoder scheme could also be used for detecting contrast, serving as a potential reason why similar areas are required for both orientation discrimination and contrast detection tasks. To dissect the sensory and cognitive contribution of each area to the visual tasks, I attempt to use the sensitivity and bias measures from the Signal Detection Theory (SDT). However, both behavioral and neuronal evidence suggests that changes in bias can result from changes in sensory encoding, decision criterion or both, limiting the usage of SDT in dissociating two key components of perceptual decision-making process: sensory encoding versus decision criterion placement. Lastly, since adaptation can induce changes in sensory encoding and thus has profound perceptual consequences, I also apply in vivo extracellular single-unit recording in the mouse visual areas to characterize adaptation profiles. I observe a cascaded increase of adaptation along the geniculate visual pathway and much heterogeneity of adaptation within any recorded visual areas. In all, these serials of studies provide rich neuronal, behavioral and computational evidence to link between sensory encoding and perceptual behaviors.

Pain relief by vibrotactile touch is a common human experience. Previous neurophysiological investigations focused on spinal mechanisms in anesthetized animals. However, whether and how cortex, especially the primary somatosensory cortex (S1), is involved in this process in behaving animals remains unknown. Here I used awake behaving mice to study this touch induced pain relief. First, I discovered that vibrotactile reafferent signals from self-generated whisking significantly reduce facial nociception. Second, I showed that specific blocking of touch transmission from thalamus to barrel cortex (S1B) abolished whisking induced analgesia. Third, I developed a neuron extraction pipeline for 1-photon based calcium imaging and used in vivo imaging to show that tactile and noxious stimuli differentially activate S1B neurons. Lastly, I applied the intrinsic manifold analysis of S1B population activity to reveal that whisking pushes the transition of neural state induced by noxious stimuli towards the state encoding non-nocifensive actions. I concluded with an awake behaving mouse model for studying S1B touch induced pain relief, and that S1B contains nociceptive representations and integrates tactile and painful signals to enable touch mediated pain relief.