Roles of Aminergic Neurons in Formation and Recall of Associative Memory in Crickets

We review recent progress in the study of roles of octopaminergic (OA-ergic) and dopaminergic (DA-ergic) signaling in insect classical conditioning, focusing on our studies on crickets. Studies on olfactory learning in honey bees and fruit-flies have suggested that OA-ergic and DA-ergic neurons convey reinforcing signals of appetitive unconditioned stimulus (US) and aversive US, respectively. Our work suggested that this is applicable to olfactory, visual pattern, and color learning in crickets, indicating that this feature is ubiquitous in learning of various sensory stimuli. We also showed that aversive memory decayed much faster than did appetitive memory, and we proposed that this feature is common in insects and humans. Our study also suggested that activation of OA- or DA-ergic neurons is needed for appetitive or aversive memory recall, respectively. To account for this finding, we proposed a model in which it is assumed that two types of synaptic connections are strengthened by conditioning and are activated during memory recall, one type being connections from neurons representing conditioned stimulus (CS) to neurons inducing conditioned response and the other being connections from neurons representing CS to OA- or DA-ergic neurons representing appetitive or aversive US, respectively. The former is called stimulus–response (S–R) connection and the latter is called stimulus–stimulus (S–S) connection by theorists studying classical conditioning in vertebrates. Results of our studies using a second-order conditioning procedure supported our model. We propose that insect classical conditioning involves the formation of S–S connection and its activation for memory recall, which are often called cognitive processes

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

Alarm Pheromone Processing in the Ant Brain: An Evolutionary Perspective

Social insects exhibit sophisticated communication by means of pheromones, one example of which is the use of alarm pheromones to alert nestmates for colony defense. We review recent advances in the understanding of the processing of alarm pheromone information in the ant brain. We found that information about formic acid and n-undecane, alarm pheromone components, is processed in a set of specific glomeruli in the antennal lobe of the ant Camponotus obscuripes. Alarm pheromone information is then transmitted, via projection neurons (PNs), to the lateral horn and the calyces of the mushroom body of the protocerebrum. In the lateral horn, we found a specific area where terminal boutons of alarm pheromone-sensitive PNs are more densely distributed than in the rest of the lateral horn. Some neurons in the protocerebrum responded specifically to formic acid or n-undecane and they may participate in the control of behavioral responses to each pheromone component. Other neurons, especially those originating from the mushroom body lobe, responded also to non-pheromonal odors and may play roles in integration of pheromonal and non-pheromonal signals. We found that a class of neurons receive inputs in the lateral horn and the mushroom body lobe and terminate in a variety of premotor areas. These neurons may participate in the control of aggressive behavior, which is sensitized by alarm pheromones and is triggered by non-pheromonal sensory stimuli associated with a potential enemy. We propose that the alarm pheromone processing system has evolved by differentiation of a part of general odor processing system

Differential Odor Processing in Two Olfactory Pathways in the Honeybee

An important component in understanding central olfactory processing and coding in the insect brain relates to the characterization of the functional divisions between morphologically distinct types of projection neurons (PN). Using calcium imaging, we investigated how the identity, concentration and mixtures of odors are represented in axon terminals (boutons) of two types of PNs – lPN and mPN. In lPN boutons we found less concentration dependence, narrow tuning profiles at a high concentration, which may be optimized for fine, concentration-invariant odor discrimination. In mPN boutons, however, we found clear rising concentration dependence, broader tuning profiles at a high concentration, which may be optimized for concentration coding. In addition, we found more mixture suppression in lPNs than in mPNs, indicating lPNs better adaptation for synthetic mixture processing. These results suggest a functional division of odor processing in both PN types

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

Knockout crickets for the study of learning and memory : Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets

