Caren B. Les, email@example.com
MARTINSRIED, Germany – Researchers at Max Planck Institute of Biochemistry have obtained
three-dimensional images of the vesicles and filaments involved in the communication
among neurons in the brains of mammals. Using cryoelectron tomography, a microscopy
technique based on the ultrafast freezing of cells, they acquired 3-D images of
synapsis, the cellular structure in which the communication takes place.
“I found particularly interesting that so little was known
on the mechanisms of synaptic transmission, which is the basic process by which
neurons communicate with each other and with muscle cells, allowing nervous and
motor function,” said team leader Rubén Fernández-Busnadiego.
“One of the key aspects in the understanding of synaptic transmission is the
structural organization of the molecular complexes and organelles that perform this
task. Only electron microscopy has sufficient resolution to image these structures,
and only cryoelectron tomography can provide a three-dimensional picture of them
in their native environment.
“Cryoelectron microscopy is based on the cryofixation of
the specimens – such fast freezing that water molecules do not have time to
rearrange into crystals and remain in a solid but vitreous state. Thus, biological
samples can be observed in the electron microscope in their fully hydrated, native
state. Conversely, conventional sample preparation methods are based on sample dehydration.
Water, the main component of cells, is completely removed by this process, introducing
artifacts that affect image interpretation. In cryoelectron tomography, micrographs
of vitrified frozen-hydrated samples are recorded at different angles and then merged
computationally to reconstruct the original specimen in 3-D.”
He said samples thinner than 0.5 to 1.0 µm can be vitrified by
plunge-freezing; i.e., immersed directly into a cryogen at 77 K with a simple apparatus.
Thicker samples must first be frozen in special machines that apply jets of 2000-bar
liquid nitrogen (high-pressure freezing); then the samples are sliced into thin
sections (“cryosectioning”). He noted that this procedure is extremely
demanding because samples are very labile and must remain at temperatures below
~100 K. The resulting vitrified samples can be imaged in microscopes equipped especially
for that task and for allowing rotation of the sample inside the microscope column
for tomographic studies.
Shown is a tomographic synapse from the mammalian hippocampus. Note
the compression of the features resulting from the cryosectioning process. The scale
bar equals 100 nm. Photos courtesy of Rubén Fernández-Busnadiego, Max
Planck Institute of Biochemistry.
“The main advantages of cryoelectron tomography are that
samples are imaged in their native, fully hydrated conditions and that three-dimensional
information is provided. The main disadvantages are high cost and difficulty, compared
to conventional electron microscopy, and the radiation sensitivity of the samples,
which results in very noisy data, complicating interpretation. Also, the difficulties
of sample preparation limit the spectrum of samples that can be analyzed,”
For this study, the researchers treated synapses from the mammalian
brain cortex with various pharmacological agents, then vitrified and examined them
in the electron microscope. In the resulting cryoelectron tomograms, they observed
the fine ultrastructure of the synapse, in which hundreds of ~40-nm synaptic vesicles
accumulate to release neurotransmitters. They found that most of these vesicles
were linked to each other and to the active zone – the area of the neuronal
cell membrane where vesicle fusion actually occurs – by small filaments, and
that these filaments suffered important rearrangements in different functional states
of the synapse.
A tomographic slice of a mammalian synapse is depicted. Scale bar equals 100 nm.
In particular, the number of filaments that interconnected synaptic
vesicles was significantly reduced upon synaptic stimulation, indicating that they
play a role in vesicle mobilization and release. The study also showed that the
exact configuration of the filaments that tether the vesicles to the active zone
indicates the availability of the vesicles to be released. In summary, their results
indicate that these small filaments have an important function in synaptic communication.
“The main implications of our study may be that the current
ultrastructural picture of the synapse, obtained from electron microscopy studies
on dehydrated samples, needs to be revised, and that the short filaments present
in the presynaptic terminal play a very important role in synaptic transmission,”
Cryoelectron tomography is now a mature technique, he added, but
improvements in the hardware could push up its resolution limit. For this goal,
cameras of higher sensitivity and microscopes allowing more elaborate sample-tilting
geometries are needed, he said, noting that it also is important to advance in the
automation of the process to reach higher throughput. As for the software, improved
3-D reconstruction algorithms also will increase resolution.
This image represents a 3-D rendering
of a mammalian synapse. The synaptic vesicles (yellow) are extensively connected
to each other and to the cell membrane (purple) by short filaments (red and blue,
respectively). The material of the synaptic cleft is shown in light green and the
postsynaptic density, in orange.
“In terms of sample preparation, new methods for thinning
down thick vitrified samples – focused ion beam milling, cryoplaning –
are needed to expand the catalog of problems that can be addressed by cryoelectron
tomography. The technique is best suited for the study of thin – <0.5 µm
– cellular structures in high resolution. Given the current advance of different
thinning methods, this thickness limitation is likely to disappear in the future,”
The study is reported in the Jan. 11, 2010, issue of the Journal of Cell Biology.