Toward elucidating diversity of neural mechanisms underlying insect learning
© Mizunami et al.; licensee BioMed Central. 2015
Received: 25 September 2014
Accepted: 7 November 2014
Published: 10 February 2015
Insects are widely used as models to study neural mechanisms of learning and memory. Our recent studies on crickets, together with reports on other insect species, suggest that some fundamental differences exist in neural and molecular mechanisms of learning and memory among different species of insects, particularly between crickets and fruit flies. First, we suggested that in crickets octopamine (OA) and dopamine (DA) neurons convey reward and punishment signals, respectively, in associated learning. On the other hand, it has been reported that in fruit flies different sets of DA neurons convey reward or punishment signals. Secondly, we have suggested that in crickets OA and DA neurons participate in the retrieval of appetitive and aversive memories, respectively, while this is not the case in fruit flies. Thirdly, cyclic AMP signaling is critical for short-term memory formation in fruit flies, but not in crickets. Finally, nitric oxide-cyclic GMP signaling and calcium-calmodulin signaling are critical for long-term memory (LTM) formation in crickets, but such roles have not been reported in fruit flies. Not all of these differences can be ascribed to different experimental methods used in studies. We thus suggest that there are unexpected diversities in basic mechanisms of learning and memory among different insect species, especially between crickets and fruit flies. Studies on a larger number of insect species will help clarify the diversity of learning and memory mechanisms in relation to functional adaptation to the environment and evolutionary history.
KeywordsCrickets Insects Olfactory learning Octopamine Dopamine Long-term memory Evolution
Insects have excellent learning and memory capabilities despite the relative simplicity of their central nervous systems, and thus they have been used as models to study basic mechanisms underlying learning and memory [1-5]. Much knowledge has been accumulated regarding neural and molecular mechanisms of learning and memory in a few species of insects, such as the fruit fly Drosophila melanogaster, the honeybee Apis mellifera, and the cricket Gryllus bimaculatus. In our studies in crickets, we noted that some of the basic features of neural mechanisms of learning and memory in crickets differ from those reported in fruit flies, although they are similar to those reported in honeybees. Such differences among insects have not been recognized in previous studies.
Here we review our major findings on learning and memory in crickets, and discuss how they are similar or different from those reported in other species. We also discuss whether the observed differences reflect species-specific features or can be explained by other factors such as the different experimental methods used. Finally, we briefly discuss the possible evolutionary perspective on diversity in learning and memory mechanisms in insects.
Procedures for conditioning
Studies of classical conditioning on fruit flies and honeybees have been performed using somewhat different procedures. In aversive classical conditioning in the fruit fly Drosophila, a group of flies was exposed to two odors in the training chamber, one of which is paired with electric shock and the other is not. The animals were then exposed to both odors in a T-maze, one odor from each side . For appetitive conditioning, a group of flies was exposed to an odor during presentation of sucrose solution and then exposed to another odor without presentation of sucrose . In appetitive conditioning in honeybees, a bee was placed in a metal tube and an odor was presented to the antennae and then sucrose solution was presented to the antennae and the mouth. After training, the bee extended its proboscis in response to the presentation of conditioned odor . For aversive conditioning, a harnessed bee was presented with an odor and then electric shock. After training, the bee exhibited sting extension in response to the conditioned odor . On account of differences in conditioning procedures, previous studies concluded that many features of learning and memory are much the same among different insects. Here we focus on our recent findings that do not support the general notion that basic features of learning and memory systems are much the same among insects.
Roles of octopamine neurons and dopamine neurons in conveying reward and punishment signals
These findings in crickets are consistent with those in honeybees. In bees, it has been reported that OA neurons play roles in appetitive olfactory conditioning with sucrose reward [20,21], whereas DA neurons play roles in aversive olfactory conditioning with electric shock .
In fruit flies, some early neurogenetic studies suggested that OA and DA neurons convey sucrose reward and electric shock punishment, respectively, in olfactory conditioning [15,22], but recent extended studies have revealed that different subsets of DA neurons projecting to the mushroom body convey reward or punishment signals in olfactory learning [23-27]. The mushroom body is a higher-order olfactory and multisensory center in the insect brain that is implicated in olfactory and other forms of learning [1,2,4,28,29,30]. In fruit flies, OA neurons have been shown to act upstream of DA neurons and send sweet taste signal to DA neurons in appetitive learning [26,27]. The critical difference between fruit flies and crickets, therefore, is that DA neurons play critical roles in appetitive learning in fruit flies, but not in crickets. In fruit flies, it has been shown that appetitive reinforcement by DA neurons is mediated by Type 1 dopamine receptor (DopR1).
It can be argued that the observed difference in the roles of DA neurons in appetitive learning may be attributed to the difference of appetitive US used in the experiments; namely, water was reward in crickets whereas sucrose reward was used in fruit flies. This argument, however, does not match the finding in honeybees that OA neurons mediate sucrose reward in appetitive learning [20,21].
Roles of octopamine neurons and dopamine neurons in appetitive and aversive memory retrieval
This is in accordance with finding in honeybees that disruption of OA-ergic transmission in the antennal lobe, the primary olfactory center, by an OA receptor antagonist (mianserin) or by RNAi of the OA receptor gene disrupted appetitive olfactory memory retrieval . The possible roles of DA neurons in aversive memory retrieval, however, have not been tested in honeybees.
