Social state gates vision using three circuit mechanisms in Drosophila.

Animals are often bombarded with visual information and must prioritize specific visual features based on their current needs. The neuronal circuits that detect and relay visual features have been well-studied. Yet, much less is known about how an animal adjusts its visual attention as its goals or environmental conditions change. During social behaviors, flies need to focus on nearby flies. Here, we study how the flow of visual information is altered when female Drosophila enter an aggressive state. From the connectome, we identified three state-dependent circuit motifs poised to selectively amplify the response of an aggressive female to fly-sized visual objects: convergence of excitatory inputs from neurons conveying select visual features and internal state; dendritic disinhibition of select visual feature detectors; and a switch that toggles between two visual feature detectors. Using cell-type-specific genetic tools, together with behavioral and neurophysiological analyses, we show that each of these circuit motifs function during female aggression. We reveal that features of this same switch operate in males during courtship pursuit, suggesting that disparate social behaviors may share circuit mechanisms. Our work provides a compelling example of using the connectome to infer circuit mechanisms that underlie dynamic processing of sensory signals.

Imaging fictive locomotor patterns in larval Drosophila.

We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques.

Female mice ultrasonically interact with males during courtship displays.

During courtship males attract females with elaborate behaviors. In mice, these displays include ultrasonic vocalizations. Ultrasonic courtship vocalizations were previously attributed to the courting male, despite evidence that both sexes produce virtually indistinguishable vocalizations. Because of this similarity, and the difficulty of assigning vocalizations to individuals, the vocal contribution of each individual during courtship is unknown. To address this question, we developed a microphone array system to localize vocalizations from socially interacting, individual adult mice. With this system, we show that female mice vocally interact with males during courtship. Males and females jointly increased their vocalization rates during chases. Furthermore, a female's participation in these vocal interactions may function as a signal that indicates a state of increased receptivity. Our results reveal a novel form of vocal communication during mouse courtship, and lay the groundwork for a mechanistic dissection of communication during social behavior.

Automated multi-day tracking of marked mice for the analysis of social behaviour.

A quantitative description of animal social behaviour is informative for behavioural biologists and clinicians developing drugs to treat social disorders. Social interaction in a group of animals has been difficult to measure because behaviour develops over long periods of time and requires tedious manual scoring, which is subjective and often non-reproducible. Computer-vision systems with the ability to measure complex social behaviour automatically would have a transformative impact on biology. Here, we present a method for tracking group-housed mice individually as they freely interact over multiple days. Each mouse is bleach-marked with a unique fur pattern. The patterns are automatically learned by the tracking software and used to infer identities. Trajectories are analysed to measure behaviour as it develops over days, beyond the range of acute experiments. We demonstrate how our system may be used to study the development of place preferences, associations and social relationships by tracking four mice continuously for five days. Our system enables accurate and reproducible characterisation of wild-type mouse social behaviour and paves the way for high-throughput long-term observation of the effects of genetic, pharmacological and environmental manipulations.

The effects of temperature on the stability of a neuronal oscillator.

The crab Cancer borealis undergoes large daily fluctuations in environmental temperature (8-24°C) and must maintain appropriate neural function in the face of this perturbation. In the pyloric circuit of the crab stomatogastric ganglion, we pharmacologically isolated the pacemaker kernel (the AB and PD neurons) and characterized its behavior in response to temperature ramps from 7°C to 31°C. For moderate temperatures, the pacemaker displayed a frequency-temperature curve statistically indistinguishable from that of the intact circuit, and like the intact circuit maintained a constant duty cycle. At high temperatures (above 23°C), a variety of different behaviors were seen: in some preparations the pacemaker increased in frequency, in some it slowed, and in many preparations the pacemaker stopped oscillating ("crashed"). Furthermore, these crashes seemed to fall into two qualitatively different classes. Additionally, the animal-to-animal variability in frequency increased at high temperatures. We used a series of Morris-Lecar mathematical models to gain insight into these phenomena. The biophysical components of the final model have temperature sensitivities similar to those found in nature, and can crash via two qualitatively different mechanisms that resemble those observed experimentally. The crash type is determined by the precise parameters of the model at the reference temperature, 11°C, which could explain why some preparations seem to crash in one way and some in another. Furthermore, even models with very similar behavior at the reference temperature diverge greatly at high temperatures, resembling the experimental observations.

Robustness of a rhythmic circuit to short- and long-term temperature changes.

