Analysis of Functional Circuitry and the Effect of Activity Across Development in the Mouse Accessory Olfactory System

The vomeronasal system (VNS) is a chemosensory system designed for the detection of chemical messengers dissolved in bodily secretions which help to identify kin, conspecifics, and predators in the animal‟s environment. Although a growing body of evidence over the last two decades has revealed a very important role for the VNS in many stereotypical behaviors which are essential to the animal‟s survival, our understanding of the detailed organization of the circuitry of this system, and its development, remains limited. Consequently, the goal of this dissertation is to examine the development of connectivity from the vomeronasal organ to the accessory olfactory bulb to determine the fundamental principles of adult connectivity in this circuit, and to manipulate sensory activity to understand the role that activity might play in anatomical development of vomeronasal projections to their targets in the accessory olfactory bulb.

There are several anatomical challenges we faced when trying to visualize connectivity in a select population of neurons in the VNS. We utilized two tools to overcome some of these barriers. First, we took advantage of a transgenic mouse line whose vomeronasal sensory neurons (VSNs) expressing the V2r1b-receptor also expressed tau-GFP to visualize a specific population of sensory neurons in the vomeronasal organ (VNO) and their projections in to the accessory olfactory bulb (AOB). Second we developed a local electroporation technique enabling us to label specific populations of mitral cells in the AOB receiving common input. This method is described in detail in Chapter 2 where we show the versatility of this method, not only for use in the AOB, but in many other brain systems and with a diverse set of dyes and calcium indicators.

We exploited these two tools to examine both the gross anatomical development of the VNO and the AOB, as well as the development of connectivity between them in Chapter 3. Our results show that the first few post-natal weeks represent a dramatic period of growth in both the
VNO and the AOB. In addition, we show that axons of VSNs expressing the V2r1b-receptor undergo a striking and rapid period of refinement and coalescence during the first four post-natal days of the animal‟s life. Subsequently, mitral cell dendrites, which initially promiscuously send out multiple dendritic branches, begin to ramify and form tufts in specific glomeruli following this period of axonal refinement into well-defined glomeruli. Finally, our results support the hypothesis that mitral cells precisely project their dendrites to target glomeruli receiving input from sensory neurons expressing the same receptor type.

Finally in Chapter 4, we explored the role of activity in modulating the development described in Chapter 3. We demonstrate that the duct connecting the VNO to the external world is indeed open and thus that external ligands can access the VNO and bind to VSNs, as early as postnatal day 0. Further, we demonstrate that VSNs are capable or releasing neurotransmitter onto mitral cells at these early postnatal ages. These results suggested that early activity in this system might help regulate development. To test this hypothesis we needed a ligand that would activate a known subset of VSNs. We determined the concentration of a major histocompatibility complex peptide known to activate V2r1b-receptors and found it to be more than 1,000 fold above what would likely activate these sensory neurons. We show that these peptides are capable of inducing immediate early genes in downstream mitral cells and then employed the use of two of these peptides to selectively manipulate the activity of sensory neurons expressing the V2r1b-receptor. Although strong sensory activity did not affect the number of VSNs expressing the V2r1b-receptor, it did significantly alter axonal refinement and coalescence in AOB, resulting in delayed pruning and formation of well-defined glomeruli.

Taken together, the results from the chapters in this dissertation suggest that connectivity between mitral cells and sensory neurons in the vomeronasal organ is specifically targeted and that early activity may influence the development of this circuit to ensure proper connectivity providing a substrate for information processing in this system.