Discrimination of bursts and tonic activity in multifunctional sensorimotor neural network using the extended hill-valley method.

Individual neurons can exhibit a wide range of activity, including spontaneous spiking, tonic spiking, bursting, or spike-frequency adaptation, and can also transition between these activity types. Manual identification of these activity patterns can be subjective and inconsistent. The extended hill-valley (EHV) analysis discriminates tonic spiking and bursts in a spike train by detecting fluctuations in a local, history-dependent analysis signal derived from the spike train. Consequently, the EHV method is not susceptible to changes in baseline firing rate and can identify different types of activity patterns. In addition, output from the EHV method can be used to identify more complex activity patterns such as phasotonic bursting, in which a burst is immediately followed by a period of tonic spiking.NEW & NOTEWORTHY Neurons exhibit diverse spiking patterns, but automated activity classification has focused mainly on detecting bursts. The novel extended hill-valley algorithm uses a smoothed, history-dependent signal to discriminate different types of activity, such as bursts and tonic spiking.

The effect of sensory feedback on crayfish posture and locomotion: I. Experimental analysis of closing the loop.

The effect of proprioceptive feedback on the control of posture and locomotion was studied in the crayfish Procambarus clarkii (Girard). Sensory and motor nerves of an isolated crayfish thoracic nerve cord were connected to a computational neuromechanical model of the crayfish thorax and leg. Recorded levator (Lev) and depressor (Dep) nerve activity drove the model Lev and Dep muscles to move the leg up and down. These movements released and stretched a model stretch receptor, the coxobasal chordotonal organ (CBCO). Model CBCO length changes drove identical changes in the real CBCO; CBCO afferent responses completed the feedback loop. In a quiescent preparation, imposed model leg lifts evoked resistance reflexes in the Dep motor neurons that drove the leg back down. A muscarinic agonist, oxotremorine, induced an active state in which spontaneous Lev/Dep burst pairs occurred and an imposed leg lift excited a Lev assistance reflex followed by a Lev/Dep burst pair. When the feedback loop was intact, Lev/Dep burst pairs moved the leg up and down rhythmically at nearly three times the frequency of burst pairs when the feedback loop was open. The increased rate of rhythmic bursting appeared to result from the positive feedback produced by the assistance reflex.

The effect of sensory feedback on crayfish posture and locomotion: II. Neuromechanical simulation of closing the loop.

Neuromechanical simulation was used to determine whether proposed thoracic circuit mechanisms for the control of leg elevation and depression in crayfish could account for the responses of an experimental hybrid neuromechanical preparation when the proprioceptive feedback loop was open and closed. The hybrid neuromechanical preparation consisted of a computational model of the fifth crayfish leg driven in real time by the experimentally recorded activity of the levator and depressor (Lev/Dep) nerves of an in vitro preparation of the crayfish thoracic nerve cord. Up and down movements of the model leg evoked by motor nerve activity released and stretched the model coxobasal chordotonal organ (CBCO); variations in the CBCO length were used to drive identical variations in the length of the live CBCO in the in vitro preparation. CBCO afferent responses provided proprioceptive feedback to affect the thoracic motor output. Experiments performed with this hybrid neuromechanical preparation were simulated with a neuromechanical model in which a computational circuit model represented the relevant thoracic circuitry. Model simulations were able to reproduce the hybrid neuromechanical experimental results to show that proposed circuit mechanisms with sensory feedback could account for resistance reflexes displayed in the quiescent state and for reflex reversal and spontaneous Lev/Dep bursting seen in the active state.

Neural circuit reconfiguration by social status.

The social rank of an animal is distinguished by its behavior relative to others in its community. Although social-status-dependent differences in behavior must arise because of differences in neural function, status-dependent differences in the underlying neural circuitry have only begun to be described. We report that dominant and subordinate crayfish differ in their behavioral orienting response to an unexpected unilateral touch, and that these differences correlate with functional differences in local neural circuits that mediate the responses. The behavioral differences correlate with simultaneously recorded differences in leg depressor muscle EMGs and with differences in the responses of depressor motor neurons recorded in reduced, in vitro preparations from the same animals. The responses of local serotonergic interneurons to unilateral stimuli displayed the same status-dependent differences as the depressor motor neurons. These results indicate that the circuits and their intrinsic serotonergic modulatory components are configured differently according to social status, and that these differences do not depend on a continuous descending signal from higher centers.

