Interactive parallel sex pheromone circuits that promote and suppress courtship behaviors in the cockroach.

Many animals use multicomponent sex pheromones for mating, but the specific function and neural processing of each pheromone component remain unclear. The cockroach Periplaneta americana is a model for studying sex pheromone communication, and an adult female emits major and minor sex pheromone components, periplanone-B and -A (PB and PA), respectively. Attraction and courtship behaviors (wing-raising and abdominal extension) are strongly expressed when adult males are exposed to PB but weakly expressed when they are exposed to PA. When major PB is presented together with minor PA, behaviors elicited by PB were impaired, indicating that PA can both promote and suppress courtship behaviors depending on the pheromonal context. In this study, we identified the receptor genes for PA and PB and investigated the effects of knocking down each receptor gene on the activities of PA- and PB-responsive sensory neurons (PA- and PB-SNs), and their postsynaptic interneurons, and as well as effects on courtship behaviors in males. We found that PB strongly and PA weakly activate PB-SNs and their postsynaptic neurons, and activation of the PB-processing pathway is critical for the expression of courtship behaviors. PA also activates PA-SNs and the PA-processing pathway. When PA and PB are simultaneously presented, the PB-processing pathway undergoes inhibitory control by the PA-processing pathway, which weakens the expression of courtship behaviors. Our data indicate that physiological interactions between the PA- and PB-processing pathways positively and negatively mediate the attraction and courtship behaviors elicited by sex pheromones.

Critical roles of nicotinic acetylcholine receptors in olfactory memory formation and retrieval in crickets.

Acetylcholine (ACh) is a major excitatory neurotransmitter in the insect central nervous system, and insect neurons express several types of ACh receptors (AChRs). AChRs are classified into two subgroups, muscarinic AChRs and nicotinic AChRs (nAChRs). nAChRs are also divided into two subgroups by sensitivity to α-bungarotoxin (α-BGT). The cricket Gryllus bimaculatus is one of the useful insects for studying the molecular mechanisms in olfactory learning and memory. However, the roles of nAChRs in olfactory learning and memory of the cricket are still unknown. In the present study, to investigate whether nAChRs are involved in cricket olfactory learning and memory, we tested the effects of two different AChR antagonists on long-term memory (LTM) formation and retrieval in a behavioral assay. The two AChR antagonists that we used are mecamylamine (MEC), an α-BGT-insensitive nAChR antagonist, and methyllycaconitine (MLA), an α-BGT-sensitive nAChR antagonist. In crickets, multiple-trial olfactory conditioning induced 1-day memory (LTM), whereas single-trial olfactory conditioning induced 1-h memory (mid-term memory, MTM) but not 1-day memory. Crickets injected with MEC 20 min before the retention test at 1 day after the multiple-trial conditioning exhibited no memory retrieval. This indicates that α-BGT-insensitive nAChRs participate in memory retrieval. In addition, crickets injected with MLA before the multiple-trial conditioning exhibited MTM but not LTM, indicating that α-BGT-sensitive nAChRs participate in the formation of LTM. Moreover, injection of nicotine (an nAChR agonist) before the single-trial conditioning induced LTM. Finally, the nitric oxide (NO)-cGMP signaling pathway is known to participate in the formation of LTM in crickets, and we conducted co-injection experiments with an agonist or inhibitor of the nAChR and an activator or inhibitor of the NO-cGMP signaling pathway. The results suggest that nAChR works upstream of the NO-cGMP signaling system in the LTM formation process.

Octopamine neurons mediate reward signals in social learning in an insect.

Social learning is found in many animals, but its mechanisms are not understood. We previously showed that a cricket that was trained to observe a conspecific staying at a drinking apparatus exhibited an increased preference for the odor of that drinking apparatus. Here we investigated a hypothesis that this learning is achieved by second-order conditioning (SOC), i.e., by associating conspecifics at a drinking bottle with water reward during group drinking in the rearing stage and then associating an odor with a conspecific in training. Injection of an octopamine receptor antagonist before training or testing impaired the learning or response to the learned odor, as we reported for SOC, thereby supporting the hypothesis. Notably, the SOC hypothesis predicts that octopamine neurons that respond to water in the group-rearing stage also respond to a conspecific in training, without the learner itself drinking water, and such mirror-like activities mediate social learning. This awaits future investigation.

