Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

The spatial and temporal structure of neural activity across the fly brain.

What are the spatial and temporal scales of brainwide neuronal activity? We used swept, confocally-aligned planar excitation (SCAPE) microscopy to image all cells in a large volume of the brain of adult Drosophila with high spatiotemporal resolution while flies engaged in a variety of spontaneous behaviors. This revealed neural representations of behavior on multiple spatial and temporal scales. The activity of most neurons correlated (or anticorrelated) with running and flailing over timescales that ranged from seconds to a minute. Grooming elicited a weaker global response. Significant residual activity not directly correlated with behavior was high dimensional and reflected the activity of small clusters of spatially organized neurons that may correspond to genetically defined cell types. These clusters participate in the global dynamics, indicating that neural activity reflects a combination of local and broadly distributed components. This suggests that microcircuits with highly specified functions are provided with knowledge of the larger context in which they operate.

A rapid and bidirectional reporter of neural activity reveals neural correlates of social behaviors in Drosophila.

Neural activity is modulated over different timescales encompassing subseconds to hours, reflecting changes in external environment, internal state and behavior. Using Drosophila as a model, we developed a rapid and bidirectional reporter that provides a cellular readout of recent neural activity. This reporter uses nuclear versus cytoplasmic distribution of CREB-regulated transcriptional co-activator (CRTC). Subcellular distribution of GFP-tagged CRTC (CRTC::GFP) bidirectionally changes on the order of minutes and reflects both increases and decreases in neural activity. We established an automated machine-learning-based routine for efficient quantification of reporter signal. Using this reporter, we demonstrate mating-evoked activation and inactivation of modulatory neurons. We further investigated the functional role of the master courtship regulator gene fruitless (fru) and show that fru is necessary to ensure activation of male arousal neurons by female cues. Together, our results establish CRTC::GFP as a bidirectional reporter of recent neural activity suitable for examining neural correlates in behavioral contexts.

A brain atlas for the camouflaging dwarf cuttlefish, Sepia bandensis.

The coleoid cephalopods (cuttlefish, octopus, and squid) are a group of soft-bodied marine mollusks that exhibit an array of interesting biological phenomena, including dynamic camouflage, complex social behaviors, prehensile regenerating arms, and large brains capable of learning, memory, and problem-solving.1,2,3,4,5,6,7,8,9,10 The dwarf cuttlefish, Sepia bandensis, is a promising model cephalopod species due to its small size, substantial egg production, short generation time, and dynamic social and camouflage behaviors.11 Cuttlefish dynamically camouflage to their surroundings by changing the color, pattern, and texture of their skin. Camouflage is optically driven and is achieved by expanding and contracting hundreds of thousands of pigment-filled saccules (chromatophores) in the skin, which are controlled by motor neurons emanating from the brain. We generated a dwarf cuttlefish brain atlas using magnetic resonance imaging (MRI), deep learning, and histology, and we built an interactive web tool (https://www.cuttlebase.org/) to host the data. Guided by observations in other cephalopods,12,13,14,15,16,17,18,19,20 we identified 32 brain lobes, including two large optic lobes (75% the total volume of the brain), chromatophore lobes whose motor neurons directly innervate the chromatophores of the color-changing skin, and a vertical lobe that has been implicated in learning and memory. The brain largely conforms to the anatomy observed in other Sepia species and provides a valuable tool for exploring the neural basis of behavior in the experimentally facile dwarf cuttlefish.

Flexible neural control of transition points within the egg-laying behavioral sequence in Drosophila.

Innate behaviors are frequently comprised of ordered sequences of component actions that progress to satisfy essential drives. Progression is governed by specialized sensory cues that induce transitions between components within the appropriate context. Here we have characterized the structure of the egg-laying behavioral sequence in Drosophila and found significant variability in the transitions between component actions that affords the organism an adaptive flexibility. We identified distinct classes of interoceptive and exteroceptive sensory neurons that control the timing and direction of transitions between the terminal components of the sequence. We also identified a pair of motor neurons that enact the final transition to egg expulsion. These results provide a logic for the organization of innate behavior in which sensory information processed at critical junctures allows for flexible adjustments in component actions to satisfy drives across varied internal and external environments.

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies' (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Individual bitter-sensing neurons in Drosophila exhibit both ON and OFF responses that influence synaptic plasticity.