Spatial segregation of excitatory and inhibitory effects of 5-HT on crayfish motoneurons.

Altering neuronal membrane properties, including input resistance, is a key modulatory mechanism for changing neural activity patterns. The effect of membrane currents generated by either synaptic or voltage-dependent channels directly depends on neuron input resistance. We found that local application of serotonin to different regions of identified motoneurons (MNs) of the postural/walking network of isolated crayfish produced different changes in input resistance. Puff-applied 5-HT in the periphery of the initial segment produced exclusively inhibitory responses. In contrast, when 5-HT was puff-applied on the central arbor of the same depressor (Dep) MN, exclusively depolarizing responses were obtained. Both inhibitory and excitatory responses were direct because they persisted in low-calcium saline. We found numerous close appositions between 5-HT-immunoreactive processes and the initial segment of dextran-rhodamine-filled Dep MNs. In contrast, almost no close apposition sites were found in Dep MN arbor. It seems that the 5-HT controls the level of excitability of postural network MNs by two mechanisms acting at two different sites: inhibitory responses (consistent with an action involving opening of K(+) channels) occur in the initial segment region and may involve classic synaptic transmission, whereas depolarizing responses (consistent with an action involving closing of K(+) channels) occur on MN branches via apparent paracrine effects.

Social interactions determine postural network sensitivity to 5-HT.

The excitability of the leg postural circuit and its response to serotonin (5-HT) were studied in vitro in thoracic nervous system preparations of dominant and subordinate male crayfishes. We demonstrate that the level of spontaneous tonic activity of depressor and levator motoneurons (MNs) (which control downward and upward movements of the leg, respectively) and the amplitude of their resistance reflex are larger in dominants than in subordinates. Moreover, we show that serotonergic neuromodulation of the postural circuit also depends on social status. Depressor and levator MN tonic firing rates and resistance reflex amplitudes were significantly modified in the presence of 10 mum 5-HT in dominants but not in subordinates. Using intracellular recording from depressor MNs, we show that their input resistance was not significantly different in dominants and subordinates in control conditions. However, 5-HT produced a marked depolarization in dominants and a significantly weaker depolarization in subordinates. Moreover, in the presence of 5-HT, the amplitude of the resistance reflex and the input resistance of MNs increased in dominants and decreased in subordinates. The peak amplitude and the decay phase of unitary EPSPs triggered by sensory spikes were significantly increased by 5-HT in dominants but not in subordinates. These observations suggest that neural networks are more reactive in dominants than in subordinates, and this divergence is even reinforced by 5-HT modulation.

Control of tumbling during the locust jump.

Locust can jump precisely to a target, yet they can also tumble during the trajectory. We propose two mechanisms that would allow the locust to control tumbling during the jump. The first is that prior to the jump, locusts adjust the pitch of their body to move the center of mass closer to the intended thrust vector. The second is that contraction of the dorsolongitudinal muscles during the jump will produce torques that counter the torque produced by thrust. We found that locusts increased their take-off angle as the initial body pitch increased, and that little tumbling occurred for jumps that observed this relationship. Simulations of locust jumping demonstrated that a pitch versus take-off angle relationship that minimized tumbling in simulated jumps was similar to the relationship observed in live locusts. Locusts were strongly biased to pitch head-upward, and performed dorsiflexions far more often than ventral flexions. The direction and magnitude of tumbling could be controlled in simulations by adjusting the tension in the dorsolongitudinal muscles. These mechanisms allowed the simulations to match the data from the live animals. Control of tumbling was also found to influence the control of jump elevation. The bias to pitch head-upwards may have an evolutionary advantage when evading a predator and so make control of tumbling important for the locust.

Ritualized submission and the reduction of aggression in an invertebrate.

Ritualized behaviors that signify acceptance of a dominance relationship and reduce aggression between rivals are a common feature of vertebrate social behavior. Although some invertebrates, including crayfish, lobsters, and ants, display dominance postures, more complex dominance rituals and their effects on fitness have not been reported. We found that crayfish display such a complex ritual, when two males engaged in pseudocopulatory behavior to signify their dominance relationship. This was followed by a reduction in aggression and an increased likelihood of the subordinate's survival. Pseudocopulation was initiated by the eventual dominant and could be accepted or refused by the eventual subordinate. The frequency of aggressive behavior declined significantly during the first hour in all pairs that pseudocopulated but remained high in pairs that did not. Whereas all the subordinate members of pairs that pseudocopulated survived the initial 24 hr of pairing, half of subordinates that did not pseudocopulate were killed during that time. This differential mortality indicates that the reduction of aggression induced by the pseudocopulatory ritual directly enhances the differential survival of male crayfish that engage in this behavior.

