Research

Determinants of Local Circuit Organization

Current Research Program

OphthalmologyA major goal of my laboratory is understanding the basic mechanisms that contribute to the establishment of orderly topographic maps within the CNS, during both normal development and during regenerative events following injury or disease. We have been focusing our efforts on the olfactory system, in part because of the complexity of the map between the olfactory epithelium and the olfactory bulb.

Axons sort into 1,000 functional subsets that are targeted with high specificity to olfactory bulb glomeruli. Using both in vivo and in vitro models, we are currently isolating several mechanisms that may contribute to this complex reorganization including laminin, tenascin and the expression of putative odor receptors in growth cones. In related studies we continue to characterize a unique population of glial cells, ensheathing cells, found in the olfactory nerve. While elsewhere in the mature CNS glia are an impediment to axon growth, the ensheathing cell glia support axon extension and targeting throughout life.

We recently demonstrated that the growth promoting effects of ensheathing cells are not limited to olfactory receptor neurons but are also seen in other populations of neurons. Particularly exciting, our recent studies demonstrate that the ensheathing cells remain pluri-potential and that when implanted into demyelinated spinal cord can adopt a myelinating phenotype which remyelinates the axons and contributes to a restoration of normal conduction velocities.

In parallel studies we are examining the molecular and synaptic organization of the olfactory bulb glomeruli. Using RT-PCR we are mapping the distribution of subsets of olfactory receptor cell axons in glomeruli to gain insights into the topography of odor-ligand maps in the olfactory bulb. In addition, working with colleagues, we are using a GFP tag to test hypotheses regarding the specificity of synaptic organization within glomeruli. Second, using antibodies synaptic vesicle related proteins and confocal microscopy we have begun to describe a hitherto unrecognized segregation of local and projection synaptic circuits in the glomeruli. It may be that this segregation underlies the lateral inhibitory systems that are believed to be operative in the olfactory system. Beyond my colleagues in Neurosurgery, I maintain active collaborations with the following Departments at Yale: Neurology, Neurobiology, Anesthesiology and Ophthalmology. In addition, I have collaborative studies with members of the faculty at Columbia University, Emory University, The Rockefeller, University of Maryland and University of Colorado.

Relationship of Research to Neurological Disease

Increasingly, the neurological sciences are focusing on intervention strategies that will both limit the debilitating consequences of injury/disease as well as increase the probability of successful regeneration of pathways and connections in the CNS. In order to facilitate these processes it is necessary for us to understand the molecular and cellular events operative during axon extension, target selection and synapse formation. The studies described above directly assess those questions and, particularly in the case of the ensheathing cells, offer the possibility of clinical application in the near future. Dr. Greer can be reached at 203-785-4034 or email to charles.greer@yale.edu.

Axon Targeting

In the mouse, olfactory sensory neurons express only 1 of ~1,200 potential odor receptors. Sensory neurons expressing any one receptor are stochastically distributed in the epithelium. However, their axons later sort into 1,200 molecularly defined subsets that target ~3,600 glomeruli in olfactory bulb with high specificity. Using both in vivo and in vitro models, an important goal of the lab is understanding the mechanisms that underlie the development and plasticity of these molecularly-defined maps in the olfactory bulb. Using conditional expression of GFP under Ascl-1 and serial block-face electron microscopy, we are probing the timeline of axon extension from the sensory neurons and their reorganization into glomerular-specific fascicles on the surface of the olfactory bulb (in collaboration with the Alain Trembleau lab at the Université Pierre et Marie Curie in Paris).

Olfactory Bulb Synaptic Circuits

There are two fundamental levels of synaptic processing in the olfactory bub: the glomeruli where primary afferent input is received and modulated by small populations of inhibitory interneurons; and 2) the external plexiform layer, where inhibitory granule cells form reciprocal dendrodendritic synapses with output neurons, mitral and tufted cells. Our prior work characterized the genesis and migration of olfactory bulb neurons and the first appearance of synaptic circuits beginning in the embryo. Currently we are using birthdating strategies to dissect the role of neurogenesis timing for the organization and topography of olfactory bulb granule cells. We are conducting this analysis both perinatally and in the adult, where ongoing neurogenesis introduces ~10,000 new granule cells to the olfactory bulb daily. We are further exploring the expression of the synaptic vesicle proteins at both primary excitatory and the reciprocal dendrodendritic synapses in olfactory bulb circuits. Our data thus far are suggesting a heterogenous expression in subsets of neurons and excitatory vs. inhibitory synapses that may regulate the dynamics of these circuits.

Adult Neurogenesis in the Olfactory System

A unique feature of the olfactory system is the ongoing genesis of neuroblasts in the subventricular zone that migrate via the rostral migratory stream, where they differentiate into granule cells and incorporate into local synaptic circuits. We previously established the timeline for this process, but the mechanisms by which neuroblasts migrate to the bulb prior to the formation of the adult cellular architecture of the rostral migratory stream is enigmatic. Microglia are well known for their developmental role in shaping the architecture of synapses with the removal of supernumerary contacts and otherwise “cleaning up” the CNS. We recently found that the distribution of microglia in the developing rostral migratory stream is unique for the central nervous system. Given the unexpected organization of microglia in association with the migratory stream we are using in utero electroporation to label neuroblasts to determine if the loss of microglia affects their migration or otherwise the organization of the migratory stream.

Development of Cortical Olfactory Bulb Targets

The projection neurons in the olfactory bulb target two primary central locations: 1) piriform cortex; and 2) the olfactory tubercle. While we have recently gained new insights into the development of the olfactory bulb and its circuits, little has been known about neurogenesis, neuroblast migration or the specificity of laminar organization in piriform cortex or the tubercle. Using in utero electroporation to label progenitor cells, we are examining the timing neurogenesis, the migratory routes of the neuroblasts and the fate of neurons targeted to the piriform cortex and tubercle. Using CRISPR technology we will next explore the mechanisms underlying those processes.