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 behavioral and genetic dissection of two forms of olfactory plasticity in Caenorhabditis elegans: adaptation and habituation

Continuous presentation of an olfactory stimulus causes a decrement of the chemotaxis response in the nematode Caenorhabditis elegans. However, the differences between the learning process of habituation (a readily reversible decrease in behavioral response) and other types of olfactory plasticity such as adaptation (a decrement in response due to sensory fatigue, which cannot be dishabituated) have not been addressed. The volatile odorant diacetyl (DA) was used within a single paradigm to assess the distinct processes of olfactory adaptation and habituation. Preexposing and testing worms to 100% DA vapors caused a chemotaxis decrement that was not reversible despite the presentation of potentially dishabituating stimuli. This DA adaptation was abolished in worms with an odr-10 mutation (encoding a high-affinity DA receptor on the AWA neuron), even though naive chemotaxis remained unaffected. Conversely, DA adaptation remained intact in odr-1 mutants (defective in AWC neuron-mediated olfactory behavior), even though naive chemotaxis to DA decreased. Surprisingly, exposure to vapors of intermediate concentrations of DA (0.01% and 25%) did not cause worms to exhibit any response decrement. In contrast to preexposure to high DA concentrations, preexposure to low DA concentrations (0.001%) produced habituation of the chemotaxis response (a dishabituating stimulus could reverse the response decrement back to baseline levels). The distinct behavioral effects produced by DA preexposure highlight a concentration-dependent dissociation between two decremental olfactory processes: adaptation at high DA concentrations versus habituation at low DA concentrations.     

Mapping of the Anatomical Circuit of CaM Kinase-Dependent Courtship Conditioning in Drosophila

Globally inhibiting CaM kinase activity in Drosophila, using a variety of genetic techniques, disrupts associative memory yet leaves visual and chemosensory perception intact. These studies implicate CaM kinase in the plastic processes underlying learning and memory but do not identify the neural circuitry that specifies the behavior. In this study, we use the GAL4/UAS binary expression system to define areas of the brain that require CaM kinase for modulation of courtship conditioning. The CaM kinase-dependent neurons that determine the response to the mated female during conditioning and those involved in formation and expression of memory were found to be located in distinct areas of the brain. This supports the idea that courtship conditioning results in two independent behavioral modifications: a decrement in courtship during the conditioning period and an associative memory of conditioning. This study has allowed us for the first time to genetically determine the circuit of information flow for a memory process in Drosophila. The map we have generated dissects the behavior into multiple components and will provide tools that allow both molecular and electrophysiological access to this circuit.     

Activity of cGMP-dependent protein kinase (PKG) affects sucrose responsiveness and habituation in Drosophila melanogaster

The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The foraging (for) gene encodes a PKG in Drosophila melanogaster. Here, we demonstrate a function for the for gene in sensory responsiveness and nonassociative learning. Larvae of the natural variant sitter (for(s)) show less locomotor activity during feeding and have a lower PKG activity than rover (for(R)) larvae. We used rover and sitter adult flies to test whether PKG activity affects (1) responsiveness to sucrose stimuli applied to the front tarsi, and (2) habituation of proboscis extension after repeated sucrose stimulation. To determine whether the differences observed resulted from variation in the for gene, we also tested for(s2), a sitter mutant produced on a rover genetic background. We found that rovers (for(R)) were more responsive to sucrose than sitters (for(s) and for(s2)) at 1-, 2-, and 3-wk old. This was true for both sexes. Differences in sucrose responsiveness between rovers and sitters were greater after 2 h of food deprivation than after 24 h. Of flies with similar sucrose responsiveness, for(R) rovers showed less habituation and generalization of habituation than for(s) and for(s2) sitters. These results show that the PKG encoded by for independently affects sensory responsiveness and habituation in Drosophila melanogaster.     

