Long-lasting and context-specific freezing preference is acquired after spaced repeated presentations of a danger stimulus in the crab Chasmagnathus

A visual danger stimulus elicits an escape response in the crab Chasmagnathus that declines after repeated presentations. Previous results report that such waning may be retained as context-signal memory (CSM) or signal memory (SM): CSM is long lasting, associative, and produced by spaced training, while SM is an intermediate memory, nonassociative, and produced by massed training. The performances of both spaced and massed trained crabs are here examined, using video analysis to determine topographic changes in the behavioral response during and after training. During spaced training, escape vanishes and is mainly replaced by freezing, while during massed training, escape decreases over trials without being replaced by any defensive response. After 24 h, the marked proclivity to freezing persists in spaced trained crabs, while a high level of escaping is shown by massed trained crabs. The long-lasting freezing preference of spaced trained crabs proves to be context-specific and apparent from the very first presentation of the danger stimulus at testing, though freezing is not triggered by the sole exposure to the context. We conclude (a) that freezing preference is the acquired response of the CSM process; (b) that CSM can be properly categorized as an instance of contextual conditioning and SM of classical habituation; (c) that CSM and SM are not two phases of a memory processing but two distinctly types of memory; and (d) that therefore, the temporal distribution of training trials has a drastic effect on crab's memory, more dramatic than that previously described. The possibility that massed and spaced presentations of the same stimulus may represent two different stimulus types is discussed.     

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.     

Contextual Pavlovian conditioning in the crab Chasmagnathus

In contextual conditioning, a complex pattern of information is processed to associate the characteristics of a particular place with incentive or aversive reinforcements. This type of learning has been widely studied in mammals, but studies of other taxa are scarce. The context-signal memory (CSM) paradigm of the crab Chasmagnathus has been extensively used as a model of learning and memory. Although initially interpreted as habituation, some characteristics of contextual conditioning have been described. However, no anticipatory response has been detected for animals exposed to the training context. Thus, CSM could be interpreted either as an associative habituation or as contextual conditioning that occurs without a context-evoked anticipatory response. Here, we describe a training protocol developed for contextual Pavlovian conditioning (CPC). For each training trial, the context (conditioned stimulus, CS) was discretely presented and finished together with the unconditioned stimulus (US). In agreement with the CSM paradigm, a robust freezing response was acquired during the 15 training trials, and clear retention was found when tested with the US presentation after short (2 and 4 h) and long (1–4 days) delays. This CPC memory showed forward but not simultaneous presentation conditioning and was context specific and protein synthesis dependent. Additionally, a weak CPC memory was enhanced during consolidation. One day after training, CPC was extinguished by repeated CS presentation, while one presentation induced a memory labilisation–reconsolidation process. Finally, we found an anticipatory conditioned response (CR) during the CS presentation for both short-term (4 h) and long-term memory (24 h). These findings support the conditioning nature of the new paradigm.     

Generalization of Habituation and Intrinsic Sensitization in the Leech

Using the shortening reflex of the medicinal leech Hirudo medicinalis we examined stimulus generalization of habituation learning. Preparations received mechanosensory stimulus at two positions on the leech body wall, one site used to carry out habituation training and a second novel site to test for generalization of habituation. After training, the specific mechanosensory neurons activated by each stimulus were assessed using intracellular recordings. As expected, the closer the two sites were to each other, the greater the degree of generalization of habituation at the novel site and the more sensory cells were shared. However, a form of behavioral facilitation was observed at the trained site that resembled behavioral sensitization, but differed from the standard sensitization process in several respects. (1) Facilitation was induced by stimulation of the novel site before habituation training at the trained site, although the stimulus intensity at the novel site was equivalent to the training stimuli and was not the strong, noxious stimuli that normally induce sensitization. (2) The magnitude of the facilitating effect was proportional to the proximity of the novel and trained stimulation sites. (3) Although behavior at the trained site was facilitated, behavior at the novel site was habituated, indicating that the induced behavioral facilitation did not generalize throughout the animal, as normally occurs during sensitization, but was limited to a single stimulus–response pathway.     

