Nested neural circuits generate distinct acoustic signals during Drosophila courtship.

Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? Here, we characterize the neuronal circuits that drive the distinct courtship songs of Drosophila melanogaster. Male flies vibrate their wings toward females to produce two different song modes-pulse and sine song-which signal species identity and male quality. Using cell-type-specific genetic reagents and the connectome, we provide a cellular and synaptic map of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. Our data reveal that the song circuit is organized into two nested feedforward pathways with extensive reciprocal and feedback connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility onto a basic movement pattern.

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

The Consistency of Gastropod Identified Neurons Distinguishes Intra-Individual Plasticity From Inter-Individual Variability in Neural Circuits.

Gastropod mollusks are known for their large, individually identifiable neurons, which are amenable to long-term intracellular recordings that can be repeated from animal to animal. The constancy of individual neurons can help distinguish state-dependent or temporal variation within an individual from actual variability between individual animals. Investigations into the circuitry underlying rhythmic swimming movements of the gastropod species, Tritonia exsulans and Pleurobranchaea californica have uncovered intra- and inter-individual variability in synaptic connectivity and serotonergic neuromodulation. Tritonia has a reliably evoked escape swim behavior that is produced by a central pattern generator (CPG) composed of a small number of identifiable neurons. There is apparent individual variability in some of the connections between neurons that is inconsequential for the production of the swim behavior under normal conditions, but determines whether that individual can swim following a neural lesion. Serotonergic neuromodulation of synaptic strength intrinsic to the CPG creates neural circuit plasticity within an individual and contributes to reorganization of the network during recovery from injury and during learning. In Pleurobranchaea, variability over time in the modulatory actions of serotonin and in expression of serotonin receptor genes in an identified neuron directly reflects variation in swimming behavior. Tracking behavior and electrophysiology over hours to days was necessary to identify the functional consequences of these intra-individual, time-dependent variations. This work demonstrates the importance of unambiguous neuron identification, properly assessing the animal and network states, and tracking behavior and physiology over time to distinguish plasticity within the same animal at different times from variability across individual animals.

Neural Evolution of Context-Dependent Fly Song.

It is unclear where in the nervous system evolutionary changes tend to occur. To localize the source of neural evolution that has generated divergent behaviors, we developed a new approach to label and functionally manipulate homologous neurons across Drosophila species. We examined homologous descending neurons that drive courtship song in two species that sing divergent song types and localized relevant evolutionary changes in circuit function downstream of the intrinsic physiology of these descending neurons. This evolutionary change causes different species to produce divergent motor patterns in similar social contexts. Artificial stimulation of these descending neurons drives multiple song types, suggesting that multifunctional properties of song circuits may facilitate rapid evolution of song types.

Threshold-Based Ordering of Sequential Actions during Drosophila Courtship.

Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. Here, we identify a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. We find evidence consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds.

Reconciling the deep homology of neuromodulation with the evolution of behavior.

The evolution of behavior seems inconsistent with the deep homology of neuromodulatory signaling. G protein coupled receptors (GPCRs) evolved slowly from a common ancestor through a process involving gene duplication, neofunctionalization, and loss. Neuropeptides co-evolved with their receptors and exhibit many conserved functions. Furthermore, brain areas are highly conserved with suggestions of deep anatomical homology between arthropods and vertebrates. Yet, behavior evolved more rapidly; even members of the same genus or species can differ in heritable behavior. The solution to the paradox involves changes in the compartmentalization, or subfunctionalization, of neuromodulation; neurons shift their expression of GPCRs and the content of monoamines and neuropeptides. Furthermore, parallel evolution of neuromodulatory signaling systems suggests a route for repeated evolution of similar behaviors.

Parallel evolution of serotonergic neuromodulation underlies independent evolution of rhythmic motor behavior.