Sites of Plasticity in the Neural Circuit Mediating Tentacle Withdrawal in the Snail Helix aspersa: Implications for Behavioral Change and Learning Kinetics

The tentacle withdrawal reflex of the snail Helix aspersa exhibits a complex combination of habituation and sensitization consistent with the dual-process theory of plasticity. Habituation, sensitization, or a combination of both were elicited by varying stimulation parameters and lesion condition. Analysis of response plasticity shows that the late phase of the response is selectively enhanced by sensitization, whereas all phases are decreased by habituation. Previous data have shown that tentacle withdrawal is mediated conjointly by parallel monosynaptic and polysynaptic pathways. The former mediates the early phase, whereas the latter mediates the late phase of the response. Plastic loci were identified by stimulating and recording at different points within the neural circuit, in combination with selective lesions. Results indicate that depression occurs at an upstream locus, before circuit divergence, and is therefore expressed in all pathways, whereas facilitation requires downstream facilitatory neurons and is selectively expressed in polysynaptic pathways. Differential expression of plasticity between pathways helps explain the behavioral manifestation of depression and facilitation. A simple mathematical model is used to show how serial positioning of depression and facilitation can explain the kinetics of dual-process learning. These results illustrate how the position of cellular plasticity in the network affects behavioral change and how forms of plasticity can interact to determine the kinetics of the net changes.     

Behavioral and neuronal attributes of short- and long-term habituation in the crab Chasmagnathus

Investigations using invertebrate species have led to a considerable progress in our understanding of the mechanisms underlying learning and memory. In this review we describe the main behavioral and neuronal findings obtained by studying the habituation of the escape response to a visual danger stimulus in the crab Chasmagnathus granulatus. Massed training with brief intertrial intervals lead to a rapid reduction of the escape response that recovers after a short term. Conversely, few trials of spaced training renders a slower escape reduction that endures for many days. As predicted by Wagner’s associative theory of habituation, long-term habituation in the crab proved to be determined by an association between the contextual environment of the training and the unconditioned stimulus. By performing intracellular recordings in the brain of the intact animal at the same time it was learning, we identified a group of neurons that remarkably reflects the short- and long-term behavioral changes. Thus, the visual memory abilities of crabs, their relatively simple and accessible nervous system, and the recording stability that can be achieved with their neurons provide an opportunity for uncovering neurophysiological and molecular events that occur in identifiable neurons during learning.     

Dishabituation of the Tritonia escape swim

When repeatedly elicited, the oscillatory escape swim of the marine mollusc Tritonia diomedea undergoes habituation of the number of cycles per swim. Although similar in most respects to habituation observed in vertebrates and other invertebrates, one key feature, dishabituation, has been surprisingly difficult to demonstrate. Here we evaluate the hypothesis that this is due to interference from short-term sensitization, which is manifested as a reduction in swim onset latency, that occurs simultaneously during habituation training. Robust dishabituation was obtained using a multisession habituation protocol designed to allow this sensitization to dissipate before the dishabituatory stimulus was applied. These results extend the similarity of habituation in Tritonia to that described in other species, strengthening the usefulness of this preparation as a model system for studies of the cellular basis of habituation.     

Natural variation in Drosophila larval reward learning and memory due to a cGMP-dependent protein kinase

Animals must be able to find and evaluate food to ensure survival. The ability to associate a cue with the presence of food is advantageous because it allows an animal to quickly identify a situation associated with a good, bad, or even harmful food. Identifying genes underlying these natural learned responses is essential to understanding this ability. Here, we investigate whether natural variation in the foraging (for) gene in Drosophila melanogaster larvae is important in mediating associations between either an odor or a light stimulus and food reward. We found that for influences olfactory conditioning and that the mushroom bodies play a role in this for-mediated olfactory learning. Genotypes associated with high activity of the product of for, cGMP-dependent protein kinase (PKG), showed greater memory acquisition and retention compared with genotypes associated with low activity of PKG when trained with three conditioning trials. Interestingly, increasing the number of training trials resulted in decreased memory retention only in genotypes associated with high PKG activity. The difference in the dynamics of memory acquisition and retention between variants of for suggests that the ability to learn and retain an association may be linked to the foraging strategies of the two variants.     

