Anatomical and functional analysis of non-retinal tectal afferents in larval zebrafish

Abstract

Processes including metabolism, movement, language, learning, and cognition are all delivered through the complex functioning of the nervous system. In the human brain, these processes are produced by an estimated 85 billion neurons (Azevedo et al., 2009), which in turn connect to each other through an immeasurable number of neural synapses. Studies describing the functional properties of the nervous system can be found dating back to ancient Egypt, where the effects of lobotomies as treatment for neurological ailments were described (Cave, 1950; Nevin et al., 2010; Loukas et al., 2011; Fanous and Couldwell, 2012). A more modern understanding of how the anatomy of the nervous system underlies behaviour began to develop in the early 19th century, when Marie-Jean-Pierre Flourens used vertebrate models to understand how the removal of brain regions affected behaviour (Flourens, 1842; Yildirim and Sarikcioglu, 2007). Since these first descriptions were carried out, numerous other studies describing how the anatomy and function of neural populations underlie behaviours have been performed in humans, however ethical and physical limitations associated with these experiments have meant that research has turned to non-human models for describing neural function. Although mammalian models have been favoured historically, they have properties that limit the incisiveness of studies, especially at the circuit level, that can be performed. Larval zebrafish, which possess a simplified nervous system and other favourable experimental properties, offer an appealing alternative for the study of the nervous system. In particular, recent advances in microscopy and optical physiology allow for simultaneous observation and manipulation of neural circuits in vivo in larval zebrafish. This, paired with modern labelling and visualization techniques for the anatomy of neural circuits, has resulted in a plethora of studies directly linking the anatomy of a circuit to both its activity and function. Because of their small size, relatively conserved neuroanatomy and transparency at larval stages, zebrafish are an ideal candidate for describing both the anatomy and function of neural circuits using optical physiology. My aim, in this thesis, is to apply these techniques to describe the anatomy and function of neural circuits formed within the teleost equivalent of the superior colliculus, the optic tectum. Historically, the teleost tectum has been viewed as a visual structure, and models of its function have focused almost exclusively on retinal inputs. In contrast, its mammalian counterpart, the superior colliculus, integrates inputs from a multitude of brain regions relaying information from multiple sensory modalities. In this thesis, I use both anatomical and functional approaches to describe projections from the thalamus, the hypothalamus, and the cerebellum into the tectum of larval zebrafish. My finding that the larval zebrafish tectum receives diverse inputs suggests that it is a more complete counterpart to the mammalian superior colliculus than has previously been recognized. Specifically, this thesis shows that the thalamus, hypothalamus, and cerebellum target specific regions of the tectal neuropil; the thalamus projects to the entirety of the neuropil, whereas the hypothalamus and cerebellum specifically target the deepest laminae. Notably, I show that the thalamus and hypothalamus target non-retinorecipient laminae of the neuropil, suggesting that the information they carry is not necessarily visual. I have also used optogenetic techniques to show that the tectal afferent information is received by tectal periventricular neurons. Through a careful analysis, I show that the thalamus sends a combination of excitatory and inhibitory signals, whereas the cerebellum sends purely excitatory, and the hypothalamus sends purely inhibitory signals. The filtering of visual information in the larval zebrafish has traditionally been attributed to circuits within the retina and tectum, with outputs to motor areas of the brain that drive behaviour. To explore whether projections from other brain regions influence tectal activity and visual behaviour, I undertook a study describing how thalamic projections to the tectum affect its processing of visual stimuli. This study has resulted in two major findings. For the first time it proves that the thalamus of larval zebrafish responds to visual cues. I also show, through ablation of the thalamic projections to the tectum, that thalamic input to the tectum is necessary for tectal responses to threatening visual stimuli, and to the larva’s eventual adaptive escape response.