Head removal enhances planarian electrotaxis.

Certain animal species utilize electric fields for communication, hunting and spatial orientation. Freshwater planarians move toward the cathode in a static electric field (cathodic electrotaxis). This planarian behavior was first described by Raymond Pearl more than a century ago. However, planarian electrotaxis has received little attention since, and the underlying mechanisms and evolutionary significance remain unknown. To close this knowledge gap, we developed an apparatus and scoring metrics for automated quantitative and mechanistic studies of planarian behavior upon exposure to a static electric field. Using this automated setup, we characterized electrotaxis in the planarian Dugesia japonica and found that this species responds to voltage instead of current, in contrast to results from previous studies using other planarian species. Surprisingly, we found differences in electrotaxis ability between small (shorter) and large (longer) planarians. To determine the cause of these differences, we took advantage of the regenerative abilities of planarians and compared electrotaxis in head, tail and trunk fragments of various lengths. We found that tail and trunk fragments electrotaxed, whereas head fragments did not, regardless of size. Based on these data, we hypothesized that signals from the head may interfere with electrotaxis when the head area/body area reached a critical threshold. In support of this hypothesis, we found that (1) smaller intact planarians that cannot electrotax have a relatively larger head-to-body-ratio than large planarians that can electrotax, and (2) the electrotaxis behavior of cut head fragments was negatively correlated with the head-to-body ratio of the fragments. Moreover, we could restore cathodic electrotaxis in head fragments via decapitation, directly demonstrating inhibition of electrotaxis by the head.

Planarian fragments behave as whole animals.

Behavioral responses of freshwater planarians have been studied for over a century.1 In recent decades, behavior has been used as a readout to study planarian development and regeneration,2-6 wound healing,7,8 molecular evolution,4,9,10 neurotoxicology,11-13 and learning and memory.14-17The planarian nervous system is among the simplest of the bilaterally symmetric animals,18 with an anterior brain attached to two ventral nerve cords interconnected by multiple commissures. We found that, in response to mechanical and near-UV stimulation, head stimulation produces turning, tail stimulation produces contraction, and trunk stimulation produces midbody elongation in the planarian Dugesia japonica. When cut into two or three pieces, the anterior end of each headless piece switched its behavior to turning instead of elongation; i.e., it responded as though it were the head. In addition, posterior ends of the head and midbody pieces sometimes produced contraction instead of elongation. Thus, each severed piece acts like an intact animal, with each midbody region having nearly complete behavioral capabilities. These observations show that each midbody region reads the global state of the organism and adapts its response to incoming signals from the remaining tissue. Selective lateral incisions showed that the changes in behavior are not due to nonselective pain responses and that the ventral nerve cords and cross-connectives are responsible for coordinating local behaviors. Our findings highlight a fast functional reorganization of the planarian nervous system that complements the slower repairs provided by regeneration. This reorganization provides needed behavioral responses for survival as regeneration proceeds.

Thermal tolerance and routine oxygen consumption of convict cichlid, Archocentrus nigrofasciatus, acclimated to constant temperatures (20 °C and 30 °C) and a daily temperature cycle (20 °C → 30 °C).

Organismal temperature tolerance and metabolic responses are correlated to recent thermal history, but responses to thermal variability are less frequently assessed. There is great interest in whether organisms that experience greater thermal variability can gain metabolic or tolerance advantages through phenotypic plasticity. We compared thermal tolerance and routine aerobic metabolism of Convict cichlid acclimated for 2 weeks to constant 20 °C, constant 30 °C, or a daily cycle of 20 → 30 °C (1.7 °C/h). Acute routine mass-specific oxygen consumption ([Formula: see text]O2) and critical thermal maxima/minima (CTMax/CTMin) were compared between groups, with cycle-acclimated fish sampled from the daily minimum (20 °C, 0900 h) and maximum (30 °C, 1600 h). Cycle-acclimated fish demonstrated statistically similar CTMax at the daily minimum and maximum (39.0 °C, 38.6 °C) but distinct CTMin values, with CTMin 2.4 °C higher for fish sampled from the daily 30 °C maximum (14.8 °C) compared to the daily 20 °C minimum (12.4 °C). Measured acutely at 30 °C, [Formula: see text]O2 decreased with increasing acclimation temperature; 20 °C acclimated fish had an 85% higher average [Formula: see text]O2 than 30 °C acclimated fish. Similarly, acute [Formula: see text]O2 at 20 °C was 139% higher in 20 °C acclimated fish compared to 30 °C acclimated fish. Chronic [Formula: see text]O2 was measured in separate fish continually across the 20 → 30 °C daily cycle for all 3 acclimation groups. Chronic [Formula: see text]O2 responses were very similar between groups between average individual hourly values, as temperatures increased or decreased (1.7 °C/h). Acute [Formula: see text]O2 and thermal tolerance responses highlight "classic" trends, but dynamic, chronic trials suggest acclimation history has little effect on the relative change in oxygen consumption during a thermal cycle. Our results strongly suggest that the minimum and maximum temperatures experienced more strongly influence fish physiology, rather than the thermal cycle itself. This research highlights the importance of collecting data in both cycling and static (constant) thermal conditions, and further research should seek to understand whether ectotherm metabolism does respond uniquely to fluctuating temperatures.

Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits.

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3's processes. Further, we traced back the processes of DE-3's presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3's branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

Behavioral analysis of substrate texture preference in a leech, Helobdella austinensis.

Leeches in the wild are often found on smooth surfaces, such as vegetation, smooth rocks or human artifacts such as bottles and cans, thus exhibiting what appears to be a "substrate texture preference". Here, we have reproduced this behavior under controlled circumstances, by allowing leeches to step about freely on a range of silicon carbide substrates (sandpaper). To begin to understand the neural mechanisms underlying this texture preference behavior, we have determined relevant parameters of leech behavior both on uniform substrates of varying textures, and in a behavior choice paradigm in which the leech is confronted with a choice between rougher and smoother substrate textures at each step. We tested two non-exclusive mechanisms which could produce substrate texture preference: (1) a Differential Diffusion mechanism, in which a leech is more likely to stop moving on a smooth surface than on a rough one, and (2) a Smoothness Selection mechanism, in which a leech is more likely to attach its front sucker (prerequisite for taking a step) to a smooth surface than to a rough one. We propose that both mechanisms contribute to the texture preference exhibited by leeches.

Verifying, Challenging, and Discovering New Synapses Among Fully EM-Reconstructed Neurons in the Leech Ganglion.

Neural circuits underpin the production of animal behavior, largely based upon the precise pattern of synaptic connectivity among the neurons involved. For large numbers of neurons, determining such "connectomes" by direct physiological means is difficult, as physiological accessibility is ultimately required to verify and characterize the function of synapses. We collected a volume of images spanning an entire ganglion of the juvenile leech nervous system via serial blockface electron microscopy (SBEM). We validated this approach by reconstructing a well-characterized circuit of motor neurons involved in the swimming behavior of the leech by locating the synapses among them. We confirm that there are multiple synaptic contacts between connected pairs of neurons in the leech, and that these synapses are widely distributed across the region of neuropil in which the neurons' arbors overlap. We verified the anatomical existence of connections that had been described physiologically among longitudinal muscle motor neurons. We also found that some physiological connections were not present anatomically. We then drew upon the SBEM dataset to design additional physiological experiments. We reconstructed an uncharacterized neuron and one of its presynaptic partners identified from the SBEM dataset. We subsequently interrogated this cell pair via intracellular electrophysiology in an adult ganglion and found that the anatomically-discovered synapse was also functional physiologically. Our findings demonstrate the value of combining a connectomics approach with electrophysiology in the leech nervous system.

Functional Connectomics: How Maggots Make Up Their Minds.

How Drosophila larvae select one behavior or a sequence of behaviors, and then persist in the final one, has been addressed by a powerful combination of electron-microscopy reconstruction of neuronal connections, genetic manipulations, electrophysiology, and neuronal modeling. Surprisingly, reciprocal inhibitory synaptic connections are major players in choosing, sequencing and maintaining behaviors.

Early evolution of neurons.