Neurogenetic approaches to habituation and dishabituation in Drosophila

We review work in the major model systems for habituation in Drosophila melanogaster, encompassing several sensory modalities and behavioral contexts: visual (giant fiber escape response, landing response); chemical (proboscis extension reflex, olfactory jump response, locomotory startle response, odor-induced leg response, experience-dependent courtship modification); electric (shock avoidance); and mechanical (leg resistance reflex, cleaning reflex). Each model system shows several of Thompson and Spencer’s [Thompson, R. F., & Spencer, W. A. (1966). Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychological Review, 73, 16–43] parametric criteria for habituation: spontaneous recovery and dishabituation have been described in almost all of them and dependence of habituation upon stimulus frequency and stimulus intensity in the majority. Stimulus generalization (and conversely, the delineation of stimulus specificity) has given insights into the localization of habituation or the neural architecture underlying sensory processing.The strength of Drosophila for studying habituation is the range of genetic approaches available. Mutations have been used to modify specific neuroanatomical structures, ion channels, elements of synaptic transmission, and second-messenger pathways. rutabaga and dunce, genes of the cAMP signal pathway that have been studied most often in the reviewed experiments, have also been implicated in synaptic plasticity and associative conditioning in Drosophila and other species including mammals. The use of the Gal4/UAS system for targeting gene expression has enabled genetic perturbation of defined sets of neurons. One clear lesson is that a gene may affect habituation differently in different behaviors, depending on the expression, processing, and localization of the gene product in specific circuits. Mutations of specific genes not only provide links between physiology and behavior in the same circuit, but also reveal common mechanisms in different paradigms of behavioral plasticity. The rich repertoire of models for habituation in the fly is an asset for combining a genetic approach with behavioral, anatomical and physiological methods with the promise of a more complete understanding.     

A new group-training procedure for habituation demonstrates that presynaptic glutamate release contributes to long-term memory in Caenorhabditis elegans

In the experiments reported here we have developed a new group-training protocol for assessing long-term memory for habituation in Caenorhabditis elegans. We have replicated all of the major findings of the original single-worm protocol using the new protocol: (1) distributed training produced long-term retention of training, massed training did not; (2) distributed training at long interstimulus intervals (ISIs) produced long-term retention, short ISIs did not; and (3) long-term memory for distributed training is protein synthesis-dependent as it could be blocked by heat shock during the inter-block interval. In addition, we have shown that long-term memory for habituation is graded, depending on the number of blocks of stimuli in training. The inter-block interval must be >40 min for long-term retention of training to occur. Finally, we have tested long-term memory for habituation training in a strain of worms with a mutation in a vesicular glutamate transporter in the sensory neurons that transduce tap (eat-4). The results from these eat-4 worms indicate that glutamate release from the sensory neurons has an important role in the formation of long-term memory for habituation.     

Behavioral and genetic characterization of habituation using Caenorhabditis elegans

This review surveys the literature that investigates the behavioral characterization and cellular and molecular mechanisms of habituation using the model organism Caenorhabditis elegans. In 1990, C. elegans was first observed to show habituation to a non-localized mechanical tap. The parameters that govern this behavioral plasticity in C. elegans were subsequently characterized, which lead to the important hypothesis that habituation is mediated by multiple mechanisms. Many tools are available to C. elegans researchers that allow for relatively easy genetic manipulation. This has lead to a number of recent genetic studies that have begun to identify key genes and molecules that play a role in the mechanisms of habituation. Some of these genes include a vesicular glutamate transporter, a glutamate receptor subunit, a dopamine receptor and downstream intracellular signaling molecules, such as G proteins and kinases. Some of these genes only affect certain parameters of habituation, but not others supporting the hypothesis that multiple mechanisms mediate habituation. The field of research has also led to the dissection of different phases of memory (short-term vs. long-term memory for habituation), which are triggered by different training paradigms. The differences in mechanism between these various forms of memory are also beginning to be revealed.     

