Drosophila Mushroom Bodies Are Dispensable for Visual, Tactile, and Motor Learning

A total of 18 associative learning/memory tests have been applied to Drosophila melanogaster flies lacking mushroom bodies. Only in paradigms involving chemosensory cues as conditioned stimuli have flies been found to be compromised by a block in the mushroom body pathway. Among the learning tasks not requiring these structures are a case of motor learning (yaw torque/heat), a test of the fly’s spatial orientation in total darkness, conditioned courtship suppression by mated females, and nine different examples of visual learning. The latter used the reinforcers of heat, visual oscillations, mechanical shaking, or sucrose, and as conditioned stimuli, color, intensity contrast, as well as stationary and moving visual patterns. No forms of consolidated memory have been tested in mushroom body-less flies. With respect to short-term memory the mushroom bodies of Drosophila are specially required for chemosensory learning tasks, but not for associative learning and memory in general.     

Second-order conditioning in Drosophila

Associative conditioning in Drosophila melanogaster has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning (SOC) protocol that further demonstrates the flexibility of fly behavior. In SOC, a previously conditioned stimulus (CS1) is used as reinforcement for a second conditioned stimulus (CS2) in associative learning. This higher-order context presents an opportunity for reassessing the roles of known learning and memory genes and neuronal networks in a new behavioral paradigm.     

Learning and memory deficits upon TAU accumulation in Drosophila mushroom body neurons

Mutations in the neuronal-specific microtubule-binding protein TAU are associated with several dementias and neurodegenerative diseases. However, the effects of elevated TAU accumulation on behavioral plasticity are unknown. We report that directed expression of wild-type vertebrate and Drosophila TAU in adult mushroom body neurons, centers for olfactory learning and memory in Drosophila, strongly compromised associative olfactory learning and memory, but olfactory conditioning-relevant osmotactic and mechanosensory responses remained intact. In addition, TAU accumulation in mushroom body neurons did not result in detectable neurodegeneration or premature death. Therefore, TAU-mediated structural or functional perturbation of the microtubular cytoskeleton in mushroom body neurons is likely causal of the behavioral deficit. These results indicate that behavioral plasticity decrements may be the earliest detectable manifestations of tauopathies.     

Roles for Drosophila mushroom body neurons in olfactory learning and memory

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. We investigated the requirements for the different MB neurons, specifically the alpha/beta versus the gamma neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. We expressed the rutabaga (rut)-encoded adenylyl cyclase in either the gamma or alpha/beta neurons and examined the effects on restoring olfactory associative learning and memory of rut mutant flies. We also expressed a temperature-sensitive shibire (shi) transgene in these neuron sets and examined the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning. Our results indicate that although we did not detect odor-pair-specific learning using GAL4 drivers that primarily express in gamma neurons, expression of the transgenes in a subset of alpha/beta neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi(ts1).     

The role of cuticular pheromones in courtship conditioning of Drosophila males

Courtship conditioning is an associative learning paradigm in Drosophila melanogaster, wherein male courtship behavior is modified by experience with unreceptive, previously mated females. While the training experience with mated females involves multiple sensory and behavioral interactions, we hypothesized that female cuticular hydrocarbons function as a specific chemosensory conditioned stimulus in this learning paradigm. The effects of training with mated females were determined in courtship tests with either wild-type virgin females as courtship targets, or with target flies of different genotypes that express distinct cuticular hydrocarbon (CH) profiles. Results of tests with female targets that lacked the normal CH profile, and with male targets that expressed typically female CH profiles, indicated that components of this CH profile are both necessary and sufficient cues to elicit the effects of conditioning. Results with additional targets indicated that the female-specific 7,11-dienes, which induce naive males to court, are not essential components of the conditioned stimulus. Rather, the learned response was significantly correlated with the levels of 9-pentacosene (9-P), a compound found in both males and females of many Drosophila strains and species. Adding 9-P to target flies showed that it stimulates courting males to attempt to copulate, and confirmed its role as a component of the conditioned stimulus by demonstrating dose-dependent increases in the expression of the learned response. Thus, 9-P can contribute significantly to the conditioned suppression of male courtship toward targets that express this pheromone.     

