Irwin Neher: Chemistry helps neuroscience: the use of caged compounds and indicator dyes for the study of neurotransmitter release

Ah, a solid science talk. It wasn’t bad, except that it was very basic—maybe if I were a real journalist instead of a fake journalist I would have appreciated it more, but as it was, it was a nice overview of some common ideas in neuroscience, with some discussion of pretty new tools on top.

He started with a little history to outline what we know, with Ramon Y Cajal showing that the brain is made up of network of neurons (which we now know to be approxiamately 1012 neurons large). He also predicted the direction of signal propagation, and was mostly right. Each neuron sends signals outwards through an axon, and receives input from thousands of other cells on its cell body and dendrites.

Signals move between neurons mostly by synaptic transmission, or the exocytosis of transmitter-loaded vesicles induced by changes in calcium concentration. That makes calcium a very interesting ion, and makes calcium concentration an extremely important parameter affecting physiological function, so we want to know more about it. Furthermore, it’s a parameter that is in constant flux, changing second by second in the cell. So how do we see an ion in real time or near real time?

The answer is to use fluorescent indicator dyes which are sensitive to changes in calcium concentration — these molecules fluoresce at different wavelenths or absorb light at different wavelengths depending on whether they are bound or not bound to calcium, making the concentration visible as changes in either the absorbed or emitted wavelength of light. There is a small battery of fluorescent compounds — Fura-2, fluo 3, indo-1 — that allow imaging of localized increases in calcium.

There’s another problem: resolution. Where the concentration of calcium matters most is in a tiny microdomain, a thin rind of the cytoplasm near the cell membrane called the cortex, which is where vesicles are lined up, ready to be triggered to fuse with the cell membrane by calcium, leading to the expulsion of their contents to the exterior. This microdomain is tiny, only 10-50nm thick, and is below the limit of resolution of your typical light microscope. If you’re interested in the calcium concentration at one thin, tiny spot, you’ve got a problem.

Most presynaptic terminals are very small and difficult to study; they can be visualized optically, but it’s hard to do simultaneous electrophysiology. One way Neher gets around this problem is to use unusually large synapses, the calyx of Held synapse, which is part of an auditory brainstem pathway. It’s an important pathway in sound localization, and the signals must be very precise. They have a pecial structure, a cup-like synapse that envelops the post-synaptic cell body — they’re spectacularly large, so large that one can insert recording electrodes both pre- and post-synaptically, and both compartments can be loaded with indicator dyes and caged compounds.

The question being addressed is the concentration of Ca2 at the microdomain of the cytoplasmic cortex, where vesicle fusion occurs. This is below the level of resolution of the light microscope, so just imaging a calcium indicator dye won’t work — they need an alternative solution. The one they came up with was to use caged molecules, in particular a reagent call Ca-DMN.

Caged molecules are cool, with one special property: when you flash UV light of just the right wavelength at them, they fall apart into a collection of inert (you hope) photoproducts, releasing the caged molecule, which is calcium in this case. So you can load up a cell with Ca-DMN, and then with one simple signal, you can trigger it to release all of its calcium, generating a uniform concentration at whatever level you desire across the entire cell. So instead of triggering an electrical potential in the synaptic terminal and asking what concentration of calcium appears at the vesicle fusion zone, they reversed the approach, generating a uniform calcium level and then asking how much transmitter was released, measured electrophysiologically at the post-synaptic cell. When they got a calcium level that produced an electrical signal mimicking the natural degree of transmitter release, they knew they’d found the right concentration.

Caged compounds don’t have to be just calcium ions: other useful probes are caged ATP, caged glutamate (a neurotransmitter), and even caged RNA. The power of the technique is that you can use light to manipulate the chemical composition of the cell at will, and observe how it responds. These are tools that can be used to modify cell states, to characterize excretory properties, or to generate extracellular signals, all with the relatively noninvasive probe of a brief focused light flash.