Duality of 5-HT Effects on Crayfish Motoneurons.

Serotonin (5-HT) is a major neuromodulator acting on the nervous system. Its various effects have been studied in vertebrates, as well as in arthropods, from the cellular and subcellular compartments up to the behavioral level, which includes the control of mood, aggression, locomotion, and anxiety. The diversity of responses of neurons to 5-HT has been related to its mode of application, the diversity of 5-HT-receptors, and the animals' social status history. In the locomotor network of socially isolated crayfish, the duality of 5-HT-evoked responses (excitatory/inhibitory) on motoneurons (MNs), sensorimotor pathways, and their consequences on motor network activity has largely been studied. The aim of the present report is to examine if this duality of exogenous 5-HT-evoked responses in the crayfish locomotor network can be reproduced by direct activation of 5-HT neurons in the case of socially isolated animals. Our previous studies have focused on the mechanisms supporting these opposite effects on MNs, pointing out spatial segregation of 5-HT receptors responsible either for positive or negative responses. Here, we report new findings indicating that excitatory and inhibitory effects can be achieved simultaneously in different leg MNs by the activation of a single 5-HT cell in the first abdominal ganglion.

Role of the olfactory pathway in agonistic behavior of crayfish, Procambarus clarkii.

Crayfish establish social dominance hierarchies through agonistic interactions, and these hierarchies are maintained through assessment of social status. Chemical signals influence several aspects of fighting behavior, but the specific chemosensory sensilla involved in detecting these signals in crayfish are unknown. The goal of our study was to examine the importance of aesthetasc sensilla--olfactory sensors on the antennules of decapod crustaceans--in regulating changes in fighting behavior in crayfish, Procambarus clarkii, over the course of repeated pairings. We selectively ablated aesthetascs from pairs of crayfish after the first day of trials and compared the behavior of these ablated animals to that of pairs of intact controls. Results show that unablated crayfish significantly decreased the number and duration of fights over repeated pairings, whereas crayfish lacking aesthetascs continued to engage in similar amounts of fighting across all three trial days. This difference shows that aesthetascs regulate fighting behavior in P. clarkii.

Direct benefits of social dominance in juvenile crayfish.

Crayfish are known for their innate aggressiveness and willingness to quickly establish dominance relationships among group members. Consequently, the formation of dominance hierarchies and the analysis of behavioral patterns displayed during agonistic encounters have mostly been tested in environments that provide no immediate resources besides space. We tested the hypothesis that social hierarchy formation in crayfish serves to determine access to future resources. Individuals within groups of three juvenile crayfish were allowed to form a social hierarchy in a featureless environment before a single food resource was presented. Higher dominance indices were significantly correlated with increased access to the food. The highest ranked crayfish spent more time in contact with the food than did medium-ranked and lowest ranked crayfish, and crayfish of medium rank spent more time in contact with the resource than did lowest ranked animals. The highest ranked crayfish consolidated their dominant status in the presence of food, indicated by a complete absence of any submissive behaviors during that period. The results of these experiments show that the disposition of crayfish to engage in fighting and formation of a dominance hierarchy in a featureless environment serves to determine future access to an emerging resource, thereby entailing greater benefits for animals of higher social rank.

The retrograde spread of synaptic potentials and recruitment of presynaptic inputs.

Lateral excitation is a mechanism for amplifying coordinated input to postsynaptic neurons that has been described recently in several species. Here, we describe how a postsynaptic neuron, the lateral giant (LG) escape command neuron, enhances lateral excitation among its presynaptic mechanosensory afferents in the crayfish tailfan. A lateral excitatory network exists among electrically coupled tailfan primary afferents, mediated through central electrical synapses. EPSPs elicited in LG dendrites as a result of mechanosensory stimulation spread antidromically back through electrical junctions to unstimulated afferents, summate with EPSPs elicited through direct afferent-to-afferent connections, and contribute to recruitment of these afferents. Antidromic potentials are larger if the afferent is closer to the initial input on LG dendrites, which could create a spatial filtering mechanism within the network. This pathway also broadens the temporal window over which lateral excitation can occur, because of the delay required for EPSPs to spread through the large LG dendrites. The delay allows subthreshold inputs to the LG to have a priming effect on the lateral excitatory network and lowers the threshold of the network in response to a second, short-latency stimulus. Retrograde communication within neuronal pathways has been described in a number of vertebrate and invertebrate species. A mechanism of antidromic passage of depolarizing current from a neuron to its presynaptic afferents, similar to that described here in an invertebrate, is also present in a vertebrate (fish). This raises the possibility that short-term retrograde modulation of presynaptic elements through electrical junctions may be common.

