Capturing Snapshots in Time to Pick Apart Synaptic Activity

As we take in the world around us, learn, and form memories, the synapses between neurons in our brains are constantly being modified. Some get stronger, while others are allowed to shrink or get weaker. The network of enzyme-regulated chemical reactions that control these modifications is complex, to say the least. Now Mary Kennedy, the Allen and Lenabelle Davis Professor of Biology at Caltech, has come up with a way to tease apart the elusive details of that network. 

Beyond the basic scientific importance of understanding the ins and outs of our brains, the work could have significant implications for the mental health field. "It's becoming increasingly clear that slight mutations in some of these pathways make people more vulnerable to many disorders, including schizophrenia, bipolar disorder, and autism," Kennedy says.

Over the past 30 years, researchers have pieced together an understanding of the regulatory pathways and enzymes involved in controlling and modifying synaptic activity, and they've created "cartoons"—maps showing how the various components of the pathways interact. Looking at the cartoons, with their tangles of crisscrossing arrows connecting proteins and enzymes, the complexity of the network becomes apparent. 

Researchers have worked out many of the players involved and how they can interact to modify synapse strength. But they still know little about the dynamics of the network and how the processes are activated over time and under different circumstances. "We know how little strings of enzymatic processes can get activated," Kennedy says. "But we don't have very good ways of asking what happens when, say, 20 of these processes are interacting and you tweak one or two."

Now Kennedy believes she's found a way to ask such questions. The key is to stop the action in a series of samples and to see what changes from one second to the next. 

Kennedy is putting together an experimental method that will enable her to capture such "snapshots in time." She recently acquired a plunge-freeze device that she plans to modify so that it can freeze brain tissue samples in liquid propane/ethane as quickly as one second after electrical stimulation. Previously, Kennedy says, “we had no way of putting recording-electrodes into a brain slice, stimulating, and then freezing it solid or stopping it within a second.” 

There really was no reason to do so until the science and technology had progressed to a level that would enable a full analysis of the frozen samples. Kennedy says now is the time to move forward with these studies. Once she has the frozen tissue samples in hand, she'll process them with a new proteomic technique. Then, with the help of the Proteome Exploration Laboratory in the Beckman Institute, she'll be able to inject the sample into a mass spectrometer for analysis.

Many of the enzyme-driven reactions that take place in the synapses involve adding a highly energetic phosphate group to an amino acid, a process called phosphorylation, which alters the function of a protein. Since scientists have already determined the sequences of amino acids surrounding many of the critical sites of regulation in these pathways, Kennedy believes she will be able to use a mass spectrometer to measure the concentration of as many as 20 to 40 known phosphorylated sites in a single small tissue sample.

"That will let us map out the changes that happen in this large network immediately after a synchronized synaptic input," Kennedy says. "That means we'll be able to measure much more globally how these complex pathways interact with each other—which ones are more important at early stages, and which ones come in later—all of which has been very difficult to understand."

She hopes that she'll be able to use her new method to identify synaptic pathways that may be relevant to mental illnesses and Alzheimer's disease. "In order to screen in a more effective way for drugs or anything that could bring a particular set of processes into range, such global measurements are really critical," she says.

Kennedy's new Leica plunge-freeze apparatus was a gift from the Allen and Lenabelle Davis Foundation. The work is also supported by a grant from the National Institute of Mental Health.