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

Abstract

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.

The complexity of learning, memory and neural processes in an evolutionary ecological context

“…Until recently, signaling of aversive and appetitive reinforcement in insects was thought to be mediated in a dichotomous manner by dopaminergic and octopaminergic neurons respectively. This view was and is still supported by studies in crickets [57,58], in the Honeybee [59][60][61] and initially also in Drosophila [62]. However, in Drosophila this view changed as octopamine signaling is only involved in appetitive learning, leading to short lasting memory formation, whereas dopamine signaling is involved in learning leading to long lasting appetitive memory [14,63,64].…”

Searching for cognitive processes underlying insect learning

“…In order to resolve the issue discussed above, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to produce crickets with knockout of the Dop1 gene (Awata, Watanabe, Hamanaka, et al, 2015). We found that Dop1 knockout crickets exhibited impairment in aversive learning with sodium chloride but exhibited no impairment in appetitive learning with water or sucrose.…”

“…Secondly, knowledge has been accumulated on the brain and behavior since they have been used as materials for the study of neural basis of behavior. Thirdly, pharmacological approaches (Unoki, Matsumoto, & Mizunami, 2005, 2006Matsumoto, Unoki, Aonuma, & Mizunami 2006,;Matsumoto, Hirashima, Terao, & Mizunami, 2013), gene knockdown by RNA interference (Takahashi, Matsumoto, & Mizunami, 2009;Awata, Wakuda, Ishimaru, Matsuoka, Terao, Katata, Matsumoto, Hamanaka, Noji, Mito, & Mizunami, 2016), and genome editing by CRISPR/cas9 system (Awata, Watanabe, Hamanaka, Mito, Noji, & Mizunami, 2015) have been well established in crickets, which allows detailed analysis of molecular basis of learning and memory. Finally, as we will discuss in this article, conditioning with different conditioned stimuli (CSs) (odor, visual pattern and color) and different unconditioned stimuli (USs) (water, sucrose and sodium chloride) can be easily performed in a very similar experimental setting, which greatly facilitates studies on stimulus parameters to achieve various forms of conditioning and its underlying neurotransmitter mechanisms.…”

Remembering Franz Huber (November 20, 1925–April 27, 2017), a pioneer of insect neuroethology

“…In his laudatio on the occasion of Franz Huber's 90th birthday, Berthold Hedwig (Hedwig 2016) pointed to more recent advances in insect neurobiology and acoustic communication, to a large extent made possible by the availability of refined and new techniques. In the meanwhile 1 3 the first knock-out crickets have been created for the study of learning and memory (Awata et al 2015). However, "Neuroethology and Behavioral Physiology -Roots and Growing Points" (Huber and Markl 1983), edited more than 30 years ago and with a thoughtful epilogue by Ted Bullock, still is a must-read for someone interested in key questions of neuroethology.…”