Abstract
Studies of vertebrate brain evolution have mainly focused on measures of brain size, particularly relative mass and its allometric scaling across lineages, commonly with the goal of identifying the substrates that underly differences in cognition. However, recent studies on birds and mammals have demonstrated that brain size is an imperfect proxy for neuronal parameters that underly function, such as the number of neurons that make up a given brain region. Here we present estimates of neuron numbers and density in two species of lizard, Anolis cristatellus and A. evermanni, representing the first such data from squamate species, and explore its implications for differences in cognitive performance and vertebrate brain evolution. The isotropic fractionator protocol outlined in this article is optimized for the unique challenges that arise when using this technique with lineages having nucleated erythrocytes and relatively small brains. The number and density of neurons and other cells we find in Anolis for the telencephalon, cerebellum, and the rest of the brain (ROB) follow similar patterns as published data from other vertebrate species. Anolis cristatellus and A. evermanni exhibited differences in their performance in a motor task frequently used to evaluate behavioral flexibility, which was not mirrored by differences in the number, density, or proportion of neurons in either the cerebellum, telencephalon, or ROB. However, the brain of A. evermanni had a significantly higher number of nonneurons and a higher nonneuron to neuron ratio across the whole brain, which could contribute to the observed differences in problem solving between A. cristatellus and A. evermanni. Although limited to two species, our findings suggest that neuron number and density in lizard brains scale similarly to endothermic vertebrates in contrast to the differences observed in brain to body mass relationships. Data from a wider range of species are necessary before we can fully understand vertebrate brain evolution at the neuronal level.
Isotropic Fractionator: A Simple, Rapid Method for the Quantification of Total Cell and Neuron Numbers in the Brain