Roles of octopamine neurons in the vertical lobe of the mushroom body for the execution of a conditioned response in cockroaches.

Aminergic neurons mediate reward signals in mammals and insects. In crickets, we showed that blockade of synaptic transmission from octopamine neurons (OANs) impairs conditioning of an odor (conditioned stimulus, CS) with water or sucrose (unconditioned stimulus, US) and execution of a conditioned response (CR) to the CS. It has not yet been established, however, whether findings in crickets can be applied to other species of insects. In this study, we investigated the roles of OANs in conditioning of salivation, monitored by activities of salivary neurons, and in execution of the CR in cockroaches (Periplaneta americana). We showed that injection of epinastine (an OA receptor antagonist) into the head hemolymph impaired both conditioning and execution of the CR, in accordance with findings in crickets. Moreover, local injection of epinastine into the vertical lobes of the mushroom body (MB), the center for associative learning and control of the CR, impaired execution of the CR, whereas injection of epinastine into the calyces of the MB or the antennal lobes (primary olfactory centers) did not. We propose that OANs in the MB vertical lobes play critical roles in the execution of the CR in cockroaches. This is analogous to the fact that midbrain dopamine neurons govern execution of learned actions in mammals.

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.

Conditioned taste aversion in the cricket Gryllus bimaculatus.

Conditioned taste aversion (CTA) is a form of classical conditioning in which animals associate the taste of a food with illness caused by toxin contained in the food. CTA in mammals is achieved with a long interval of up to several hours between food ingestion and illness induced by LiCl injection. Insects also exhibit CTA, but not much is known about its features. We investigated whether the cricket Gryllus bimaculatus exhibits CTA when ingestion of a sugar solution is followed by LiCl injection. Crickets that ingested sucrose solution 5-10 min before LiCl injection exhibited reduction of sucrose consumption tested 24 or 48 h after injection compared to that tested 24 h before injection. In contrast, crickets that ingested sucrose solution 5-10 min after LiCl injection or 1 h or 8 h before or after injection did not exhibit reduction of sucrose consumption, indicating that reduction of sucrose consumption by CTA training is pairing-specific. We conclude that CTA in crickets is similar to that in mammals in that one-trial pairing is sufficient to achieve memory retention for days, but it differs in that it is achieved with a relatively short interval (< 1 h) between food ingestion and toxin injection.

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.

Silencing the odorant receptor co-receptor impairs olfactory reception in a sensillum-specific manner in the cockroach.

Insects detect odors via a large variety of odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). The insect OR is a heteromeric complex composed of a ligand-specific receptor and the co-receptor (ORco). In this study, we identified the ORco gene of the cockroach, Periplaneta americana (PameORco), and performed RNAi-based functional analysis of PameORco. All OSNs in the basiconic sensilla expressed PameORco and received a large variety of odors including sex pheromones. In trichoid sensilla, a PameORco-positive OSN was consistently paired with a PameORco-negative OSN tuned to acids. In adult cockroaches injected with PameORco dsRNA at the nymphal stage, the expression of PameORco, odor receptions via ORs, and its central processing were strongly suppressed. These results provide new insights into the molecular basis of olfactory reception in the cockroach. The long-lasting and irreversible effects of PameORco RNAi would be an effective method for controlling the household pest.

What Is Learned in Pavlovian Conditioning in Crickets? Revisiting the S-S and S-R Learning Theories.

