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.

Neural Representations of Unconditioned Stimuli in Basolateral Amygdala Mediate Innate and Learned Responses.

Stimuli that possess inherently rewarding or aversive qualities elicit emotional responses and also induce learning by imparting valence upon neutral sensory cues. Evidence has accumulated implicating the amygdala as a critical structure in mediating these processes. We have developed a genetic strategy to identify the representations of rewarding and aversive unconditioned stimuli (USs) in the basolateral amygdala (BLA) and have examined their role in innate and learned responses. Activation of an ensemble of US-responsive cells in the BLA elicits innate physiological and behavioral responses of different valence. Activation of this US ensemble can also reinforce appetitive and aversive learning when paired with differing neutral stimuli. Moreover, we establish that the activation of US-responsive cells in the BLA is necessary for the expression of a conditioned response. Neural representations of conditioned and unconditioned stimuli therefore ultimately connect to US-responsive cells in the BLA to elicit both innate and learned responses.

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.

Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing.

Behavior cannot be predicted from a "connectome" because the brain contains a chemical "map" of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. Here we describe a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms.

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.

Driving opposing behaviors with ensembles of piriform neurons.

Anatomic and physiologic studies have suggested a model in which neurons of the piriform cortex receive convergent input from random collections of glomeruli. In this model, odor representations can only be afforded behavioral significance upon experience. We have devised an experimental strategy that permits us to ask whether the activation of an arbitrarily chosen subpopulation of neurons in piriform cortex can elicit different behavioral responses dependent upon learning. Activation of a small subpopulation of piriform neurons expressing channelrhodopsin at multiple loci in the piriform cortex, when paired with reward or shock, elicits either appetitive or aversive behavior. Moreover, we demonstrate that different subpopulations of piriform neurons expressing ChR2 can be discriminated and independently entrained to elicit distinct behaviors. These observations demonstrate that the piriform cortex is sufficient to elicit learned behavioral outputs in the absence of sensory input. These data imply that the piriform does not use spatial order to map odorant identity or behavioral output.

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.

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.

The participation of cortical amygdala in innate, odour-driven behaviour.

Innate behaviours are observed in naive animals without prior learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically determined and stereotyped. The neural circuits that convey olfactory information from the sense organ to the cortical and subcortical olfactory centres have been anatomically defined, but the specific pathways responsible for innate responses to volatile odours have not been identified. Here we devise genetic strategies that demonstrate that a stereotyped neural circuit that transmits information from the olfactory bulb to cortical amygdala is necessary for innate aversive and appetitive behaviours. Moreover, we use the promoter of the activity-dependent gene arc to express the photosensitive ion channel, channelrhodopsin, in neurons of the cortical amygdala activated by odours that elicit innate behaviours. Optical activation of these neurons leads to appropriate behaviours that recapitulate the responses to innate odours. These data indicate that the cortical amygdala plays a critical role in generating innate odour-driven behaviours but do not preclude its participation in learned olfactory behaviours.

Random convergence of olfactory inputs in the Drosophila mushroom body.

The mushroom body in the fruitfly Drosophila melanogaster is an associative brain centre that translates odour representations into learned behavioural responses. Kenyon cells, the intrinsic neurons of the mushroom body, integrate input from olfactory glomeruli to encode odours as sparse distributed patterns of neural activity. We have developed anatomic tracing techniques to identify the glomerular origin of the inputs that converge onto 200 individual Kenyon cells. Here we show that each Kenyon cell integrates input from a different and apparently random combination of glomeruli. The glomerular inputs to individual Kenyon cells show no discernible organization with respect to their odour tuning, anatomic features or developmental origins. Moreover, different classes of Kenyon cells do not seem to preferentially integrate inputs from specific combinations of glomeruli. This organization of glomerular connections to the mushroom body could allow the fly to contextualize novel sensory experiences, a feature consistent with the role of this brain centre in mediating learned olfactory associations and behaviours.

Distinct representations of olfactory information in different cortical centres.

