The Use of Pseudorabies Virus to Define
Neural Circuitry in Rat Brain
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Durba Mitra |
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The mammalian brain contains networks of neural circuits that specialize in functions ranging from autonomic control to conscious thought. Within each part of the brain, there are neuron modules that perform these specific tasks. The anatomy of these important neuron connections is not fully determined, creating an essential opportunity in neuroanatomy. The definition of these pathways will provide the basis for determining the physiology, and therefore, function of specific regions of the brain. With this basic knowledge of location and circuitry, neurologists have the ability to identify and understand the complexity of the brain. The rat brain serves as the prototype for the mammalian brain. By defining pathways within the rat brain, researchers will create a model for further research on the mammalian brain.
Different techniques are used to identify the neural pathways; however, the use of viral tracers has proved to be one of the most effective tracking devices in rats. The virus is micro-injected into a specific site in the rat and is taken up by axonal terminals, or synapses, of the neuron. The virus then travels against the gradient of the axon, or retrogradely, eventually establishing a viral "factory" within the nucleus. As the virus is released from the initial (first order) neuron, it is taken up by surrounding across synapses to label a neural circuit. The virus is very effective because researchers are able to track products of the virus, including the protein coat. Viruses also act as a self-amplifying marker, replicating within the nucleus, and are transported through a specific neural circuit.
In Dr. AD Loewy's lab, viruses are used as neural tracers to identify nerve innervating the cerebral cortex and hypothalamus. The Pseudorabies Virus (PRV) most effectively demonstrated the tracing method of circuits, wit ha weak version of PRV actively traveling within sympathetic pathways of rats. The locations of the virus are detected by immunohistochemistry, the use of antibodies and fluorescent dyes. The virus and other neuron substances, such as neuropeptides and proteins can be simultaneously labeled.
During my six weeks in Dr. AD Loewy's lab, I had the opportunity for a hands-on learning experience. My participation ranged from rat surgery to immunohistochemical procedures to the mapping of virally-labeled neurons. In my experiment, I dealt mainly with the location of neuron modules within the hypothalamus and cerebral cortex. The hypothalamus contains cell groups, or nuclei, that serve as control centers for autonomic functions. The cerebral cortex is involved with autonomic function as well as sensory and motivational states. In my reading, I explored the significance of a specific neurotransmitter, orexin. This neuropeptide has been widely researched recently, implicated for various functions of the hypothalamus. Recent studies emphasize the idea that orexin is a vital component for feeding behavior and sleep-wake cycles in mammals, as well as a genetic component of the disease Narcolepsy. Narcolepsy is a sleep disorder which leads to sudden loss of consciousness, associated with disorientation and disruption of hypothalamic functions. My experiments investigated the anatomical significance of orexin, determining the pathway of neurons to orexin cells within the hypothalamus.
We hypothesized that orexin cells of the hypothalamus project to, and thus can physiologically affect neurons of the prefrontal cortex. The injection of PRV was made in the prefrontal cortex, taken up by axon terminals, and the infection allowed to spread for three days. Cross sections of the rat brain were then cut, and sections nearest to the optic chiasm were tested with double immunohistochemical procedures to detect both PRV cells and orexin cells. Flourescent markers were attached to both PRV and orexin, and cells that contained both substances fluoresced red and green. These double-labeled cells were then mapped out on a drawing of the rat brain section.
The double-labeled cells found within the hypothalamus support the hypothesis that there is a connection between the orexin cells of the hypothalamus and the cerebral cortex. By tracking PRV, we were able to determine that orexin cells must connect to the prefrontal cortex through first, second, or third order neurons. Further tests will help determine the specific neural circuits connecting orexin cells to the prefrontal cortex.
The implications of this research extend beyond anatomy. As mentioned before, anatomical definitions provide the basis for further research into the function of each neural circuit in the brain. The role of orexin in feeding and sleep-wake cycles may have a direct correlation to the motivational control of the cerebral cortex. This research creates more questions about the role of orexin cells and their connections to other circuits in the mammalian brain.
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Washington University - Biology
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