What Can Drosophila Tell Us About the Engram?

The search for memory traces or the engram (Semon, 1904) in the brain is one of the key aspects in neurosciences investigating learning and memory. The engram is located to the brain in which specific neurons integrate external cues that cause plastic changes in these neurons to form a memory. External or internal triggers can retrieve this memory, eliciting behavioral outputs for which these neurons are sufficient and required (Gerber et al., 2004; Josselyn et al., 2015; Semon, 1904). By visualizing neuronal plasticity using calcium imaging, several engrams could be found in Drosophila residing mainly in the MB circuit (Davis, 2011). MB intrinsic KCs exhibit several forms of memory traces that depend on the KC type. STM traces were found in the γ-lobe (Boto et al., 2014) and the α'/β'-lobe (Wang et al., 2008), both showing increased calcium responses for the CS+ condition. LTM traces were found in the γ-lobe (Akalal et al., 2010, 2011; Bouzaiane et al., 2015) and the α-lobe (Yu et al., 2006) showing increased calcium responses to the CS+

presentation as well. However, memory traces are not only stored in KCs. They can be found in MB extrinsic neurons such as MBONs (Hige et al., 2015a; Owald et al., 2015; Pai et al., 2013;

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Perisse et al., 2016; Séjourné et al., 2011), the APL neuron (Liu and Davis, 2009), the DPM neuron (Yu et al., 2005), TH-GAL4 positive DANs (Riemensperger et al., 2005), and, interestingly, already at the level of PNs (Ashraf et al., 2006; Yu et al., 2004). Here I demonstrate another so far never shown form of calcium transient-dependent plasticity that is not explained by mere response intensities but the synchrony of single synaptic sites (boutons). As the above mentioned studies lack single cell and single synapse resolution, they might have missed memory traces. Even though insects are somewhat different from vertebrates, they share many common principles and anatomical similarities involved in learning and memory.

In vertebrates the cerebellum was shown to be the main brain structure involved in classical conditioning of reflexes (e.g., eye-blink conditioning; see review: Thompson and Steinmetz, 2009), showing remarkable similarities to the insect MB circuit (see e.g., Schürmann, 1974; Yasuyama et al., 2002). In brief: glutamatergic mossy fibers convey the CS information (conforming the PN input to MBs) to granule cells with its parallel fibers (conforming MB intrinsic KCs). Glutamatergic climbing fibers convey the US information (resembling dopaminergic PPL1/PAM input to the MB) to the cortical Purkinje cells (can be regarded as the MBONs) that are also connected to granule cells. Purkinje cells, as well as mossy and climbing fibers, project onto deep cerebellar nuclei (e.g., Interpositus) that are required for the CR expression (Schürmann, 1974; Thompson and Steinmetz, 2009; Yasuyama et al., 2002). Two main memory traces were found in the cerebellar circuit: one in the Interpositus nucleus showing increased responses to the CS presentation predicting the US and another one in Purkinje cells showing synaptic depression and facilitation.

Both memory traces were found to work somehow independently of each other though the role of the Purkinje cell layer-related memory trace supposedly has more a modulatory role, acting on the Interpositus-related memory trace (see review: Thompson and Steinmetz, 2009).

Besides the cerebellum, other brain areas in vertebrates show memory traces in the course of classical conditioning, e.g., the amygdala in fear conditioning (see review: Maren, 2001). Here, the basolateral complex (BLA) was shown to be required for the acquisition and storage of fear conditioning, whereas the central amygdaloid nucleus is needed for the generation of conditioned fear responses (CR). Visual, auditory, as well as shock stimuli enter the amygdala through thalamic and cortical tracts conveying both the CS and the US. The coincidence of both alters synaptic plasticity in the form of increased responses in neurons of the BLA which show a short term component and a protein synthesis dependent long term potentiation that also involves voltage-gated calcium channels (Maren, 2001). Together with the amygdala, the hippocampus is involved in fear conditioning. However, the hippocampus exhibits a more contextual conditioning component that is e.g., the behavioral set up in which the animal is trained. This means, that the placement of the animal into this set up can elicit the CR that is conditioned through the actual

93 CS-US pairing (Holland and Bouton, 1999; Maren, 2001). The hippocampus is furthermore a key structure for place learning in which the so-called place cells play the major role and might be related to contextual learning as place cells can store information about location in a certain context (see review: Moser et al., 2015).

The importance of the dopaminergic system in classical conditioning in Drosophila – relaying aversive and appetitive valence – was already described previously (see also: Kaun and Rothenfluh, 2017). The tiling of the Drosophila brain by DAN clusters is a conserved feature that also exists in vertebrates and mainly involves the basal ganglia relaying punishment and reward in learning paradigms (Scaplen and Kaun, 2016). Even though insect (e.g., Drosophila) brain structures resemble those of vertebrates, the insect brain doesn’t have this elaborated distribution of forebrain centers involved in different learning and memory tasks. In the insect brain, the MBs are the key structures that serve the many functions involved in learning and memory. This aspect of simplicity and the physiological similarities makes the investigation of learning and memory principles in Drosophila a suitable approach.

In this study, only γ-KC activity was measured. To investigate what happens on the complete circuit level is a very challenging task as the MB circuit was recently shown to be enormously complex (Eichler et al., 2017; Takemura et al., 2017) and remains to be elucidated. However, this study contributes an important aspect into the field of synaptic plasticity in MB intrinsic KCs that was not shown so far. First, it shows that synaptic plasticity in the MB of Drosophila resides in the pre synapse, where the memory trace is embodied in form of the change in synaptic weights.

Second, giving the findings that single spines in Purkinje cells of the vertebrate cerebellum can be activated by single parallel fibers of granule cells (Denk et al., 1995) and that localized, clustered as well as dispersed calcium influx is integrated over time and space in dendrites (Stuart and Spruston, 2015) indicates that an uncorrelated parallel fiber input reduces the response of the Purkinje cell. As parallel fibers resemble the MB KCs and Purkinje cells the MBONs, this mechanism of synchronized or de-synchronized plasticity is likely a shared mechanism. This kind of synaptic plasticity likely influences learning and memory performance in vertebrates and is now shown for the first time in the invertebrate MB. It furthermore fulfills one major criterion of defining a memory trace: synaptic plasticity within the neuronal substrate (Gerber et al., 2004;

Martin et al., 2000; Thompson, 2005). Future experiments have to further elucidate the exact circuit mechanism for this kind of synaptic plasticity.

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