Closer to a functional atlas of the brain

The different types of neuroreceptors and their location in the nervous system are well characterised. However, their function can vary depending on their location within the neuron or in different regions of the brain. Up to now, no method has enabled experts to distinguish these differences.

Now, scientists from the Institute for Bioengineering of Catalonia (IBEC) have developed a technique to determine the role of a neuroreceptor with high spatial and temporal precision. This method, transferrable to other receptors and proteins, provides a new tool to understand how the brain works.

This innovative methodology opens the door to mapping a functional atlas of the brain. “There are already atlases of the brain, three-dimensional maps that allow us to know its anatomy and what neuroreceptors there are in each region”, explains Pau Gorostiza, ICREA research professor and head of the Nanoprobes and Nanoswitches Group at IBEC that led the study. “But we still don’t know what roles the same type of receptor can play in different regions of the brain. Our study takes new steps in the development of a functional atlas of the brain, a tool as powerful as it is necessary for neurobiology.”

Photopharmacology and positive logic

To understand the physiological function of a receptor, it is necessary to know where, how and for how long it carries out its activity. Generally, this challenge is approached using techniques based on genetic modification and pharmacology.

However, these methodologies have several shortcomings. Firstly, they involve the inhibition of the receptor throughout the whole organism, which prevents the observation of the specific function of the receptor at each site. Furthermore, it isn’t possible to study essential receptors in detail, since inhibiting them can result in the death of the animal.

But the most notable shortcoming is inherent to the technique itself: can we identify the function of a receptor based solely on what happens when it is not active in an organism? This is where the technique developed by Gorostiza’s team comes into play. “We have tried to apply an inverse, positive logic. We have used a drug that specifically inhibits the neuroreceptor mGlu5, in addition to being photoswitchable. This means that we can activate the receptors where we light them up. Using a pulsed near-infrared laser, we have managed to activate them with micrometer precision (a thousandth of a milllimeter) and at great depth. In this way, we have been able to see how the neuroreceptor acts in different regions of the same neuron”, explains the expert.

In addition to allowing the spatial control of the neuroreceptor activity, this technique provides great control over the duration of activity. “For the first time, we can apply pharmacology in 4D: not only can we control where the drug is activated on a three-dimensional level, we can also decide for how long. We simply have to turn off the laser.”

The key to the study lies in the use of two-photon excitation using pulsed near-infrared lasers. Until recently, techniques based on photoswitchable molecules used blue or violet continuous-wave lasers (one-photon excitation) to activate these compounds, but the stimulus can’t be studied on a three-dimensional scale using this method. Two-photon excitation, predicted by Maria Göppert-Mayer and demonstrated by the winners of the Nobel Prize in Physics in 2018, Donna Strickland and Gérard Mourou, has represented a revolution for the visualisation and manipulation of neuronal activity.

“The brain is the most complex organ that exists. Although its anatomy and the genes that are expressed in it have been characterised with great resolution, until now there was no method to characterise the function of neuronal receptors in situ with spatial and temporal resolution”, adds Silvia Pittolo, first-author of the study and current Marie Skłodowska-Curie Fellow at the University of California in San Francisco. “Our study could serve as the key to unlock some of the mysteries hidden in the brain and the networks that form it. If we can manage to decipher the function of each receptor depending on its location, we will be closer to understanding the entire neural network.”

The study was carried out in collaboration with scientists from the Institute for Research in Biomedicine (IRB Barcelona), a centre that, like IBEC, forms part of the Barcelona Institute of Science and Technology (BIST); as well as researchers from the Cajal Institute, the University of Barcelona (UB), the Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), the Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED-ISCIII) and the Network Center for Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN).

Reference article: Pittolo et al. Reversible silencing of endogenous receptors in intact brain tissue using two-photon pharmacology. PNAS, 2019. DOI: