A herpes simplex viral vector expressing green fluorescent protein can be used to visualize morphological changes in high-density neuronal culture
Rebecca S. Borders
Ramsey K. Kilani
Andrea J. Yool
Scott J. Sherman*
NIH grants 5 K08 NS 02015-03 (S.J.S.) and 1 R01 MH59747-01A1 (A.J.Y.); Epilepsy
Foundation of America Fellowship (S.J.S.); and Small Grants Program funding
from The University of Arizona (S.J.S.).
With the completion of the human genome project, there are great expectations for the development of gene therapies for neurological disease. Before the realization of such therapies, there remain several technological challenges to be met. One major obstacle is the development of gene transfer methods, which will enable scientists to place selected genes into the principal type of brain cells known as neurons. A second technological obstacle is reliably identifying those neurons in which successful gene transfer has occurred. Once a new gene has been placed in a neuron, it will be important to track changes in the growth, anatomical shape, and other characteristics of the genetically-modified neuron.
One of the most promising techniques for introducing new genes into neurons is the use of genetically-engineered viruses. In nature, viruses transfer genes into many different cell types, including brain cells. Often this process is pathological and leads to a viral infection; however, many viruses can be modified to be less harmful and their gene transfer abilities potentially used as a powerful therapeutic tool. In our studies, we have employed a disarmed herpes virus to transfer genes into neurons that are maintained in a culture dish under laboratory conditions. The herpes virus has been modified so that it does not reproduce in the cells and thus cannot cause a spreading infection. The neurons that we employ are initially derived from the immature brains of laboratory rats. Under the proper conditions, they undergo many of the same early developmental processes that occur in the intact brain of animals including humans.
The standard technique of identifying genetically-altered neurons is to introduce a "marker gene" which produces a colored pigment in the neuron to be studied. Prior research has employed a gene which produces a blue color, indicating successful gene transfer. One drawback of this method is that the chemical process used to develop the blue color cannot be performed on living neurons. Our studies explore an alternative to this method, which allows identification of genetically-altered neurons in the living state. Our method uses a disarmed herpes virus to introduce a luminescent marker gene, known as "green fluorescent protein". The gene for this protein was initially derived from a species of jellyfish that glows in the dark.
Figure 1 shows photographs of four different neurons that have been genetically altered by a herpes virus capable of transferring the green fluorescent protein gene. The photographs were taken with a microscope at high magnification using ultraviolet light to activate the green fluorescence shown. These neurons shown were initially removed from an area of the brain known as the hippocampus, which is important for short-term memory. They were then grown under laboratory conditions for up to two weeks while they developed the features of more mature neurons. After two weeks, they were treated with the disarmed herpes virus in order to transfer the green fluorescent protein gene. One day later, we were able to detect this gene and obtain photographs such as shown in Figure 1, which give a detailed image of the cell shape. The center portion of the neuron is known as the "soma" or cell body. In the photograph labeled A, the soma has a triangular shape and identifies this neuron as a "pyramidal" cell. The neurons shown in B, C, D have different shapes corresponding to other types of neurons. The branching structures that radiate out from the soma are known as dendrites. These dendrites are used to collect information from other neurons that together form the neuronal circuitry that allows the brain to process information.
If we were obtain the photographs shown in Figure 1 using regular white light we would see that many more neurons are actually present. Using ultraviolet light, only the genetically altered neurons are visible. Using white light, however, the detailed shape of the branching dendrites of a single neuron would be lost amid the tangle of dendrites from these other neurons. Thus, our new technique allows us to identify single neurons that have been genetically altered and study their individual shapes. Moreover, this new technique can be used in living and growing neurons since our process does not require destroying the cell.
We have shown that changes in the shape of the neuron continue to occur in these genetically-altered cells. Indeed, the green fluorescent protein enables us to make detailed measurements of the changing shape of living neurons. Using this technique, we were able to demonstrate that neurons maintained under laboratory culture conditions continue to undergo changes in the shape and position of the dendrites. Figure 2 shows a series of photographs of a single genetically-altered neuron taken at three hour intervals (labeled A through D). In these digital photographs, the black area corresponds to the green fluorescence illustrated in Figure 1. Changes in shape and position can be detected from close inspection of this series of photographs. In order to more easily demonstrate these changes, the bottom row (labeled E, F, G) compares the shape and position in digitally superimposed images. In this series, the white outline indicates the initial position of the neuron, while the black images indicate the shift that has occurred with time. Photograph E, F, and G demonstrate the changes that occur after 3, 6, 9 hours respectively.
A similar example of this phenomenon is shown in Figure 3. Here we show a different type of neuron that was taken from a portion of the brain known as the cerebellum (involved in coordinating movements and balance). This neuron is labeled PN for its technical designation of being a Purkinje neuron. In this series, the fluorescence is shown in white against a blue background. A second cell type found in the brain is the glial cell which provides a supporting role for neurons. Our gene transfer virus can also induce fluorescence in this cell type. An example of a genetically-altered glial cell is shown in Figure 3 and labeled "G". Note that over a three hour time period, one of the Purkinje cell dendrites (see arrow) moves closer to the glial cell (see box), again demonstrating a relatively rapid change in neuron shape.
These results demonstrate that rapid changes in the shape of neurons and position of dendrites continue to occur in laboratory culture conditions. These changes in the dendrites are important for the development of proper connections within the brain, and are critical for learning and memory. In the developing brain, the intertwined dendrites of thousands of neurons form a dense tangle. Tracking the changes in structure of the dendrites of a single living neuron is a daunting task. Our studies have demonstrated a new method for studying these changes under laboratory conditions.
Overall relevance of the results
In summary, the current report is important for several reasons:
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