In a landmark study, scientists at the Massachusetts Institute of Technology, using new genetic and multiple-cell monitoring technologies, have demonstrated how animals form memory for places, which may directly relate to the same ability in humans.
This latest "regional gene knockout" technology, through which scientists can develop a breed of mice in which a gene is eliminated in a specific area or only in one particular type of cell, will be valuable in the study of neurological diseases such as Alzheimer's, Huntington's, Parkinson's and drug addictions.
The study, in three related scientific papers, is being published in the December 27 issue of the scientific journal, Cell. It involves a molecular, behavioral and electrical study of how animals develop spatial memory of a new environment after just a few minutes of exposure. The scientists believe humans use the same mechanisms to remember the location of objects and to navigate between places.
The group has finally obtained evidence showing that strengthened connections between groups or ensembles of neurons enable the formation of internal "maps" of a space which allow the animals to remember that environment, whether it is a room or a pond.
This technology is much more precise than the first generation of gene-knockout technology, in which, through cross-breeding, animals inherit a genetic makeup that totally eliminates a specific gene throughout the body, in whatever organ or cell type where those genes function. The earlier technology, developed in 1989, limited the conclusions scientists could definitively make, because the same gene can have different functions in different regions within organs, and may function at different times during the maturation process.
"We have developed a method to create mice in which the deletion (knockout) of virtually any gene of interest is restricted to a sub region or a specific cell type in the brain. The brain sub region-restricted gene knockout should allow a more precise analysis of the impact of a gene mutation on animal behaviors," wrote the authors of the first paper. The nine-person scientific team at MIT's Center for Learning and Memory was headed by Assistant Professor Matthew Wilson and the Director of the Center, Nobel laureate Susumu Tonegawa.
"This work proves once and for all time that this part of the brain is the crucial part for this kind of memory," said Dr. Charles F. Stevens, a Howard Hughes Medical Investigator and Professor at the Salk Institute.
In an interview, Dr. Stevens, who commented on the paper for Cell, said: "It is a dream of neurobiologists to understand some interesting cognitive phenomena like memory from the molecular level right up through behavior. The articles in Cell are a big step in that direction."
Commenting on the regional gene knockout technology, Professor Emilio Bizzi, head of the MIT Department of Brain and Cognitive Sciences, said: "The new genetic technique utilized by Tonegawa and Wilson will revolutionize the field of brain research. With this technique, scientists will be able to study how the brain encodes different types of memory, that is, memory of facts, of objects, and of motor skills."
The research scientists, as described in the first paper, created a strain of mice in which the gene for a specific neurotransmitter receptor called the NMDA receptor is knocked out only in one type of nerve cells (CA1 pyramidal cells) in the hippocampus, the brain area that plays a crucial role in some forms of memory such as "spatial memory." Earlier other scientists have hypothesized that this and many other types of memory are stored as strengthened neuronal connections or synapses.
This strengthening of synapses is termed "synaptic plasticity." But hard evidence for this hypothesis has been difficult to find.
The scientists in the second paper discovered a critical link between synaptic strengthening and spatial memory by having the new knockout mice perform spatial memory tasks and by measuring the ability of cellular connections to change.
In order to further understand how animals and humans acquire spatial information, MIT's scientists in the third paper analyzed these knockout mice by a highly sophisticated electrophysiological technique that precisely monitors the electrical activity of a large number of the individual nerve cells in the CA1 region of the hippocampus, as the mice explore an environment freely. They discovered that, unlike the normal mice, these new knockout mice have a deficiency in establishing an internal spatial "map" among an ensemble of CA1 nerve cells because of the inability to strengthen synaptic connections.
