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.

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.

The melatonin metabolite N1-acetyl-5-methoxykynuramine facilitates long-term object memory in young and aging mice.

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL's metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post-training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age-associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK's memory-enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL-to-AMK metabolic pathway disrupted object memory at 24 hours post-training, suggesting that endogenous AMK might play an important role in long-term memory formation. This is the first study to report that AMK facilitates long-term object memory performance in mice, and that MEL crosses the blood-brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.

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.

Interaction of inhibitory and facilitatory effects of conditioning trials on long-term memory formation.

Animals learn through experience and consolidate the memories into long-time storage. Conditioning parameters to induce protein synthesis-dependent long-term memory (LTM) have been the subject of extensive studies in many animals. Here we found a case in which a conditioning trial inhibits or facilitates LTM formation depending on the intervals from preceding trials. We studied the effects of conditioning parameters on LTM formation in olfactory conditioning of maxillary-palpi extension response with sucrose reward in the cockroach Periplaneta americana We found, at first, that translation- and transcription-dependent LTM forms 1 h after training, the fastest so far reported in insects. Second, we observed that multiple-trial training with an intertrial interval (ITI) of 20 or 30 sec, often called massed training, is more effective than spaced training for LTM formation, an observation that differs from the results of most studies in other animals. Third, we found that a conditioning trial inhibits LTM formation when the intervals from preceding trials were in the range of 10-16 min. This inhibitory effect is pairing-specific and is not due to decreased motivation for learning (overtraining effect). To our knowledge, no similar inhibition of LTM formation by a conditioning trial has been reported in any animals. We propose a model to account for the effects of trial number and ITIs on LTM formation. Olfactory conditioning in cockroaches should provide pertinent materials in which to study neuronal and molecular mechanisms underlying the inhibitory and facilitatory processes for LTM formation.

Effects of Caffeine on Olfactory Learning in Crickets.

Caffeine is a plant-derived alkaloid that is generally known as a central nervous system (CNS) stimulant. In order to examine the effects of caffeine on higher CNS functions in insects, we used an appetitive olfactory learning paradigm for the cricket Gryllus bimaculatus. Crickets can form significant long-term memories (LTMs) after repetitive training sessions, during which they associate a conditioned stimulus (CS: odor) with an unconditioned stimulus (US: reward). Administration of hemolymphal injections of caffeine established LTM after only single-trial conditioning over a wide range of caffeine dosages (1.6 µµg/kg to 39 mg/kg). We investigated the physiological mechanisms underlying this enhancement of olfactory learning performance pharmacologically, focusing on three major physiological roles of caffeine: 1) inhibition of phosphodiesterase (PDE), 2) agonism of ryanodine receptors, and 3) antagonism of adenosine receptors. Application of drugs relevant to these actions resulted in significant effects on LTM formation. These results suggest that externally applied caffeine enhances LTM formation in insect olfactory learning via multiple cellular mechanisms.

Cyclic nucleotide-gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee.

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

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.

Analysis and modeling of neural processes underlying sensory preconditioning.

Sensory preconditioning (SPC) is a procedure to demonstrate learning to associate between relatively neutral sensory stimuli in the absence of an external reinforcing stimulus, the underlying neural mechanisms of which have remained obscure. We address basic questions about neural processes underlying SPC, including whether neurons that mediate reward or punishment signals in reinforcement learning participate in association between neutral sensory stimuli. In crickets, we have suggested that octopaminergic (OA-ergic) or dopaminergic (DA-ergic) neurons participate in memory acquisition and retrieval in appetitive or aversive conditioning, respectively. Crickets that had been trained to associate an odor (CS2) with a visual pattern (CS1) (phase 1) and then to associate CS1 with water reward or quinine punishment (phase 2) exhibited a significantly increased or decreased preference for CS2 that had never been paired with the US, demonstrating successful SPC. Injection of an OA or DA receptor antagonist at different phases of the SPC training and testing showed that OA-ergic or DA-ergic neurons do not participate in learning of CS2-CS1 association in phase 1, but that OA-ergic neurons participate in learning in phase 2 and memory retrieval after appetitive SPC training. We also obtained evidence suggesting that association between CS2 and US, which should underlie conditioned response of crickets to CS2, is formed in phase 2, contrary to the standard theory of SPC assuming that it occurs in the final test. We propose models of SPC to account for these findings, by extending our model of classical conditioning.

Context-dependent olfactory learning monitored by activities of salivary neurons in cockroaches.