In fruit flies, DA neurons participate in formation, but not retrieval, of electric shock-induced aversive memory . In addition, it has been concluded that a subset of DA neurons projecting to the mushroom body (called PAM neurons) participates in formation, but not retrieval, of sugar-induced appetitive memory . Thus, it appears that OA and DA neurons participate in memory retrieve in crickets but not in fruit flies. We discuss the implications of these findings in the next section.
Models of classical conditioning in insects
We have proposed a new model (Figure 5B), with minimal modifications to the previous one by Schwaerzel et al. . In our model, we assumed that (1) co-activation of “OA/DA” neurons and “CS” neurons are needed to activate “CR” neurons after conditioning (AND gate) and (2) synaptic connection from “CS” neurons to “OA/DA” neurons representing US (“CS–OA/DA” synapse) is strengthened by coincident activation of “CS” neurons and “OA/DA” neurons by pairing of a CS with a US. Strengthening of this synapse allows activation of “OA/DA” neurons by CS presentation and by subsequent activation of “CS” neurons, which then allows activation of “CR” neurons. In short, our model assumes enhancement of two synapses, “CS-CR” synapses and “CS-OA/DA” synapses, by conditioning; namely, it assumes formation of multiple memory traces [12,31]. It also assumes Kandelian synaptic plasticity for “CS–CR” synapses and Hebbian plasticity for “CS–OA/DA” synapses.
We also showed participation of OA and DA neurons in appetitive and aversive forms of second-order conditioning  and sensory preconditioning , and showed that these high-order learning phenomena can be accounted for by our new models with some modification of the above model.
Roles of cyclic AMP signaling in formation of short-term memory
In fruit flies, cyclic AMP (cAMP) signaling plays critical roles in formation of olfactory short-term memory (STM), a memory phase that lasts a few minutes after conditioning and is sensitive to amnestic treatment . For example, rutabaga mutants, with defects in adenylyl cyclase, an enzyme producing cAMP, and dunce mutants, with defects in phosphodiesterase (PDE), which degrades cAMP, both exhibit deficiency in STM. In crickets, on the other hand, pharmacological intervention of cAMP signaling by an inhibitor of adenylyl cyclase or cAMP-dependent protein kinase (PKA) impairs formation of LTM but neither STM nor MTM . A recent study in honeybees also showed that inhibitors of adenylyl cyclase do not block STM and MTM , indicating that biochemical processes underlying STM in crickets and honeybees differ from those in fruit flies.
It has been shown that rutabaga mutants exhibit a low but significant level of olfactory STM, and thus fruit flies possess a cAMP-independent component of STM . Whether this minor component of STM in flies is based on biochemical processes similar to those underlying STM in crickets and honeybees remains a subject for future studies.
Roles of nitric oxide-cyclic GMP signaling in formation of long-term memory
We also demonstrated that RNAi of the NOS gene impairs olfactory LTM formation in crickets . In situ hybridization demonstrated a high level of NOS mRNA expression in outer Kenyon cells of the mushroom body, in addition to some neurons around the antennal lobe and the base of the optic lobe. We thus assume that olfactory LTM is formed in the mushroom body, by interaction of outer and inner Kenyon cells.
We also demonstrated participation of NO signaling in LTM formation in cockroaches. Cockroaches exhibit an increased level of salivation in response to an odor paired with sucrose reward , which can be monitored by changes in responses of salivary neurons to odors [40,41]. Injection of an NOS inhibitor impairs formation of LTM, but not that of STM or MTM, in olfactory conditioning of activities of salivary neurons .
Studies in honeybees also suggested that the NO-cGMP signaling pathway and cAMP pathway in the antennal lobe act in parallel and are complementary for the formation of LTM [43,44]. This is in contrast to our conclusion that the NO-cGMP system and the cAMP system are serially arranged for LTM formation in crickets . Thus, the manner by which NO signaling contributes to LTM formation may not be identical between crickets and honeybees. Participation of the CNG channel, calcium-calmodulin and CaMKII in LTM formation has also been demonstrated in honeybees .
Interestingly, so far no report has suggested the involvement of NO-cGMP signaling, or calcium-calmodulin signaling in biochemical cascades underlying LTM formation in fruit flies, although there have been extensive studies on biochemical cascades underlying LTM formation . Biochemical cascades for LTM formation in fruit flies may differ from those in crickets and honeybees.
Comparison of neural processing underlying learning in three insect species
Crickets ( Gryllus bimaculatus )
Fruit flies ( Drosophila melanogaster )
Honeybees ( Apis mellifera ) (A or B?)
A: OA and DA neurons participate in appetitive and aversive memory retrieval, respectively .
B: cAMP signaling participates in STM formation .
B: There is no report on participation of NO-cGMP signaling in LTM formation.
We conclude that there are unexpected diversities in basic mechanisms of learning and memory among different insect species, especially between crickets and fruit-flies. We recently found that the discrepancy, or error, between the actual US and the predicted US determines whether learning occurs in crickets , indicating that the prediction error theory is applicable to crickets. It is of great significance to study whether this finding is applicable to other species of insects. Insects are one of the most successful animals group in terms of species richness and diversity of life styles [46,47]. Elucidation of diversity of learning and memory mechanisms in insects in relation to functional adaptation to the environments and evolutionary history should emerge as a fascinating future subject.
Cyclic adenosine monophosphate
Cyclic guanosine monophosphate
Type 1 dopamine receptor
Cyclic AMP-dependent protein kinase
- CNG channel:
Cyclic nucleotide-gated channel
Calcium/calmodulin-dependent kinase II
Soluble guanylyl cyclase
Cyclic AMP-responsive element-binding protein
This study was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan to MM (Grant # 21370028, 24657049, 24370030).
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