Recent computational and experimental work has shown that similar network performance can result from variable sets of synaptic and intrinsic properties. Because temperature is a global perturbation that differentially influences every biological process within the nervous system, one might therefore expect that individual animals would respond differently to temperature. Nonetheless, the phase relationships of the pyloric rhythm of the stomatogastric ganglion (STG) of the crab, Cancer borealis, are remarkably invariant between 7 and 23°C (Tang et al., 2010). Here, we report that, when isolated STG preparations were exposed to more extreme temperature ranges, their networks became nonrhythmic, or "crashed", in a reversible fashion. Animals were acclimated for at least 3 weeks at 7, 11, or 19°C. When networks from the acclimated animals were perturbed by acute physiologically relevant temperature ramps (11-23°C), the network frequency and phase relationships were independent of the acclimation group. At high acute temperatures (>23°C), circuits from the cold-acclimated animals produced less-regular pyloric rhythms than those from warm-acclimated animals. At high acute temperatures, phase relationships between pyloric neurons were more variable from animal to animal than at moderate acute temperatures, suggesting that individual differences across animals in intrinsic circuit parameters are revealed at high temperatures. This shows that individual and variable neuronal circuits can behave similarly in normal conditions, but their behavior may diverge when confronted with extreme external perturbations.

What we talk about when we talk about capacitance measured with the voltage-clamp step method.

Capacitance is a fundamental neuronal property. One common way to measure capacitance is to deliver a small voltage-clamp step that is long enough for the clamp current to come to steady state, and then to divide the integrated transient charge by the voltage-clamp step size. In an isopotential neuron, this method is known to measure the total cell capacitance. However, in a cell that is not isopotential, this measures only a fraction of the total capacitance. This has generally been thought of as measuring the capacitance of the "well-clamped" part of the membrane, but the exact meaning of this has been unclear. Here, we show that the capacitance measured in this way is a weighted sum of the total capacitance, where the weight for a given small patch of membrane is determined by the voltage deflection at that patch, as a fraction of the voltage-clamp step size. This quantifies precisely what it means to measure the capacitance of the "well-clamped" part of the neuron. Furthermore, it reveals that the voltage-clamp step method measures a well-defined quantity, one that may be more useful than the total cell capacitance for normalizing conductances measured in voltage-clamp in nonisopotential cells.

Multiple models to capture the variability in biological neurons and networks.

How tightly tuned are the synaptic and intrinsic properties that give rise to neuron and circuit function? Experimental work shows that these properties vary considerably across identified neurons in different animals. Given this variability in experimental data, this review describes some of the complications of building computational models to aid in understanding how system dynamics arise from the interaction of system components. We argue that instead of trying to build a single model that captures the generic behavior of a neuron or circuit, it is beneficial to construct a population of models that captures the behavior of the population that provided the experimental data. Studying a population of models with different underlying structure and similar behaviors provides opportunities to discover unsuspected compensatory mechanisms that contribute to neuron and network function.

Precise temperature compensation of phase in a rhythmic motor pattern.

Most animal species are cold-blooded, and their neuronal circuits must maintain function despite environmental temperature fluctuations. The central pattern generating circuits that produce rhythmic motor patterns depend on the orderly activation of circuit neurons. We describe the effects of temperature on the pyloric rhythm of the stomatogastric ganglion of the crab, Cancer borealis. The pyloric rhythm is a triphasic motor pattern in which the Pyloric Dilator (PD), Lateral Pyloric (LP), and Pyloric (PY) neurons fire in a repeating sequence. While the frequency of the pyloric rhythm increased about 4-fold (Q(10) approximately 2.3) as the temperature was shifted from 7 degrees C to 23 degrees C, the phase relationships of the PD, LP, and PY neurons showed almost perfect temperature compensation. The Q(10)'s of the input conductance, synaptic currents, transient outward current (I(A)), and the hyperpolarization-activated inward current (I(h)), all of which help determine the phase of LP neuron activity, ranged from 1.8 to 4. We studied the effects of temperature in >1,000 computational models (with different sets of maximal conductances) of a bursting neuron and the LP neuron. Many bursting models failed to monotonically increase in frequency as temperature increased. Temperature compensation of LP neuron phase was facilitated when model neurons' currents had Q(10)'s close to 2. Together, these data indicate that although diverse sets of maximal conductances may be found in identified neurons across animals, there may be strong evolutionary pressure to restrict the Q(10)'s of the processes that contribute to temperature compensation of neuronal circuits.

Slow and persistent postinhibitory rebound acts as an intrinsic short-term memory mechanism.

