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
Water forms 'spine of hydration' around DNA, group finds
26.05.2017 | Cornell University
How herpesviruses win the footrace against the immune system
26.05.2017 | Helmholtz-Zentrum für Infektionsforschung
Staphylococcus aureus is a feared pathogen (MRSA, multi-resistant S. aureus) due to frequent resistances against many antibiotics, especially in hospital infections. Researchers at the Paul-Ehrlich-Institut have identified immunological processes that prevent a successful immune response directed against the pathogenic agent. The delivery of bacterial proteins with RNA adjuvant or messenger RNA (mRNA) into immune cells allows the re-direction of the immune response towards an active defense against S. aureus. This could be of significant importance for the development of an effective vaccine. PLOS Pathogens has published these research results online on 25 May 2017.
Staphylococcus aureus (S. aureus) is a bacterium that colonizes by far more than half of the skin and the mucosa of adults, usually without causing infections....
Physicists from the University of Würzburg are capable of generating identical looking single light particles at the push of a button. Two new studies now demonstrate the potential this method holds.
The quantum computer has fuelled the imagination of scientists for decades: It is based on fundamentally different phenomena than a conventional computer....
An international team of physicists has monitored the scattering behaviour of electrons in a non-conducting material in real-time. Their insights could be beneficial for radiotherapy.
We can refer to electrons in non-conducting materials as ‘sluggish’. Typically, they remain fixed in a location, deep inside an atomic composite. It is hence...
Two-dimensional magnetic structures are regarded as a promising material for new types of data storage, since the magnetic properties of individual molecular building blocks can be investigated and modified. For the first time, researchers have now produced a wafer-thin ferrimagnet, in which molecules with different magnetic centers arrange themselves on a gold surface to form a checkerboard pattern. Scientists at the Swiss Nanoscience Institute at the University of Basel and the Paul Scherrer Institute published their findings in the journal Nature Communications.
Ferrimagnets are composed of two centers which are magnetized at different strengths and point in opposing directions. Two-dimensional, quasi-flat ferrimagnets...
An Australian-Chinese research team has created the world's thinnest hologram, paving the way towards the integration of 3D holography into everyday...
24.05.2017 | Event News
23.05.2017 | Event News
22.05.2017 | Event News
26.05.2017 | Life Sciences
26.05.2017 | Life Sciences
26.05.2017 | Physics and Astronomy