Habituation in Aplysia: The Cheshire Cat of neurobiology

The marine snail, Aplysia californica, is a valuable model system for cell biological studies of learning and memory. Aplysia exhibits a reflexive withdrawal of its gill and siphon in response to weak or moderate tactile stimulation of its skin. Repeated tactile stimulation causes this defensive withdrawal reflex to habituate. Both short-term habituation, lasting <30 min, and long-term habituation, which can last >24 h, have been reported in Aplysia. Habituation of the withdrawal reflex correlates with, and is in part due to, depression of transmission at the monosynaptic connection between mechanoreceptive sensory neurons and motor neurons within the abdominal ganglion. Habituation-related short-term depression of the sensorimotor synapse appears to be due exclusively to presynaptic changes. However, changes within the sensory neuron, by themselves, do not account for more persistent depression of the sensorimotor synapse. Recent behavioral work suggests that long-term habituation in Aplysia critically involves postsynaptic processes, specifically, activation of AMPA- and NMDA-type receptors. In addition, long-term habituation requires activity of protein phosphatases, including protein phosphatases 1, 2A, and 2B, as well as activity of voltage-dependent Ca²⁺ channels. Cellular work has succeeded in demonstrating long-term, homosynaptic depression (LTD) of the sensorimotor synapse in dissociated cell culture and, more recently, LTD of the glutamate response of isolated motor neurons in culture (“hemisynaptic” LTD). These in vitro forms of LTD have mechanistic parallels to long-term habituation. In particular, homosynaptic LTD of the sensorimotor synapse requires elevated intracellular Ca²⁺ within the motor neuron, and hemisynaptic LTD requires activity of AMPA- and NMDA-type receptors. In addition, activation of group I and II metabotropic glutamate receptors (mGluRs) can induce hemisynaptic LTD. The demonstration of LTD in vitro opens up a promising new avenue for attempts to relate long-term habituation to cellular changes within the nervous system of Aplysia.     

Habituation in children within a behavior suppression paradigm

Using an instrumental response suppression paradigm, Experiment 1 demonstrated habituation and long-term retention of habituation to a tone stimulus in third-grade children, but specificity of habituation was not obtained when a new tone stimulus was presented. In Experiment 2 first graders showed specificity of habituation to auditory stimuli on the response suppression measure whereas fifth graders did not. However, both age groups evidenced specificity when a skin conductance measure was employed. In addition, specificity of habituation was not affected by amount of training at either age level. Experiment 3 demonstrated specificity of habituation in fifth graders on the response suppression measure when a cross-modality stimulus change was introduced. Implications of these data for analyses of habituation and traditional learning are discussed.     

Extinction memory in the crab Chasmagnathus: recovery protocols and effects of multi-trial extinction training

A decline in the frequency or intensity of a conditioned behavior following the withdrawal of the reinforcement is called experimental extinction. However, the experimental manipulation necessary to trigger memory reconsolidation or extinction is to expose the animal to the conditioned stimulus in the absence of reinforcement. Recovery protocols were used to reveal which of these two processes was developed. By using the crab contextual memory model (a visual danger stimulus associated with the training context), we investigated the dynamics of extinction memory in Chasmagnathus. Here, we reveal the presence of three recovery protocols that restore the original memory: the old memory comes back 4 days after the extinction training, or when a weak training is administered later, or once the VDS is presented in a novel context 24 h after the extinction session. Another objective was to evaluate whether the administration of multi-trial extinction training could trigger an extinction memory in Chasmagnathus. The results evince that the extinction memory appears only when the total re-exposure time is around 90 min independently of the number of trials employed to accumulate it. Thus, it is feasible that the mechanisms described for the case of the extinction memory acquired through a single training trial are valid for multi-trial extinction protocols. Finally, these results are in agreement with those reports obtained with models phylogenetically far apart from the crab. Behind this attempt is the idea that in the domain of studies on memory, some principles of behavior organization and basic mechanisms have universal validity.     

Memoirs of a locust: Density-dependent behavioral change as a model for learning and memory

A locust outbreak is a stupendous natural phenomenon that remains in the memory of whoever has been lucky (or unlucky) enough to witness it. Recent years have provided novel and important insights into the neurobiology of locust swarming. However, the central nervous system processes that accompany and perhaps even lie at the basis of locust phase transformation are still far from being fully understood. Our current work deals with the memory of a locust outbreak from a new perspective: that of the individual locust. We take locust density-dependent phase transformation – a unique example of extreme behavioral plasticity, and place it within the context of the accepted scheme of learning and memory. We confirm that a short time period of exposure to a small crowd of locusts is sufficient to induce a significant behavioral change in a previously solitary locust. Our results suggest that part of the behavioral change is due to long-term habituation of evasive and escape responses. We further demonstrate that the memory of a crowding event lasts for at least 24 h, and that this memory is sensitive to a protein synthesis blocker. These findings add much to our understanding of locust density-dependent phase polyphenism. Furthermore, they offer a novel and tractable model for the study of learning and memory-related processes in a very distinctive behavioral context.     

Competitive short-term and long-term memory processes in spatial habituation

Exposure to a spatial location leads to habituation of exploration such that, in a novelty preference test, rodents subsequently prefer exploring a novel location to the familiar location. According to Wagner's (1981) theory of memory, short-term and long-term habituation are caused by separate and sometimes opponent processes. In the present study, this dual-process account of memory was tested. Mice received a series of exposure training trials to a location before receiving a novelty preference test. The novelty preference was greater when tested after a short, rather than a long, interval. In contrast, the novelty preference was weaker when exposure training trials were separated by a short, rather than a long interval. Furthermore, it was found that long-term habituation was determined by the independent effects of the amount of exposure training and the number of exposure training trials when factors such as the intertrial interval and the cumulative intertrial interval were controlled. A final experiment demonstrated that a long-term reduction of exploration could be caused by a negative priming effect due to associations formed during exploration. These results provide evidence against a single-process account of habituation and suggest that spatial habituation is determined by both short-term, recency-based memory and long-term, incrementally strengthened memory.     

Associative learning versus fear habituation as predictors of long-term extinction retention

Violation of unconditioned stimulus (US) expectancy during extinction training may enhance associative learning and result in improved long-term extinction retention compared to within-session habituation. This experiment examines variation in US expectancy (i.e., expectancy violation) as a predictor of long-term extinction retention. It also examines within-session habituation of fear-potentiated startle (electromyography, EMG) and fear of conditioned stimuli (CS) throughout extinction training as predictors of extinction retention. Participants (n?=?63) underwent fear conditioning, extinction and retention and provided continuous ratings of US expectancy and EMG, as well as CS fear ratings before and after each phase. Variation in US expectancy throughout extinction and habituation of EMG and fear was entered into a regression as predictors of retention and reinstatement of levels of expectancy and fear. Greater variation in US expectancy throughout extinction training was significantly predictive of enhanced extinction performance measured at retention test, although not after reinstatement test. Slope of EMG and CS fear during extinction did not predict retention of extinction. Within-session habituation of EMG and self-reported fear is not sufficient for long-term retention of extinction learning, and models emphasizing expectation violation may result in enhanced outcomes.     

Habituation to repeated stress: get used to it

Habituation, as described in the landmark paper by Thompson et al. [Thompson, R. F., & Spencer, W. A. (1966). Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychological Review, 73(1), 16–43], is a form of simple, nonassociative learning in which the magnitude of the response to a specific stimulus decreases with repeated exposure to that stimulus. A variety of neuronal and behavioral responses have been shown to be subject to habituation based on the criteria presented in that paper. It has been known for several decades that the magnitude of hypothalamic–pituitary–adrenal (HPA) activation occurring in response to a stressor declines with repeated exposure to that same stressor. For some time this decline has been referred to as “habituation” in the stress neurobiology literature. However, how this usage compares to the definition proposed by Thompson and Spencer has not been systematically addressed. For this special issue, we review the stress neurobiology literature and examine the support available for considering declines in HPA response to repeated stress to be response habituation in the sense defined by Thompson and Spencer. We conclude that habituation of HPA activity meets many, but not all, important criteria for response habituation, supporting the use of this term within the context of repeated stress. However, we also propose that response habituation can, at best, only partially explain the phenomenon of HPA habituation, which also involves well-known negative feedback mechanisms, activation of broad stress-related neural circuitry and potentially more complex associative learning mechanisms.     

Escape from fear: a detailed behavioral analysis of two atypical responses reinforced by CS termination

Escape from fear (EFF) is a controversial paradigm according to which animals learn to actively escape a fear-eliciting conditioned stimulus (CS) if the escape response (R-sub(e)) is paired with CS termination. Some theories posit that EFF learning is responsible for instrumental avoidance conditioning. However, EFF learning has typically been weaker than avoidance learning and difficult to reproduce. The authors examined EFF learning and memory with 2 atypical R-sub(e)s: rearing and nose-poking. The data suggest that rearing, but not nose-poking, can be learned as an instrumental EFF response. Further, EFF memory was response specific, aversively motivated, and controlled by the CS. Successful EFF learning also resulted in better long-term elimination of a passive fear reaction (freezing). Factors important for EFF experiments and theoretical considerations are discussed.     