Many neurons exhibit postinhibitory rebound (PIR), in which neurons display enhanced excitability following inhibition. PIR can strongly influence the timing of spikes on rebound from an inhibitory input. We studied PIR in the lateral pyloric (LP) neuron of the stomatogastric ganglion of the crab Cancer borealis. The LP neuron is part of the pyloric network, a central pattern generator that normally oscillates with a period of approximately 1 s. We used the dynamic clamp to create artificial rhythmic synaptic inputs of various periods and duty cycles in the LP neuron. Surprisingly, we found that the strength of PIR increased slowly over multiple cycles of synaptic input. Moreover, this increased excitability persisted for 10-20 s after the rhythmic inhibition was removed. These effects are considerably slower than the rhythmic activity typically observed in LP. Thus this slow postinhibitory rebound allows the neuron to adjust its level of excitability to the average level of inhibition over many cycles, and is another example of an intrinsic "short-term memory" mechanism.

Functional consequences of animal-to-animal variation in circuit parameters.

How different are the neuronal circuits for a given behavior across individual animals? To address this question, we measured multiple cellular and synaptic parameters in individual preparations to see how they correlated with circuit function, using neurons and synapses in the pyloric circuit of the stomatogastric ganglion of the crab Cancer borealis. There was considerable preparation-to-preparation variability in the strength of two identified synapses, in the amplitude of a modulator-evoked current and in the expression of six ion channel genes. Nonetheless, we found strong correlations across preparations among these parameters and attributes of circuit performance. These data illustrate the importance of making multidimensional measurements from single preparations for understanding how variability in circuit output is related to the variability of multiple circuit parameters.

Membrane capacitance measurements revisited: dependence of capacitance value on measurement method in nonisopotential neurons.

During growth or degeneration neuronal surface area can change dramatically. Measurements of membrane protein concentration, as in ion channel or ionic conductance density, are often normalized by membrane capacitance, which is proportional to the surface area, to express changes independently from cell surface variations. Several electrophysiological protocols are used to measure cell capacitance, all based on the assumption of membrane isopotentiality. Yet, most neurons violate this assumption because of their complex anatomical structure, raising the question of which protocol yields measurements that are closest to the actual total membrane capacitance. We measured the capacitance of identified neurons from crab stomatogastric ganglia using three different protocols: the current-clamp step, the voltage-clamp step, and the voltage-clamp ramp protocols. We observed that the current-clamp protocol produced significantly higher capacitance values than those of either voltage-clamp protocol. Computational models of various anatomical complexities suggest that the current-clamp protocol can yield accurate capacitance estimates. In contrast, the voltage-clamp protocol estimates rapidly deteriorate as isopotentiality is reduced. We provide a mathematical description of these results by analyzing a simple two-compartment model neuron to facilitate an intuitive understanding of these methods. Together, the experiments, modeling, and mathematical analysis indicate that accurate total membrane capacitance measurements cannot be obtained with voltage-clamp protocols in nonisopotential neurons. Furthermore, although current-clamp steps can theoretically yield accurate measurements, experimentalists should be aware of limitations imposed by step duration and numerical errors during fitting procedures to obtain the membrane time constant.

How multiple conductances determine electrophysiological properties in a multicompartment model.

Most neurons have large numbers of voltage- and time-dependent currents that contribute to their electrical firing patterns. Because these currents are nonlinear, it can be difficult to determine the role each current plays in determining how a neuron fires. The lateral pyloric (LP) neuron of the stomatogastric ganglion of decapod crustaceans has been studied extensively biophysically. We constructed approximately 600,000 versions of a four-compartment model of the LP neuron and distributed 11 different currents into the compartments. From these, we selected approximately 1300 models that match well the electrophysiological properties of the biological neuron. Interestingly, correlations that were seen in the expression of channel mRNA in biological studies were not found across the approximately 1300 admissible LP neuron models, suggesting that the electrical phenotype does not require these correlations. We used cubic fits of the function from maximal conductances to a series of electrophysiological properties to ask which conductances predominantly influence input conductance, resting membrane potential, resting spike rate, phasing of activity in response to rhythmic inhibition, and several other properties. In all cases, multiple conductances contribute to the measured property, and the combinations of currents that strongly influence each property differ. These methods can be used to understand how multiple currents in any candidate neuron interact to determine the cell's electrophysiological behavior.

Spectral analyses reveal the presence of adult-like activity in the embryonic stomatogastric motor patterns of the lobster, Homarus americanus.

The stomatogastric nervous system (STNS) of the embryonic lobster is rhythmically active prior to hatching, before the network is needed for feeding. In the adult lobster, two rhythms are typically observed: the slow gastric mill rhythm and the more rapid pyloric rhythm. In the embryo, rhythmic activity in both embryonic gastric mill and pyloric neurons occurs at a similar frequency, which is slightly slower than the adult pyloric frequency. However, embryonic motor patterns are highly irregular, making traditional burst quantification difficult. Consequently, we used spectral analysis to analyze long stretches of simultaneous recordings from muscles innervated by gastric and pyloric neurons in the embryo. This analysis revealed that embryonic gastric mill neurons intermittently produced pauses and periods of slower activity not seen in the recordings of the output from embryonic pyloric neurons. The slow activity in the embryonic gastric mill neurons increased in response to the exogenous application of Cancer borealis tachykinin-related peptide 1a (CabTRP), a modulatory peptide that appears in the inputs to the stomatogastric ganglion (STG) late in larval development. These results suggest that the STG network can express adult-like rhythmic behavior before fully differentiated adult motor patterns are observed, and that the maturation of the neuromodulatory inputs is likely to play a role in the eventual establishment of the adult motor patterns.

