Tracing Brain Pathways: Mapping the Neurons that Make the Eyes Work Benedict Ifedi and Christine Kiruthu

James W. Gnadt, PhD (Principal Investigator)

Chuan-Jiang Yu, PhD (Senior Research Fellow) Physiology & Biophysics and Specialized Neuroscience Research Program

Howard University College of Medicine, Washington, DC

Abstract By the process of retrograde, trans-neuronal tracing with a genetically engineered pseudorabies virus (PRV), our goal is to construct a detailed map outlining the extensive network of nerves that function to make the eyes work. PRV is one of only a few tracers that will cross synapses in the “backwards” direction, allowing us to inject the agent in the peripheral muscles and follow the sequence of neural connections back into the brain. One variation of the PRV used to track the location of neurons expresses green fluorescent protein (GFP), while the other expresses red fluorescent protein (RFP); both serve as markers used to identify the location of neurons related to eye functionality. Tracing the neuronal pathways linked to oculomotor function will allow us to develop a better understanding for the basis of the behavior exhibited by the activity and movement of the eyes.

Introduction PRV is injected into the peripheral muscles of the rodent eye and passed back neuron by neuron, an effective trans-neuronal tracing technique. The highly selective PRV is taken up by neurons responsible for the function and activity of the eyes, specifically omnipause neurons (OPNs) and excitatory burst neurons (EPNs). By revealing specific, targeted sets of neurons labeled by GFP (green fluorescent protein) and/or RFP (red fluorescent protein), those neurons’ electrical properties can be examined in a brain slice preparation. This helps us to understand how neurons communicate with one another by way of electric currents in a controlled way known as in vitro, or “in the dish”.

Methods & Materials Rodent injection and abstraction and processing of brain tissue After the injected PRV took its effect in the rodents, the rodents underwent perfusion where upon completion of this procedure, the brain was removed and placed into 4% paraformaldehyde for approimately 24 hours. Fixation The brain was then processed with 0.1 M Phosphate Buffer 3x for 10min and 3x for 30min. It was immersed in 15% Sucrose solution (for 2-3 days) and then into 30% sucrose solution prior to the sectioning and mounting of the brain sections. Sectioning The brain was cut using a microtome. It was placed onto a microtome stage that was frozen with dry ice. When firmly in place, frozen sections were cut into sections 40µm thick from rostal to caudal. The sections were stored in cryo-protected buffer. Visualization Every sixth tissue section was mounted on a subbed slide out of 0.1M phosphate buffer and was left to dry. We used glycerol to put the cover slip on the slide and was ready for microscopy with bright field and fluorescent microscope. Nissl Stain Selected mounted sections were stained using cresyl violet, a Nissl stain.

Conclusions 1.  Neurons responsible for oculomotor function

and activity can be successfully labeled and indentified via the injection of PRV and the utilization of a high powered microscope which can image fluorescent light, respectively.

2.  In the majority of cases where rodents were injected with PRV, the targeted neurons expressed RFP, while very few cases exhibited neurons expressing GFP. This implies that the PRV 614 strain (red) is more effective than PRV 152 (green) in expressing itself in neurons, which in turn allows us to better construct a map detailing the brain’s neural circuitry in relation to eye function.

Future Projections Future goals of this project include, but are not

limited to: 1. Recording and analyzing membrane properties

of the neurons in brain slices from rodents in order to determine the cellular properties of specific neurons within eye movement circuits

2.  Recording the electrical activity from neurons as they communicate through electric currents.

3.  Determine possible correlation(s) between the cellular properties of the specifically identified neurons and the physiological behaviors/actions exhibited by the eyes

References 1. Graf, Werner, Nicolaas Gerrits, Najiya Yatim-Dhiba,

and Gabriella Ugolini. "Mapping the oculomotor system: the power of." European Journal of Neuroscience. 15 (2002): 1557-562.

2.  Gnadt, James W. "Cellular Properties of Neuron Types in the Saccade Circuit." The Laboratory of James W. Gnadt, PhD. 16 Nov. 2006. 24 July 2008 <http://www9.georgetown.edu///faculty/jwg37/labwebsite/project_08.htm>.

Acknowledgements Amgen Foundation

James W. Gnadt, PhD Chuan-Jiang Yu, PhD

Stephanie A. Johnson, Esq. Eva K. Polston, PhD

Dexter Lee, PhD Justin Wilson Figure 1: Interconnecting neurons illuminating

RFP under fluorescent light microscopy

Figure 2: Labeled neurons of rat brain illuminating RFP and GFP under fluorescent light microscopy

Figure 4: Labeled neurons illuminating RFP under fluorescent light microscopy

Figure 3: Labeled neurons of rat brain illuminating GFP under fluorescent light microscopy

4th Ventricle

Injected with PRV 152

Injected with PRV 614

Predorsal Bundle