Aversive Olfactory Conditioning Research Articles

Glia transmit negative valence information during aversive learning in Drosophila.

During Drosophila aversive olfactory conditioning, aversive shock information needs to be transmitted to the mushroom bodies (MBs) to associate with odor information. We report that aversive information is transmitted by ensheathing glia (EG) that surround the MBs. Shock induces vesicular exocytosis of glutamate from EG. Blocking exocytosis impairs aversive learning, whereas activation of EG can replace aversive stimuli during conditioning. Glutamate released from EG binds to N-methyl-d-aspartate receptors in the MBs, but because of Mg2+ block, Ca2+ influx occurs only when flies are simultaneously exposed to an odor. Vesicular exocytosis from EG also induces shock-associated dopamine release, which plays a role in preventing formation of inappropriate associations. These results demonstrate that vesicular glutamate released from EG transmits negative valence information required for associative learning.

Forgotten memory storage and retrieval in Drosophila

Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαβ and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαβ, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαβ, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval.

IMD signaling in the gut and the brain modulates Amyloid-beta-induced deficits in Drosophila

AimsEvidence indicates accumulating Aβ peptides in brain activates immune responses in neuronal and peripheral system, which may collaboratively influence pathogenesis of Alzheimer's disease (AD). We aim to investigate whether regulating intestinal innate immune signaling ameliorates Aβ-induced impairments in Drosophila melanogaster. Main methodsQuantitative polymerase chain reaction (qPCR) was used to observe expression changes of innate immune responses related genes in brain and in gut under the circumstance of Aβ overexpressing in nerve system. Aversive olfactory conditioning and survival assay were used to investigate effects of modulating Attacin-A (AttA) and Dpitercin-A (DptA). Fluorometric assays of respiratory burst activity was introduced to explore whether reducing oxidative stress enables overexpressing intestinal AttA and DptA to reverse Aβ-induced deficits. Key findingsIn vivo genetic analysis revealed that accumulating Aβ42 in neurons modulates innate immune signaling of the IMD pathway both in the brain and in the gut. Increased expression levels of the intestinal AttA and DptA improved learning performance and extended the lifespan of Aβ42 flies. The administration of apramycin led to alleviations of Aβ-induced behavioral changes, indicating that gram-negative bacteria are associated with the development of Aβ-induced pathologies. Further analysis showed that the neural expression of Aβ42 increased oxidative stress in the gut, which disrupted intestinal integrity and decreased learning performance. In addition, increased levels of AMPs targeting gram-negative bacteria and antioxidants reduced oxidative stress in the gut and reversed Aβ-induced behavioral damage. SignificanceThese findings suggest that innate immune responses in the gut play a pivotal role in modulating Aβ-induced pathologies.

Transient active zone remodeling in the Drosophila mushroom body supports memory

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. We here provide evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1day). Our data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories.

Input-timing-dependent plasticity at incoming synapses of the mushroom body facilitates olfactory learning in Drosophila

Aversive olfactory conditioning in Drosophila is a valuable model for elucidating the mechanism of associative learning. Much effort has centered around the role of neuroplasticity at the mushroom body (MB)-mushroom body output neuron (MBON) synapses in mapping odors to specific behaviors. By electrophysiological recordings from MB neurons, we discovered a form of input-timing-dependent plasticity at the incoming synapses from projection neurons that controls the efficacy of aversive olfactory memory formation. Importantly, this plasticity is facilitated by the neural activity of PPL1, the neuronal cluster that also modulates MB-MBON connections at the output stage of MB. Unlike the MB-MBON synapses that probably utilize dopamine D1-like receptors, this neuroplasticity is dependent on D2-like receptors that are expressed mainly by γ Kenyon cells noticeably in their somato-dendritic region. The D2-like receptors recruit voltage-gated calcium channels, leading to calcium influx in the soma and dendrites of γ neurons. Together, our results reveal a previously unrecognized synaptic component of the MB circuit architecture that not only could increase the salience of a conditioning odor but also couples the process of memory encoding and valency mapping to drive-associative learning.

Dopamine activity in projection neurons regulates short-lasting olfactory approach memory in Drosophila.