How did a structure as complex as our own brain ever evolve? Although biologists have pondered this question since Charles Darwin, the explosion of molecular information in recent years has provided new insights into this question, particularly its first step: the evolution of neurons. Meshing information about genomes with insights from more classical anatomical, physiological, and developmental approaches has led to some remarkable insights and surprises. Because 'phylogenomics' is still a young field, however, there are arguments about which genes to include in comparisons, how much to weigh genetic versus 'classical' features, and which algorithms to use in making such comparisons. One source of serious discussion is the explanation for a feature being present in one clade (a group of animals with a common ancestor) but absent in a second clade. Does the feature's absence in clade 2 mean that the feature was never present in the ancestors of clade 2, or was it present in clade 2's ancestors but subsequently lost? A second phylogenomic problem is posed by convergent evolution (or 'homoplasy' in genetic terminology): a feature or a molecule that is present in two clades might have evolved independently in each clade. Both of these problems, secondary loss and homoplasy, confound the interpretation of evolutionary relationships. For the moment, the only solution to these problems is to compare more genes in more animals to see whether the features that are missing from one species, for instance, can be found in other closely-related species. The purpose of this primer is not to consider the evolution of brains, however, but the more modest goal of determining the evolution of neurons, the information processing cells that compose brains. Even this more limited goal is, at this juncture, beyond our reach, but the journey to this goal has already uncovered some remarkable relationships and has made clearer what are the key questions and how they can be approached.

Patterns and distribution of presynaptic and postsynaptic elements within serial electron microscopic reconstructions of neuronal arbors from the medicinal leech Hirudo verbana.

Microscale connectomics involves the large-scale acquisition of high-resolution serial electron micrographs from which neuronal arbors can be reconstructed and synapses can be detected. In addition to connectivity information, these data sets are also rich with structural information, including vesicle types, number of postsynaptic partners at a given presynaptic site, and spatial distribution of synaptic inputs and outputs. This study uses serial block-face scanning electron microscopy (EM) to collect two volumes of serial EM data from ganglia of the medicinal leech. For the first volume, we sampled a small fraction of the neuropil belonging to an adult ganglion. From this data set we measured the proportion of arbors that contained vesicles and the types of vesicles contained and developed criteria to identify synapses and to measure the number of apparent postsynaptic partners in apposition to presynaptic boutons. For the second data set, we sampled an entire juvenile ganglion, which included the somata and arbors of all the neurons. We used this data set to placd our findings from mature tissue in the context of fully reconstructed arbors and to explore the spatial distribution of synaptic inputs and outputs on these arbors. We observed that some neurons segregated their arbors into input only and mixed input/output zones, that other neurons contained exclusively mixed input/output zones, and that still others contained only input zones. These results provide the groundwork for future behavioral studies. J. Comp. Neurol. 524:3677-3695, 2016. © 2016 Wiley Periodicals, Inc.

Scalable Semisupervised Functional Neurocartography Reveals Canonical Neurons in Behavioral Networks.

Large-scale data collection efforts to map the brain are underway at multiple spatial and temporal scales, but all face fundamental problems posed by high-dimensional data and intersubject variability. Even seemingly simple problems, such as identifying a neuron/brain region across animals/subjects, become exponentially more difficult in high dimensions, such as recognizing dozens of neurons/brain regions simultaneously. We present a framework and tools for functional neurocartography-the large-scale mapping of neural activity during behavioral states. Using a voltage-sensitive dye (VSD), we imaged the multifunctional responses of hundreds of leech neurons during several behaviors to identify and functionally map homologous neurons. We extracted simple features from each of these behaviors and combined them with anatomical features to create a rich medium-dimensional feature space. This enabled us to use machine learning techniques and visualizations to characterize and account for intersubject variability, piece together a canonical atlas of neural activity, and identify two behavioral networks. We identified 39 neurons (18 pairs, 3 unpaired) as part of a canonical swim network and 17 neurons (8 pairs, 1 unpaired) involved in a partially overlapping preparatory network. All neurons in the preparatory network rapidly depolarized at the onsets of each behavior, suggesting that it is part of a dedicated rapid-response network. This network is likely mediated by the S cell, and we referenced VSD recordings to an activity atlas to identify multiple cells of interest simultaneously in real time for further experiments. We targeted and electrophysiologically verified several neurons in the swim network and further showed that the S cell is presynaptic to multiple neurons in the preparatory network. This study illustrates the basic framework to map neural activity in high dimensions with large-scale recordings and how to extract the rich information necessary to perform analyses in light of intersubject variability.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

Pinning down randomness.