The brain generates internal representations that translate sensory stimuli into appropriate behavior. In the taste system, different tastes activate distinct populations of sensory neurons. We investigated the temporal properties of taste responses in Drosophila and discovered that different types of taste sensory neurons show striking differences in their response dynamics. Strong responses to stimulus onset (ON responses) and offset (OFF responses) were observed in bitter-sensing neurons in the labellum, whereas bitter neurons in the leg and other classes of labellar taste neurons showed only an ON response. Individual labellar bitter neurons generate both ON and OFF responses through a cell-intrinsic mechanism that requires canonical bitter receptors. A single receptor complex likely generates both ON and OFF responses to a given bitter ligand. These ON and OFF responses in the periphery are propagated to dopaminergic neurons that mediate aversive learning, and the presence of the OFF response impacts synaptic plasticity when bitter is used as a reinforcement cue. These studies reveal previously unknown features of taste responses that impact neural circuit function and may be important for behavior. Moreover, these studies show that OFF responses can dramatically influence timing-based synaptic plasticity, which is thought to underlie associative learning.

Evolving the olfactory system with machine learning.

The convergent evolution of the fly and mouse olfactory system led us to ask whether the anatomic connectivity and functional logic of olfactory circuits would evolve in artificial neural networks trained to perform olfactory tasks. Artificial networks trained to classify odor identity recapitulate the connectivity inherent in the olfactory system. Input units are driven by a single receptor type, and units driven by the same receptor converge to form a glomerulus. Glomeruli exhibit sparse, unstructured connectivity onto a larger expansion layer of Kenyon cells. When trained to both classify odor identity and to impart innate valence onto odors, the network develops independent pathways for identity and valence classification. Thus, the defining features of fly and mouse olfactory systems also evolved in artificial neural networks trained to perform olfactory tasks. This implies that convergent evolution reflects an underlying logic rather than shared developmental principles.

Olfactory landmarks and path integration converge to form a cognitive spatial map.

The convergence of internal path integration and external sensory landmarks generates a cognitive spatial map in the hippocampus. We studied how localized odor cues are recognized as landmarks by recording the activity of neurons in CA1 during a virtual navigation task. We found that odor cues enriched place cell representations, dramatically improving navigation. Presentation of the same odor at different locations generated distinct place cell representations. An odor cue at a proximal location enhanced the local place cell density and also led to the formation of place cells beyond the cue. This resulted in the recognition of a second, more distal odor cue as a distinct landmark, suggesting an iterative mechanism for extending spatial representations into unknown territory. Our results establish that odors can serve as landmarks, motivating a model in which path integration and odor landmarks interact sequentially and iteratively to generate cognitive spatial maps over long distances.

Representational drift in primary olfactory cortex.

Perceptual constancy requires the brain to maintain a stable representation of sensory input. In the olfactory system, activity in primary olfactory cortex (piriform cortex) is thought to determine odour identity1-5. Here we present the results of electrophysiological recordings of single units maintained over weeks to examine the stability of odour-evoked responses in mouse piriform cortex. Although activity in piriform cortex could be used to discriminate between odorants at any moment in time, odour-evoked responses drifted over periods of days to weeks. The performance of a linear classifier trained on the first recording day approached chance levels after 32 days. Fear conditioning did not stabilize odour-evoked responses. Daily exposure to the same odorant slowed the rate of drift, but when exposure was halted the rate increased again. This demonstration of continuous drift poses the question of the role of piriform cortex in odour perception. This instability might reflect the unstructured connectivity of piriform cortex6-12, and may be a property of other unstructured cortices.

Embryonic development of the camouflaging dwarf cuttlefish, Sepia bandensis.

BACKGROUND: The dwarf cuttlefish Sepia bandensis, a camouflaging cephalopod from the Indo-Pacific, is a promising new model organism for neuroscience, developmental biology, and evolutionary studies. Cuttlefish dynamically camouflage to their surroundings by altering the color, pattern, and texture of their skin. The skin's "pixels" (chromatophores) are controlled by motor neurons projecting from the brain. Thus, camouflage is a visible representation of neural activity. In addition to camouflage, the dwarf cuttlefish uses dynamic skin patterns for social communication. Despite more than 500 million years of evolutionary separation, cuttlefish and vertebrates converged to form limbs, camera-type eyes and a closed circulatory system. Moreover, cuttlefish have a striking ability to regenerate their limbs. Interrogation of these unique biological features will benefit from the development of a new set of tools. Dwarf cuttlefish reach sexual maturity in 4 months, they lay dozens of eggs over their 9-month lifespan, and the embryos develop to hatching in 1 month. RESULTS: Here, we describe methods to culture dwarf cuttlefish embryos in vitro and define 25 stages of cuttlefish development. CONCLUSION: This staging series serves as a foundation for future technologies that can be used to address a myriad of developmental, neurobiological, and evolutionary questions.