Survival in many animals requires the ability to associate certain cues with danger and others with safety. In a Drosophila melanogaster aversive olfactory conditioning paradigm, flies are exposed to two odours, one presented coincidentally with electrical shocks, and a second presented 45 s after shock cessation. When flies are later given a choice between these two odours, they avoid the shock‐paired odour and prefer the unpaired odour. While many studies have examined how flies learn to avoid the shock‐paired odour through formation of odour‐fear associations, here we demonstrate that conditioning also causes flies to actively approach the second odour. In contrast to fear memories, which are longer lasting and requires activity of D1‐like dopamine receptors only in the mushroom bodies, approach memory is short‐lasting and requires activity of D1‐like dopamine receptors in projection neurons originating from the antennal lobes, primary olfactory centers. Further, while recall of fear memories requires activity of the mushroom bodies, recall of approach memories does not. Our data suggest that olfactory approach memory is formed using different mechanisms in different brain locations compared to aversive and appetitive olfactory memories.

Mutants of the white ABCG Transporter in Drosophila melanogaster Have Deficient Olfactory Learning and Cholesterol Homeostasis.

Drosophila’s white gene encodes an ATP-binding cassette G-subfamily (ABCG) half-transporter. White is closely related to mammalian ABCG family members that function in cholesterol efflux. Mutants of white have several behavioral phenotypes that are independent of visual defects. This study characterizes a novel defect of white mutants in the acquisition of olfactory memory using the aversive olfactory conditioning paradigm. The w1118 mutants learned slower than wildtype controls, yet with additional training, they reached wildtype levels of performance. The w1118 learning phenotype is also found in the wapricot and wcoral alleles, is dominant, and is rescued by genomic white and mini-white transgenes. Reducing dietary cholesterol strongly impaired olfactory learning for wildtype controls, while w1118 mutants were resistant to this deficit. The w1118 mutants displayed higher levels of cholesterol and cholesterol esters than wildtype under this low-cholesterol diet. Increasing levels of serotonin, dopamine, or both in the white mutants significantly improved w1118 learning. However, serotonin levels were not lower in the heads of the w1118 mutants than in wildtype controls. There were also no significant differences found in synapse numbers within the w1118 brain. We propose that the w1118 learning defect may be due to inefficient biogenic amine signaling brought about by altered cholesterol homeostasis.

CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. Here, we demonstrate enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. Our experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins.

Insulin signaling represents a gating mechanism between different memory phases in Drosophila larvae.

The ability to learn new skills and to store them as memory entities is one of the most impressive features of higher evolved organisms. However, not all memories are created equal; some are short-lived forms, and some are longer lasting. Formation of the latter is energetically costly and by the reason of restricted availability of food or fluctuations in energy expanses, efficient metabolic homeostasis modulating different needs like survival, growth, reproduction, or investment in longer lasting memories is crucial. Whilst equipped with cellular and molecular pre-requisites for formation of a protein synthesis dependent long-term memory (LTM), its existence in the larval stage of Drosophila remains elusive. Considering it from the viewpoint that larval brain structures are completely rebuilt during metamorphosis, and that this process depends completely on accumulated energy stores formed during the larval stage, investing in LTM represents an unnecessary expenditure. However, as an alternative, Drosophila larvae are equipped with the capacity to form a protein synthesis independent so-called larval anaesthesia resistant memory (lARM), which is consolidated in terms of being insensitive to cold-shock treatments. Motivated by the fact that LTM formation causes an increase in energy uptake in Drosophila adults, we tested the idea of whether an energy surplus can induce the formation of LTM in the larval stage. Suprisingly, increasing the metabolic state by feeding Drosophila larvae the disaccharide sucrose directly before aversive olfactory conditioning led to the formation of a protein synthesis dependent longer lasting memory. Moreover, formation of this memory component is accompanied by the suppression of lARM. We ascertained that insulin receptors (InRs) expressed in the mushroom body Kenyon cells suppresses the formation of lARM and induces the formation of a protein synthesis dependent longer lasting memory in Drosophila larvae. Given the numerical simplicity of the larval nervous system this work offers a unique prospect to study the impact of insulin signaling on the formation of protein synthesis dependent memories on a molecular level.