Analysis of the catnip reaction: mediation by olfactory system, not vomeronasal organ

Pet owners and behavioral scientists alike are fascinated by unique behavioral reactions that cats show in the presence of catnip. These experiments explored the possibility that the catnip reaction might be triggered by chemosensory stimulation of the vomeronasal organ. In the chewing and mouthing of the catnip source, substances might be dissolved in saliva and transported to the vomeronasal organ. The rolling and rubbing during a catnip reaction might be a sexual response activated by the accessory olfactory system since the system projects to parts of the brain involved in mediation of sexual behavior. However, removal of the vomeronasal organ did not attenuate any of the behavioral reactions to catnip. Olfactory bulbectomy immediately eliminated catnip responding, revealing that the chemosensory stimulus evoking the catnip reaction is undoubtedly mediated through the main olfactory system. Catnip activates behavioral elements associated with several species-specific behaviors, including sniffing and chewing as associated with oral appetitive behavior, rolling and rubbing characteristic of female sexual behavior, batting the catnip source characteristic of play behavior, and a type of kicking associated with predatory behavior. These behavioral reactions occur randomly and intermittently.     

Sociochemosensory and Emotional Functions: Behavioral Evidence for Shared Mechanisms

ABSTRACT— Olfaction and emotion are distinctively different systems. Nevertheless, there are reasons to suspect that they influence each other on the social level. Functionally, olfactory chemosensory communication is used by a wide range of animals to convey individual and group identity, as well as attraction or repulsion. Anatomically, the olfactory brain overlaps with the socioemotional brain, and is believed to have contributed to the evolution of the latter. Little is known about how the functional and anatomical links are manifested in behavior, however. Using human olfaction as a model, we demonstrate that chemosensory recognition of individuals—one of the most ubiquitous forms of social communication—is interconnected with both the cognitive and the visual processing of emotion. Our results provide the first behavioral evidence for mechanisms being shared by a sensory system and emotion.     

Average group behavior does not represent individual behavior in classical conditioning of the honeybee

Conditioned behavior as observed during classical conditioning in a group of identically treated animals provides insights into the physiological process of learning and memory formation. However, several studies in vertebrates found a remarkable difference between the group-average behavioral performance and the behavioral characteristics of individual animals. Here, we analyzed a large number of data (1640 animals) on olfactory conditioning in the honeybee (Apis mellifera). The data acquired during absolute and differential classical conditioning differed with respect to the number of conditioning trials, the conditioned odors, the intertrial intervals, and the time of retention tests. We further investigated data in which animals were tested for spontaneous recovery from extinction. In all data sets we found that the gradually increasing group-average learning curve did not adequately represent the behavior of individual animals. Individual behavior was characterized by a rapid and stable acquisition of the conditioned response (CR), as well as by a rapid and stable cessation of the CR following unrewarded stimuli. In addition, we present and evaluate different model hypotheses on how honeybees form associations during classical conditioning by implementing a gradual learning process on the one hand and an all-or-none learning process on the other hand. In summary, our findings advise that individual behavior should be recognized as a meaningful predictor for the internal state of a honeybee--irrespective of the group-average behavioral performance.     

Psychology of learning: a new approach to study behavior of Rhodnius prolixus stal under laboratory conditions

Conditioning methodologies associated with the psychology of learning are suggested as a new strategy to investigate behavior of the assassin bug Rhodnius prolixus, which is the main vector of Chagas disease in Venezuela. Chagas disease is the fourth leading cause of death in Latin America, as it causes severe chronic illness and approximately 43,000 deaths per year. To illustrate this strategy, two preliminary experiments are reported. In the first, Pavlovian conditioning was examined by pairing an olfactory conditioned stimulus with a temperature unconditioned stimulus. A temperature of 42 degrees C elicits a complex behavioral sequence in R. prolixus consisting of proboscis extension and crawling. Over the course of 12 training trials, this behavioral sequence was not elicited by an olfactory conditioned stimulus. In the second experiment, a latent inhibition paradigm was used to pre-expose R. prolixus to an olfactory conditioned stimulus before pairing the odor with temperature. Over the course of training, an effect of pre-exposure was found. Suggestions for research are discussed and potential conditioned and unconditioned stimuli identified.     

