oskar acts with the transcription factor Creb to regulate long-term memory in crickets.

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for oskar. We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus. In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb, which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar's original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar's later co-option to the germ line in holometabolous insects.

Spontaneous recovery from overexpectation in an insect.

In associative learning in mammals, it is widely accepted that learning is determined by the prediction error, i.e., the error between the actual reward and the reward predicted by the animal. However, it is unclear whether error-based learning theories are applicable to the learning occurring in other non-mammalian species. Here, we examined whether overexpectation, a phenomenon that supports error-based learning theories, occurs in crickets. Crickets were independently trained with two different conditioned stimuli (CSs), an odour and a visual pattern, that were followed by an appetitive unconditioned stimulus (US). Then the two CSs were presented simultaneously as a compound, followed by the same US. This treatment resulted in a reduced conditioned response to the odour CS when tested immediately after training. However, the response to the CS was partially recovered after 1 day. These results are the first to show overexpectation and its spontaneous recovery in an invertebrate species. While the results showing overexpectation are in agreement with the prediction by the Rescorla-Wagner model, a major form of error-based learning theories, the ones showing spontaneous recovery are not. Our results suggest that conventional error-based learning models account for some, but not for all essential features of Pavlovian conditioning in crickets.

Development of behavioural automaticity by extended Pavlovian training in an insect.

The effect of repetitive training on learned actions has been a major subject in behavioural neuroscience. Many studies of instrumental conditioning in mammals, including humans, suggested that learned actions early in training are goal-driven and controlled by outcome expectancy, but they become more automatic and insensitive to reduction in the value of the outcome after extended training. It was unknown, however, whether the development of value-insensitive behaviour also occurs by extended training of Pavlovian conditioning in any animals. Here we show that crickets Gryllus bimaculatus that had received minimal training to associate an odour with water (unconditioned stimulus, US) did not exhibit conditioned response (CR) to the odour when they were given water until satiation before the test, but those that had received extended training exhibited CR even when they were satiated with water. Further pharmacological experiments suggested that octopamine neurons, the invertebrate counterparts of noradrenaline neurons, mediate US value signals and control execution of CR after minimal training, but the control diminishes with the progress of training and hence the CR becomes insensitive to US devaluation. The results suggest that repetitive sensory experiences can lead to a change from a goal-driven response to a more automatic one in crickets.

Courtship behavior induced by appetitive olfactory memory.

Reinforcement signals such as food reward and noxious punishment can change diverse behaviors. This holds true in fruit flies, Drosophila melanogaster, which can be conditioned by an odor and sugar reward or electric shock punishment. Despite a wide variety of behavior modulated by learning, conditioned responses have been traditionally measured by altered odor preference in a choice, and other memory-guided behaviors have been only scarcely investigated. Here, we analyzed detailed conditioned odor responses of flies after sugar associative learning by employing a video recording and semi-automated processing pipeline. Trajectory analyses revealed that multiple behavioral components were altered along with conditioned approach to the rewarded odor. Notably, we found that lateral wing extension, a hallmark of courtship behavior of D. melanogaster, was robustly increased specifically in the presence of the rewarded odor. Strikingly, genetic disruption of the mushroom body output did not impair conditioned courtship increase, while markedly weakening conditioned odor approach. Our results highlight the complexity of conditioned responses and their distinct regulatory mechanisms that may underlie coordinated yet complex memory-guided behaviors in flies.

Application of a Prediction Error Theory to Pavlovian Conditioning in an Insect.

Elucidation of the conditions in which associative learning occurs is a critical issue in neuroscience and comparative psychology. In Pavlovian conditioning in mammals, it is thought that the discrepancy, or error, between the actual reward and the predicted reward determines whether learning occurs. This theory stems from the finding of Kamin's blocking effect, in which after pairing of a stimulus with an unconditioned stimulus (US), conditioning of a second stimulus is blocked when the two stimuli are presented in compound and paired with the same US. Whether this theory is applicable to any species of invertebrates, however, has remained unknown. We first showed blocking and one-trial blocking of Pavlovian conditioning in the cricket Gryllus bimaculatus, which supported the Rescorla-Wagner model but not attentional theories, the major competitive error-correction learning theories to account for blocking. To match the prediction error theory, a neural circuit model was proposed, and prediction from the model was tested: the results were consistent with the Rescorla-Wagner model but not with the retrieval theory, another competitive theory to account for blocking. The findings suggest that the Rescorla-Wagner model best accounts for Pavlovian conditioning in crickets and that the basic computation rule underlying Pavlovian conditioning in crickets is the same to those suggested in mammals. Moreover, results of pharmacological studies in crickets suggested that octopamine and dopamine mediate prediction error signals in appetitive and aversive conditioning, respectively. This was in contrast to the notion that dopamine mediates appetitive prediction error signals in mammals. The functional significance and evolutionary implications of these findings are discussed.