Study of the release of endogenous amines in Drosophila brain in vivo in response to stimuli linked to aversive olfactory conditioning.

A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. Here, we report a procedure to record for the first time the release of endogenous amines -dopamine, serotonin and octopamine- in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, we carried out recordings in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, we detect an increase in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and shed new light on the contribution of these amines to olfactory processing in this animal model.

Evolutionary conserved role of neural cell adhesion molecule-1 in memory

The neural cell adhesion molecule 1 (NCAM-1) has been implicated in several brain-related biological processes, including neuronal migration, axonal branching, fasciculation, and synaptogenesis, with a pivotal role in synaptic plasticity. Here, we investigated the evolutionary conserved role of NCAM-1 in learning and memory. First, we investigated sustained changes in ncam-1 expression following aversive olfactory conditioning in C. elegans using molecular genetic methods. Furthermore, we examined the link between epigenetic signatures of the NCAM1 gene and memory in two human samples of healthy individuals (N = 568 and N = 319) and in two samples of traumatized individuals (N = 350 and N = 463). We found that olfactory conditioning in C. elegans induced ncam-1 expression and that loss of ncam-1 function selectively impaired associative long-term memory, without causing acquisition, sensory, or short-term memory deficits. Reintroduction of the C. elegans or human NCAM1 fully rescued memory impairment, suggesting a conserved role of NCAM1 for memory. In parallel, DNA methylation of the NCAM1 promoter in two independent healthy Swiss cohorts was associated with memory performance. In two independent Sub-Saharan populations of conflict zone survivors who had faced severe trauma, DNA methylation at an alternative promoter of the NCAM1 gene was associated with traumatic memories. Our results support a role of NCAM1 in associative memory in nematodes and humans, and might, ultimately, be helpful in elucidating diagnostic markers or suggest novel therapy targets for memory-related disorders, like PTSD.

Long-term memory is formed immediately without the need for protein synthesis-dependent consolidation in Drosophila

It is believed that long-term memory (LTM) cannot be formed immediately because it must go through a protein synthesis-dependent consolidation process. However, the current study uses Drosophila aversive olfactory conditioning to show that such processes are dispensable for context-dependent LTM (cLTM). Single-trial conditioning yields cLTM that is formed immediately in a protein-synthesis independent manner and is sustained over 14 days without decay. Unlike retrieval of traditional LTM, which requires only the conditioned odour and is mediated by mushroom-body neurons, cLTM recall requires both the conditioned odour and reinstatement of the training-environmental context. It is mediated through lateral-horn neurons that connect to multiple sensory brain regions. The cLTM cannot be retrieved if synaptic transmission from any one of these centres is blocked, with effects similar to those of altered encoding context during retrieval. The present study provides strong evidence that long-term memory can be formed easily without the need for consolidation.

Aversive Training Induces Both Presynaptic and Postsynaptic Suppression in Drosophila.

The α'β' subtype of Drosophila mushroom body neurons (MBn) is required for memory acquisition, consolidation and early memory retrieval after aversive olfactory conditioning. However, in vivo functional imaging studies have failed to detect an early forming memory trace in these neurons as reflected by an enhanced G-CaMP signal in response to presentation of the learned odor. Moreover, whether cellular memory traces form early after conditioning in the mushroom body output neurons (MBOn) downstream of the α'β' MBn remains unknown. Here, we show that aversive olfactory conditioning suppresses the calcium responses to the learned odor in both α'3 and α'2 axon segments of α'β' MBn and in the dendrites of α'3 MBOn immediately after conditioning using female flies. Notably, the cellular memory traces in both α'3 MBn and α'3 MBOn are short-lived and persist for <30 min. The suppressed response in α'3 MBn is accompanied by a reduction of acetylcholine (ACh) release, suggesting that the memory trace in postsynaptic α'3 MBOn may simply reflect the suppression in presynaptic α'3 MBn. Furthermore, we show that the α'3 MBn memory trace does not occur from the inhibition of GABAergic neurons via GABAA receptor activation. Because activation of the α'3 MBOn drives approach behavior of adult flies, our results demonstrate that aversive conditioning promotes avoidance behavior through suppression of the α'3 MBn-MBOn circuit.SIGNIFICANCE STATEMENTDrosophila learn to avoid an odor if that odor is repeatedly paired with electric shock. Mushroom body neurons (MBns) are known to be major cell types that mediate this form of aversive conditioning. Here we show that aversive conditioning causes a reduced response to the conditioned odor in an axon branch of one subtype of the MBn for no more than 30 min after conditioning, and in the dendrites of postsynaptic, MB output neurons (MBOns). Because experimenter-induced activation of the MBOn induces approach behavior by the fly, our data support a model that aversive learning promotes avoidance by suppressing the MBn-MBOn synapses that normally promote attraction.