In Pavlovian conditioning in mammals, two theories have been proposed for associations underlying conditioned responses (CRs). One theory, called S-S theory, assumes an association between a conditioned stimulus (CS) and internal representation of an unconditioned stimulus (US), allowing the animal to adjust the CR depending on the current value of the US. The other theory, called S-R theory, assumes an association or connection between the CS center and the CR center, allowing the CS to elicit the CR. Whether these theories account for Pavlovian conditioning in invertebrates has remained unclear. In this article, results of our studies in the cricket Gryllus bimaculatus are reviewed. We showed that after a standard amount of Pavlovian training, crickets exhibited no response to odor CS when water US was devalued by providing it until satiation, whereas after extended training, they exhibited a CR after US devaluation. An increase of behavioral automaticity by extended training has not been reported in Pavlovian conditioning in any other animals, but it has been documented in instrumental conditioning in mammals. Our pharmacological analysis suggested that octopamine neurons mediate US (water) value signals and control execution of the CR after standard training. The control, however, diminishes with extension of training and hence the CR becomes insensitive to the US value. We also found that the nature of the habitual response after extended Pavlovian training in crickets is not the same as that after extended instrumental training in mammals concerning the context specificity. Adaptive significance and evolutionary implications for our findings are discussed.

Reduction of contextual control of conditioned responses by extended Pavlovian training in an insect.

The effect of repetitive training on learned behavior has been an important subject in neuroscience. In instrumental conditioning in mammals, learned action early in training is often goal-driven and controlled by outcome expectancy, but as training progresses, it becomes more habitual and insensitive to outcome devaluation. Similarly, we recently showed in Pavlovian conditioning in crickets (Gryllus bimaculatus) that a conditioned response (CR) is initially sensitive to devaluation of the unconditioned stimulus but becomes insensitive to it after extended training. It is known that habitual responses after extended instrumental training are characterized by a higher context specificity than are initial goal-directed actions in mammals. In this study, we investigated whether this is applicable to Pavlovian conditioning in crickets. In crickets that received a standard amount of training to associate an odor with water reward under illumination, CR under illumination was stronger than that in the dark. In crickets that received extended training under illumination, on the other hand, the level of CR did not differ in different light conditions. Further experiments confirmed that context specificity decreases with the development of behavioral automaticity by extended training, as opposed to findings in instrumental training in mammals. We conclude that the nature of habitual behaviors after extended training differs in different learning systems of animals.

Appetitive and aversive social learning with living and dead conspecifics in crickets.

Many animals acquire biologically important information from conspecifics. Social learning has been demonstrated in many animals, but there are few experimental paradigms that are suitable for detailed analysis of its associative processes. We established procedures for appetitive and aversive social learning with living and dead conspecifics in well-controlled stimulus arrangements in crickets, Gryllus bimaculatus. A thirsty demonstrator cricket was released in a demonstrator room and allowed to visit two drinking apparatuses that contained water or saltwater and emitted apple or banana odour, and a thirsty learner was allowed to observe the demonstrator room through a net. In the post-training test, the learner preferred the odour of the water-containing apparatus at which the demonstrator stayed. When a dead cricket was placed on one of the two apparatuses, the learner avoided the odour of that apparatus. Further experiments suggested that a living conspecific can be recognized by either visual or olfactory cues for appetitive social learning, whereas olfactory cues are needed to recognize a dead conspecific for aversive social learning, and that different associative processes underlie social learning with living and dead conspecifics. The experimental paradigms described here will pave the way for detailed research on the neural basis of social learning.

Separate But Interactive Parallel Olfactory Processing Streams Governed by Different Types of GABAergic Feedback Neurons in the Mushroom Body of a Basal Insect.

