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In JoVE (1)
Other Publications (11)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Microscopy Research and Technique
- The Journal of Experimental Biology
- The Journal of Experimental Biology
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Experimental Biology
- The Biological Bulletin
- The Journal of Experimental Biology
- Journal of Neurophysiology
- Proceedings. Biological Sciences / The Royal Society
- Frontiers in Behavioral Neuroscience
Articles by Jens Herberholz in JoVE
JoVE General
Recordings of Neural Circuit Activation in Freely Behaving Animals
Department of Psychology, Neuroscience and Cognitive Science Program , University of Maryland
Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.
Other articles by Jens Herberholz on PubMed
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.
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.
Escape Behavior and Escape Circuit Activation in Juvenile Crayfish During Prey-predator Interactions
The neural systems that control escape behavior have been studied intensively in several animals, including mollusks, fish and crayfish. Surprisingly little is known, however, about the activation and the utilization of escape circuits during prey-predator interactions. To complement the physiological and anatomical studies with a necessary behavioral equivalent, we investigated encounters between juvenile crayfish and large dragonfly nymphs in freely behaving animals using a combination of high-speed video-recordings and measurements of electric field potentials. During attacks, dragonfly nymphs rapidly extended their labium, equipped with short, sharp palps, to capture small crayfish. Crayfish responded to the tactile stimulus by activating neural escape circuits to generate tail-flips directed away from the predator. Tail-flips were the sole defense mechanism in response to an attack and every single strike was answered by tail-flip escape behavior. Crayfish used all three known types of escape tail-flips during the interactions with the dragonfly nymphs. Tail-flips generated by activity in the giant neurons were predominantly observed to trigger the initial escape responses to an attack, but non-giant mediated tail-flips were often generated to attempt escape after capture. Attacks to the front of the crayfish triggered tail-flips mediated either by the medial giant neuron or by non-giant circuitry, whereas attacks to the rear always elicited tail-flips mediated by the lateral giant neuron. Overall, tail flipping was found to be a successful behavior in preventing predation, and only a small percentage of crayfish were killed and consumed.
Anatomy of a Live Invertebrate Revealed by Manganese-enhanced Magnetic Resonance Imaging
Non-invasive imaging technologies such as Magnetic Resonance Imaging (MRI) are increasingly in demand by researchers in many biological disciplines. However, when imaging small animals such as invertebrates, not only is the use of high-field magnets necessary to gain satisfactory spatial resolution, but the achievement of adequate contrast between tissues also requires the identification of applicable imaging parameters by means of expensive and time-consuming procedures. Here we report that systemically administered manganese can act as an effective MRI contrast agent for quick and non-invasive imaging of the nervous system and other complex anatomical structures in a small aquatic animal. Due to the tendency of manganese ions to differentially accumulate in most soft tissues, higher overall signal intensity and strongly improved contrast between structures yield data well suited for digital post-processing into three-dimensional models. Within a few hours this technique can efficiently generate anatomical images that are not obtainable with conventional methods, thus demonstrating a new and exciting approach to invertebrate research.
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.
The Effects of Social Experience on the Behavioral Response to Unexpected Touch in Crayfish
Crayfish fight and form a dominance hierarchy characterized by a pattern of repeated agonistic interactions between animals with a consistent outcome of winner and loser. Once a dominance hierarchy is established, dominant animals display an elevated posture with both claws held laterally and forward, whereas subordinate animals display a more prone posture with both claws extended forward and down. Dominant animals behave aggressively towards the subordinate opponent, often approaching and attacking, whereas subordinate animals behave submissively by tailflipping and retreating. To evaluate whether the differences in social behavior are accompanied by differences in responses to non-social stimuli, we exposed socially naïve and experienced crayfish (Procambarus clarkii) to an unexpected touch in different social conditions. Socially naïve animals turned to confront the source of a unilateral touch with raised claws and elevated posture. Dominant animals also turned to face the stimulus source with raised claws and elevated posture, both when tested alone and in the presence of a subordinate opponent. Subordinate animals displayed this orienting response only while separated from their dominant partners. When paired with their dominant partners, subordinates avoided the stimulus source by walking rapidly forwards or backwards. When the subordinate animals were later tested again, first while semi-separated from the dominant and later while fully separated, they displayed a mixed pattern of avoidance and orienting responses. These results indicate that the behavioral responses of subordinate crayfish to touch depend on their social status, their current social conditions and their recent social history.
