Beyond the response-High throughput behavioral analyses to link genome to phenome in Caenorhabditis elegans.

The development and application of methods for automated behavioral analysis have revolutionized behavioral genetics across model organisms. In this review we summarize the history of automated behavioral analysis in the nematode Caenorhabditis elegans. We highlight recent studies of learning and memory to exemplify just how complex the genetic and neural circuit mechanisms underlying a seemingly simple single behavioral response can be. We finish by looking forward at the exciting prospects of combing genomic technologies with connectomic and phenomic level measurements.

Genetic dissection of memory for associative and non-associative learning in Caenorhabditis elegans.

The distinction between non-associative and associative forms of learning has historically been based on the behavioral training paradigm. Through discovering the molecular mechanisms that mediate learning, we can develop a deeper understanding of the relationships between different forms of learning. Here, we genetically dissect short- and long-term memory for a non-associative form of learning, habituation and an associative form of learning, context conditioning for habituation, in the nematode Caenorhabditis elegans. In short-term chemosensory context conditioning for habituation, worms trained and tested in the presence of either a taste (sodium acetate) or smell (diacetyl) context cue show greater retention of habituation to tap stimuli when compared with animals trained and tested without a salient cue. Long-term memory for olfactory context conditioning was observed 24 h after a training procedure that does not normally induce 24 h memory. Like long-term habituation, this long-term memory was dependent on the transcription factor cyclic AMP-response element-binding protein. Worms with mutations in glr-1 [a non-N-methyl-d-aspartate (NMDA)-type glutamate receptor subunit] showed short-term but not long-term habituation or short- or long-term context conditioning. Worms with mutations in nmr-1 (an NMDA-receptor subunit) showed normal short- and long-term memory for habituation but did not show either short- or long-term context conditioning. Rescue of nmr-1 in the RIM interneurons rescued short- and long-term olfactory context conditioning leading to the hypothesis that these interneurons function to integrate information from chemosensory and mechanosensory systems for associative learning.

Early patterned stimulation leads to changes in adult behavior and gene expression in C. elegans.

Across phylogeny, early experience plays a critical role in nervous system development. In these experiments, we investigated the long-term effects that specific patterns of sensory experience during development had on the biology and function of the Caenorhabditis elegans nervous system. The delivery of a specific pattern of mechanosensory stimulation in the first larval stage (L1) produced significant enhancement in the tap withdrawal behavioral response, expression patterns of an ionotropic glutamate receptor (iGluR) subunit and mRNA levels for that receptor in 3-day-old adult worms and a depression of these same three measures in 5-day-old adult worms. A critical period for the 3-day enhanced behavior and GLR distribution was observed in L1, whereas there was no critical period for the depressed effects observed in 5-day-old worms. The spaced pattern of stimulation was essential for expression of this effect: Various forms of massed training produced neither the enhancement at 3 days nor the depression at 5 days. The 5-day depressed behavioral response had many features in common with long-term memory, including sensitivity to disruption following retrieval. The different behavioral and molecular effects that early patterned mechanosensory stimulation produced in 3 and 5-day-old worms led us to hypothesize that separate cellular phenomena produced the enhanced 3-day and depressed 5-day behaviors and molecular effects.

Analyses of habituation in Caenorhabditis elegans.

Although the nonassociative form of learning, habituation, is often described as the simplest form of learning, remarkably little is known about the cellular processes underlying its behavioral expression. Here, we review research on habituation in the nematode Caenorhabditis elegans that addresses habituation at behavioral, neural circuit, and genetic levels. This work highlights the need to understand the dynamics of a behavior before attempting to determine its underlying mechanism. In many cases knowing the characteristics of a behavior can direct or guide a search for underlying cellular mechanisms. We have highlighted the importance of interstimulus interval (ISI) in both short- and long-term habituation and suggested that different cellular mechanisms might underlie habituation at different ISIs. Like other organisms, C. elegans shows both accumulation of habituation with repeated training blocks and long-term retention of spaced or distributed training, but not for massed training. Exposure to heat shock during the interblock intervals eliminates the long-term memory for habituation but not the accumulation of short-term habituation over blocks of training. Analyses using laser ablation of identified neurons, and of identified mutants have shown that there are multiple sites of plasticity for the response and that glutamate plays a role in long-term retention of habituation training.