Elucidation of reinforcement mechanisms in associative learning is an important subject in neuroscience. In mammals, dopamine neurons are thought to play critical roles in mediating both appetitive and aversive reinforcement. Our pharmacological studies suggested that octopamine and dopamine neurons mediate reward and punishment, respectively, in crickets, but recent studies in fruit-flies concluded that dopamine neurons mediates both reward and punishment, via the type 1 dopamine receptor Dop1. To resolve the discrepancy between studies in different insect species, we produced Dop1 knockout crickets using the CRISPR/Cas9 system and found that they are defective in aversive learning with sodium chloride punishment but not appetitive learning with water or sucrose reward. The results suggest that dopamine and octopamine neurons mediate aversive and appetitive reinforcement, respectively, in crickets. We suggest unexpected diversity in neurotransmitters mediating appetitive reinforcement between crickets and fruit-flies, although the neurotransmitter mediating aversive reinforcement is conserved. This study demonstrates usefulness of the CRISPR/Cas9 system for producing knockout animals for the study of learning and memory

Pavlov's Cockroach: Classical Conditioning of Salivation in an Insect

Secretion of saliva to aid swallowing and digestion is an important physiological function found in many vertebrates and invertebrates. Pavlov reported classical conditioning of salivation in dogs a century ago. Conditioning of salivation, however, has been so far reported only in dogs and humans, and its underlying neural mechanisms remain elusive because of the complexity of the mammalian brain. We previously reported that, in cockroaches Periplaneta americana, salivary neurons that control salivation exhibited increased responses to an odor after conditioning trials in which the odor was paired with sucrose solution. However, no direct evidence of conditioning of salivation was obtained. In this study, we investigated the effects of conditioning trials on the level of salivation. Untrained cockroaches exhibited salivary responses to sucrose solution applied to the mouth but not to peppermint or vanilla odor applied to an antenna. After differential conditioning trials in which an odor was paired with sucrose solution and another odor was presented without pairing with sucrose solution, sucrose-associated odor induced an increase in the level of salivation, but the odor presented alone did not. The conditioning effect lasted for one day after conditioning trials. This study demonstrates, for the first time, classical conditioning of salivation in species other than dogs and humans, thereby providing the first evidence of sophisticated neural control of autonomic function in insects. The results provide a useful model system for studying cellular basis of conditioning of salivation in the simpler nervous system of 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

Roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in an insect

<p>Abstract</p> <p>Background</p> <p>In insect classical conditioning, octopamine (the invertebrate counterpart of noradrenaline) or dopamine has been suggested to mediate reinforcing properties of appetitive or aversive unconditioned stimulus, respectively. However, the roles of octopaminergic and dopaminergic neurons in memory recall have remained unclear.</p> <p>Results</p> <p>We studied the roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in olfactory and visual conditioning in crickets. We found that pharmacological blockade of octopamine and dopamine receptors impaired aversive memory recall and appetitive memory recall, respectively, thereby suggesting that activation of octopaminergic and dopaminergic neurons and the resulting release of octopamine and dopamine are needed for appetitive and aversive memory recall, respectively. On the basis of this finding, we propose a new model in which it is assumed that two types of synaptic connections are formed by conditioning and are activated during memory recall, one type being connections from neurons representing conditioned stimulus to neurons inducing conditioned response and the other being connections from neurons representing conditioned stimulus to octopaminergic or dopaminergic neurons representing appetitive or aversive unconditioned stimulus, respectively. The former is called 'stimulus-response connection' and the latter is called 'stimulus-stimulus connection' by theorists studying classical conditioning in higher vertebrates. Our model predicts that pharmacological blockade of octopamine or dopamine receptors during the first stage of second-order conditioning does not impair second-order conditioning, because it impairs the formation of the stimulus-response connection but not the stimulus-stimulus connection. The results of our study with a cross-modal second-order conditioning were in full accordance with this prediction.</p> <p>Conclusion</p> <p>We suggest that insect classical conditioning involves the formation of two kinds of memory traces, which match to stimulus-stimulus connection and stimulus-response connection. This is the first study to suggest that classical conditioning in insects involves, as does classical conditioning in higher vertebrates, the formation of stimulus-stimulus connection and its activation for memory recall, which are often called cognitive processes.</p

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 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 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