| Abstract: | Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. Stäubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.It is widely believed that memories are formed as a result of alterations in synaptic connections between axons and dendrites, an idea first proposed by Ramon y Cajal (1–4). Although synapse changes have been extensively studied in brain slices in the context of long-term potentiation (5, 6), less is known about how synapses in a living vertebrate are modified when a memory is formed.Memory formation has been widely studied using classical conditioning (CC), a robust and straightforward form of learning in which an animal is exposed to a neutral stimulus (conditioned stimulus, CS) paired with an appetitive or aversive stimulus (unconditioned stimulus, US) that evokes a specific behavioral response (UR, unconditioned response) (7, 8). As a result of the pairing, animals learn to associate the CS with the US, causing them to respond to the CS with a conditioned response (CR) identical to the UR, signifying memory retrieval (9, 10). Memory retrieval is also evoked by activating a cellular engram, a group of neurons active during memory formation and retrieval (11–18). The central locus of CC in mammals, the amygdala (19), is located in a relatively inaccessible area beneath the cortex (20). Thus, although numerous longitudinal imaging studies have documented experience-dependent changes in the structure of spines of cortical and hippocampal neurons (21, 22), few imaging studies have directly examined synaptic changes that occur in the amygdala during associative memory formation.Instead, synaptic changes that occur in the amygdala during CC (23) have been studied primarily using indirect measures of synaptic strength, such as the ratio of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor/N-methyl D-aspartate (AMPA/NMDA) currents in excitatory postsynaptic currents (EPSCs). Increases in AMPA/NMDA ratio in amygdalar neurons following auditory fear conditioning (FC), a type of CC (24–27), indicate that associative memory formation coincides with increases in synaptic strength. In addition, imaging experiments in brain regions beyond the amygdala have shown diverse effects following CC. For example, following contextual fear conditioning, engram neurons in the CA1 region of the hippocampus that receive inputs from CA3 engram neurons displayed spines that were larger and more densely packed than nonengram cells (28). Furthermore, experiments in which neuronal morphology was directly observed before and after FC found that neurons in the frontal association (29) and primary motor cortex (30) showed a decrease in the number of spines, whereas neurons in the auditory cortex showed an increase in spine number with memory formation (31).To obtain previously unavailable insight into memory formation within the central locus of associative memory storage, we developed a paradigm combining in vivo labeling and imaging with informatics and analysis tools. We used this paradigm to map synaptic changes that occur over time in the intact brain of a living vertebrate during memory formation. We imaged the pallium of teleost fish, which contains the putative homolog of the mammalian amygdala based on anatomy (32), gene expression (33), and function (34). The pallium is on the surface of the brain (35), and zebrafish larvae are highly transparent, allowing for intact, whole-brain imaging using selective plane illumination microscopy (SPIM) without the need for invasive intervention (36). In addition, while most studies of learning in zebrafish have used adults (37–40), at least one study showed that larval zebrafish can learn to associate a place with a positive valence US (41). These attributes suggest that larval zebrafish may be an ideal model organism for studying synaptic changes during memory formation due to CC. We have engaged this challenge by combining purpose-built experimental tools with data management software that enables transparent analyses of large and heterogeneous datasets. All data were characterized and stored at the time of creation in a customized data management system designed to conform to findability, accessibility, interoperability, and reusability (i.e., FAIR principles) (see Materials and Methods) (42). |