Newborn habituation to visual stimuli: a sex difference in novelty detection

Thirty-six newborn human infants were repeatedly exposed to one of two visual stimuli, a four-square or 144-square black and white checkerboard target, until a set criterion of habituation was demonstrated, as measured by a decrement in visual fixation time. When the habituation criterion was reached, independent groups of Ss were either presented with the same target or with a target of either moderate or large discrepancy from the standard habituation stimulus. Results indicated that (1) there was habituation of visual attention, which replicated previous findings, and suggests that some infants soon after birth are capable of storing simple visual information, and (2) following habituation female infants displayed greater recovery of attention than male infants when the moderate stimulus change was introduced.     

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ～20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. Our observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation.     

Using habituation in machine learning

Habituation, a decrement in response to a stimulus that is presented repeatedly without ill effect, can be identified in almost all animals. It can also be used in machine learning to provide a variety of different applications, such as novelty detection, recency encoding, and temporal signal pre-processing. This paper examines how habituation can be mathematically modelled, and discusses how well these models fit the revised characteristics of habituation. It then demonstrates how the models can be combined with neural networks in order to realise the various applications. Finally, some simple experimental results are presented that demonstrate the effectiveness of the methods.     

A role for Synapsin in associative learning: the Drosophila larva as a study case

Synapsins are evolutionarily conserved, highly abundant vesicular phosphoproteins in presynaptic terminals. They are thought to regulate the recruitment of synaptic vesicles from the reserve pool to the readily-releasable pool, in particular when vesicle release is to be maintained at high spiking rates. As regulation of transmitter release is a prerequisite for synaptic plasticity, we use the fruit fly Drosophila to ask whether Synapsin has a role in behavioral plasticity as well; in fruit flies, Synapsin is encoded by a single gene (syn). We tackled this question for associative olfactory learning in larval Drosophila by using the deletion mutant syn^97CS, which had been backcrossed to the Canton-S wild-type strain (CS) for 13 generations. We provide a molecular account of the genomic status of syn^97CS by PCR and show the absence of gene product on Western blots and nerve-muscle preparations. We found that olfactory associative learning in syn^97CS larvae is reduced to ~50% of wild-type CS levels; however, responsiveness to the to-be-associated stimuli and motor performance in untrained animals are normal. In addition, we introduce two novel behavioral control procedures to test stimulus responsiveness and motor performance after “sham training.” Wild-type CS and syn^97CS perform indistinguishably also in these tests. Thus, larval Drosophila can be used as a case study for a role of Synapsin in associative learning.     

Interactions between Depression and Facilitation within Neural Networks: Updating the Dual-Process Theory of Plasticity

Repetitive stimulation often results in habituation of the elicited response. However, if the stimulus is sufficiently strong, habituation may be preceded by transient sensitization or even replaced by enduring sensitization. In 1970, Groves and Thompson formulated the dual-process theory of plasticity to explain these characteristic behavioral changes on the basis of competition between decremental plasticity (depression) and incremental plasticity (facilitation) occurring within the neural network. Data from both vertebrate and invertebrate systems are reviewed and indicate that the effects of depression and facilitation are not exclusively additive but, rather, that those processes interact in a complex manner. Serial ordering of induction of learning, in which a depressing locus precedes the modulatory system responsible for inducing facilitation, causes the facilitation to wane. The parallel and/or serial expression of depression and waning facilitation within the stimulus–response pathway culminates in the behavioral changes that characterize dual-process learning. A mathematical model is presented to formally express and extend understanding of the interactions between depression and facilitation.     

Habituation in normal and Down's syndrome fetuses.

