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14.12.2015

New method visualizes the hidden language of the brain

Gold particles allow to stain neuropeptides and visualize neurons in the brain’s wiring diagram


Tübingen, December 17, 2015. Dr. Gáspár Jékely and his team at the Max Planck Institute for Developmental Biology in Tübingen have developed an innovative method called siGOLD for the complete three dimensional reconstruction of neurons and their surrounding network. Antibodies coupled to gold particles are used to stain neuronal signaling molecules specific for one subset of nerve cells. siGOLD combines this molecular information with electron microscopy imaging, allowing a more complete understanding of how neurons communicate.

To understand how an organism's nervous system functions, a circuit map of all its neuronal connections, known as the connectome, can be generated. A connectome is similar to an electrical wiring diagram of a circuit board and includes information about all the wires of neurons (axons and dendrites) and how these connect to each other by synapses. Besides the wiring, neurons talk to each other through a diverse set of small signaling molecules such as neurotransmitters or neuropeptides. The type of small molecule produced by different types of neurons will determine how neurons influence each other.

To directly assign small neuropeptide molecules to neurons in a connectome in a high-resolution, high-throughput manner, Jékely and his team have developed a new method. Very thin slices of nervous tissue have to be imaged by high-resolution electron microscopy (EM). By combining these slices, the neurons can be followed throughout the sections.

The research team identified small neuropeptides suitable for EM. These neuropeptides are widely distributed throughout the nervous system and each occurs in a specific subset of neurons. The team developed antibodies that each recognize one neuropeptide. These antibodies can be coupled to tiny gold particles so that they are easily recognizable on EM sections as black dots. The scientists stained various sections with different antibodies, allowing the tagging of several different neurons. The team calls this method serial-multiplex immunogold, or siGOLD for short. siGOLD allows the complete three dimensional reconstruction of neurons to which the small neuropeptides have been directly assigned. It is also possible to reconstruct the partner neurons of these nerve cells, and obtain wiring diagrams that contain the distribution of neuropeptides in the circuit.

Jékely´s team worked with the marine annelid Platynereis dumerilii, which qualifies as a model due to its small size. "Size matters", says Dr. Jékely, "the Platynereis larva is about 12,000 times smaller in volume than the mouse brain, so it can be completely reconstructed by serial EM much faster". The widespread nature and functional importance of neuropeptides in animal nervous systems makes the antibody labeling approach used in siGOLD convenient and applicable in many organisms. "We are confident that siGOLD will also work in other organisms", says Shahidi, first author of the study "and we are rather excited by this prospect. Many neuropeptides in vertebrates, for example, enkephalin, are also related to the neuropeptides of the Platynereis nervous system."

The siGOLD approach enables the direct overlaying of information about small molecules present in neurons onto neuronal circuit maps and could be adapted to enrich connectome data with molecular information in many other organisms. 

Insights into the method

An understanding of how the nervous system functions requires not only the knowledge of precise anatomical structure of the neuronal connections but also of the molecules expressed by each neuron in the connectome. Since synapses are very small (in the nanometer range), the discernible examination of neuronal connections in a nervous system requires high-resolution, only obtainable by electron microscopy (EM). For EM imaging, nervous tissue has to be fixed, embedded in plastic resin, and sliced with a diamond knife into ultrathin sections, usually only 40 nm thick. The first author of the study, Réza Shahidi, sectioned an entire larva of the marine annelid Platynereis dumerilii into 5,000 sections. Each section is then imaged at a very high resolution, allowing the identification of all membranes and synapses. The neurons can be followed throughout the sections, allowing the reconstruction of entire neuronal circuits. This recontsruction does not yet contain information about the signaling molecules the neurons communicate with.

There are some techniques that allow researchers to assign small molecules to neurons at EM resolution. However, these techniques require the expression of special 'tags' in defined neurons by means of transgenesis, limiting the use of these techniques to one or a few markers per individual. Some of these technologies also lack optimal resolution and proper contrast for EM studies. Jékely and his team identified a diverse class of neuropeptides that survive the fixation and processing treatment for EM. These neuropeptides can be tagged by specific antibodies coupled to gold particles. Dr. Jékely's team demonstrated the utility of the siGOLD method by identifying and reconstructing over 80 neurons using 11 different antibodies on the Platynereis larval sections.


Original Publication:
Réza Shahidi, Elizabeth A. Williams, Markus Conzelmann, Albina Asadulina, Csaba Verasztó, Sanja Jasek, Luis A. Bezares-Calderón and Gáspár Jékely: A Serial Multiplex Immunogold Labeling Method for Identifying Peptidergic Neurons in Connectomes. Published December 15, 2015
Cite as eLife 2015;10.7554/eLife.11147
http://elifesciences.org/content/early/2015/12/15/eLife.11147

Contact:
Gáspár Jékely
Max-Planck-Institut für Entwicklungsbiologie
Tel.: 07071 601-1310
E-Mail: gaspar.jekely(at)tuebingen.mpg.de

Nadja Winter Press officer (Public Relations)
Phone: +49 7071 601- 444
E-Mail: presse-eb(at)tuebingen.mpg.de


Scanning electron microscopic image of a Platynereis larva. Such a larva was sectioned to 5000 sections and its neuronal circuits containing neuropeptides were reconstructed after siGOLD labelling. Credit: Réza Shahidi/Max Planck Institute for Developmental Biology

Scanning electron microscopic image of a Platynereis larva. Such a larva was sectioned to 5000 sections and its neuronal circuits containing neuropeptides were reconstructed after siGOLD labelling. Credit: Réza Shahidi/Max Planck Institute for Developmental Biology

Reconstructed neurons in a Platynereis larva. The neurons shown in color were reconstructed from electron microscopic sections and the neuropeptide signalling molecules contained within these neurons were identified using the siGOLD labelling method. Each color represents a different neuropeptide. Credit: Réza Shahidi/ Max Planck Institute for Developmental Biology

Reconstructed neurons in a Platynereis larva. The neurons shown in color were reconstructed from electron microscopic sections and the neuropeptide signalling molecules contained within these neurons were identified using the siGOLD labelling method. Each color represents a different neuropeptide. Credit: Réza Shahidi/ Max Planck Institute for Developmental Biology