Genetics, molecular control and clinical relevance of habituation learning.

Habituation is the most fundamental form of learning. As a firewall that protects our brain from sensory overload, it is indispensable for cognitive processes. Studies in humans and animal models provide increasing evidence that habituation is affected in autism and related monogenic neurodevelopmental disorders (NDDs). An integrated application of habituation assessment in NDDs and their animal models has unexploited potential for neuroscience and medical care. With the aim to gain mechanistic insights, we systematically retrieved genes that have been demonstrated in the literature to underlie habituation. We identified 258 evolutionarily conserved genes across species, describe the biological processes they converge on, and highlight regulatory pathways and drugs that may alleviate habituation deficits. We also summarize current habituation paradigms and extract the most decisive arguments that support the crucial role of habituation for cognition in health and disease. We conclude that habituation is a conserved, quantitative, cognition- and disease-relevant process that can connect preclinical and clinical work, and hence is a powerful tool to advance research, diagnostics, and treatment of NDDs.

cacna2d3, a voltage-gated calcium channel subunit, functions in vertebrate habituation learning and the startle sensitivity threshold.

BACKGROUND: The ability to filter sensory information into relevant versus irrelevant stimuli is a fundamental, conserved property of the central nervous system and is accomplished in part through habituation learning. Synaptic plasticity that underlies habituation learning has been described at the cellular level, yet the genetic regulators of this plasticity remain poorly understood, as do circuits that mediate sensory filtering. METHODS: To identify genes critical for plasticity, a forward genetic screen for zebrafish genes that mediate habituation learning was performed, which identified a mutant allele, doryp177, that caused reduced habituation of the acoustic startle response. In this study, we combine whole-genome sequencing with behavioral analyses to characterize and identify the gene affected in doryp177 mutants. RESULTS: Whole-genome sequencing identified the calcium voltage-gated channel auxiliary subunit alpha-2/delta-3 (cacna2d3) as a candidate gene affected in doryp177 mutants. Behavioral characterization of larvae homozygous for two additional, independently derived mutant alleles of cacna2d3, together with failure of these alleles to complement doryp177, confirmed a critical role for cacna2d3 in habituation learning. Notably, detailed analyses of the acoustic response in mutant larvae also revealed increased startle sensitivity to acoustic stimuli, suggesting a broader role for cacna2d3 in controlling innate response thresholds to acoustic stimuli. CONCLUSIONS: Taken together, our data demonstrate a critical role for cacna2d3 in sensory filtering, a process that is disrupted in human CNS disorders, e.g. ADHD, schizophrenia, and autism.

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila.

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.

What can a worm learn in a bacteria-rich habitat?

With a nervous system that has only a few hundred neurons, Caenorhabditis elegans was initially not regarded as a model for studies on learning. However, the collective effort of the C. elegans field in the past several decades has shown that the worm displays plasticity in its behavioral response to a wide range of sensory cues in the environment. As a bacteria-feeding worm, C. elegans is highly adaptive to the bacteria enriched in its habitat, especially those that are pathogenic and pose a threat to survival. It uses several common forms of behavioral plasticity that last for different amounts of time, including imprinting and adult-stage associative learning, to modulate its interactions with pathogenic bacteria. Probing the molecular, cellular and circuit mechanisms underlying these forms of experience-dependent plasticity has identified signaling pathways and regulatory insights that are conserved in more complex animals.

The role of neuropeptides in learning: Insights from C. elegans.

Learning is critical for survival as it provides the capacity to adapt to a changing environment. At the molecular and cellular level, learning leads to alterations within neural circuits that include synaptic rewiring and synaptic plasticity. These changes are mediated by signalling molecules known as neuromodulators. One such class of neuromodulators are neuropeptides, a diverse group of short peptides that primarily act through G protein-coupled receptors. There has been substantial progress in recent years on dissecting the role of neuropeptides in learning circuits using compact yet powerful invertebrate model systems. We will focus on insights gained using the nematode Caenorhabditis elegans, with its unparalleled genetic tractability, compact nervous system of ∼300 neurons, high level of conservation with mammalian systems and amenability to a suite of behavioural analyses. Specifically, we will summarise recent discoveries in C. elegans on the role of neuropeptides in non-associative and associative learning.