Context-dependent discrimination learning, a sophisticated form of nonelemental associative learning, has been found in many animals, including insects. The major purpose of this research is to establish a method for monitoring this form of nonelemental learning in rigidly restrained insects for investigation of underlying neural mechanisms. We report context-dependent olfactory learning (occasion-setting problem solving) of salivation, which can be monitored as activity changes of salivary neurons in immobilized cockroaches, Periplaneta americana. A group of cockroaches was trained to associate peppermint odor (conditioned stimulus, CS) with sucrose solution reward (unconditioned stimulus, US) while vanilla odor was presented alone without pairing with the US under a flickering light condition (1.0 Hz) and also trained to associate vanilla odor with sucrose reward while peppermint odor was presented alone under a steady light condition. After training, the responses of salivary neurons to the rewarded peppermint odor were significantly greater than those to the unrewarded vanilla odor under steady illumination and those to the rewarded vanilla odor was significantly greater than those to the unrewarded peppermint odor in the presence of flickering light. Similar context-dependent responses were observed in another group of cockroaches trained with the opposite stimulus arrangement. This study demonstrates context-dependent olfactory learning of salivation for the first time in any vertebrate and invertebrate species, which can be monitored by activity changes of salivary neurons in restrained cockroaches.

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.

General anesthesia with remimazolam for a pediatric patient with MELAS and recurrent epilepsy: a case report.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disease. We report here the safe use of remimazolam in a pediatric MELAS patient. CASE PRESENTATION: A 10-year-old girl (118 cm, 16 kg) was scheduled for an open gastrostomy to improve nutrition and epileptic seizure control. We induced and maintained general anesthesia with remimazolam, remifentanil, fentanyl, and rocuronium. We also performed a bilateral subcostal transversus abdominis plane block before the surgery. The surgery finished uneventfully. After we discontinued remimazolam administration, the patient woke up immediately but calmly without flumazenil. Epileptic seizures did not occur during intra- and early post-operative periods. CONCLUSION: Remimazolam enabled us to provide a pediatric MELAS patient with general anesthesia without causing delayed emergence or epileptic seizures.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Early calcium increase triggers the formation of olfactory long-term memory in honeybees.

BACKGROUND: Synaptic plasticity associated with an important wave of gene transcription and protein synthesis underlies long-term memory processes. Calcium (Ca2+) plays an important role in a variety of neuronal functions and indirect evidence suggests that it may be involved in synaptic plasticity and in the regulation of gene expression correlated to long-term memory formation. The aim of this study was to determine whether Ca2+ is necessary and sufficient for inducing long-term memory formation. A suitable model to address this question is the Pavlovian appetitive conditioning of the proboscis extension reflex in the honeybee Apis mellifera, in which animals learn to associate an odor with a sucrose reward. RESULTS: By modulating the intracellular Ca2+ concentration ([Ca2+]i) in the brain, we show that: (i) blocking [Ca2+]i increase during multiple-trial conditioning selectively impairs long-term memory performance; (ii) conversely, increasing [Ca2+]i during single-trial conditioning triggers long-term memory formation; and finally, (iii) as was the case for long-term memory produced by multiple-trial conditioning, enhancement of long-term memory performance induced by a [Ca2+]i increase depends on de novo protein synthesis. CONCLUSION: Altogether our data suggest that during olfactory conditioning Ca2+ is both a necessary and a sufficient signal for the formation of protein-dependent long-term memory. Ca2+ therefore appears to act as a switch between short- and long-term storage of learned information.

Why the carrot is more effective than the stick: different dynamics of punishment memory and reward memory and its possible biological basis.

One of the most extensively debated topics in educational psychology is whether punishment or reward is more effective for producing short-term and long-term behavioral changes, and it has been proposed that the effect of punishment is less durable than the effect of reward. However, no conclusive evidence to support this proposal has been obtained in any animals. We recently found that punishment memory decayed much faster than reward memory in olfactory learning and visual pattern learning in crickets. We also found that neurotransmitters conveying punishment and reward signals differ in crickets: dopaminergic and octopaminergic neurons play critical roles in conveying punishment and reward signals, respectively. In this study, we investigated whether these features are general features of cricket learning or are specific to olfactory and visual pattern learning. We found that crickets have the capability of color learning and that their color learning has the same features. Based on our findings in crickets and those reported in other species of insects, we conclude that these two features are conserved in many forms of insect learning. In mammals, aminergic neurons are known to convey reward and punishment signals in learning of a variety of sensory stimuli. We propose that the faster decay of punishment memory than reward memory observed in insects and humans reflects different cellular and biochemical processes after activation of receptors for amines conveying punishment and reward signals. The possible adaptive significance of relatively limited durability of punishment memory is proposed.

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.

Roles of Octopamine and Dopamine Neurons for Mediating Appetitive and Aversive Signals in Pavlovian Conditioning in Crickets.

