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.
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.
Réza Shahidi/Max Planck Institute for Developmental Biology
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.
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
Max-Planck-Institut für Entwicklungsbiologie
Tel.: 07071 601-1310
Press officer (Public Relations)
Phone: +49 7071 601- 444
Printable images can be obtained at the Public Relations Office. Please send a proof upon publication.
The Max Planck Institute for Developmental Biology conducts basic research in the fields of biochemistry, genetics and evolutionary biology. It employs about 360 people and is located at the Max Planck Campus in Tübingen. The Max Planck Institute for Developmental Biology is one of 83 research institutes that the Max Planck Society for the Advancement of Science maintains in Germany.
Nadja Winter | Max-Planck-Institut für Entwicklungsbiologie
Two Group A Streptococcus genes linked to 'flesh-eating' bacterial infections
25.09.2017 | University of Maryland
Rainbow colors reveal cell history: Uncovering β-cell heterogeneity
22.09.2017 | DFG-Forschungszentrum für Regenerative Therapien TU Dresden
At the productronica trade fair in Munich this November, the Fraunhofer Institute for Laser Technology ILT will be presenting Laser-Based Tape-Automated Bonding, LaserTAB for short. The experts from Aachen will be demonstrating how new battery cells and power electronics can be micro-welded more efficiently and precisely than ever before thanks to new optics and robot support.
Fraunhofer ILT from Aachen relies on a clever combination of robotics and a laser scanner with new optics as well as process monitoring, which it has developed...
Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.
A warming planet
Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.
The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...
Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...
Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!
When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...
19.09.2017 | Event News
12.09.2017 | Event News
06.09.2017 | Event News
25.09.2017 | Power and Electrical Engineering
25.09.2017 | Health and Medicine
25.09.2017 | Physics and Astronomy