Context conditioning in habituation in the nematode Caenorhabditis elegans.

Habituation has traditionally been considered a nonassociative form of learning. However, recent research suggests that retention of this nonassociative form of learning may be aided by associations formed during training. An example of this is context conditioning, in which animals that are trained and tested in the presence of a contextual cue show greater retention than animals trained and tested in different environments. This article reports context conditioning in habituation in the nematode Caenorhabditis elegans. The results showed that retention of habituation to tap at both 10- and 60-s interstimulus intervals was significantly greater if training and testing occurred in the presence of the same chemosensory cue (NaCH3COO). This context conditioning showed both extinction and latent inhibition, demonstrating that these simple worms with only 302 neurons are capable of associative context conditioning.

Developmental analysis of habituation in the Nematode C. elegans.

Habituation and spontaneous recovery from habituation to tap were studied across development in C. elegans. Unlike adult worms, larval worms do not consistently swim backwards to tap, but reverse half of the time and accelerate forward half of the time. In adult worms, the tap response is produced by the integration of two competing circuits: The head touch circuit, mediated by ALM and AVM sensory neurons, produces backward movement (reversals); the tail touch circuit, mediated by PLM neurons, produces forward movement (accelerations). Because the response type changes over development, habituation of each of the subcircuits was studied separately. Habituation of the head touch circuit was studied by laser ablating PLM, and habituation of the tail circuit was studied by ablating ALM. Worms were tested at six stages of development at either 10- or 60-s interstimulus intervals. All stages of development showed normal habituation and spontaneous recovery at both interstimulus intervals.

Mutations of the caenorhabditis elegans brain-specific inorganic phosphate transporter eat-4 affect habituation of the tap-withdrawal response without affecting the response itself.

The studies reported here were designed to investigate the role of the mutation eat-4 in the response to tap and in habituation in the nematode Caenorhabditis elegans. In C. elegans eat-4 has been found to affect a number of glutamatergic pathways. It has been hypothesized to positively regulate glutaminase activity and therefore glutamatergic neurotransmission. In the eat-4(ky5) loss-of-function worms, there is presumably insufficient glutamate available for sustained transmission. In the experiments reported here eat-4 worms showed no differences from wild-type in the magnitude of response to a single tap, indicating that the neural circuit underlying the response was intact and functional in the mutant worms. However, when eat-4 worms were given repeated taps the resulting habituation was different from that seen in wild-type worms: eat-4 worms habituate more rapidly and recover more slowly than wild-type worms at all interstimulus intervals tested. In addition, eat-4 worms do not show dishabituation. The same transgene rescues pharyngeal activity defects and both the habituation and dishabituation deficits seen in the eat-4 worms. Our results suggest that neurotransmitter regulation plays a role in habituation and may play a role in dishabituation.

Effects of tap withdrawal response habituation on other withdrawal behaviors: the localization of habituation in the nematode Caenorhabditis elegans.

Four experiments were conducted to identify the possible loci of habituation of the nematode tap withdrawal response (TWR) by characterizing the effects of TWR habituation on other nonmechanosensory withdrawal behaviors that are mediated by overlapping sets of neurons. Experiments 1-2 established behavioral and anatomical relationships between spontaneous and tap-induced backward locomotion in the worm. Experiment 3 demonstrated that habituation of the TWR affected neither the magnitude nor frequency of spontaneous reversal activity. Experiment 4 extended this result to an evoked response: Habituation of the TWR had no effect on reversals evoked by a thermal stimulus. These studies, which show that the loci of change associated with habituation of the TWR are presynaptic to the interneurons and motor neurons that control locomotion, probably distributed among the mechanosensory neurons, illustrate that a complete understanding of plasticity requires a knowledge of both the anatomical and molecular substrates of change.

