Evolution of neural circuitry and cognition.

Neural circuits govern the interface between the external environment, internal cues and outwardly directed behaviours. To process multiple environmental stimuli and integrate these with internal state requires considerable neural computation. Expansion in neural network size, most readily represented by whole brain size, has historically been linked to behavioural complexity, or the predominance of cognitive behaviours. Yet, it is largely unclear which aspects of circuit variation impact variation in performance. A key question in the field of evolutionary neurobiology is therefore how neural circuits evolve to allow improved behavioural performance or innovation. We discuss this question by first exploring how volumetric changes in brain areas reflect actual neural circuit change. We explore three major axes of neural circuit evolution-replication, restructuring and reconditioning of cells and circuits-and discuss how these could relate to broader phenotypes and behavioural variation. This discussion touches on the relevant uses and limitations of volumetrics, while advocating a more circuit-based view of cognition. We then use this framework to showcase an example from the insect brain, the multi-sensory integration and internal processing that is shared between the mushroom bodies and central complex. We end by identifying future trends in this research area, which promise to advance the field of evolutionary neurobiology.

A modified method to analyse cell proliferation using EdU labelling in large insect brains.

The study of neurogenesis is critical to understanding of the evolution of nervous systems. Within invertebrates, this process has been extensively studied in Drosophila melanogaster, which is the predominant model thanks to the availability of advanced genetic tools. However, insect nervous systems are extremely diverse, and by studying a range of taxa we can gain additional information about how nervous systems and their development evolve. One example of the high diversity of insect nervous system diversity is provided by the mushroom bodies. Mushroom bodies have critical roles in learning and memory and vary dramatically across species in relative size and the type(s) of sensory information they process. Heliconiini butterflies provide a useful snapshot of this diversity within a closely related clade. Within Heliconiini, the genus Heliconius contains species where mushroom bodies are 3-4 times larger than other closely related genera, relative to the rest of the brain. This variation in size is largely explained by increases in the number of Kenyon cells, the intrinsic neurons which form the mushroom body. Hence, variation in mushroom body size is the product of changes in cell proliferation during Kenyon cell neurogenesis. Studying this variation requires adapting labelling techniques for use in less commonly studied organisms, as methods developed for common laboratory insects often do not work. Here, we present a modified protocol for EdU staining to examine neurogenesis in large-brained insects, using Heliconiini butterflies as our primary case, but also demonstrating applicability to cockroaches, another large-brained insect.

An atlas of the developing Tribolium castaneum brain reveals conservation in anatomy and divergence in timing to Drosophila melanogaster.

Insect brains are formed by conserved sets of neural lineages whose fibers form cohesive bundles with characteristic projection patterns. Within the brain neuropil, these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed, for example, in size, shape, and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood, although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, we present an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage, and the adult, by combining wholemount immunohistochemical labeling of fiber bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in Drosophila melanogaster, we confirm an overall high degree of conservation. Fiber tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like antiacetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed us to describe differences between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.

Sequence heterochrony led to a gain of functionality in an immature stage of the central complex: A fly-beetle insight.

Animal behavior is guided by the brain. Therefore, adaptations of brain structure and function are essential for animal survival, and each species differs in such adaptations. The brain of one individual may even differ between life stages, for instance, as adaptation to the divergent needs of larval and adult life of holometabolous insects. All such differences emerge during development, but the cellular mechanisms behind the diversification of brains between taxa and life stages remain enigmatic. In this study, we investigated holometabolous insects in which larvae differ dramatically from the adult in both behavior and morphology. As a consequence, the central complex, mainly responsible for spatial orientation, is conserved between species at the adult stage but differs between larvae and adults of one species as well as between larvae of different taxa. We used genome editing and established transgenic lines to visualize cells expressing the conserved transcription factor retinal homeobox, thereby marking homologous genetic neural lineages in both the fly Drosophila melanogaster and the beetle Tribolium castaneum. This approach allowed us for the first time to compare the development of homologous neural cells between taxa from embryo to the adult. We found complex heterochronic changes including shifts of developmental events between embryonic and pupal stages. Further, we provide, to our knowledge, the first example of sequence heterochrony in brain development, where certain developmental steps changed their position within the ontogenetic progression. We show that through this sequence heterochrony, an immature developmental stage of the central complex gains functionality in Tribolium larvae.

The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development.

With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.

Immunohistochemistry and Fluorescent Whole Mount RNA In Situ Hybridization in Larval and Adult Brains of Tribolium.

Arthropod brains are fascinating structures that exhibit great complexity but also contain conserved elements that can be recognized between species. There is a long tradition of research in insect neuroanatomy, cell biology, and in studying the genetics of insect brain development. Recently, the beetle Tribolium castaneum has gained attention as a model for insect head and brain development, and many anterior patterning genes have so far been characterized in beetle embryos. The outcome of embryonic anterior development is the larval and, subsequently, the adult brain. A basic requirement to understand genetic cell type diversity within these structures is the ability to localize mRNA and protein of neural genes. Here we detail our protocols for RNA in situ hybridization in combination with immunohistochemistry, optimized for dissected brains of larval and adult beetles.