Much of the brain remains only loosely charted terrain, and one idea for unravelling some of its many mysteries is to map each and every neuron. One region that has received a lot of attention in this regard is the cerebral cortex, which has a key role in processing incoming 'sensory' signals — that is, those from the senses, such as vision and hearing. It is also essential to higher functions like reasoning and planning. Leopoldo Petreanu, a postdoc at the Janelia Farm Research Campus of the Howard Hughes Medical Institute in Ashburn, Virginia, and his colleagues have now found a way to locate the connections, known as synapses, that neurons in other brain regions make onto a group of highly branched cortical cells called pyramidal neurons.

When channelrhodopsin-2 (ChR2) — a light-sensitive ion-channel protein found in green algae — was sequenced in 2003, Petreanu was keen to try it as a new tool with which to map neuronal circuits. He hoped to use light to electrically excite one neuron at a time and then watch others respond. “We expected to be able to get single-cell resolution in a circuit map of the cortex,” he says.

He wanted to establish the source of cells stimulating the pyramidal neurons, but he encountered a glitch. When ChR2 is expressed in a neuron, it is spread across the cell surface, and because axons are so intermingled in the brain, even a focused laser would set off more than just the target cell under study. “That was bad news,” says Petreanu.

Petreanu and his colleagues realized that, rather than isolating a single neuron, they could instead isolate just the very tips of ChR2-expressing neurons. They turned to an old, reliable constituent of the neuroscience toolbox: tetrodotoxin, a neurotoxin that blocks the propagation of nerve signals from the neuron's cell body down the axon. Now, only functional connections to other neurons would react to light. This is because, even in the absence of nerve signals, the light-activated ChR2 at the very ends of the nerve terminals would still release chemical neurotransmitters, thus activating any neighbouring pyramidal neuron. Using this method, the team was able to identify from which neurons in other brain areas pyramidal neurons receive input.

Petreanu expressed ChR2 in areas of the mouse brain outside the cortex, such as the thalamus, a relay station for incoming sensory signals. He took brain slices and bathed them in tetrodotoxin, then shone a light on them. At the same time, he recorded the activity of the cortical pyramidal neurons. Only those pyramidal cells that came into contact with ChR2-expressing nerve terminals generated a reading (see page 1142).

“We have a technique by which you can rapidly map where the connections are made,” says Petreanu. “Before, the only way to see a connection was with electron microscopy.”

One surprise was the orderliness of neuronal connections in the cortex. Anatomically, synapses with specific types of input were segregated within certain branches of the pyramidal neurons. How this translates into function is not known, although Petreanu and his co-authors speculate that spatially clustered inputs might rally stronger responses. The next step, says Petreanu, is “to go from this anatomical observation to a functional understanding”.