This is a landmark study because, for the first time, it was demonstrated beyond doubt that synaptic plasticity in a specific region of the brain is essential for spatial memory and, in addition, the scientists could demonstrate that synaptic plasticity gives rise to spatial memory through the establishment of internal "maps" of the space. In addition, the new gene knockout technology established by MIT scientists will also be extremely valuable for medical researchers to study the precise functions of disease genes in neurological disease processes such as in Alzheimer's, Huntington's, and Parkinson's diseases as well as in drug addictions.
The lead authors were Joe Z. Tsien, a post-doctoral associate with Dr. Tonegawa, on the first two papers, and Thomas J. McHugh, a biology graduate student in Dr. Tonegawa's lab who has been supervised principally by Dr. Wilson for this work.
FURTHER EXPLANATION FOR THE DISCOVERY
Memory storage in the mammalian brain involves strengthening the synaptic connections between neurons (synaptic plasticity). D.O. Hebb, in a hypothesis first advanced in 1949, said that associative memory (such as spatial memory) requires correlated activity - the connections become strengthened when pre synaptic neurons and post synaptic neurons are firing their electrical charges at the same time. Spatial memory involves the hippocampus, a ridge of the brain located just behind each ear. Although the hippocampus has been recognized for the past 25 years as the likely area for spatial memory, research involving genetic or pharmacological changes in animals have been unable, precisely, to demonstrate how the process works.
The crucial elements for inducing synaptic plasticity that is dependent on activity are neural receptors known as N-methyl-D-aspartate receptors (NMDARs). Active NMDAR channels allow for an influx of calcium into the post synaptic cell which triggers a cascade of biochemical events resulting in synaptic strengthening.
The scientists noted that past experiments in which NMDAR1 genes were knocked out of the entire body resulted in death within a day or so.
The scientists at the MIT Center for Learning and Memory, however, developed a mouse strain (Paper #1) in which the deletion of the NMDAR1 gene is restricted to just the pyramidal cells in the CA1 region of the hippocampus. The knockout mice grow into adulthood without obvious abnormalities.
Mice are natural swimmers but they prefer to be on land or a solid surface. A test of memory used in Paper #2 is the Morris water maze, a round pool in milky water where there is a platform that is just below the level of the water. The platform can be easily spotted by all mice, including the new knockout mice, when there is a flag that extends above the water attached to the platform. But it's different when the flag is gone.
The mice are placed in the Morris water maze three or four times a day for several days. They explore the maze and swim into the platform. By the fourth day, normal mice make a beeline for the submerged platform, knowing precisely where it is.
But the knockout mice swim all over the place, and show clear deficits in their ability to find the underwater platform. Clearly, they are unable to form a map of the space in their brain. Why?
The answer was found by monitoring the electrical activity in the hippocampus's pyramidal cells of ordinary mice and the knockout mice. The authors of the third paper found that each pyramidal cell has its own region of heightened firing of neurons ("place field"). Large numbers of hippocampal place cells will tile each environment with overlapping place fields, allowing the mouse to remember and navigate the space or maze.
The firing patterns are so clear that the location of the animal can be well estimated just by looking at the firing patterns on the computer screen, rather than at the animal.
The great difference between knockout mice and ordinary mice were shown when the software developed by Dr. Wilson's laboratory tracked the coordinated firing of pairs of place cells.
The place cells of knockout mice exhibited completely non-correlated firing while the place cells of ordinary mice showed significant correlation.
Because 30 to 50 percent of the cells within the hippocampus become active within a given environment, the scientists concluded that rodents use information from ensembles of cells to calculate location, rather than mapping places to individual cells.
Dr. Stevens, of the Salk Institute, commented, "This is the first time these two very high tech, cutting edge technologies have been combined to make the connection between the molecule triggering long term potentiation (a kind of memory) right through observing the behavior of the animal and monitoring the cells that are responsible for that behavior."
"Downstream brain regions will fail to learn anything about place" from the CA1 region of the hippocampus in the knockout mice, the authors of Paper #3 wrote.