SA55 - NEURAL CELL ADHESION MOLECULE 1 (NCAM-1) IS ASSOCIATED TO LONG-TERM AVERSIVE MEMORY IN NEMATODES AND HUMANS

Background The Neural Cell Adhesion Molecule 1 (NCAM-1) has been implicated in several brain-related biological processes, including neuronal migration, axonal branching, fasciculation, and synaptogenesis, with a pivotal role in synaptic plasticity. Here, using an interdisciplinary approach we investigated the evolutionary conserved role of Neural Cell Adhesion Molecule 1 (NCAM-1) in learning and memory. Methods First, we examined the temporal transcriptome profiles during C. elegans aversive olfactory long-term memory in a large-scale gene expression study (up to 24 hours upon conditioning). We observed sustained changes in NCAM-1 expression and thus we further investigated its association with aversive memory by using molecular genetics methods. Following, we examined the link between molecular signatures of the NCAM-1 gene and aversive memory in two independent human samples of European descent, by using genome-wide expression and methylation profiling. Finally, in a population of genocide survivors of African descent we investigated the link between NCAM-1 promoter DNA methylation and PTSD. Results NCAM-1 expression showed sustained expression changes up to 24 hours upon aversive olfactory conditioning. Loss of NCAM-1 function selectively impaired long-term memory in a negative but not in positive olfactory associative memory paradigm, without causing acquisition, sensory, motor or short-term memory deficits. Finally, both C. elegans and human NCAM-1 expression in nematodes rescued loss of C. elegans NCAM-1 gene function. Considering the conserved role of NCAM-1 in higher complex organisms, we next expanded our C. elegans findings in human. In two samples of European descent, we showed a negative correlation between DNA methylation of the NCAM-1 promoter and aversive memory (i.e., recognition of negative pictures) as well as a strong positive correlation between NCAM-1 expression and the same recognition performance. In the second sample, we could also show a strong correlation between NCAM-1 expression and long-delayed recall of negative pictures. Finally, in a population of genocide survivors DNA methylation at NCAM-1 gene promoter was inversely correlated with intrusive memories symptoms and Posttraumatic Stress Disorder (PTSD) risk. Discussion Taken together, our results support a conserved role of NCAM-1 in negative associative memory from nematodes to humans and might, ultimately, prove to be helpful in elucidating diagnostic markers and suggest novel therapy targets for memory-related disorders, like PTSD.

Bayesian updating during development predicts genotypic differences in plasticity.

Interactions between genotypes and environments are central to evolutionary genetics, but such interactions are typically described, rather than predicted from theory. Recent Bayesian models of development generate specific predictions about genotypic differences in developmental plasticity (changes in the value of a given trait as a result of a given experience) based on genotypic differences in the value of the trait that is expressed by naïve subjects. We used these models to make a priori predictions about the effects of an aversive olfactory conditioning regime on the response of Drosophila melanogaster larvae to the odor of ethyl acetate. As predicted, across 116 genotypes initial trait values were related to plasticity. Genotypes most strongly attracted to the odor of ethyl acetate when naïve reduced their attraction scores more as a result of the aversive training regime than those less attracted to the same odor when naïve. Thus, as predicted, the variance across genotypes in attraction scores was higher before than after the shared experience. These results support predictions generated by Bayesian models of development and indicate that such models can be successfully used to investigate how variation across genotypes in information derived from ancestors combines with personal experience to differentially affect developmental plasticity in response to specific types of experience.