The integration of antagonistic reflexes revealed by laser ablation of identified neurons determines habituation kinetics of the Caenorhabditis elegans tap withdrawal response.

Previously, we described the circuitry that underlies the tap withdrawal response of the nematode Caenorhabditis elegans. In response to a light mechanosensory stimulus a worm will withdraw, usually by initiating backward locomotion, but occasionally with increased forward locomotion. The form of an animal's response is a product of the balance between two antagonistic reflexes: backward locomotion (reversals) triggered by anterior mechanosensory input and forward locomotion (accelerations) triggered by posterior mechanosensory input. During habituation of this reflex, the frequency of forward and backward locomotion in response to tap is modulated by both experience and interstimulus interval; reversals are more frequent early in a habituation series and at longer Inter stimulus intervals. Single-cell laser microsurgery was used to study each of the subcomponents of the intact behavior during habituation training. Groups of intact or laser-ablated worms were habituated at either a 10-s or a 60-s inter stimulus interval and the kinetics of habituation in each group was analyzed. We demonstrate that each component of the behavior habituates and does so with kinetics that are consistent with the decrement observed in the intact animal.

Recovery from habituation in Caenorhabditis elegans is dependent on interstimulus interval and not habituation kinetics.

The habituation of the tap withdrawal reflex of Caenorhabditis elegans was assessed to determine whether the kinetics of recovery from habituation were dependent on the interstimulus interval (ISI) used during habituation training, or alternately, on the rate and asymptotic level of habituation produced at a given ISI. Two groups of intact animals were trained at either a 10-s (CON10) or a 60-s (CON60) ISI. Laser ablation was used to alter the habituation kinetics in one further group of animals (PLM10), independent of ISI. Although the PLM10 animals trained at a 10-s ISI habituated like CON60 worms, the recovery from habituation of the PLM10 animals very closely resembled the recovery of the CON10 worms. Thus recovery kinetics are dictated by consequences of a given ISI, which do not impact upon habituation rate and asymptote. This suggests the recruitment of multiple ISI-dependent processes during habituation in C. elegans.

A dynamic network simulation of the nematode tap withdrawal circuit: predictions concerning synaptic function using behavioral criteria.

The nematode tap withdrawal reflex demonstrates several forms of behavioral plasticity. Although the neural connectivity that supports this behavior is identified (Integration of mechanosensory stimuli in Caenorhabditis elegans, Wicks and Rankin, 1995, J Neurosci 15:2434-2444), the neurotransmitter phenotypes, and hence whether the synapses in the circuit are excitatory or inhibitory, remain uncharacterized. Here we use a novel strategy to predict the polarity configuration, i.e., the array of excitatory and inhibitory connections, of the nematode tap withdrawal circuit using an anatomically and physiologically justifiable dynamic network simulation of that circuit. The output of the modeled circuit was optimized to the behavior of animals, which possessed circuits altered by surgical ablation by exhaustively enumerating an array of synaptic signs that constituted the modeled circuit. All possible polarity configurations were then compared, and a statistical analysis was used to determine whether, for a given synaptic class, a particular polarity was associated with a good fit to behavioral data. The results from four related experiments were used to predict the polarities of seven of the nine cell classes of the tap withdrawal circuit. In addition, the model was used to assess possible roles for two novel mechanosensory integration neurons: DVA and PVD.

Heat shock disrupts long-term memory consolidation in Caenorhabditis elegans.