Structure and visualization of high-dimensional conductance spaces.

Neurons, and realistic models of neurons, typically express several different types of voltage-gated conductances. These conductances are subject to continual regulation. Therefore it is essential to understand how changes in the conductances of a neuron affect its intrinsic properties, such as burst period or delay to firing after inhibition of a particular duration and magnitude. Even in model neurons, it can be difficult to visualize how the intrinsic properties vary as a function of their underlying maximal conductances. We used a technique, called clutter-based dimension reordering (CBDR), which enabled us to visualize intrinsic properties in high-dimensional conductance spaces. We applied CBDR to a family of models with eight different types of voltage- and calcium-dependent channels. CBDR yields images that reveal structure in the underlying conductance space. CBDR can also be used to visualize the results of other types of analysis. As examples, we use CBDR to visualize the results of a connected-components analysis, and to visually evaluate the results of a separating-hyperplane (i.e., linear classifier) analysis. We believe that CBDR will be a useful tool for visualizing the conductance spaces of neuronal models in many systems.

Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion.

Neuronal firing patterns can contain different temporal information. It has long been known that the fast pyloric and the slower gastric motor patterns in the stomatogastric ganglion of decapod crustaceans interact. However, the bidirectional influences between the pyloric rhythm and the gastric mill rhythm have not been quantified in detail from preparations that spontaneously express both patterns in vitro. We found regular and stable spontaneous gastric and pyloric activity in 71% of preparations of the isolated stomatogastric nervous system of the lobster, Homarus americanus. The gastric [cycle period: 10.96 +/- 2.67 (SD) s] and pyloric (cycle period: 1.35 +/- 0.18 s) patterns showed bidirectional interactions and coordination. Gastric neuron firing showed preferred phases within the reference frame of the pyloric cycle. The relative timing and burst parameters of the pyloric neurons systematically changed within the reference frame of the gastric cycle. The gastric rhythm showed a tendency to run at cycle periods that were integer multiples of the pyloric periods, but coupling and coordination between the two rhythms were variable. We used power spectra to quantify the gastric and pyloric contributions to the firing pattern of each individual neuron. This provided us with a way to analyze the firing pattern of each gastric and pyloric neuron type individually without reference to either gastric or pyloric phase. Possible functional consequences of these network interactions for motor output are discussed.

Invertebrate central pattern generation moves along.

Central pattern generators (CPGs) are circuits that generate organized and repetitive motor patterns, such as those underlying feeding, locomotion and respiration. We summarize recent work on invertebrate CPGs which has provided new insights into how rhythmic motor patterns are produced and how they are controlled by higher-order command and modulatory interneurons.

Imaging reveals synaptic targets of a swim-terminating neuron in the leech CNS.

In the leech, the command-like neuron called cell Tr2 is known to stop swimming, but the connections from cell Tr2 to the swim central pattern generator have not been identified. We used fluorescence resonance energy transfer voltage-sensitive dyes to identify three neurons that are synaptic targets of cell Tr2. We then used electrophysiological techniques to show that these connections are monosynaptic, chemical, and excitatory. Two of the novel targets, cell 256 and cell 54, terminate swimming when stimulated. These neurons are likely to mediate swim cessation caused by cell Tr2 activity, and thus play the role of intermediate control cells in the leech CNS.

Analysis of oscillations in a reciprocally inhibitory network with synaptic depression.

We present and analyze a model of a two-cell reciprocally inhibitory network that oscillates. The principal mechanism of oscillation is short-term synaptic depression. Using a simple model of depression and analyzing the system in certain limits, we can derive analytical expressions for various features of the oscillation, including the parameter regime in which stable oscillations occur, as well as the period and amplitude of these oscillations. These expressions are functions of three parameters: the time constant of depression, the synaptic strengths, and the amount of tonic excitation the cells receive. We compare our analytical results with the output of numerical simulations and obtain good agreement between the two. Based on our analysis, we conclude that the oscillations in our network are qualitatively different from those in networks that oscillate due to postinhibitory rebound, spike-frequency adaptation, or other intrinsic (rather than synaptic) adaptational mechanisms. In particular, our network can oscillate only via the synaptic escape mode of Skinner, Kopell, and Marder (1994).