Revealing neural systems that mediate appetite and aversive signals in associative learning is critical for understanding the brain mechanisms controlling adaptive behavior in animals. In mammals, it has been shown that some classes of dopamine neurons in the midbrain mediate prediction error signals that govern the learning process, whereas other classes of dopamine neurons control execution of learned actions. In this review, based on the results of our studies on Pavlovian conditioning in the cricket Gryllus bimaculatus and by referring to the findings in honey bees and fruit-flies, we argue that comparable aminergic systems exist in the insect brain. We found that administrations of octopamine (the invertebrate counterpart of noradrenaline) and dopamine receptor antagonists impair conditioning to associate an olfactory or visual conditioned stimulus (CS) with water or sodium chloride solution (appetitive or aversive unconditioned stimulus, US), respectively, suggesting that specific octopamine and dopamine neurons mediate appetitive and aversive signals, respectively, in conditioning in crickets. These findings differ from findings in fruit-flies. In fruit-flies, appetitive and aversive signals are mediated by different dopamine neuron subsets, suggesting diversity in neurotransmitters mediating appetitive signals in insects. We also found evidences of "blocking" and "auto-blocking" phenomena, which suggested that the prediction error, the discrepancy between actual US and predicted US, governs the conditioning in crickets and that octopamine neurons mediate prediction error signals for appetitive US. Our studies also showed that activations of octopamine and dopamine neurons are needed for the execution of an appetitive conditioned response (CR) and an aversive CR, respectively, and we, thus, proposed that these neurons mediate US prediction signals that drive appetitive and aversive CRs. Our findings suggest that the basic principles of functioning of aminergic systems in associative learning, i.e., to transmit prediction error signals for conditioning and to convey US prediction signals for execution of CR, are conserved among insects and mammals, on account of the fact that the organization of the insect brain is much simpler than that of the mammalian brain. Further investigation of aminergic systems that govern associative learning in insects should lead to a better understanding of commonalities and diversities of computational rules underlying associative learning in animals.

Contextual olfactory learning in cockroaches.

We investigated the capability of context-dependent olfactory learning in cockroaches. One group of cockroaches received training to associate peppermint odor (conditioning stimulus) with sucrose solution (appetitive unconditioned stimulus) and vanilla odor with saline solution under illumination and to associate peppermint with aversive unconditioned stimulus and vanilla with appetitive unconditioned stimulus in the dark. Another group received training with the opposite stimulus arrangement. Before training, both groups exhibited preference for vanilla over peppermint. After training, the former group preferred peppermint over vanilla under illumination but preferred vanilla over peppermint in the dark, and the latter group exhibited the opposite odor preference. We conclude that cockroaches are capable of disambiguating the meaning of conditioning stimuli according to visual context.

[Usefulness of intraoperative transesophageal echocardiography in stent graft implantation for thoracic descending aorta].

BACKGROUND: Stent graft implantation for thoracic descending aorta is a promising alternative to open repair. Transesophageal echocardiography (TEE) is a sensitive imaging modality for aortic disease. We reviewed our experience with TEE in stent graft implantation for thoracic descending aorta. METHOD: Five patients underwent stent graft implantation for thoracic descending aorta under general anesthesia. Intraoperative angiography and TEE were used to identify the extent of the aneurysm and the placement of the stent. RESULTS: TEE showed stent graft configuration and presence of leakage in all cases. In three cases, additional stent graft placement or bypass was performed. CONCLUSIONS: Useful information was obtained by TEE in enhancing the accuracy of stent graft positioning potentially improving outcomes. TEE may facilitate repair by confirming aortic pathology, identifying endograft placement, and assessing the adequacy of aneurysm sack isolation, presence of leakage, as well as dynamic intraoperative cardiac performance.

Activation of NO-cGMP Signaling Rescues Age-Related Memory Impairment in Crickets.

Age-related memory impairment (AMI) is a common feature and a debilitating phenotype of brain aging in many animals. However, the molecular mechanisms underlying AMI are still largely unknown. The cricket Gryllus bimaculatus is a useful experimental animal for studying age-related changes in learning and memory capability; because the cricket has relatively short life-cycle and a high capability of olfactory learning and memory. Moreover, the molecular mechanisms underlying memory formation in crickets have been examined in detail. In the present study, we trained male crickets of different ages by multiple-trial olfactory conditioning to determine whether AMI occurs in crickets. Crickets 3 weeks after the final molt (3-week-old crickets) exhibited levels of retention similar to those of 1-week-old crickets at 30 min or 2 h after training; however they showed significantly decreased levels of 1-day retention, indicating AMI in long-term memory (LTM) but not in anesthesia-resistant memory (ARM) in olfactory learning of crickets. Furthermore, 3-week-old crickets injected with a nitric oxide (NO) donor, a cyclic GMP (cGMP) analog or a cyclic AMP (cAMP) analog into the hemolymph before conditioning exhibited a normal level of LTM, the same level as that in 1-week-old crickets. The rescue effect by NO donor or cGMP analog injection was absent when the crickets were injected after the conditioning. For the first time, an NO donor and a cGMP analog were found to antagonize the age-related impairment of LTM formation, suggesting that deterioration of NO synthase (NOS) or molecules upstream of NOS activation is involved in brain-aging processes.