Download Tracing Brain Pathways: Mapping the Neurons

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.
4th Ventricle
Figure 2: Labeled neurons of rat brain illuminating
RFP and GFP under fluorescent light microscopy
Injected with PRV 152
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
Figure 3: Labeled neurons of rat brain illuminating
GFP under fluorescent light microscopy
Injected with PRV 614
Predorsal Bundle
Figure 1: Interconnecting neurons illuminating
RFP under fluorescent light microscopy
Figure 4: Labeled neurons illuminating RFP under
fluorescent light microscopy
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