The ability of ten normal and two Down's syndrome fetuses to habituate to a repeated auditory stimulus was assessed. The normal fetuses were assessed on two separate occasions during the final trimester of pregnancy. The number of trials to habituate and latency to respond were recorded. The results demonstrated that all normal fetuses exhibited a decrement in response to repeated presentations of the stimulus, a 250-Hz sine wave. This was the result of habituation and not fatigue since fetuses recovered responding upon presentation of a new (500 Hz) stimulus and habituated faster on re-presentation of the original stimulus. The performance of fetuses remained stable over the two testing periods. The response of the Down's syndrome fetuses was different to that of normal fetuses rarely overlapping indices of habituation exhibited by normal fetuses. The study concludes that habituation may be a powerful tool to examine the behavioural and sensory development of the fetus and may be of use in the antenatal assessment of the existence and severity of neural abnormality.     

Habituation in normal and down's syndrome fetuses

The ability of ten normal and two Down's syndrome fetuses to habituate to a repeated auditory stimulus was assessed. The normal fetuses were assessed on two separate occasions during the final trimester of pregnancy. The number of trials to habituate and latency to respond were recorded. The results demonstrated that all normal fetuses exhibited a decrement in response to repeated presentations of the stimulus, a 250-Hz sine wave. This was the result of habituation and not fatigue since fetuses recovered responding upon presentation of a new (500 Hz) stimulus and habituated faster on re-presentation of the original stimulus. The performance of fetuses remained stable over the two testing periods. The response of the Down's syndrome fetuses was different to that of normal fetuses rarely overlapping indices of habituation exhibited by normal fetuses. The study concludes that habituation may be a powerful tool to examine the behavioural and sensory development of the fetus and may be of use in the antenatal assessment of the existence and severity of neural abnormality.     

Visual input regulates circuit configuration in courtship conditioning of Drosophila melanogaster

Courtship and courtship conditioning are behaviors that are regulated by multiple sensory inputs, including chemosensation and vision. Globally inhibiting CaMKII activity in Drosophila disrupts courtship plasticity while leaving visual and chemosensory perception intact. Light has been shown to modulate CaMKII-dependent memory formation in this paradigm and the circuitry for the nonvisual version of this behavior has been investigated. In this paradigm, volatile and tactile pheromones provide the primary driving force for courtship, and memory formation is dependent upon intact mushroom bodies and parts of the central complex. In the present study, we use the GAL4/UAS binary expression system to define areas of the brain that require CaMKII for modulation of courtship conditioning in the presence of visual, as well as chemosensory, information. Visual input suppressed the ability of mushroom body- and central complex-specific CaMKII inhibition to disrupt memory formation, indicating that the cellular circuitry underlying this behavior can be remodeled by changing the driving sensory modality. These findings suggest that the potential for plasticity in courtship behavior is distributed among multiple biochemically and anatomically distinct cellular circuits.     

Embodied infant attention

Does real time coupling between mental and physical activity early in development have functional significance? To address this question, we examined the habituation of visual attention and the subsequent response to change in two groups of 3‐month‐olds with different patterns of movement–attention coupling. In suppressors, the typical decrease in body movement at the onset of looks persists into the looks. In rebounders, the initial decrease is more transient and movement quickly returns above baseline. Suppressors and rebounders did not differ on measures of looking during habituation, but when the stimulus changed rebounders looked more than suppressors. When it did not change, they looked less. In addition, during habituation rebounders spent more time looking away from the stimulus. Rapid motor reactivation soon after gaze locks onto a target, characteristic of rebounders, may influence visual foraging and the response to change by keeping attention near a threshold of engagement.     

Ontogenetic dissociation of two classes of habituation.

The rates of decrement of two classes of response (an elicited startle reflex and emitted exploratory behavior) were determined in rats of two different ages (15 and 36 days). The rate of decrement in the startle reflex was not clearly differentiated as a function of age. In contrast, there was no evidence of habituation of exploration in the younger animals, whereas the older rats uniformly showed profound response decrements. This ontogenetic dissociation of the two instances of response decrement indicates that accounts of both instances in terms of a common process called habituation may be unwarranted. In addition, these data, in conjunction with earlier findings, indirectly support the possibility that reflex decrements may be relatively more dependent on brain serotonin whereas decrements in exploration may be more dependent on normal cholinergic activity in brain.     