A lateral excitatory network in the escape circuit of crayfish.

A phasic stimulus directed to the rear of a crayfish (Procambarus clarkii) creates mechanosensory input to the lateral giant (LG) interneuron, a command neuron for escape. A single LG spike is necessary and sufficient to produce a highly stereotyped tail flip that thrusts the animal away from the source of stimulation. Here we describe a lateral excitatory network among primary afferent axons in the last abdominal ganglion of crayfish that produces nonlinear amplification of the sensory input to the command circuitry for escape. The lateral excitation is mediated by electrical synapses between central terminals of primary mechanosensory afferents. The network enables stimulated afferents to recruit unstimulated afferents that contribute additional input to LG and to mechanosensory interneurons that also converge on LG. When depolarized, the LG neuron increases its own inputs from primary afferents and primary interneurons by facilitating the recruitment of both. Conversely, hyperpolarization of LG reduces the excitability of primary afferents and primary interneurons. The crayfish's decision to escape, previously thought to lie exclusively in the synaptic integrative properties of LG, is now seen to depend on the interactions between LG dendritic postsynaptic potentials and the responses of primary afferent terminals in the lateral excitatory network.

Immunocytochemical mapping and quantification of expression of a putative type 1 serotonin receptor in the crayfish nervous system.

Serotonin is an important neurotransmitter that is involved in modulation of sensory, motor, and higher functions in many species. In the crayfish, which has been developed as a model for nervous system function for over a century, serotonin modulates several identified circuits. Although the cellular and circuit effects of serotonin have been extensively studied, little is known about the receptors that mediate these signals. Physiological data indicate that identified crustacean cells and circuits are modulated via several different serotonin receptors. We describe the detailed immunocytochemical localization of the crustacean type 1 serotonin receptor, 5-HT1crust, throughout the crayfish nerve cord and on abdominal superficial flexor muscles. 5-HT1crust is widely distributed in somata, including those of several identified neurons, and neuropil, suggesting both synaptic and neurohormonal roles. Individual animals show very different levels of 5-HT1crust immunoreactivity (5-HT(1crust)ir) ranging from preparations with hundreds of labeled cells per ganglion to some containing only a handful of 5-HT(1crust)ir cells in the entire nerve cord. The interanimal variability in 5-HT(1crust)ir is great, but individual nerve cords show a consistent level of labeling between ganglia. Quantitative RT-PCR shows that 5-HT1crust mRNA levels between animals are also variable but do not directly correlate with 5-HT(1crust)ir levels. Although there is no correlation of 5-HT1crust expression with gender, social status, molting or feeding, dominant animals show significantly greater variability than subordinates. Functional analysis of 5-HT1crust in combination with this immunocytochemical map will aid further understanding of this receptor's role in the actions of serotonin on identified circuits and cells.

Differential dye coupling reveals lateral giant escape circuit in crayfish.

The lateral giant (LG) escape circuit of crayfish mediates a coordinated escape triggered by strong attack to the abdomen. The LG circuit is one of the best understood of small systems, but models of the circuit have mostly been limited to simple ball-and-stick representations, which ignore anatomical details of contacts between circuit elements. Many of the these contacts are electrical; here we use differential dye coupling, a technique which could help reveal connection patterns in many neural circuits, to reveal in detail the circuit within the terminal abdominal ganglion. Sensory input from the tailfan forms a somatotopic map on the projecting LG dendrites, which together with interafferent coupling mediates a lateral excitatory network that selectively amplifies strong, phasic, converging input to LG. Mechanosensory interneurons contact LG at sites distinct from the primary afferents and so maximize their summated effect on LG. Motor neurons and premotor interneurons are excited near the initial segments of the LGs and innervate muscles for generating uropod flaring and telson flexion. Previous research has shown that spatial patterns of input are important for signal integration in LG; this map of electrical contact points will help us to understand synaptic processing in this system.