While some behavioral responses to a stimulus are invariant in animals, most are more likely to be variable or stochastic. In this issue, Gordus et al. illuminate a set of combinatorial neuronal activities that control the variability of a chemotactic behavior in response to an odor, providing a tractable system for understanding how stochastic circuit dynamics affect behavior.

Improved PeT molecules for optically sensing voltage in neurons.

VoltageFluor (VF) dyes have the potential to measure voltage optically in excitable membranes with a combination of high spatial and temporal resolution essential to better characterize the voltage dynamics of large groups of excitable cells. VF dyes sense voltage with high speed and sensitivity using photoinduced electron transfer (PeT) through a conjugated molecular wire. We show that tuning the driving force for PeT (ΔGPeT + w) through systematic chemical substitution modulates voltage sensitivity, estimate (ΔGPeT + w) values from experimentally measured redox potentials, and validate the voltage sensitivities in patch-clamped HEK cells for 10 new VF dyes. VF2.1(OMe).H, with a 48% ΔF/F per 100 mV, shows approximately 2-fold improvement over previous dyes in HEK cells, dissociated rat cortical neurons, and medicinal leech ganglia. Additionally, VF2.1(OMe).H faithfully reports pharmacological effects and circuit activity in mouse olfactory bulb slices, thus opening a wide range of previously inaccessible applications for voltage-sensitive dyes.

Behavioral sequencing: competitive queuing in the fly CNS.

A study of grooming behaviors in Drosophila suggests a neuronal mechanism for how animals produce complex motor patterns from ordered interactions among modules of different motor acts. This mechanism may be a common one in many nervous systems.

Multiplexed modulation of behavioral choice.

Stimuli in the environment, as well as internal states, influence behavioral choice. Of course, animals are often exposed to multiple external and internal factors simultaneously, which makes the ultimate determinants of behavior quite complex. We observed the behavioral responses of European leeches, Hirudo verbana, as we varied one external factor (surrounding water depth) with either another external factor (location of tactile stimulation along the body) or an internal factor (body distention following feeding). Stimulus location proved to be the primary indicator of behavioral response. In general, anterior stimulation produced shortening behavior, midbody stimulation produced local bending, and posterior stimulation usually produced either swimming or crawling but sometimes a hybrid of the two. By producing a systematically measured map of behavioral responses to body stimulation, we found wide areas of overlap between behaviors. When we varied the surrounding water depth, this map changed significantly, and a new feature - rotation of the body along its long axis prior to swimming - appeared. We found additional interactions between water depth and time since last feeding. A large blood meal initially made the animals crawl more and swim less, an effect that was attenuated as water depth increased. The behavioral map returned to its pre-feeding form after approximately 3 weeks as the leeches digested their blood meal. In summary, we found multiplexed impacts on behavioral choice, with the map of responses to tactile stimulation modified by water depth, which itself modulated the impact that feeding had on the decision to swim or crawl.

NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.

NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.

Sexual attraction: sex-specific wiring of neural circuitry.

Two recent studies describe mechanisms by which sexually dimorphic responses to pheromones in the nematode worm Caenorhabditis elegans are driven by differences in the balance of neural circuits that control attraction and repulsion behaviors.

Decision points: the factors influencing the decision to feed in the medicinal leech.

The decision to feed is a complex task that requires making several small independent choices. Am I hungry? Where do I look for food? Is there something better I'd rather be doing? When should I stop? With all of these questions, it is no wonder that decisions about feeding depend on several sensory modalities and that the influences of these sensory systems would be evident throughout the nervous system. The leech is uniquely well suited for studying these complicated questions due to its relatively simple nervous system, its exceptionally well-characterized behaviors and neural circuits, and the ease with which one can employ semi-intact preparations to study the link between physiology and decision-making. We will begin this review by discussing the cellular substrates that govern the decision to initiate and to terminate a bout of feeding. We will then discuss how feeding temporarily blocks competing behaviors from being expressed while the animal continues to feed. Then we will review what is currently known about how feeding affects long-term behavioral choices of the leech. Finally, we conclude with a short discussion of the advantages of the leech's decision-making circuit's design and how this design might be applicable to all decision circuits.