Fantasy formation: a child analyst's perspective

Unconscious fantasy is the principal unit of psychoanalytic investigation. Though individual fantasies, either conscious or unconscious, may emphasize drive, defense, or superego interests, all fantasy life develops from a limited number of themes; these themes concern drive-related issues, experiences of helplessness, or combinations of both. Fantasy formation and fantasy content undergo developmental change. Sensorimotor, behavioral memories occur prior to fantasy and are influential in determining repetitive behavioral enactments. The complexities of infant behavior do not require the postulation of fantasy or representational memory. A complex, innate, instinctual organization of the newborn, similar in many respects to that of other newborn mammals, and distinct from the psychological organization of the older infant, is suggested as an explanation of these phenomena.     

Flexibility in a single behavioral variable of Drosophila

The flexibility of behavior is so rich, and its components are so exquisitely interwoven, that one may be well advised to turn to an isolated behavioral module for study. Gill withdrawal in Aplysia, the proboscis extension reflex in the honeybee, and lid closure in mammals are such examples. We have chosen yawing, a single component of flight orientation in Drosophila melanogaster, for this approach. A specialty of this preparation is that the behavioral output can be reduced beyond the single module by one further step. It can be studied in tethered animals in which all turns are blocked while the differentially beating wings still provide the momentum. These intended yaw turns are measured by a torque meter to which the fly is hooked. The fly is held horizontally as if cruising at high speed. The head is glued to the thorax. It can bend its abdomen, extend its proboscis, and move its legs but cannot shift its direction of gaze or its orientation in space. Evidently, a fly hardly ever encounters this bizarre situation in the wild. We describe here the flexibility in this single behavioral variable. It provides insights into the relation between classical and operant conditioning, the processing of and interactions between the conditioned visual stimuli, early visual memory, visual pattern recognition, selective attention, and several other experience-dependent properties of visual orientation behavior. We start with a brief summary of visual flight control at the torque meter.     

Social modulation of associative fear learning by pheromone communication

Mice communicate through visual, vocal, and olfactory cues that influence innate, nonassociative behavior. We here report that exposure to a recently fear-conditioned familiar mouse impairs acquisition of conditioned fear and facilitates fear extinction, effects mimicked by both an olfactory chemosignal emitted by a recently fear-conditioned familiar mouse and by the putative stress-related anxiogenic pheromone β-phenylethylamine (β-PEA). Together, these findings suggest social modulation of higher-order cognitive processing through pheromone communication and support the concurrent excitor hypothesis of extinction learning.Social communication in mammals has evolved to facilitate reproductive behavior and for protection against environmental threat and predation. Mice communicate information about imminent danger through vocal (Seyfarth and Cheney 2003), visual (Kavaliers et al. 2001; Langford et al. 2006), and odor or pheromone cues (Rottman and Snowdon 1972), each with profound influences on defensive responding. There is also evidence of social empathy in mice (Langford et al. 2006). Mice will sensitize to pain-inducing stimuli simply by observing a conspecific that is currently experiencing pain. Importantly, sensitization occurs only when the conspecific is familiar with the observer (i.e., sibling or cage mate), a clear example of social modulation of an innate behavior. Müller-Velten (1966) provided the first evidence of a functional alarm chemosignal in mice by showing that animals would avoid a pathway in which the odor of a stressed mouse was present. Subsequent studies have shown effects of mammalian olfactory chemosignals on a variety of defensive behaviors such as analgesia, vigilance, and avoidance (Rottman and Snowdon 1972; Mackay-Sim and Laing 1981; Fanselow 1985; Zalaquett and Thiessen 1991). To date, research on social modulation of behavior has focused primarily on observational learning and innate or nonassociative processes. Two recent studies have demonstrated an influence of fear-related chemosignals on associative learning in humans (Chen et al. 2006; Prehn et al. 2006), evidence that supports the hypothesis that social modulation of behavior extends to higher-order cognitive processing.In the following experiments, we asked whether exposure to a familiar mouse recently fear conditioned or trained for fear extinction would influence associative fear learning in a conspecific. We find that exposure to a recently fear-conditioned mouse impairs acquisition of conditioned fear, while the same experience facilitates the extinction of conditioned fear; effects mimicked by exposure to an olfactory chemosignal emitted from fear-conditioned mice and by the putative anxiogenic pheromone, β-phenylethylamine (β-PEA). Interestingly, we find that exposure to a recently extinction-trained mouse results in an inhibition of fear extinction learning, an effect not related to an olfactory chemosignal emitted by a recently extinguished mouse or by exposure to β-PEA. These data suggest that mice communicate information about their experience, in part through pheromone communication, with different effects on associative learning depending on the valence of the task.     

