S E C O M

application note

Neuroscience: Synaptic
connectivity in the songbird brain

application note

S E C O M

Sample courtesy of T. Templier & R.H.R. Hahnloser,
University of Zürich & ETH Zurichapplication note

Neuroscience: Synaptic
connectivity in the songbird brain
In order to study the neurobiological mecha-

then analyzed on an ultrastructural levelnisms of language acquisition, it is essential

using electron microscopy to visualize theto understand synaptic connectivity. Vocal

circuit context of the labeled structures.

learning, which is the modification of vocal
output by reference to auditory information,
iscriticalfor

spokenlanguage.

Despitevariations in the vocal learning phenotype,theneurallearningiscircuitry

necessaryconserved

infor

vocal

humans,bats,cetaceans, songbirds, parrots, and hummingbirds. The songbird brain in particular proves
to be a good example for studying neuralpathways because the vocal control areasthat correspond to the language learning
brain structures are segregated into separate
nuclei. This makes it relatively straightforward
to study synaptic connectivity by labeling theprojection neurons in the vocal control area
using neural tracers.

CORRELATIVE MICROSCOPY
The method of choice for understanding
synaptic connectivity is electron microscopy(EM). The advantage of EM is its high spatialresolution. EM allows for the detection ofsynapses by being able to resolve synaptic
vesicles and post-synaptic densities at highresolution. Electron microscopy however, has
the inherent drawback of extremely long
imaging times when tracing neurons, which isnecessary for studying projection neurons
over a long range. It is therefore difficult toanalyze

andassociatetheseobserved

synapses with specific neuron types using EM

In this study, fluorescent tracers are injected

methods only.

into neurons connecting to the HVC. The HVC

On the other hand, fluorescence microscopy

region is the main premotor area for vocal
production and is involved in song learningand production. Connections to the HVC are(FM) is a powerful technique for studying
large-scale interactions in the brain. The
multi-color

DELMIC BV | +31 (0)15 744 0158 | info@delmic.com | www.delmic.com

labellingcapabilities

combinedapplication note

S E C O Mwiththelarge

field -of-view

providestheflexibility to label many components overlong distances.
The combination of the labelling power overlarge distances of FM and the high resolution
structural information provided by EM thus
makes correlative microscopy the perfect tool
for studying synaptic brain connectivity.

METHODS
The sample was prepared in a similar mannerto the protocol described in detail in Oberti
et al. 1 Brain area X in the songbird brain waslocated and injected with Alexa Fluor 647.
After 5 days the bird was sacrificed and thebrain was removed. Areas containing the HVC
were localized and relevant sections wereimagedinaconfocalmicroscope.

Afterverifying the sections contained fluorescence,
theywerepost-fixedwith1%osmium

tetroxide and 1% uranyl acetate. Following
dehydration, the sections were embedded in
Durcupan ACM resin and cured for 48 h at
52°C. Once cured, the HVC was localized withthe light microscope, resected and attached

Figure 1 Projection neurons in songbird brain. Imaging was performed
using the SECOM platform (DELMIC) mounted on a Quanta 250 FEG SEM(FEI). A, B) Overlay of fluorescence and electron images with the injectedtracer clearly visible. SEM image shows the ultrastructure of the region ofinterest.

RESULTS
Figure 1 shows an automated overlay image
that was taken with the SECOM platform. The
results clearly show the successful labellingof the injected tracer. The EM image provides
the ultrastructural context to examine theto a blank resin block and serially sectioned

synapse in detail.

at a thickness of 60 -90nm and collected on

It is noteworthy that the EM staining used

ITO

on

here quenched the initial fluorescence of thecoverslips were treated with primary antibody

tracers. However, the tracer was able to beand secondary antibodies to visualize therelabeled after sectioning using fluorescent

Alexa Fluor 647 tracer.

antibodies, demonstrating that the protocol

Imaging was performed using the SECOM

preserved antigenicity well enough to allow

coatedcoverslips.

The

sections

platform mounted on a Quanta 250 FEG SEM.

on-section immunolabelling.

REFERENCES[1] Oberti, D., Kirschmann, M.A., & Hahnloser, R.H.R. “Correlative microscopy of densely labeled projection
neurons using neural tracers.’’ F ron t ie rs in ne u ro an at omy 4, 2010.

DELMIC BV | +31 (0)15 744 0158 | info@delmic.com | www.delmic.com

DELMIC B.V. is a company based in Delft, the Netherlands that
produces correlative light and electron microscopy solutions.
DELMIC's systems cater to a broad range of researchers infields ranging from nanophotonics to cell biology.
The SECOM platform is a fluorescence microscope made to be
integrated with a scanning electron microscope produced byDELMIC, that enables extremely fast correlative microscopy,
with the highest optical quality and overlay accuracy.
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