Optogenetics captures neuronal transmission in live mammalian brain

This is a reconstruction of a pair of synaptically connected neurons.

Neurons, the cells of the nervous system, communicate by transmitting chemical signals to each other through junctions called synapses. This "synaptic transmission" is critical for the brain and the spinal cord to quickly process the huge amount of incoming stimuli and generate outgoing signals. However, studying synaptic transmission in living animals is very difficult, and researchers have to use artificial conditions that don't capture the real-life environment of neurons. Now, EPFL scientists have observed and measured synaptic transmission in a live animal for the first time, using a new approach that combines genetics with the physics of light. Their breakthrough work is published in Neuron.

Aurélie Pala and Carl Petersen at EPFL's Brain Mind Institute used a novel technique, "optogenetics," that has been making significant inroads in the field of neuroscience in the past ten years. This method uses light to precisely control the activity of specific neurons in living, even moving, animals in real time. Such precision is critical in being able to study the hundreds of different neuron types, and understand higher brain functions such as thought, behavior, language, memory -- or even mental disorders.

Activating neurons with light

Optogenetics works by inserting the gene of a light-sensitive protein into live neurons, from a single cell to an entire family of them. The genetically modified neurons then produce the light-sensitive protein, which sits on their outside, the membrane. There, it acts as an electrical channel -- something like a gate. When light is shone on the neuron, the channel opens up and allows electrical ions to flow into the cell; a bit like a battery being charged by a solar cell.

The addition of electrical ions changes the voltage balance of the neuron, and if the optogenetic stimulus is sufficiently strong it generates an explosive electrical signal in the neuron. And that is the impact of optogenetics: controlling neuronal activity by switching a light on and off.

Recording neuronal transmissions

Pala used optogenetics to stimulate single neurons of anesthetized mice and see if this approach could be used to record synaptic transmissions. The neurons she targeted were located in a part of the mouse's brain called the barrel cortex, which processes sensory information from the mouse's whiskers.

When Pala shone blue light on the neurons that contained the light-sensitive protein, the neurons activated and fired signals. At the same time, she measured electrical signals in neighboring neurons using microelectrodes that can record small voltage changes across a neuron's membrane.

Using these approaches, the researchers looked at how the light-sensitive neurons connected to some of their neighbors: small, connector neurons called "interneurons." In the brain, interneurons are usually inhibitory: when they receive a signal, they make the next neuron down the line less likely to continue the transmission.

The researchers recorded and analyzed synaptic transmissions from light-sensitive neurons to interneurons. In addition, they used an advanced imaging technique (two-photon microscopy) that allowed them to look deep into the brain of the live mouse and identify the type of each interneuron they were studying. The data showed that the neuronal transmissions from the light-sensitive neurons differed depending on the type of interneuron on the receiving end.

"This is a proof-of-concept study," says Aurélie Pala, who received her PhD for this work. "Nonetheless, we think that we can use optogenetics to put together a larger picture of connectivity between other types of neurons in other areas of the brain."

The scientists are now aiming to explore other neuronal connections in the mouse barrel cortex. They also want to try this technique on awake mice, to see how switching neuronal activity on and off with a light can affect higher brain functions.

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Neurons listen to glia cells

Oligodendrocyte progenitor cells in the brain (OPC, green) influence synaptic signaling between neurones (red) integrated in the neuronal network.

Scientists at Johannes Gutenberg University Mainz (JGU) have discovered a new signal pathway in the brain that plays an important role in learning and the processing of sensory input. It was already known that distinct glial cells receive information from neurons. However, it was unknown that these same glial cells also transmit information to neurons. The glia release a specific protein fragment that influences neuronal cross-talk, most likely by binding to the synaptic contacts that neurons use for communication. Disruption of this information flow from the glia results in changes in the neural network, for example during learning processes. The team composed of Dr. Dominik Sakry, Dr. Angela Neitz, Professor Jacqueline Trotter, and Professor Thomas Mittmann unravelled the underlying mechanism, from the molecular and cellular level to the network and finally the resulting behavioral consequences. Their findings constitute major progress in understanding complex pathways of signal transmission in the brain.

In mammalian brains glial cells outnumber nerve cells, but their functions are still largely unelucidated. A group of glial cells, so-called oligodendrocyte precursor cells (OPC), develop into the oligodendrocytes which ensheathe neuronal axons with a protective myelin layer thus promoting the rapid transmission of signals along the axon. Interestingly, these OPCs are present as a stable proportion -- some five to eight percent of all cells in all brain regions, including adult brains. The Mainz-based researchers decided to take a closer look at these OPCs.

In 2000 it was discovered that OPCs receive signals from the neural network via synaptic contacts that they make with neurons. "We have now discovered that the precursor cells do not only receive information via the synapses, but in their turn use these to transmit signals to adjacent nerve cells. They are thus an essential component of the network," explained Professor Jacqueline Trotter from the Institute of Molecular Cell Biology at Mainz University. Classically, neurons have been considered as the major players in the brain. Over the past few years, however, increasing evidence has come to light that glial cells may play an equally important role. "Glial cells are enormously important for our brains and we have now elucidated in detail a novel important role for glia in signal transmission," explained Professor Thomas Mittmann of the Institute of Physiology of the Mainz University Medical Center.

The chain of communication starts with signals traveling from the neurons to the OPCs across the synaptic cleft via the neurotransmitter glutamate. This results in a stimulation of the activity of a specific protease, the alpha-secretase ADAM 10 in OPCs, which acts on the NG2 protein expressed by the precursor cells releasing a NG2 fragment into the extracellular space, where it influences neighboring neuronal synapses. The neurons react to this in the form of altered electrical activity. "We can use patch-clamp techniques to hear, as it were, how the cells talk to one another," said Mittmann.

"The process starts with the reception of signals coming from the neurons by the OPCs. This means that the feedback to the neurons cannot be seen as separated from the signal reception," explained Dr. Dominik Sakry, joint first author of the study, describing the cascade of events. The role of NG2 in this process became apparent when the researchers removed the protein: neuronal synaptic function is altered, modifying learning and disrupting the processing of sensory input that manifests in the form of behavioral changes in test animals.

The evidence that the communication between the two cell types in the brain is not a one-way system but a complex mechanism involving feedback loops was obtained in a collaborative project involving physiologists and molecular biologists. Participating in the project at Mainz University were the Faculties of Biology and Medicine and the Focus Program Translational Neurosciences (FTN) in the form of platform technology provided by the Mouse Behavioral Unit (MBU). The project was additionally supported by two Mainz Collaborative Research Centers (CRC 1080 and CRC-TR 128) and involved participation of the Leibniz Institute for Neurobiology in Magdeburg. Scientists from seven countries participated in the study.

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