By David N. Leff

Why should there be a receptor in the human body for a commercial insecticide?

That bug-clobbering compound, ryanodine, is an alkaloid derived from a tropical plant, Ryania speciosa, which is distantly related to plums and violets. In people, ryanodine activates calcium stores in cardiac and skeletal muscles, wherein it produces sustained contractions.

The ryanodine receptor is a husky 450-kiloDalton molecule, said neuroscientist Daniel Alkon, at the National Institute of Neurological Disorders and Stroke (NINDS), in Bethesda, Md. "It has 10 membrane-spanning domains and is virtually a giant calcium channel in the cell's endoplasmic reticulum.

"The ryanodine receptor," Alkon observed, "is becoming more and more understood as a major player in mobilizing calcium in the cell for its manifold functions, such as secretion, development, or learning and memory."

Alkon, who heads NINDS's Laboratory of Adaptive Systems, showed many years ago that calcium mobilization initiates many changes in brain cells "that eventually result in a more permanent memory record."

A paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Sept. 2, 1997, reports his latest finding on this subject. The article is titled: "Late memory-related genes in the hippocampus revealed by RNA fingerprinting."

It describes, Alkon told BioWorld Today, "a critical molecular step that occurs during learning and memory, which is the activation and increased synthesis of the ryanodine receptor. We had found a memory-signaling protein for it in the 1980s," he added, "but we didn't know until this year that our learning protein was the natural signal for the ryanodine receptor. Now we see why, because that receptor itself participates in memory storage in the brain's hippocampus."

The hippocampus is a brain area critically involved in spatial learning.

The story of how Alkon and his co-authors tracked down the gene that encodes this protein begins with a large colony of rats which were taught to memorize the location of a small island hidden in a sizable swimming pool.

What those 36 adult test animals confronted was a round basin five feet in diameter and two feet deep. Submerged in its depths was a transparent platform about five inches square, with its top a quarter-inch below the water level.

The rats' Memory & Learning 101 course consisted of swimming around in the pool and trying to get out until they found the platform, onto which they could climb out of the water's reach. Like ancient mariners navigating by the stars, the rodent swimmers could get a fix on their island refuge by noticing and committing to memory the various visual cues around the room, such as a door, a table, a computer, a curtain.

For Control Rodents, A No-Brainer

This mental process of memorization, the NINDS team knew, would create memory traces in the hippocampal cells of the rats' brains. To identify these changes, they would compare those brain slices with those of nine control mice, deprived of a chance to memorize the island refuge because it had been removed from the pool, unbeknownst to them.

Both cohorts of rats were sacrificed at two, six, 12 and 24 hours after training, and their hippocampal messenger RNA analyzed by PCR fingerprinting. "This methodology," Alkon pointed out, "is a fairly recent modification of screening mRNAs, but it's never been used for learning and memory. It's really the first time we had a way of screening large numbers of proteins and looking at their gene expression.

"After harvesting the RNA in the rats' brains," he continued, "we used this technique to display many thousands of random RNAs. From about 2,000 in our first run," he recounted, "we generated 10 genes that were memory-related candidates. The product of one was markedly upregulated in the rats' hippocampal cells; another signaled the ryanodine receptor."

"To our knowledge," his PNAS paper observed, "no changes in late effector genes have been previously demonstrated during associative memory."

Alkon's results also found that "the ryanodine receptor remained elevated for many, many hours, so you now have a relay of events through time into something that is more and more permanent for a memory trace. For a rat," he went on, "this memory consolidation takes a day, but the memory can last for months."

Search For Related Genes Under Way

His lab now has a series of experiments under way, looking at other genes, "and trying to put together a kind of cascade that will take us not only from seconds to minutes to hours, but also to days and weeks. For example, we can now look inside the dendritic compartments of neuronal cells, which are a critical site for synaptic contacts. There we see molecular changes for many days, and biophysical changes even months afterwards."

Alkon sees his current and near-future findings as having potential application in diagnosing, and perhaps treating, Alzheimer's disease (AD), which by definition involves memory deficits.

"A few years ago, we showed that the signaling molecule for the ryanodine receptor is deficient in AD. This may play a role," he said.

He and his group are close to announcing identification of "the first messenger protein for the neuronal ryanodine receptor. I think this is important," Alkon observed, "not only for memory scientists but for endocrinolgists, cardiologists, cell biology in general." He has pinpointed this molecule in human skin fibroblasts and brain tissue.

"Recently," the NINDS neuroscientist mentioned, "the U.S. has issued a series of patents covering our inventions for using rather sensitive and specific parameters to make the diagnosis of Alzheimer's. And in fact my understanding is that a company has been formed to work with us to start to scale up this diagnostic and has put substantial resources into this project."

Alkon's longer-range goal is to develop his newly identified pathways "for designing cognitive enhancers — targeting drugs into specific steps of memory formation. The more specific information we can get," he concluded, "like the ryanodine receptor, which is really a new insight, the more likely we'll be able to develop pharmacology that can facilitate relearning or learning in clinical contexts." *