The hippocampus and memory for "what," "where," and "when"

Previous studies have indicated that nonhuman animals might have a capacity for episodic-like recall reflected in memory for "what" events that happened "where" and "when". These studies did not identify the brain structures that are critical to this capacity. Here we trained rats to remember single training episodes, each composed of a series of odors presented in different places on an open field. Additional assessments examined the individual contributions of odor and spatial cues to judgments about the order of events. The results indicated that normal rats used a combination of spatial ("where") and olfactory ("what") cues to distinguish "when" events occurred. Rats with lesions of the hippocampus failed in using combinations of spatial and olfactory cues, even as evidence from probe tests and initial sampling behavior indicated spared capacities for perception of spatial and odor cues, as well as some form of memory for those individual cues. These findings indicate that rats integrate "what," "where," and "when" information in memory for single experiences, and that the hippocampus is critical to this capacity.     

Neural substrates of olfactory discrimination learning with auditory secondary reinforcement. I. Contributions of the basolateral amygdaloid complex and orbitofrontal cortex

The basolateral amygdaloid complex (BLA) and orbitofrontal cortex (OFC) share extensive reciprocal connections, and interactions between these regions likely contribute to both mnemonic and affective processes. The present study examined the potential differential contributions of the BLA and OFC to performance of an olfactory discrimination task that incorporates auditory conditioned reinforcement and to expression of immediate post-shock freezing behavior. Damage to the BLA had little effect on performance of the conditioned reinforcement task but abolished immediate post-shock freezing behavior. In contrast, damage to OFC resulted in both a mild but significant performance decrement in the conditioned reinforcement task and a significant attenuation of immediate post-shock freezing behavior. These findings suggest that immediate post-shock freezing behavior is likely critically dependent upon interactions between the BLA and OFC. However, although mnemonic processes underlying accurate performance of the conditioned reinforcement task might be supported by OFC in part, such processes are independent of either the BLA or interactions between these two regions.     

Is an epigenetic switch the key to persistent extinction?

Many studies of learning have demonstrated that conditioned behavior can be eliminated when previously established relations between stimuli are severed. This extinction process has been extremely important for the development of learning theories and, more recently, for delineating the neurobiological mechanisms that underlie memory. A key finding from behavioral studies of extinction is that extinction eliminates behavior without eliminating the original memory; extinguished behavior often returns with time or with a return to the context in which the original learning occurred. This persistence of the original memory after extinction creates a challenge for clinical applications that use extinction as part of a treatment intervention. Consequently, a goal of recent neurobiological research on extinction is to identify potential pharmacological targets that may result in persistent extinction. Drugs that promote epigenetic changes are particularly promising because they can result in a long-term molecular signal that, combined with the appropriate behavioral treatment, can cause persistent changes in behavior induced by extinction. We will review evidence demonstrating extinction enhancements by drugs that target epigenetic mechanisms and will describe some of the challenges that epigenetic approaches face in promoting persistent suppression of memories.     