Roles of dopamine neurons in mediating the prediction error in aversive learning in insects.

In associative learning in mammals, it is widely accepted that the discrepancy, or error, between actual and predicted reward determines whether learning occurs. The prediction error theory has been proposed to account for the finding of a blocking phenomenon, in which pairing of a stimulus X with an unconditioned stimulus (US) could block subsequent association of a second stimulus Y to the US when the two stimuli were paired in compound with the same US. Evidence for this theory, however, has been imperfect since blocking can also be accounted for by competitive theories. We recently reported blocking in classical conditioning of an odor with water reward in crickets. We also reported an "auto-blocking" phenomenon in appetitive learning, which supported the prediction error theory and rejected alternative theories. The presence of auto-blocking also suggested that octopamine neurons mediate reward prediction error signals. Here we show that blocking and auto-blocking occur in aversive learning to associate an odor with salt water (US) in crickets, and our results suggest that dopamine neurons mediate aversive prediction error signals. We conclude that the prediction error theory is applicable to both appetitive learning and aversive learning in insects.

Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies.

Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects.

Critical evidence for the prediction error theory in associative learning.

In associative learning in mammals, it is widely accepted that the discrepancy, or error, between actual and predicted reward determines whether learning occurs. Complete evidence for the prediction error theory, however, has not been obtained in any learning systems: Prediction error theory stems from the finding of a blocking phenomenon, but blocking can also be accounted for by other theories, such as the attentional theory. We demonstrated blocking in classical conditioning in crickets and obtained evidence to reject the attentional theory. To obtain further evidence supporting the prediction error theory and rejecting alternative theories, we constructed a neural model to match the prediction error theory, by modifying our previous model of learning in crickets, and we tested a prediction from the model: the model predicts that pharmacological intervention of octopaminergic transmission during appetitive conditioning impairs learning but not formation of reward prediction itself, and it thus predicts no learning in subsequent training. We observed such an "auto-blocking", which could be accounted for by the prediction error theory but not by other competitive theories to account for blocking. This study unambiguously demonstrates validity of the prediction error theory in associative learning.

Roles of calcium/calmodulin-dependent kinase II in long-term memory formation in crickets.

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key molecule in many systems of learning and memory in vertebrates, but roles of CaMKII in invertebrates have not been characterized in detail. We have suggested that serial activation of NO/cGMP signaling, cyclic nucleotide-gated channel, Ca(2+)/CaM and cAMP signaling participates in long-term memory (LTM) formation in olfactory conditioning in crickets, and here we show participation of CaMKII in LTM formation and propose its site of action in the biochemical cascades. Crickets subjected to 3-trial conditioning to associate an odor with reward exhibited memory that lasts for a few days, which is characterized as protein synthesis-dependent LTM. In contrast, animals subjected to 1-trial conditioning exhibited memory that lasts for only several hours (mid-term memory, MTM). Injection of a CaMKII inhibitor prior to 3-trial conditioning impaired 1-day memory retention but not 1-hour memory retention, suggesting that CaMKII participates in LTM formation but not in MTM formation. Animals injected with a cGMP analogue, calcium ionophore or cAMP analogue prior to 1-trial conditioning exhibited 1-day retention, and co-injection of a CaMKII inhibitor impaired induction of LTM by the cGMP analogue or that by the calcium ionophore but not that by the cAMP analogue, suggesting that CaMKII is downstream of cGMP production and Ca(2+) influx and upstream of cAMP production in biochemical cascades for LTM formation. Animals injected with an adenylyl cyclase (AC) activator prior to 1-trial conditioning exhibited 1-day retention. Interestingly, a CaMKII inhibitor impaired LTM induction by the AC activator, although AC is expected to be a downstream target of CaMKII. The results suggest that CaMKII interacts with AC to facilitate cAMP production for LTM formation. We propose that CaMKII serves as a key molecule for interplay between Ca(2+) signaling and cAMP signaling for LTM formation, a new role of CaMKII in learning and memory.

Roles of NO signaling in long-term memory formation in visual learning in an insect.

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.