Synaptic Organization of Anterior Olfactory Nucleus Inputs to Piriform Cortex.

Odors activate distributed ensembles of neurons within the piriform cortex, forming cortical representations of odor thought to be essential to olfactory learning and behaviors. This odor response is driven by direct input from the olfactory bulb, but is also shaped by a dense network of associative or intracortical inputs to piriform, which may enhance or constrain the cortical odor representation. With optogenetic techniques, it is possible to functionally isolate defined inputs to piriform cortex and assess their potential to activate or inhibit piriform pyramidal neurons. The anterior olfactory nucleus (AON) receives direct input from the olfactory bulb and sends an associative projection to piriform cortex that has potential roles in the state-dependent processing of olfactory behaviors. Here, we provide a detailed functional assessment of the AON afferents to piriform in male and female C57Bl/6J mice. We confirm that the AON forms glutamatergic excitatory synapses onto piriform pyramidal neurons; and while these inputs are not as strong as piriform recurrent collaterals, they are less constrained by disynaptic inhibition. Moreover, AON-to-piriform synapses contain a substantial NMDAR-mediated current that prolongs the synaptic response at depolarized potentials. These properties of limited inhibition and slow NMDAR-mediated currents result in strong temporal summation of AON inputs within piriform pyramidal neurons, and suggest that the AON could powerfully enhance activation of piriform neurons in response to odor.SIGNIFICANCE STATEMENT Odor information is transmitted from olfactory receptors to olfactory bulb, and then to piriform cortex, where ensembles of activated neurons form neural representations of the odor. While these ensembles are driven by primary bulbar afferents, and shaped by intracortical recurrent connections, the potential for another early olfactory area, the anterior olfactory nucleus (AON), to contribute to piriform activity is not known. Here, we use optogenetic circuit-mapping methods to demonstrate that AON inputs can significantly activate piriform neurons, as they are coupled to NMDAR currents and to relatively modest disynaptic inhibition. The AON may enhance the piriform odor response, encouraging further study to determine the states or behaviors through which AON potentiates the cortical response to odor.

Transient and Persistent Representations of Odor Value in Prefrontal Cortex.

The representation of odor in olfactory cortex (piriform) is distributive and unstructured and can only be afforded behavioral significance upon learning. We performed 2-photon imaging to examine the representation of odors in piriform and in two downstream areas, the orbitofrontal cortex (OFC) and the medial prefrontal cortex (mPFC), as mice learned olfactory associations. In piriform, we observed that odor responses were largely unchanged during learning. In OFC, 30% of the neurons acquired robust responses to conditioned stimuli (CS+) after learning, and these responses were gated by internal state and task context. Moreover, direct projections from piriform to OFC can be entrained to elicit learned olfactory behavior. CS+ responses in OFC diminished with continued training, whereas persistent representations of both CS+ and CS- odors emerged in mPFC. Optogenetic silencing indicates that these two brain structures function sequentially to consolidate the learning of appetitive associations.

Context-Dependent Decision Making in a Premotor Circuit.

Cognitive capacities afford contingent associations between sensory information and behavioral responses. We studied this problem using an olfactory delayed match to sample task whereby a sample odor specifies the association between a subsequent test odor and rewarding action. Multi-neuron recordings revealed representations of the sample and test odors in olfactory sensory and association cortex, which were sufficient to identify the test odor as match or non-match. Yet, inactivation of a downstream premotor area (ALM), but not orbitofrontal cortex, confined to the epoch preceding the test odor led to gross impairment. Olfactory decisions that were not context-dependent were unimpaired. Therefore, ALM does not receive the outcome of a match/non-match decision from upstream areas. It receives contextual information-the identity of the sample-to establish the mapping between test odor and action. A novel population of pyramidal neurons in ALM layer 2 may mediate this process.