Sensory information is transmitted to the brain where it must be processed to translate stimulus features into appropriate behavioural output. In the olfactory system, distributed neural activity in the nose is converted into a segregated map in the olfactory bulb. Here we investigate how this ordered representation is transformed in higher olfactory centres in mice. We have developed a tracing strategy to define the neural circuits that convey information from individual glomeruli in the olfactory bulb to the piriform cortex and the cortical amygdala. The spatial order in the bulb is discarded in the piriform cortex; axons from individual glomeruli project diffusely to the piriform without apparent spatial preference. In the cortical amygdala, we observe broad patches of projections that are spatially stereotyped for individual glomeruli. These projections to the amygdala are overlapping and afford the opportunity for spatially localized integration of information from multiple glomeruli. The identification of a distributive pattern of projections to the piriform and stereotyped projections to the amygdala provides an anatomical context for the generation of learned and innate behaviours.

A dimorphic pheromone circuit in Drosophila from sensory input to descending output.

Drosophila show innate olfactory-driven behaviours that are observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically programmed. Despite the numerical simplicity of the fly nervous system, features of the anatomical organization of the fly brain often confound the delineation of these circuits. Here we identify a neural circuit responsive to cVA, a pheromone that elicits sexually dimorphic behaviours. We have combined neural tracing using an improved photoactivatable green fluorescent protein (PA-GFP) with electrophysiology, optical imaging and laser-mediated microlesioning to map this circuit from the activation of sensory neurons in the antennae to the excitation of descending neurons in the ventral nerve cord. This circuit is concise and minimally comprises four neurons, connected by three synapses. Three of these neurons are overtly dimorphic and identify a male-specific neuropil that integrates inputs from multiple sensory systems and sends outputs to the ventral nerve cord. This neural pathway suggests a means by which a single pheromone can elicit different behaviours in the two sexes.

The Drosophila pheromone cVA activates a sexually dimorphic neural circuit.

Courtship is an innate sexually dimorphic behaviour that can be observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate this behaviour are developmentally programmed. In Drosophila, courtship involves a complex yet stereotyped array of dimorphic behaviours that are regulated by Fru(M), a male-specific isoform of the fruitless gene. Fru(M) is expressed in about 2,000 neurons in the fly brain, including three subpopulations of olfactory sensory neurons and projection neurons (PNs). One set of Fru(+) olfactory neurons expresses the odorant receptor Or67d and responds to the male-specific pheromone cis-vaccenyl acetate (cVA). These neurons converge on the DA1 glomerulus in the antennal lobe. In males, activation of Or67d(+) neurons by cVA inhibits courtship of other males, whereas in females their activation promotes receptivity to other males. These observations pose the question of how a single pheromone acting through the same set of sensory neurons can elicit different behaviours in male and female flies. Anatomical or functional dimorphisms in this neural circuit might be responsible for the dimorphic behaviour. We therefore developed a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoactivatable green fluorescent protein. Here we show, using this technique, that the projections from the DA1 glomerulus to the protocerebrum are sexually dimorphic. We observe a male-specific axonal arbor in the lateral horn whose elaboration requires the expression of the transcription factor Fru(M) in DA1 projection neurons and other Fru(+) cells. The observation that cVA activates a sexually dimorphic circuit in the protocerebrum suggests a mechanism by which a single pheromone can elicit different behaviours in males and in females.

Generating sparse and selective third-order responses in the olfactory system of the fly.

In the antennal lobe of Drosophila, information about odors is transferred from olfactory receptor neurons (ORNs) to projection neurons (PNs), which then send axons to neurons in the lateral horn of the protocerebrum (LHNs) and to Kenyon cells (KCs) in the mushroom body. The transformation from ORN to PN responses can be described by a normalization model similar to what has been used in modeling visually responsive neurons. We study the implications of this transformation for the generation of LHN and KC responses under the hypothesis that LHN responses are highly selective and therefore suitable for driving innate behaviors, whereas KCs provide a more general sparse representation of odors suitable for forming learned behavioral associations. Our results indicate that the transformation from ORN to PN firing rates in the antennal lobe equalizes the magnitudes of and decorrelates responses to different odors through feedforward nonlinearities and lateral suppression within the circuitry of the antennal lobe, and we study how these two components affect LHN and KC responses.

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.

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.

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.

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.