The evolutionary significance of habituation and sensitization across phylogeny: A behavioral homeostasis model

The phenomenon of habituation may be interpreted as a process that has evolved for filtering out iterative stimuli of little present relevance. That habituation is seen in aneural as well as neural organisms throughout phylogeny with remarkably similar characteristics suggests that its role is an important one in animal survival. If habituation is to be viewed as a process to filter out iterative stimuli that have no significant consequences, then how is sensitization to be viewed? One way of viewing these two behavioral changes, i.e. habituation and sensitization, is that they are homeostatic processes which optimize an organism’s likelihood of detecting and assessing the significance of a stimulus in a new iterative series or a change in it. If one views the level of initial responsiveness to a new stimulus as a function of an organism’s threshold just prior to stimulus occurrence, then “high responders” (i.e. those who initially react more strongly) are assumed to have a lower threshold for detecting and assessing the significance of this stimulus than are the “low responders” (i.e. those who initially react more weakly). Thus, high-responders would initially receive more sensory input and progressively decrease their responsiveness to a non-threatening stimulus (habituation). Likewise, initial low-responders would receive less sensory input followed by a decreased threshold and an increased response to the next stimulus occurrence (sensitization). The level of responsiveness achieved in both habituaters and sensitizers, as an asymptote is approached, is a balance between being too sensitive to an unimportant stimulus (and possibly missing other significant stimuli) and being too insensitive, and missing a change in the relevance of the present stimulus. These response changes can be taken as indices of the organism’s mechanisms for achieving an appropriate threshold level to an iterative stimulus in order to accurately assess its present significance and then eventually to asymptote at an optimal stable response level. This approach toward an asymptote is a behavioral homeostatic process that reflects the accumulated significance of the iterative stimulus at each occurrence. The purpose of adding “behavioral” to the term “homeostasis” is to extend the usual meaning of the concept from primarily internal processes to also include (a) iterative external stimulation, (b) the organism’s initial threshold to the initial stimulus as well as (c) the behavior which results from it. Since we are discussing organisms that range from intact, single-celled protozoa to intact mammals, as well as surgically simplified preparations, the termsstimulus, response andbehavior will be used broadly. While other investigators have focused on specific cellular mechanisms underlying habituation and sensitization in a given organism, this paper focuses on the adaptive significance of these two behavioral processes viewed across phylogeny.     

Auditory habituation in the fetus and neonate: an fMEG study

Habituation – the most basic form of learning – is used to evaluate central nervous system (CNS) maturation and to detect abnormalities in fetal brain development. In the current study, habituation, stimulus specificity and dishabituation of auditory evoked responses were measured in fetuses and newborns using fetal magnetoencephalography (fMEG). An auditory habituation paradigm consisting of 100 trains of five 500 Hz tones, one 750 Hz tone (dishabituator) and two more 500 Hz tones, respectively, were presented to 41 fetuses (gestational age 30–39 weeks) and 22 newborns or babies (age 6–89 days). A response decrement between the first and fifth tones (habituation), an increment between the fifth tone and the dishabituator (stimulus specificity) and an increment between the fifth (last tone before the dishabituator) and seventh tones (first tone after the dishabituator) (dishabituation) were expected. Fetuses showed weak responses to the first tone. However, a significant response decrement between the second and fifth tones (habituation) and a significant increment between the fifth tone and the dishabituator (stimulus specificity) were found. No significant difference was found for dishabituation nor was a developmental trend found at the group level. From the neonatal data, significant values for stimulus specificity were found. Sensory fatigue or adaptation was ruled out as a reason for the response decrement due to the strong reactions to the dishabituator. Taken together, the current study used fMEG to directly show fetal habituation and provides evidence of fetal learning in the last trimester of pregnancy.