The basic organization of the olfactory system has been the subject of extensive studies in vertebrates and invertebrates. In many animals, GABA-ergic neurons inhibit spike activities of higher-order olfactory neurons and help sparsening of their odor representations. In the cockroach, two different types of GABA-immunoreactive interneurons (calyceal giants [CGs]) mainly project to the base and lip regions of the calyces (input areas) of the mushroom body (MB), a second-order olfactory center. The base and lip regions receive axon terminals of two different types of projection neurons, which receive synapses from different classes of olfactory sensory neurons (OSNs), and receive dendrites of different classes of Kenyon cells, MB intrinsic neurons. We performed intracellular recordings from pairs of CGs and MB output neurons (MBONs) of male American cockroaches, the latter receiving synapses from Kenyon cells, and we found that a CG receives excitatory synapses from an MBON and that odor responses of the MBON are changed by current injection into the CG. Such feedback effects, however, were often weak or absent in pairs of neurons that belong to different streams, suggesting parallel organization of the recurrent pathways, although interactions between different streams were also evident. Cross-covariance analysis of the spike activities of CGs and MBONs suggested that odor stimulation produces synchronized spike activities in MBONs and then in CGs. We suggest that there are separate but interactive parallel streams to process odors detected by different OSNs throughout the olfactory processing system in cockroaches.SIGNIFICANCE STATEMENT Organizational principles of the olfactory system have been the subject of extensive studies. In cockroaches, signals from olfactory sensory neurons (OSNs) in two different classes of sensilla are sent to two different classes of projection neurons, which terminate in different areas of the mushroom body (MB), each area having dendrites of different classes of MB intrinsic neurons (Kenyon cells) and terminations of different classes of GABAergic neurons. Physiological and morphological assessments derived from simultaneous intracellular recordings/stainings from GABAergic neurons and MB output neurons suggested that GABAergic neurons play feedback roles and that odors detected by OSNs are processed in separate but interactive processing streams throughout the central olfactory system.

Functional unification of sex pheromone-receptive glomeruli in the invasive Turkestan cockroach derived from the genus Periplaneta.

Female Periplaneta americana cockroaches emit two cooperatively working pheromone components, periplanone-B (PB) as a long-range attractant and periplanone-A (PA) as a short-range arrestant, and males develop enlarged glomeruli for processing them separately in the first-order olfactory center. Using intracellular recordings and neuronal labelings, we found that the Turkestan cockroach, Blatta lateralis, which is phylogenetically close to P. americana but having adapted to inground habitats, has an extraordinary large glomerulus. This is caused by drastic enlargement of the PB-responsive glomerulus but not the PA-responsive glomerulus during the late nymphal stage. The output neuron from the macroglomerulus is sensitive to both PA and PB, at a dose of only 0.1 fg. Nevertheless, B. lateralis males never exhibited courtship rituals in response to the presentation of periplanones or natural sex pheromone but exhibited courtship rituals in response to antennal contact with females. Our findings indicate that the unique behavioral ecology and habitats of B. lateralis are related to the functional unification of the pheromone processing system, opposite to the functional differentiation that often underlies species diversification.

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.

Tyrosine hydroxylase-immunoreactive neurons in the mushroom body of the field cricket, Gryllus bimaculatus.

The mushroom body of the insect brain participates in processing and integrating multimodal sensory information and in various forms of learning. In the field cricket, Gryllus bimaculatus, dopamine plays a crucial role in aversive memory formation. However, the morphologies of dopamine neurons projecting to the mushroom body and their potential target neurons, the Kenyon cells, have not been characterized. Golgi impregnations revealed two classes of Kenyon cells (types I and II) and five different types of extrinsic fibers in the mushroom body. Type I cells, which are further divided into two subtypes (types I core and I surface), extend their dendrites into the anterior calyx, whereas type II cells extend many bushy dendritic branches into the posterior calyx. Axons of the two classes bifurcate between the pedunculus and lobes to form the vertical, medial and γ lobes. Immunocytochemistry to tyrosine hydroxylase (TH), a rate-limiting enzyme in dopamine biosynthesis, revealed the following four distinct classes of neurons: (1) TH-SLP projecting to the distal vertical lobe; (2) TH-IP1 extending to the medial and γ lobes; (3) TH-IP2 projecting to the basal vertical lobe; and (4) a multiglomerular projection neuron invading the anterior calyx and the lateral horn (TH-MPN). We previously proposed a model in the field cricket in which the efficiency of synapses from Kenyon cells transmitting a relevant sensory stimulus to output neurons commanding an appropriate behavioral reaction can be modified by dopaminergic neurons mediating aversive signals and here, we provide putative neural substrates for the cricket's aversive learning. These will be instrumental in understanding the principle of aversive memory formation in this model species.