Previous work has demonstrated that memory for habituation training is retained for > 24 hr in Caenorhabditis elegans. In this study the timing of memory consolidation was investigated by introducing heat shock (32 degrees C, 45 min) either before training, long after training, or during training. It was found that memory consolidation was disrupted by heat shock during training but not before or after training. In addition, heat shock before training failed to induce thermal tolerance to the effects of heat shock during training on long-term memory formation. When brief heat shock (32 degrees C, 15 min) was presented during training at different intervals, the results suggested that a narrow critical period for memory consolidation of habituation may exist. These findings demonstrate that in C. elegans long-term memory for habituation is disrupted by a temporally defined agent, heat shock. Therefore, heat shock can be used as a fine-grained tool to investigate the dynamics of memory consolidation.

Integration of mechanosensory stimuli in Caenorhabditis elegans.

The tap withdrawal reflex in Caenorhabditis elegans demonstrates various forms of nonassociative learning. A first step in determining the cellular mechanisms of this learning is to identify the neuronal circuitry that underlies this reflex. Studies by Chalfie et al. (1985) have defined the touch-circuit that mediates the response to a stimulus related to tap--a light touch. We used the touch circuit as a starting point in the identification of the tap withdrawal circuitry. Here we report the effects of lesions of identified neurons on the tap withdrawal reflex. Ablations of the sensory neurons and interneurons of the touch circuit produce effects on the tap withdrawal response that generally confirm and expand upon the roles of these cells in mechanosensory integration as proposed by Chalfie et al. (1985). However, no role for the LUA interneurons could be identified in the production of the tap withdrawal response. Furthermore, the effects of ablating some neurons outside the touch circuit suggest roles for two of these cells in the integration of the tap withdrawal response. Ablation of either the midline neuron DVA or the PVD neurons resulted in a decrease in both the frequency and magnitude of reversals that were elicited by tap. Additionally, the ablation of either cell decreased the magnitude of accelerations produced by animals in response to tap.

Effects of changing interstimulus interval during habituation in Caenorhabditis elegans.

The role of the interstimulus interval (ISI) in habituation in Caenorhabditis elegans was explored by examining the effect of changing the ISI on habituation and on spontaneous recovery from habituation. When habituation stimuli were delivered at variable ISIs with an average of 10 s, recovery was slower than when habituation stimuli were delivered at fixed 10-s intervals. There were no differences in recovery following either fixed or variable stimulation at a 60-s ISI. The effect of shifting to a different ISI during habituation training was also explored. A 60-s ISI affected habituation at a 10-s ISI, but a 10-s ISI did not influence habituation at a 60-s ISI. Therefore, habituation must be viewed as an ongoing equilibrium of a number of cellular processes--some decrementing some facilitating--that are differentially activated at different ISIs.

Effects of aging on habituation in the nematode Caenorhabditis elegans.

The effects of aging on spontaneous locomotor behavior and habituation in a mechanosensory reflex were examined in the nematode Caenorhabditis elegans. Worms were tested at 4 days (at the peak of egg laying), at 7 days (when egg laying ends) and at 12 days post-hatching. Both spontaneous and reflexive movements were smaller in older worms than in younger worms. In addition the magnitude of these movements was related to life span; the shorter an animal's life span, the smaller its reversal movements while still young. Worms at all ages expressed habituation and dishabituation at a 10 s interstimulus interval (ISI); thus even aged worms were capable of non-associative learning. However, older worms showed greater habituation than did 4-day-old worms to stimuli delivered at a 60 s ISI. There was also an age-related change in the recovery from habituation. At days 4 and 7, worms had recovered from habituation by 30 min after training; However, responses of day 12 worms were still significantly smaller than baseline at 30 min after training. Further behavioral tests with normal and mutant worms may help elucidate the nature of the age-related changes in the learning and memory processes of C. elegans and the genetic mechanisms which underlie them.

An analysis of behavioral plasticity in male Caenorhabditis elegans.

