Central Cardiovascular Regulation Group
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Techniques
Neurophysiology attempts to understand the function of the most complex organ known and to place this in the overall function of the body. This fascinating task is, in many respects, beyond our abilities using currently available methods. However, by employing all of our knowledge, significant gains can be achieved. Like no other discipline, a neuroscientist needs to cross all boundaries between traditional disciplines in order to comprehend the behavior of the brain. Consequently this laboratory uses a multitude of methods and technologies that might have been considered part of the disciplines of anatomy, pharmacology or biochemistry. These include:
- Measurement of blood pressure and other cardiovascular parameters in conscious freely-moving rats, using radiotelemetry
- Intracellular and extracellular electrophysiological methods to record the activity of peripheral sympathetic nerves and neurons in the brain in various rodent preparations (as well as cardiovascular function)
- Anatomical methods to examine the cellular distribution of various substances in the brain, including receptors, enzymes and peptides
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Figure 2. Phenotypic characterization of neurons in the RVLM. In various experiments we record the activity of neurons in the RVLM that are involved in the regulation of blood pressure. We use standard tests to show that we are recording from the “correct” type of cell. After we have finished our experimental manipulations we can also fill the cell with a dye (neurobiotin- green) and look for other chemicals that the cell might contain. This gives us more information about the type of cell we have been examining. In this example the cell we recorded from contains an enzyme involved in the manufacture of catecholamines such as adrenaline. This enzyme is tyrosine hydroxylase and is stained red. |
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Figure 3. Adenoviral-induced expression of genes in the brain. The upper panels show the distribution of green fluorescent protein (GFP- green) and dopamine-ß hydroxylase (DBH- red) in the rostral ventrolateral medulla (RVLM) of a rat. These images are merged in the middle panel and the white boxes shown at higher power in the bottom panels. Within the RVLM neurons critical for the generation of nervous activity to blood vessels make catecholamines and thus contain enzymes such as DBH. (This nervous activity controls how constricted or dilated blood vessels are and thus how much blood goes to each organ and what the blood pressure is.) However, cells in this region do not normally make GFP. We injected a modified, replication-deficient adenovirus into the RVLM to cause the cells to make GFP (and other important proteins that might change cellular activity). In this experiment the virus caused GFP expression to occur in cells surrounding the RVLM neurons- these are astrocytes. Figures 3 and 4 show some other examples of cells affected by the virus. We are using this method to examine the effect of changing RVLM neuron activity on the control of blood pressure. |
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We are constantly striving to introduce and extend our repertoire of methods to answer specific questions. To this end we are developing methods to measure changes in gene expression in particular brain regions, using real-time PCR. Most excitingly we are also now using replication-deficient viruses to deliver novel genes to neurons with defined function. This method enables us to take advantage of the huge wealth of genetic information derived from sequencing the genome as well as molecular biological approaches which are used to alter the function of specific proteins.
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Figure 4. Expression of GFP in astrocytes. The protein glial fibrillary acidic protein (GFAP) is used as a marker of glia. This shows that the GFP (green) and GFAP (red) occur in the same cell. |
Figure 5. Expression of angiotensin receptors. In addition to expression of GFP, we can cause cells to make other proteins that might affect cell activity. In this example the glial cell is making GFP (green) and angiotensin AT 1 receptors (red). |
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One recent experiment that highlights the power of this method is the introduction of a mutated angiotensin receptor, which constantly signals, into the rostral ventrolateral medulla. This manipulation led to a sustained increase in blood pressure and, unexpectedly and excitingly, led us into a new area of research- namely the interaction between neurons and astrocytes.