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.

Signaling Pathways for Long-Term Memory Formation in the Cricket.

Unraveling the molecular mechanisms underlying memory formation in insects and a comparison with those of mammals will contribute to a further understanding of the evolution of higher-brain functions. As it is for mammals, insect memory can be divided into at least two distinct phases: protein-independent short-term memory and protein-dependent long-term memory (LTM). We have been investigating the signaling pathway of LTM formation by behavioral-pharmacological experiments using the cricket Gryllus bimaculatus, whose olfactory learning and memory abilities are among the highest in insect species. Our studies revealed that the NO-cGMP signaling pathway, CaMKII and PKA play crucial roles in LTM formation in crickets. These LTM formation signaling pathways in crickets share a number of attributes with those of mammals, and thus we conclude that insects, with relatively simple brain structures and neural circuitry, will also be beneficial in exploratory experiments to predict the molecular mechanisms underlying memory formation in mammals.

Spatial Receptive Fields for Odor Localization.

Animals rely on olfaction to navigate through complex olfactory landscapes, but the mechanisms that allow an animal to encode the spatial structure of an odorous environment remain unclear. To acquire information about the spatial distribution of an odorant, animals may rely on bilateral olfactory organs and compare side differences of odor intensity and timing [1-6] or may perform spatial and temporal signal integration of subsequent samplings [7]. The American cockroach can efficiently locate a source of sex pheromone even after the removal of one antenna, suggesting that bilateral comparison is not a prerequisite for odor localization in this species [8, 9]. Cognate olfactory sensory neurons (OSNs) originating from different locations on the flagellum, but bearing the same olfactory receptor, converge onto the same glomerulus within the antennal lobe, which is thought to result in a loss of spatial information. Here, we identified 12 types of pheromone-responsive projection neurons (PNs), each with spatially tuned receptive field. The combination of (1) the antennotopic organization of OSNs terminals and (2) the stereotyped compartmentalization of PNs' dendritic arborization within the macroglomerulus (MG), allows encoding the spatial position of the pheromone. Furthermore, each PN type innervates a different compartment of the mushroom body, providing the means for encoding spatial olfactory information along the olfactory circuit. Finally, MG PNs exhibit both excitatory and inhibitory spatial receptive fields and modulate their responses based on changes in stimulus geometry. In conclusion, we propose a mechanism for encoding information on the spatial distribution of a pheromone, expanding both our understanding of odor coding and of the strategies insects adopt to localize a sexual mate.

Calcium imaging method to visualize the spatial patterns of neural responses in the pygmy squid, Idiosepius paradoxus, central nervous system.

BACKGROUND: Cephalopods exhibit unique behaviors such as camouflage and tactile learning. The brain functions correlated to these behaviors have long been analyzed through behavioral observations of animals subject to surgical manipulation or electrical stimulation of brain lobes. However, physiological methods have rarely been introduced to investigate the functions of each individual lobe, though physiological work on giant axons and slices of the vertical lobe system of the cephalopods have provided deep insights into ion conductance of nerves and long-term synaptic plasticity. The lack of in vivo physiological work is partly due to difficulties in immobilizing the brain which is contained within the soft body and applying calcium indicators to the cephalopod central nervous system. NEW METHOD: We here present a calcium imaging method to visualize neural responses in the central nervous system of the smallest squid, Idiosepius paradoxus. RESULTS: We injected calcium indicator Cal-520 into the brachial lobes and revealed a spatiotemporal pattern of neural responses to the electrical stimulations of the axial nerve cord in the first arm. COMPARISON WITH EXISTING METHODS: We established a method to immobilize the central nervous system which is contained within the soft body and record the calcium responses from the intact central nervous system. CONCLUSIONS: Our method provides a novel approach to investigate the mechanisms of how the characteristic organization of the cephalopod brain functions to induce their unique behaviors.