Acute Regulation of Habituation Learning via Posttranslational Palmitoylation.

Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K+ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.

An in-depth neurobehavioral characterization shows anxiety-like traits, impaired habituation behavior, and restlessness in male Cryptochrome-deficient mice.

Many psychiatric disorders, for example, anxiety, are accompanied by disturbances of circadian rhythms, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Conversely, alternations of circadian rhythms are a risk factor for the development of psychiatric disorders. This assumption is supported by animals with clock gene mutations which often display behaviors that resemble human psychiatric disorders. In this study, we performed an in-depth behavioral analysis with male mice lacking the central clock genes Cryptochrome 1 and 2 (Cry1/2-/- ), which are thus unable to express endogenous circadian rhythms. With wild-type and Cry1/2-/- mice, we performed an extensive behavioral analysis to study their cognitive abilities, social behavior, and their expression of depression-like and anxiety-like behavior. While Cry1/2-/- mice showed only mild abnormalities at cognitive and social behavioral levels, they were consistently more anxious than wildtype mice. Anxiety-like behavior was particularly evident in reduced mobility in new environments, altered ability to habituate, compensatory behavior, and consistent restless behavior across many behavioral tests. In line with their anxiety-like behavioral phenotype, Cry1/2-/- mice have higher c-Fos activity in the amygdala after exposure to an anxiogenic stressor than wild-type mice. In our study, we identified Cry1/2-/- mice as animals that qualify as a translational mouse model for anxiety disorder in humans because of its consistent behavior of restlessness, increased immobility, and dysfunctional habituation in new environments.

Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation.

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

Habituation Learning Is a Widely Affected Mechanism in Drosophila Models of Intellectual Disability and Autism Spectrum Disorders.

BACKGROUND: Although habituation is one of the most ancient and fundamental forms of learning, its regulators and its relevance for human disease are poorly understood. METHODS: We manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and we tested these models in light-off jump habituation. We dissected neuronal substrates underlying the identified habituation deficits and integrated genotype-phenotype annotations, gene ontologies, and interaction networks to determine the clinical features and molecular processes that are associated with habituation deficits. RESULTS: We identified >100 genes required for habituation learning. For 93 of these genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly and/or overgrowth and comorbid ASD. Moreover, individuals with ASD from the Simons Simplex Collection carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras/mitogen-activated protein kinase (Ras/MAPK) signaling. Both increased Ras/MAPK signaling in gamma-aminobutyric acidergic (GABAergic) neurons and decreased Ras/MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response. CONCLUSIONS: Our work supports the relevance of habituation learning to ASD, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation, and reveals an opposing, circuit-level-based mechanism for Ras/MAPK signaling. These findings establish habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD.

Latency to startle is increased in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that results in cognitive decline and a number of other neuropsychiatric symptoms. One area that is often affected by neuropsychiatric disease is the response to sudden, loud noises, as measured by the acoustic startle response (ASR), and prepulse inhibition (PPI), which indicates sensory-gating abilities. Evidence suggests AD patients, even early in the disease, show alteration in ASR. Studies have also shown changes in this measure in transgenic mouse models of AD. To assess the homology of 5xFAD mice to AD patients, the current study analyzed several aspects of the startle response in these mice using a protocol with fewer trials than previous studies. It was found that the 5xFAD mice had a delayed startle response, similar to what has been observed in AD sufferers. These results suggest the ASR may be a useful tool in assessing the efficacy of potential therapeutics, and that a simplified protocol may be more sensitive to between-groups differences for this task.

Insights into the roles of CMK-1 and OGT-1 in interstimulus interval-dependent habituation in Caenorhabditis elegans.