Aversive learning-induced plasticity throughout the adult mammalian olfactory system: insights across development.

Experiences, such as sensory learning, are known to induce plasticity in mammalian sensory systems. In recent years aversive olfactory learning-induced plasticity has been identified at all stages of the adult olfactory pathway; however, the underlying mechanisms have yet to be identified. Much of the work regarding mechanisms of olfactory associative learning comes from neonates, a time point before which the brain or olfactory system is fully developed. In addition, pups and adults often express different behavioral outcomes when subjected to the same olfactory aversive conditioning paradigm, making it difficult to directly attribute pup mechanisms of plasticity to adults. Despite the differences, there is evidence of similarities between pups and adults in terms of learning-induced changes in the olfactory system, suggesting at least some conserved mechanisms. Identifying these conserved mechanisms of plasticity would dramatically increase our understanding of how the brain is able to alter encoding and consolidation of salient olfactory information even at the earliest stages following aversive learning. The focus of this review is to systematically examine literature regarding olfactory associative learning across developmental stages and search for similarities in order to build testable hypotheses that will inform future studies of aversive learning-induced sensory plasticity in adults.

Active Protection: Learning-Activated Raf/MAPK Activity Protects Labile Memory from Rac1-Independent Forgetting

Active forgetting explains the intrinsic instability of a labile memory lasting for hours. However, how such memory maintains stability against unwanted disruption is not completely understood. Here, we report alearning-activated active protection mechanism that enables labile memory to resist disruptive sensory experiences in Drosophila. Aversive olfactory conditioning activates mitogen-activated protein kinase (MAPK) transiently in the mushroom-body γlobe, where labile-aversive memory is stored. This increased MAPK activity significantly prolongs labile memory retention and enhances its resistance to disruption induced by heat shock, electric shock, or odor reactivation. Such experience-induced forgetting cannot be prevented by inhibition of Rac1 activity. Instead, protection of Rac1-independent forgetting correlates with non-muscle myosin II activity and persistence of learning-induced presynaptic structural changes. Increased Raf/MAPK activity, together with suppressed Rac1 activity, completely blocks labile memory decay. Thus, learning not onlyleads to memory formation, but also activates activeprotection and active forgetting to regulate the formed memory.

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation.

In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, we integrated Trojan exons encoding T2A-GAL4 into genes encoding putative kinases and selected for genes expressed in MB. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. We identified a novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human), the loss of which severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. We show that PKA phosphorylates MP and that both proteins synergize in a feedforward loop to control CREBB levels and LTM. key words: Drosophila, Mushroom bodies, SBK1, deGradFP, T2A-GAL4, MiMIC.

The clock gene period differentially regulates sleep and memory in Drosophila.

Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. Here, we show that mutations in the clock gene period result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. We further describe distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when we reintroduce a form of the period gene in glia, lateral neurons, and the fan-shaped body. Our results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena.

Electro-olfactogram Responses Before and After Aversive Olfactory Conditioning in Humans

The aim of the present study was to investigate whether repetitive aversive odor conditioning induced changes at the level of the peripheral olfactory system in humans. A total of 51 volunteers participated. A pair of indistinguishable odor enantiomers [(+)-rose oxide and (−)-rose oxide] were used as stimuli. During the pre-conditioning, participants’ ability to discriminate between the two odors was assessed using a three-alternative, forced-choice discrimination test. In addition, electro-olfactograms (EOG) from the olfactory epithelium were recorded. Participants underwent three conditioning sessions on consecutive days. The experimental group received an electrical stimulus to the forearm only following (+)-rose oxide presentation, whereas its enantiomer sibling was never paired with the aversive stimulus; the control group did not receive any electrical stimulation. During the post-conditioning session, their ability to discriminate the two enantiomers was assessed again using the discrimination test and EOG recordings were obtained similarly to the pre-conditioning session. Results showed significant differences in the peripheral electrophysiological responses between the conditioned and the unconditioned stimulus, demonstrating contextually induced changes at the level of the first neuron in the olfactory system.