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.
Behavioral and Neural Responses of Juvenile Crayfish to Moving Shadows
One of the most important decisions any animal has to make is how to respond to sensory cues that suggest an imminent attack by a predator. We measured behavioral and neural responses of juvenile crayfish to moving shadows of different velocities while the animals were searching for food. In all experiments, and independent of shadow velocity, each crayfish produced one of two discrete behavioral outputs: it either tail-flipped backwards by rapid flexion of its abdomen or it immediately stopped its forward locomotion. The probability of each behavioral response was dependent on the velocity of the shadows that were presented. While most animals responded with tail-flips to slow-moving shadows and stops were rarely observed, the number of tail-flips decreased as shadow velocity increased. Tail-flips were almost absent for very fast-moving shadows and stopping behavior became the dominating response. By using a non-invasive technique to record neural activity, we were able to identify the underlying neural circuit that controlled the observed tail-flips. All tail-flips were mediated by activation of the medial giant neurons, which are part of a hardwired neural circuit previously described to produce reflexive responses to tactile stimulation.
Sensory Activation and Receptive Field Organization of the Lateral Giant Escape Neurons in Crayfish
Crayfish (Procambarus clarkii) have bilateral pairs of giant interneurons that control rapid escape movements in response to predatory threats. The medial giant neurons (MGs) can be made to fire an action potential by visual or tactile stimuli directed to the front of the animal and this leads to an escape tail-flip that thrusts the animal directly backward. The lateral giant neurons (LGs) can be made to fire an action potential by strong tactile stimuli directed to the rear of the animal, and this produces flexions of the abdomen that propel the crayfish upward and forward. These observations have led to the notion that the receptive fields of the giant neurons are locally restricted and do not overlap with each other. Using extra- and intracellular electrophysiology in whole animal preparations of juvenile crayfish, we found that the receptive fields of the LGs are far more extensive than previously assumed. The LGs receive excitatory inputs from descending interneurons originating in the brain; these interneurons can be activated by stimulation of the antenna II nerve or the protocerebral tract. In our experiments, descending inputs alone could not cause action potentials in the LGs, but when paired with excitatory postsynaptic potentials elicited by stimulation of tail afferents, the inputs summed to yield firing. Thus the LG escape neurons integrate sensory information received through both rostral and caudal receptive fields, and excitatory inputs that are activated rostrally can bring the LGs' membrane potential closer to threshold. This enhances the animal's sensitivity to an approaching predator, a finding that may generalize to other species with similarly organized escape systems.
Neural Control of Behavioural Choice in Juvenile Crayfish
Natural selection leads to behavioural choices that increase the animal's fitness. The neuronal mechanisms underlying behavioural choice are still elusive and empirical evidence connecting neural circuit activation to adaptive behavioural output is sparse. We exposed foraging juvenile crayfish to approaching shadows of different velocities and found that slow-moving shadows predominantly activated a pair of giant interneurons, which mediate tail-flips that thrust the animals backwards and away from the approaching threat. Tail-flips also moved the animals farther away from an expected food source, and crayfish defaulted to freezing behaviour when faced with fast-approaching shadows. Under these conditions, tail-flipping, an ineffective and costly escape strategy was suppressed in favour of freezing, a more beneficial choice. The decision to freeze also dominated in the presence of a more desirable resource; however, the increased incentive was less effective in suppressing tail-flipping when paired with slow-moving visual stimuli that reliably evoked tail-flips in most animals. Together this suggests that crayfish make value-based decisions by weighing the costs and benefits of different behavioural options, and they select adaptive behavioural output based on the activation patterns of identifiable neural circuits.
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.