Caenorhabditis elegans is a simple soil-dwelling nematode which has two sexes, hermaphrodite and male. The male C. elegans is differentiated from the hermaphrodite by the presence of 14 sensory structures in the tail. In this study, we compared the behavioral responses of males and hermaphrodites to head-touch and to tap. We hypothesized that the anatomical difference in sensory structures might result in behavioral differences in the reversal response to vibratory stimulation (a tap to the side of the holding dish). In the response to increasing intensities of tap, both sexes showed an increase in response magnitude, with the males showing larger responses than hermaphrodites. In addition, the male was shown to be capable of simple nonassociative learning: it demonstrated habituation and recovery from habituation in a similar manner as the hermaphrodite. Tail-touch-induced inhibition of the reversal response appeared to be similar in males and hermaphrodites. The evidence suggests that the touch withdrawal circuit in hermaphrodites is also present in the male C. elegans, and that the subtle differences in response to tap seen in males may result from the additional sensory receptors of the copulatory bursa of the tail. It seems clear from these studies that these structures do not play a key role in the male worm's response to tap.

Factors affecting habituation and recovery from habituation in the nematode Caenorhabditis elegans.

In four experiments, the factors that affect the rate of habituation, the degree of habituation, and the rate of recovery from habituation in a simple reflex circuit in Caenorhabditis elegans were investigated. The results showed that habituation was more pronounced and faster, and that recovery from habituation was more rapid, with short interstimulus intervals (ISIs) than with longer ISIs. Rate of recovery differed in animals that had reached asymptotic response levels when compared with animals still in the descending portion of the habituation curve. Once animals reached asymptotic response levels, rate of recovery appeared to be determined by ISI and not by additional stimuli.

Interactions between two antagonistic reflexes in the nematode Caenorhabditis elegans.

1. Antagonistic reflexes that use the same final common path cannot be activated simultaneously; as a consequence one reflex often inhibits the expression of the other. Results of experiments with two antagonistic reflexes in Caenorhabditis elegans showed that the reflex inhibition in this simple animal is the same as in more complex organisms. Thus C. elegans can serve as a model system for studying the neural mechanisms underlying these behavioral patterns. 2. In adult C. elegans tail-touch normally elicits forward movement, while tap normally elicits backward movement. When tail-touch is delivered 1 s before a tap, reversals to the tap are inhibited and the magnitude of any reversal that does occur is reduced. 3. The relative magnitude of the 2 stimuli, tail-touch and tap, affects the amount of inhibition observed. 4. The effectiveness of tail-touch as an inhibitory stimulus can be varied as a result of experience. Habituating the response to tail-touch decreased the inhibition of reversal to tap following a tail-touch. 4. The tail-touch induced inhibition of reversal to tap diminishes over an interval of at least 10 s; however, following the inhibition an enhancement of responding to tap is seen. 6. Inhibition of reversal to tap is present in worms of all stages of development including newly hatched worms.

A developmental analysis of spontaneous and reflexive reversals in the nematode Caenorhabditis elegans.

Reversals of forward locomotion in the nematode Caenorhabditis elegans are thought to be mediated by a common neural circuit, the touch withdrawal circuit. Despite substantial neuroanatomical changes over post-embryonic development, one reversal behavior, the head-touch withdrawal reflex, does not appear to change over development (Chalfie and Sulston, 1981). The experiments reported here indicate that two other reversal behaviors, spontaneous reversals and the tap reversal reflex to vibratory stimuli, show developmental changes. Young adult animals showed higher frequencies of spontaneous reversals than all other developmental stages, while larval stages differed from adults in their pattern of responses to tap. Although animals of all stages reversed in response to touch, taps elicited both reversals and accelerations of forward movement. In response to single taps, larval stages reversed on approximately half the occasions; young adult and 4-day-old adults almost always reversed. Increasing stimulus magnitudes increased the probability of accelerations at all developmental stages, but larval stages showed fewer reversals and more accelerations than adults. The behavioral changes observed coincide with known periods of neuroanatomical change in the touch withdrawal circuit. The addition of a late-developing sensory neuron, AVM, is implicated in the behavioral differences between juveniles and adults.