Chronically increased Gsalpha signaling disrupts associative and spatial learning

The cAMP/PKA pathway plays a critical role in learning and memory systems in animals ranging from mice to Drosophila to Aplysia. Studies of olfactory learning in Drosophila suggest that altered expression of either positive or negative regulators of the cAMP/PKA signaling pathway beyond a certain optimum range may be deleterious. Here we provide genetic evidence of the behavioral and physiological effects of increased signaling through the cAMP/PKA pathway in mice. We have generated transgenic mice in which the expression of a constitutively active form of Gsalpha (Gsalpha* Q227L), the G protein that stimulates adenylyl cyclase activity, is driven in neurons within the forebrain by the promoter from the CaMKIIalpha gene. Despite significantly increased adenylyl cyclase activity, Gsalpha* transgenic mice exhibit PKA-dependent decreases in levels of cAMP due to a compensatory up-regulation in phosphodiesterase activity. Interestingly, Gsalpha* transgenic mice also exhibit enhanced basal synaptic transmission. Consistent with a role for the cAMP/PKA pathway in learning and memory, Gsalpha* transgenic mice show impairments in spatial learning in the Morris water maze and in contextual and cued fear conditioning tasks. The learning deficits observed in these transgenic mice suggest that associative and spatial learning requires regulated Gsalpha protein signaling, much as does olfactory learning in Drosophila.     

Neural Substrates of Olfactory Discrimination Learning with Auditory Secondary Reinforcement. I. Contributions of the Basolateral Amygdaloid Complex and Orbitofrontal Cortex

The basolateral amygdaloid complex (BLA) and orbitofrontal cortex (OFC) share extensive reciprocal connections, and interactions between these regions likely contribute to both mnemonic and affective processes. The present study examined the potential differential contributions of the BLA and OFC to performance of an olfactory discrimination task that incorporates auditory conditioned reinforcement and to expression of immediate post-shock freezing behavior. Damage to the BLA had little effect on performance of the conditioned reinforcement task but abolished immediate post-shock freezing behavior. In contrast, damage to OFC resulted in both a mild but significant performance decrement in the conditioned reinforcement task and a significant attenuation of immediate post-shock freezing behavior. These findings suggest that immediate post-shock freezing behavior is likely critically dependent upon interactions between the BLA and OFC. However, although mnemonic processes underlying accurate performance of the conditioned reinforcement task might be supported by OFC in part, such processes are independent of either the BLA or interactions between these two regions.     

Selective attention to the chemosensory modality

Previous studies have shown that behavioral responses to auditory, visual, and tactile stimuli are modulated by expectancies regarding the likely modality of an upcoming stimulus (see Spence & Driver, 1997). In the present study, we investigated whether people can also selectively attend to the chemosensory modality (involving responses to olfactory, chemical, and painful stimuli). Participants made speeded spatial discrimination responses (left vs. right) to an unpredictable sequence of odor and tactile targets. Odor stimuli were presented to either the left or the right nostril, embedded in a birhinally applied constant airstream. Tactile stimuli were presented to the left or the right hand. On each trial, a symbolic visual cue predicted the likely modality for the upcoming target (the cue was a valid predictor of the target modality on the majority of trials). Response latencies were faster when targets were presented in the expected modality than when they were presented in the unexpected modality, showing for the first time that behavioral responses to chemosensory stimuli can be modulated by selective attention.     

Unilateral olfactory conditioning in 6-day-old rat pups

The potential that early olfactory learning might be laterally organized in the brain was investigated in 6-day-old rats. This hypothesis is based on the finding that the commissural systems that subserve bilateral olfactory communication do not mature until the second week of postnatal life. Pups were trained with pairings of cedar odor and intraoral infusions of milk while one nostril was occluded. Animals expressed a conditioned orientation towards cedar if tested with the trained nostril open. No such conditioning was observed if the untrained nostril was open during testing. Further, when individual pups received cedar odor/milk pairings with one nostril open and orange odor/milk pairings with the other open, they expressed a conditioned preference for orange when tested with the orange-trained nostril open, and a preference for cedar when tested with the cedar-trained nostril open. Classically conditioned oral responses (mouthing) also appeared to be lateralized. However, no such unilateral conditioning occurred with respect to behavioral activation, which is also conditioned in this paradigm. Increases in activity to the odor CS were observed regardless of whether the trained or untrained nostril was open during testing. These results suggest that in developing rodents, olfactory memories may be partly represented in structures that can be unilaterally accessed during training and testing. They provide a starting point for isolation of neural substrates of the olfactory conditioning process.