Habituation is a ubiquitous form of non-associative learning observed as a decrement in responding to repeated stimulation that cannot be explained by sensory adaptation or motor fatigue. One of the defining characteristics of habituation is its sensitivity to the rate at which training stimuli are presented-animals habituate faster in response to more rapid stimulation. The molecular mechanisms underlying this interstimulus interval (ISI)-dependent characteristic of habituation remain unknown. In this article, we use behavioural neurogenetic and bioinformatic analyses in the nematode Caenorhabiditis elegans to identify the first molecules that modulate habituation in an ISI-dependent manner. We show that the Caenorhabditis elegans orthologues of Ca2+/calmodulin-dependent kinases CaMK1/4, CMK-1 and O-linked N-acetylglucosamine (O-GlcNAc) transferase, OGT-1, both function in primary sensory neurons to inhibit habituation at short ISIs and promote it at long ISIs. In addition, both cmk-1 and ogt-1 mutants display a rare mechanosensory hyper-responsive phenotype (i.e. larger mechanosensory responses than wild-type). Overall, our work identifies two conserved genes that function in sensory neurons to modulate habituation in an ISI-dependent manner, providing the first insights into the molecular mechanisms underlying the universally observed phenomenon that habituation has different properties when stimuli are delivered at different rates.

Human Apolipoprotein E Genotype Differentially Affects Olfactory Behavior and Sensory Physiology in Mice.

Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE ε4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the ε2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, ε3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. Although motivated behavior is influenced by many processes, hyper-excitability of ApoE4 mice may contribute to impaired odor habituation, while hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate how ApoE affects the olfactory system at early stages, prior to the development of AD.

Acid-sensing ion channel 1 contributes to normal olfactory function.

Acid-sensing ion channels (ASICs) are cation channels activated by protons. ASIC1a, a primary ASIC subunit in the brain, was recently characterized in the olfactory bulb. The present study tested the hypothesis that ASIC1a is essential for normal olfactory function. Olfactory behavior of wild-type (WT) and ASIC1-/- mice was evaluated by using three standard olfactory tests: (1) the buried food test, (2) the olfactory habituation test, and (3) the olfactory preference test. In buried food test, ASIC1-/- mice had significantly longer latency to uncover buried food than WT mice. In olfactory habituation test, ASIC1-/- mice had increased sniffing time with acidic odorants. In olfactory preference test, ASIC1-/- mice did not exhibit normal avoidance behavior for 2, 5- dihydro-2, 4, 5-trimethylthiazoline (TMT). Consistent with ASIC1 knockout, ASIC1 inhibition by nasal administration of PcTX1 increased the latency for WT mice to uncover the buried food. Together, these findings suggest a key role for ASIC1a in normal olfactory function.

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.

Behavioral and stereological characterization of Hdc KO mice: Relation to Tourette syndrome.

A premature termination codon in the human histidine decarboxylase (Hdc) gene has been identified in a family suffering from Guilles de la Tourette syndrome (GTS). In the current study we investigated if mice lacking the histamine producing enzyme HDC share the morphological and cytological phenotype with GTS patients by using magnetic resonance (MRI) and diffusion tensor imaging (DTI), unbiased stereology and immunohistochemistry. Behavior of Hdc knock-out (Hdc KO) mice was assessed in an open field test. The results of stereological, volumetric and DTI analysis measurements showed no significant differences between control and Hdc KO mice. The numbers and distribution of GABAergic parvalbumin or nitric oxide-expressing and cholinergic interneurons were normal in Hdc KO mice. Cortical morphology and layering in adult Hdc KO mice were also preserved. In open field test Hdc KO mice showed impaired exploratory activity and habituation when introduced to novel environment. Our results indicate that Hdc deficiency in mice does not disturb the development of striatal and cortical interneurons and does not lead to the morphological and cytological phenotypes characterized by humans with GTS. Nevertheless, histamine deficiency leads to behavioral alterations probably due to neurotransmitter dysbalance on the level of the striatum.

Brain-derived neurotrophic factor Val66Met genotype modulates amygdala habituation.