The pheromone darcin drives a circuit for innate and reinforced behaviours.

Organisms have evolved diverse behavioural strategies that enhance the likelihood of encountering and assessing mates1. Many species use pheromones to communicate information about the location, sexual and social status of potential partners2. In mice, the major urinary protein darcin-which is present in the urine of males-provides a component of a scent mark that elicits approach by females and drives learning3,4. Here we show that darcin elicits a complex and variable behavioural repertoire that consists of attraction, ultrasonic vocalization and urinary scent marking, and also serves as a reinforcer in learning paradigms. We identify a genetically determined circuit-extending from the accessory olfactory bulb to the posterior medial amygdala-that is necessary for all behavioural responses to darcin. Moreover, optical activation of darcin-responsive neurons in the medial amygdala induces both the innate and the conditioned behaviours elicited by the pheromone. These neurons define a topographically segregated population that expresses neuronal nitric oxide synthase. We suggest that this darcin-activated neural circuit integrates pheromonal information with internal state to elicit both variable innate behaviours and reinforced behaviours that may promote mate encounters and mate selection.

Acetic acid activates distinct taste pathways in Drosophila to elicit opposing, state-dependent feeding responses.

Taste circuits are genetically determined to elicit an innate appetitive or aversive response, ensuring that animals consume nutritious foods and avoid the ingestion of toxins. We have examined the response of Drosophila melanogaster to acetic acid, a tastant that can be a metabolic resource but can also be toxic to the fly. Our data reveal that flies accommodate these conflicting attributes of acetic acid by virtue of a hunger-dependent switch in their behavioral response to this stimulus. Fed flies show taste aversion to acetic acid, whereas starved flies show a robust appetitive response. These opposing responses are mediated by two different classes of taste neurons, the sugar- and bitter-sensing neurons. Hunger shifts the behavioral response from aversion to attraction by enhancing the appetitive sugar pathway as well as suppressing the aversive bitter pathway. Thus a single tastant can drive opposing behaviors by activating distinct taste pathways modulated by internal state.

A virtual burrow assay for head-fixed mice measures habituation, discrimination, exploration and avoidance without training.

We have designed an assay that measures approach and avoidance behaviors in head-fixed mice at millisecond timescale, is compatible with standard electrophysiological and optical methods for measuring neuronal activity, and requires no training. The Virtual Burrow Assay simulates a scenario in which a mouse, poised at the threshold of its burrow, evaluates whether to exit the enclosure or to retreat inside. The assay provides a sensitive readout of habituation, discrimination and exploration, as well as avoidance of both conditioned and innately aversive cues.

Odor Perception on the Two Sides of the Brain: Consistency Despite Randomness.

Neurons in piriform cortex receive input from a random collection of glomeruli, resulting in odor representations that lack the stereotypic organization of the olfactory bulb. We have performed in vivo optical imaging and mathematical modeling to demonstrate that correlations are retained in the transformation from bulb to piriform cortex, a feature essential for generalization across odors. Random connectivity also implies that the piriform representation of a given odor will differ among different individuals and across brain hemispheres in a single individual. We show that these different representations can nevertheless support consistent agreement about odor quality across a range of odors. Our model also demonstrates that, whereas odor discrimination and categorization require far fewer neurons than reside in piriform cortex, consistent generalization may require the full complement of piriform neurons.

Representations of Novelty and Familiarity in a Mushroom Body Compartment.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.

Optimal Degrees of Synaptic Connectivity.

Synaptic connectivity varies widely across neuronal types. Cerebellar granule cells receive five orders of magnitude fewer inputs than the Purkinje cells they innervate, and cerebellum-like circuits, including the insect mushroom body, also exhibit large divergences in connectivity. In contrast, the number of inputs per neuron in cerebral cortex is more uniform and large. We investigate how the dimension of a representation formed by a population of neurons depends on how many inputs each neuron receives and what this implies for learning associations. Our theory predicts that the dimensions of the cerebellar granule-cell and Drosophila Kenyon-cell representations are maximized at degrees of synaptic connectivity that match those observed anatomically, showing that sparse connectivity is sometimes superior to dense connectivity. When input synapses are subject to supervised plasticity, however, dense wiring becomes advantageous, suggesting that the type of plasticity exhibited by a set of synapses is a major determinant of connection density.