"We are convinced that for the first time, controlled changes in synaptic plasticity can be linked to electrophysiological changes that can explain a behavioral impairment." The scientists added that "we propose that the non-correlated activity of CA1 place cells during exploration causes downstream navigational learning impairments."
The research was supported by the National Institutes of Health grant #NS 32925, gifts from the Shionogi Institute of Medical Science in Japan, and Amgen, Inc., all to Dr. Tonegawa; and by awards from the Seaver Institute and the Sloan Foundation to Dr. Wilson.
The papers, and their authors, are as follows:
1. "Sub region and cell type-restricted gene knockout in mouse brain," by Joe Z. Tsien, Dong Feng Chen, David Gerber, Cindy Tom, Eric H. Mercer, David J. Anderson, Mark Mayford, Eric R. Kandel, and Susumu Tonegawa. Dr. Tsien. Dr. Chen and Dr. Gerber are postdoctoral fellows, and Ms. Tom is a senior in biology, all in Dr. Tonegawa's lab at the Center for Learning and Memory. Mercer and Anderson, who contributed genetic material, are with the Howard Hughes Medical Institute, Division of Biology, at the California Institute of Technology. Mayford and Kandel, who also contributed genetic material, are with the Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University.
Dr. Tonegawa is a Howard Hughes Medical Investigator at MIT; the Director of the MIT Center for Learning and Memory; the Amgen Professor of Biology and Neuroscience in the Departments of Biology and Brain and Cognitive Sciences; and a member of the MIT Center for Cancer Research.
2. "The essential role of hippocampal CAI NMDA receptor-dependent synaptic plasticity in spatial memory," by Joe Z. Tsien, Patricio T. Huerta and Susumu Tonegawa. Dr. Huerta is a Pew Latin American Postdoctoral Fellow in Dr. Tonegawa's lab.
3. "Impaired hippocampal representation of space in CA1-specific NMDAR1 knockout mice," by Thomas J. McHugh, Kenneth I. Blum, Joe Z. Tsien, Susumu Tonegawa, and Matthew A. Wilson. McHugh is a biology graduate student in Dr. Tonegawa's lab in the MIT Center for Learning and Memory who has been supervised principally by Dr. Wilson for this work. Dr. Blum is a postdoctoral fellow in Dr. Wilson's lab in the MIT Center for Learning and Memory. Dr. Wilson is the Edward J. Poitras Assistant Professor in Human Biology and Experimental Medicine in the Department of Brain and Cognitive Sciences, and Biology, and head of his laboratory in the Center for Learning and Memory.
Dr. JOE Z. Tsien was born and raised in Wuxi, China, where he graduated from high school. He lives in Malden, Mass.
Dr. DONG FENG CHEN lives in Chestnut Hill, Mass.
Dr. David GERBER was born in Baltimore, Md.; raised in Lake Oswego near Portland, Or., and graduated from high school in Worthington, Ohio. He lives in Somerville, Mass.
CINDY TOM, an MIT senior, was born and raised in San Francisco. She graduated from high school near San Jose in Milpitas, Calif., where her family now resides. She lives on campus at MIT.
DR. PATRICIO HUERTA was born and raised in Ovalle, Chile, .and graduated from high school in Santiago, Chile, He lives in Concord, Mass.
THOMAS McHUGH, a graduate student, was born in Evergreen Park, Ill., raised in Oak Lawn, Ill. and graduated from Illinois Math and Science Academy in Aurora, Ill. He lives in Somerville, Mass.
DR. Kenneth BLUM was born and raised in Chicago.and graduated from Kenwood Academy in Chicago. He lives in Belmont, Mass.
PROFESSOR SUSUMU TONEGAWA was born in Nagova, Japan, raised in the Osaka area and graduated from Hibiya High School in Tokyo. He lives in Newton, MA.
PROFESSOR MATTHEW WILSON was born in Seoul, Korea, and raised in DePere, Wis., where he graduated from high school. He lives in Acton, Mass.