A deficit in amygdala habituation to repeated emotional stimuli may be an endophenotype of disorders characterized by emotion dysregulation, such as borderline personality disorder (BPD). Amygdala reactivity to emotional stimuli is genetically modulated by brain-derived neurotrophic factor (BDNF) variants. Whether amygdala habituation itself is also modulated by BDNF genotypes remains unknown. We used imaging-genetics to examine the effect of BDNF Val66Met genotypes on amygdala habituation to repeated emotional stimuli. We used functional magnetic resonance imaging (fMRI) in 57 subjects (19 BPD patients, 18 patients with schizotypal personality disorder [SPD] and 20 healthy controls [HC]) during a task involving viewing of unpleasant, neutral, and pleasant pictures, each presented twice to measure habituation. Amygdala responses across genotypes (Val66Met SNP Met allele-carriers vs. Non-Met carriers) and diagnoses (HC, BPD, SPD) were examined with ANOVA. The BDNF 66Met allele was significantly associated with a deficit in amygdala habituation, particularly for emotional pictures. The association of the 66Met allele with a deficit in habituation to unpleasant emotional pictures remained significant in the subsample of BPD patients. Using imaging-genetics, we found preliminary evidence that deficient amygdala habituation may be modulated by BDNF genotype.

Striatopallidal Neuron NMDA Receptors Control Synaptic Connectivity, Locomotor, and Goal-Directed Behaviors.

UNLABELLED: The basal ganglia (BG) control action selection, motor programs, habits, and goal-directed learning. The striatum, the principal input structure of BG, is predominantly composed of medium-sized spiny neurons (MSNs). Arising from these spatially intermixed MSNs, two inhibitory outputs form two main efferent pathways, the direct and indirect pathways. Striatonigral MSNs give rise to the activating, direct pathway MSNs and striatopallidal MSNs to the inhibitory, indirect pathway (iMSNs). BG output nuclei integrate information from both pathways to fine-tune motor procedures and to acquire complex habits and skills. Therefore, balanced activity between both pathways is crucial for harmonious functions of the BG. Despite the increase in knowledge concerning the role of glutamate NMDA receptors (NMDA-Rs) in the striatum, understanding of the specific functions of NMDA-R iMSNs is still lacking. For this purpose, we generated a conditional knock-out mouse to address the functions of the NMDA-R in the indirect pathway. At the cellular level, deletion of GluN1 in iMSNs leads to a reduction in the number and strength of the excitatory corticostriatopallidal synapses. The subsequent scaling down in input integration leads to dysfunctional changes in BG output, which is seen as reduced habituation, delay in goal-directed learning, lack of associative behavior, and impairment in action selection or skill learning. The NMDA-R deletion in iMSNs causes a decrease in the synaptic strength of striatopallidal neurons, which in turn might lead to a imbalanced integration between direct and indirect MSN pathways, making mice less sensitive to environmental change. Therefore, their ability to learn and adapt to the environment-based experience was significantly affected. SIGNIFICANCE STATEMENT: The striatum controls habits, locomotion, and goal-directed behaviors by coordinated activation of two antagonistic pathways. Insofar as NMDA receptors (NMDA-Rs) play a key role in synaptic plasticity essential for sustaining these behaviors, we generated a mouse model lacking NMDA-Rs specifically in striatopallidal neurons. To our knowledge, this is the first time that a specific deletion of inhibitory, indirect pathway medium-sized spiny neuron (iMSN) NMDA-Rs has been used to address the role of these receptors in the inhibitory pathway. Importantly, we found that this specific deletion led to a significant reduction in the number and strength of the cortico-iMSN synapses, which resulted in the significant impairments of behaviors orchestrated by the basal ganglia. Our findings indicate that the NMDA-Rs of the indirect pathway are essential for habituation, action selection, and goal-directed learning.

Evidence of CNIH3 involvement in opioid dependence.