A crustacean serotonin receptor: cloning and distribution in the thoracic ganglia of crayfish and freshwater prawn.

Serotonin (5-HT) is involved in regulating important aspects of behavior and a variety of systemic physiological functions in both vertebrates and invertebrates. These functions are mediated through binding to 5-HT receptors, of which approximately 13 have been characterized in mammals. In crustaceans, important model systems for the study of the neural basis of behaviors, 5-HT is also linked with higher-order behaviors, associated with different 5-HT receptors that have been identified at the physiological and pharmacological levels. However, no crustacean 5-HT receptors have been identified at the molecular level. We have cloned a putative 5-HT(1) receptor (5-HT(1crust)) from crayfish, prawn, and spiny lobster and have raised antibodies that recognize this protein in all three organisms. 5-HT(1crust) immunoreactivity (5-HT(1crust)ir) was observed surrounding the somata of specific groups of neurons and as punctate staining within the neuropil in all thoracic ganglia of crayfish and prawn. In the crayfish, 5-HT(1crust)ir was also found in boutons surrounding the first and second nerves of each ganglion and on the 5-HT cells of T1-4. In the prawn, 5-HT(1crust)ir was also found in axons that project across the ganglia and along the connectives. We found examples of colocalization of 5-HT(1crust) with 5-HT, consistent with the short-term modulatory role of 5-HT, as well as cases of serotonergic staining in the absence of a 5-HT(1crust) signal, which might imply that other 5-HT receptors are found at these locations. We also observed receptors that did not possess counterpart 5-HT staining, suggesting that these may also mediate long-term neurohormonal functions of serotonin.

The neural basis of dominance hierarchy formation in crayfish.

Fifty years of study of the nervous system and behavior of crayfish have revealed neural circuits for movements that are similar to those seen during formation of a dominance hierarchy. Given this background, it is of interest to ask what is understood about the neural substrates of dominance hierarchy formation. Here we will consider the social behavior that crayfish display in the wild and in the laboratory, and its relationship to movements released by activation of specific neural circuits. We will consider how these movements might be knit together to produce the behavior patterns that are characteristic of dominant and subordinate animals.

Non-invasive imaging of neuroanatomical structures and neural activation with high-resolution MRI.

Several years ago, manganese-enhanced magnetic resonance imaging (MEMRI) was introduced as a new powerful tool to image active brain areas and to identify neural connections in living, non-human animals. Primarily restricted to studies in rodents and later adapted for bird species, MEMRI has recently been discovered as a useful technique for neuroimaging of invertebrate animals. Using crayfish as a model system, we highlight the advantages of MEMRI over conventional techniques for imaging of small nervous systems. MEMRI can be applied to image invertebrate nervous systems at relatively high spatial resolution, and permits identification of stimulus-evoked neural activation non-invasively. Since the selection of specific imaging parameters is critical for successful in vivo micro-imaging, we present an overview of different experimental conditions that are best suited for invertebrates. We also compare the effects of hardware and software specifications on image quality, and provide detailed descriptions of the steps necessary to prepare animals for successful imaging sessions. Careful consideration of hardware, software, experiments, and specimen preparation will promote a better understanding of this novel technique and facilitate future MEMRI studies in other laboratories.

Neuromechanical simulation.

The importance of the interaction between the body and the brain for the control of behavior has been recognized in recent years with the advent of neuromechanics, a field in which the coupling between neural and biomechanical processes is an explicit focus. A major tool used in neuromechanics is simulation, which connects computational models of neural circuits to models of an animal's body situated in a virtual physical world. This connection closes the feedback loop that links the brain, the body, and the world through sensory stimuli, muscle contractions, and body movement. Neuromechanical simulations enable investigators to explore the dynamical relationships between the brain, the body, and the world in ways that are difficult or impossible through experiment alone. Studies in a variety of animals have permitted the analysis of extremely complex and dynamic neuromechanical systems, they have demonstrated that the nervous system functions synergistically with the mechanical properties of the body, they have examined hypotheses that are difficult to test experimentally, and they have explored the role of sensory feedback in controlling complex mechanical systems with many degrees of freedom. Each of these studies confronts a common set of questions: (i) how to abstract key features of the body, the world and the CNS in a useful model, (ii) how to ground model parameters in experimental reality, (iii) how to optimize the model and identify points of sensitivity and insensitivity, and (iv) how to share neuromechanical models for examination, testing, and extension by others.