A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila

The Drosophila giant fiber jump-and-flight escape response is a model for genetic analysis of both the physiology and the plasticity of a sensorimotor behavioral pathway. We previously established the electrically induced giant fiber response in intact tethered flies as a model for habituation, a form of nonassociative learning. Here, we show that the rate of stimulus-dependent response decrement of this neural pathway in a habituation protocol is correlated with PKG (cGMP-Dependent Protein Kinase) activity and foraging behavior. We assayed response decrement for natural and mutant rover and sitter alleles of the foraging (for) gene that encodes a Drosophila PKG. Rover larvae and adults, which have higher PKG activities, travel significantly farther while foraging than sitters with lower PKG activities. Response decrement was most rapid in genotypes previously shown to have low PKG activities and sitter-like foraging behavior. We also found differences in spontaneous recovery (the reversal of response decrement during a rest from stimulation) and a dishabituation-like phenomenon (the reversal of response decrement evoked by a novel stimulus). This electrophysiological study in an intact animal preparation provides one of the first direct demonstrations that PKG can affect plasticity in a simple learning paradigm. It increases our understanding of the complex interplay of factors that can modulate the sensitivity of the giant fiber escape response, and it defines a new adult-stage phenotype of the foraging locus. Finally, these results show that behaviorally relevant neural plasticity in an identified circuit can be influenced by a single-locus genetic polymorphism existing in a natural population of Drosophila.     

Influence of long-term sensitization on long-term habituation of the acoustic startle response in rats: central gray lesions, preexposure, and extinction

The relation between long-term decrements of the acoustic startle response in rats and the development of freezing behavior during habituation training was examined. Freezing behavior developed over the initial trials of habituation training, and the rate of long-term response decrements was found to be inversely related to the development of freezing. Manipulations (neurological or behavioral) that either reduced the level of freezing or retarded its development promoted startle response decrements. In Experiment 1, rats receiving electrolytic lesions of the ventrolateral periaqueductal gray demonstrated both accelerated long-term startle response decrements and retarded development of freezing behavior. In Experiment 2, preexposure to the startle apparatus (i.e., latent inhibition) accelerated long-term startle decrements and inhibited development of freezing. In Experiment 3, exposure to the startle apparatus following initial habituation training (i.e., extinction) reduced both freezing behavior and startle response amplitudes. The results are discussed in terms of the influence of Pavlovian fear conditioning on long-term habituation of the acoustic startle response.     

Protection from premature habituation requires functional mushroom bodies in Drosophila

Diminished responses to stimuli defined as habituation can serve as a gating mechanism for repetitive environmental cues with little predictive value and importance. We demonstrate that wild-type animals diminish their responses to electric shock stimuli with properties characteristic of short- and long-term habituation. We used spatially restricted abrogation of neurotransmission to identify brain areas involved in this behavioral response. We find that the mushroom bodies and, in particular, the α/β lobes appear to guard against habituating prematurely to repetitive electric shock stimuli. In addition to protection from premature habituation, the mushroom bodies are essential for spontaneous recovery and dishabituation. These results reveal a novel modulatory role of the mushroom bodies on responses to repetitive stimuli in agreement with and complementary to their established roles in olfactory learning and memory.     

Regulation of low-threshold afferent activity may contribute to short-term habituation in Aplysia californica

The goal of this study was to characterize the contribution of a population of low-threshold mechanoreceptors to short-term habituation of siphon-elicited reflex responses in Aplysia californica. Since the location of their somata is unknown, we refer to them as the Unidentified Low-Threshold mechanoreceptors (ULTs). The ULTs operate in parallel to the higher-threshold and well-characterized LE sensory neurons, yet little is known regarding their contribution to behavioral plasticity. Using extracellular recordings from the siphon nerve, we found that habituation training that favors ULT activation resulted in a significant decrease in afferent activity at training intervals up to 1 min per stimulus (1 min ISI). To determine how this reduction impacts responses at other sites of the reflex network, we used intracellular recordings to measure training-induced changes in either L29 excitatory interneurons or LFS siphon motor neurons. With a 30s ISI, changes at both locations were training site-specific and matched the rate of change of afferent activity, implicating regulated sensory activity as a primary mechanism. With a shorter training interval (1s ISI), site-specificity of training was not observed in the L29s, but was still preserved in the motor neurons. For both, the rate of change during training was faster than the rate of change of afferent activity. Taken together, we conclude that regulation of low-threshold sensory neuron activity can play a significant role in short-term habituation, but other network processes may be recruited at more rapid training intervals.