Opioid dependence, a severe addictive disorder and major societal problem, has been demonstrated to be moderately heritable. We conducted a genome-wide association study in Comorbidity and Trauma Study data comparing opioid-dependent daily injectors (N=1167) with opioid misusers who never progressed to daily injection (N=161). The strongest associations, observed for CNIH3 single-nucleotide polymorphisms (SNPs), were confirmed in two independent samples, the Yale-Penn genetic studies of opioid, cocaine and alcohol dependence and the Study of Addiction: Genetics and Environment, which both contain non-dependent opioid misusers and opioid-dependent individuals. Meta-analyses found five genome-wide significant CNIH3 SNPs. The A allele of rs10799590, the most highly associated SNP, was robustly protective (P=4.30E-9; odds ratio 0.64 (95% confidence interval 0.55-0.74)). Epigenetic annotation predicts that this SNP is functional in fetal brain. Neuroimaging data from the Duke Neurogenetics Study (N=312) provide evidence of this SNP's in vivo functionality; rs10799590 A allele carriers displayed significantly greater right amygdala habituation to threat-related facial expressions, a phenotype associated with resilience to psychopathology. Computational genetic analyses of physical dependence on morphine across 23 mouse strains yielded significant correlations for haplotypes in CNIH3 and functionally related genes. These convergent findings support CNIH3 involvement in the pathophysiology of opioid dependence, complementing prior studies implicating the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate system.

Candidate genes for individual recognition in Polistes fuscatus paper wasps.

Few animals are known to individually recognize conspecifics, i.e. learn and recall unique individuals during subsequent encounters, and nearly all are social vertebrates. Remarkably, the social paper wasp Polistes fuscatus has recently been discovered to possess this ability, which is useful for remembering identities during competitive social interactions. We analyzed brain gene expression in staged encounters between pairs of individuals to explore potential mechanisms underlying wasps' ability to recall familiar individuals using real-time qRT-PCR. We identified four candidate genes (IP3K, IP3R, Nckx30C and Su(var)2-10) that were down-regulated in the presence of familiar individuals compared to single wasps and pairs of wasps meeting for the first time. These candidate genes are related to calcium signaling, therefore, we treated wasps with lithium chloride, a pharmacological agent that inhibits calcium signaling in neurons. This treatment decreased aggression in paper wasps, but did not affect expression of genes related to calcium signaling. The results suggest calcium signaling differences may be related to individual memory recall in wasps, and we present four promising candidate genes for future study. These data suggest genes associated with dominance behavior may be co-opted for individual recognition, but further work is needed to establish a causal association with the behavior.

Developmental Function of the PHR Protein RPM-1 Is Required for Learning in Caenorhabditis elegans.

The PAM/Highwire/RPM-1 (PHR) proteins are signaling hubs that function as important regulators of neural development. Loss of function in Caenorhabditis elegans rpm-1 and Drosophila Highwire results in failed axon termination, inappropriate axon targeting, and abnormal synapse formation. Despite broad expression in the nervous system and relatively dramatic defects in synapse formation and axon development, very mild abnormalities in behavior have been found in animals lacking PHR protein function. Therefore, we hypothesized that large defects in behavior might only be detected in scenarios in which evoked, prolonged circuit function is required, or in which behavioral plasticity occurs. Using quantitative approaches in C. elegans, we found that rpm-1 loss-of-function mutants have relatively mild abnormalities in exploratory locomotion, but have large defects in evoked responses to harsh touch and learning associated with tap habituation. We explored the nature of the severe habituation defects in rpm-1 mutants further. To address what part of the habituation circuit was impaired in rpm-1 mutants, we performed rescue analysis with promoters for different neurons. Our findings indicate that RPM-1 function in the mechanosensory neurons affects habituation. Transgenic expression of RPM-1 in adult animals failed to rescue habituation defects, consistent with developmental defects in rpm-1 mutants resulting in impaired habituation. Genetic analysis showed that other regulators of neuronal development that function in the rpm-1 pathway (including glo-4, fsn-1, and dlk-1) also affected habituation. Overall, our findings suggest that developmental defects in rpm-1 mutants manifest most prominently in behaviors that require protracted or plastic circuit function, such as learning.