Neuromodulation can dynamically alter neuronal and synaptic properties, thereby changing the behavioral output of a neural circuit. It is therefore conceivable that natural selection might act upon neuromodulation as a mechanism for sculpting the behavioral repertoire of a species. Here we report that the presence of neuromodulation is correlated with the production of a behavior that most likely evolved independently in two species: Tritonia diomedea and Pleurobranchaea californica (Mollusca, Gastropoda, Opisthobranchia, Nudipleura). Individuals of both species exhibit escape swimming behaviors consisting of repeated dorsal-ventral whole-body flexions. The central pattern generator (CPG) circuits underlying these behaviors contain homologous identified neurons: DSI and C2 in Tritonia and As and A1 in Pleurobranchaea. Homologs of these neurons also can be found in Hermissenda crassicornis where they are named CPT and C2, respectively. However, members of this species do not exhibit an analogous swimming behavior. In Tritonia and Pleurobranchaea, but not in Hermissenda, the serotonergic DSI homologs modulated the strength of synapses made by C2 homologs. Furthermore, the serotonin receptor antagonist methysergide blocked this neuromodulation and the swimming behavior. Additionally, in Pleurobranchaea, the robustness of swimming correlated with the extent of the synaptic modulation. Finally, injection of serotonin induced the swimming behavior in Tritonia and Pleurobranchaea, but not in Hermissenda. This suggests that the analogous swimming behaviors of Tritonia and Pleurobranchaea share a common dependence on serotonergic neuromodulation. Thus, neuromodulation may provide a mechanism that enables species to acquire analogous behaviors independently using homologous neural circuit components.

Homology and homoplasy of swimming behaviors and neural circuits in the Nudipleura (Mollusca, Gastropoda, Opisthobranchia).

How neural circuit evolution relates to behavioral evolution is not well understood. Here the relationship between neural circuits and behavior is explored with respect to the swimming behaviors of the Nudipleura (Mollusca, Gastropoda, Opithobranchia). Nudipleura is a diverse monophyletic clade of sea slugs among which only a small percentage of species can swim. Swimming falls into a limited number of categories, the most prevalent of which are rhythmic left-right body flexions (LR) and rhythmic dorsal-ventral body flexions (DV). The phylogenetic distribution of these behaviors suggests a high degree of homoplasy. The central pattern generator (CPG) underlying DV swimming has been well characterized in Tritonia diomedea and in Pleurobranchaea californica. The CPG for LR swimming has been elucidated in Melibe leonina and Dendronotus iris, which are more closely related. The CPGs for the categorically distinct DV and LR swimming behaviors consist of nonoverlapping sets of homologous identified neurons, whereas the categorically similar behaviors share some homologous identified neurons, although the exact composition of neurons and synapses in the neural circuits differ. The roles played by homologous identified neurons in categorically distinct behaviors differ. However, homologous identified neurons also play different roles even in the swim CPGs of the two LR swimming species. Individual neurons can be multifunctional within a species. Some of those functions are shared across species, whereas others are not. The pattern of use and reuse of homologous neurons in various forms of swimming and other behaviors further demonstrates that the composition of neural circuits influences the evolution of behaviors.

Physiological state influences the social interactions of two honeybee nest mates.

Physiological state profoundly influences the expression of the behaviour of individuals and can affect social interactions between animals. How physiological state influences food sharing and social behaviour in social insects is poorly understood. Here, we examined the social interactions and food sharing behaviour of honeybees with the aim of developing the honeybee as a model for understanding how an individual's state influences its social interactions. The state of individual honeybees was manipulated by either starving donor bees or feeding them sucrose or low doses of ethanol to examine how a change in hunger or inebriation state affected the social behaviours exhibited by two closely-related nestmates. Using a lab-based assay for measuring individual motor behaviour and social behaviour, we found that behaviours such as antennation, willingness to engage in trophallaxis, and mandible opening were affected by both hunger and ethanol intoxication. Inebriated bees were more likely to exhibit mandible opening, which may represent a form of aggression, than bees fed sucrose alone. However, intoxicated bees were as willing to engage in trophallaxis as the sucrose-fed bees. The effects of ethanol on social behaviors were dose-dependent, with higher doses of ethanol producing larger effects on behaviour. Hungry donor bees, on the other hand, were more likely to engage in begging for food and less likely to antennate and to display mandible opening. We also found that when nestmates received food from donors previously fed ethanol, they began to display evidence of inebriation, indicating that ethanol can be retained in the crop for several hours and that it can be transferred between honeybee nestmates during trophallaxis.

Neurochemical and neuroanatomical identification of central pattern generator neuron homologues in Nudipleura molluscs.

