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Xue lab develops a better optogenetic tool to study neuronal function

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The scientists in the laboratory of Dr. Mingshan Xue, assistant professor at Baylor College of Medicine and an investigator at the Cain Foundation Laboratories and the  Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, have modified an existing light-sensitive tool to produce fewer undesired effects, making it a better tool to study neuronal function. Moreover, this study shows that different areas of a neuron may have distinct chloride (other ion) channel gradients and therefore, may respond differently to optogenetic manipulations. The study was published in the eLife journal.

Optogenetic tools such as GtACR2 are being used increasingly to study how neurons functions. This study shows that different types of neurons and different parts of the same neuron may respond differently to these genetic manipulations. Also, differential ion gradients within a neuron could also prove beneficial for neuroscientists to selectively study axonal projections to a particular area of the brain over other areas.

Xue and his colleagues initially set out to study the effect of inhibiting a specific group of neurons in the visual cortex of the mice by genetically introducing a light-gated chloride channel GtACR2, a potent inhibitor of neural activity. They expected that in the presence of light, GtACR2 will inhibit the activity of those neurons, which would be evident by the dramatically reduced levels of released neurotransmitters.

Surprisingly, however, in the presence of light, GtACR2-expressing neurons continue to release significant amount of neurotransmitters, indicating they were still functional. This was a completely unexpected and so, the team decided to investigate the cause for this unusual neuronal activity.

Neurons receive signals from other neurons at the command center, called the cell body. If the neurons receive an excitatory (“fire”) signal, the cell body passes it down to the axons, the long threadlike extension, to release neurotransmitters that will activate the neighboring neuron and so forth, allowing the signal to propagate through the neural network. If, on the other hand, the cell body receives an inhibitory (‘stop’) signal, then it is not communicated further.

In addition to this on/off switch, neuronal activity is also finely regulated by ion-gated channels that control the flow of ions inside and outside the neurons. When the neuron is activated, GtACR2 allows negatively charged chloride ions to flow across the neuronal membranes from a region of higher concentration to lower concentration. The flow of chloride ions from inside the neuron toward the outside triggers a “fire” signal, while the opposite, results in a ‘stop’ signal. Usually, chloride concentration is higher outside of the cell than in the inside, so when GtACR2 opens, ions flow toward the inside of the cell, which results in a ‘stop’ signal. That’s why chloride channels usually inhibit neuronal activity.

Researchers expected that irrespective of whether the recipient neurons received turn on/off signal from the neighbors, when GtACR2 is activated with light, chloride ions will floods into the neuron silencing the cell body. But,they found that although the cell body was being silenced, signals still ran through the axon and neurotransmitters were still being released at the nerve terminals.

The reason for that was that in the particular neurons they were studying the cell body and axons had different chloride ion gradients. In the cell body, the concentration of chloride ions was lower than the concentration outside but in the axons, it was opposite - the concentration of chloride ions was higher inside. This difference in chloride ion concentration between the cell body and axons meant that when GtACR2 was activated with light, although it triggered a “stop” response in the cell body, it still resulted in a “fire” response in the axon.

To reduce the “fire” signal in the axon, the researchers modified the expression of GtACR2 channel so it would be mostly expressed in the cell body, and not in the axons. Relocating most of the GtACR2 channel subunits to the cell body significantly enhanced the inhibitory effect in the cell body and also, reduced the “fire” signal from axons which significantly reduced the amount of neurotransmitter released from the synapses of these neurons, acting as an improved version of the original GtACR2 channel to inhibit neural activity.

Thus, precise understanding of the effects of each light-gated chloride (or any other ion-gated) channel on each part of the neuron will help researchers better design their experiments, accurate interpret their results and even lead to exciting new findings about neuronal function.

Rajalaxmi Natarajan, PhD
Monday, August 20, 2018