Certain invertebrate neurons can be identified by their behavioral functions. However, evolutionary divergence can cause some species to not display particular behaviors, thereby making it impossible to use physiological characteristics related to those behaviors for identifying homologous neurons across species. Therefore, to understand the neural basis of species-specific behavior, it is necessary to identify homologues using characteristics that are independent of physiology. In the Nudipleura mollusc Tritonia diomedea, Cerebral Neuron 2 (C2) was first described as being a member of the swim central pattern generator (CPG). Here we demonstrate that neurochemical markers, in conjunction with previously known neuroanatomical characteristics, allow C2 to be uniquely identified without the aid of electrophysiological measures. Specifically, C2 had three characteristics that, taken together, identified the neuron: 1) a white cell on the dorsal surface of the cerebral ganglion, 2) an axon that projected to the contralateral pedal ganglion and through the pedal commissure, and 3) immunoreactivity for the peptides FMRFamide and Small Cardioactive Peptide B. These same anatomical and neurochemical characteristics also uniquely identified the C2 homologue in Pleurobranchaea californica (called A1), which was previously identified by its analogous role in the Pleurobranchaea swim CPG. Furthermore, these characteristics were used to identify C2 homologues in Melibe leonina, Hermissenda crassicornis, and Flabellina iodinea, species that are phylogenetically closer to Tritonia than Pleurobranchaea, but do not display the same swimming behavior as Tritonia or Pleurobranchaea. These identifications will allow future studies comparing and contrasting the physiological properties of C2 across species that can and cannot produce the type of swimming behavior exhibited by Tritonia.

Different roles for homologous interneurons in species exhibiting similar rhythmic behaviors.

It is often assumed that similar behaviors in related species are produced by similar neural mechanisms. To test this, we examined the neuronal basis of a simple swimming behavior in two nudibranchs (Mollusca, Opisthobranchia), Melibe leonina and Dendronotus iris. The side-to-side swimming movements of Dendronotus [1] strongly resemble those of Melibe [2, 3]. In Melibe, it was previously shown that the central pattern generator (CPG) for swimming is composed of two bilaterally symmetric pairs of identified interneurons, swim interneuron 1 (Si1) and swim interneuron 2 (Si2), which are electrically coupled ipsilaterally and mutually inhibit both contralateral counterparts [2, 4]. We identified homologs of Si1 and Si2 in Dendronotus. (Henceforth, homologous neurons in each species will be distinguished by the subscripts (Den) and (Mel).) We found that Si2(Den) and Si2(Mel) play similar roles in generating the swim motor pattern. However, unlike Si1(Mel), Si1(Den) was not part of the swim CPG, was not strongly coupled to the ipsilateral Si2(Den), and did not inhibit the contralateral neurons. Thus, species differences exist in the neuronal organization of the swim CPGs despite the similarity of the behaviors. Therefore, similarity in species-typical behavior is not necessarily predictive of common neural mechanisms, even for homologous neurons in closely related species.

Acute ethanol ingestion impairs appetitive olfactory learning and odor discrimination in the honey bee.

Invertebrates are valuable models for increasing our understanding of the effects of ethanol on the nervous system, but most studies on invertebrates and ethanol have focused on the effects of ethanol on locomotor behavior. In this work we investigate the influence of an acute dose of ethanol on appetitive olfactory learning in the honey bee (Apis mellifera), a model system for learning and memory. Adult worker honey bees were fed a range of doses (2.5%, 5%, 10%, or 25%) of ethanol and then conditioned to associate an odor with a sucrose reward using either a simple or differential conditioning paradigm. Consumption of ethanol before conditioning significantly reduced both the rate of acquisition and the asymptotic strength of the association. Honey bees also exhibited a dose dependent reduction in arousal/attention during conditioning. Consumption of ethanol after conditioning did not affect recall 24h later. The observed deficits in acquisition were not due to the affect of ethanol on gustatory sensitivity or motor function. However, honey bees given higher doses of ethanol had difficulty discriminating amongst different odors suggesting that ethanol consumption influences olfactory processing. Taken together, these results demonstrate that an acute dose of ethanol affects appetitive learning and olfactory perception in the honey bee.