GFP producing inhibitory interneurons in
mouse brain tissue
in the sample that fluoresce, we can
see them, even when surrounded by
non-fluorescent tissue. The figure
above shows inhibitory interneurons
in mouse brain tissue. These cells are
genetically engineered to produce the
fluorescent molecule GFP intracel-
lularly, causing whole cells to glow
green. We can trace the fine morphol-
ogy of individual axons even though
they are deep in the tissue.
The combination of confocal micros-
copy with digital cameras for image
acquisition and the discovery of fluo-
rescent proteins like GFP turned mi-
croscopy from a qualitative to a highly
quantitative tool. The discovery of bio-
compatible fluorescent molecules, like
GFP, paved the way for engineered
sensors capable of fluorescing only
during neuronal communication or
‘firing’. This makes it easy to see which
neurons are active and when, both
temporal and spatial resolution. All
you need is genetically engineered liv-
ing brain tissue under the microscope!
Which is actually not so easy.
Translating neuronal conversations
You see, communication between
neurons consists of two components.
(1) An electrical charge that accumu-
lates at one end of the neuron can
rapidly traverse down the fine den-
drites and axon. (2) This triggers the
release of chemical messengers into
the synaptic cleft, the tiny junction
between communicating neurons.
This chemical component requires a
local and temporally restricted influx
of calcium ions at the point of contact
between the two neurons, which is a
smoking gun for synaptic transmis-
sion, aka neuronal communication.
With a great deal of knowledge about
the mechanics of both action poten-
tial generation and synaptic transmis-
sion scientists have been able to (as
is so often the case) modify proteins
from nature that are already involved
in neuronal communication. With a
little genetic tweaking, fluorescent
molecules like GFP can be attached to
these proteins in such a way that light
is only emitted from neurons during
either a local increase in calcium con-
centration or a change in their electric
charge which results from synaptic
transmission or action potential firing
respectively.
Being able to simultaneously image
the electrical and chemical activ-
ity of multiple connected neurons is a
dream of neuroscientists as it would
allow them to link morphology with
function when studying neuronal net-
works. When done well, direct visuali-
Differentially labeled cerebellum (Source:
open)
zation of neuronal communication can
grant huge insights into how networks
of neurons respond to stimuli, process
information and ultimately give rise to
aspects of behaviour and cognition.
So-called genetically encoded calcium
indicators (GECIs) have seen wide-
spread use in neuroscience, particu-
larly in studying sensory systems. In
a typical experiment, recording the
intensity of a fluorescent pulse over
time and from a single synapse is in-
dicative of how that synapse commu-
nicates, allowing neuroscientists to
watch neuronal communication, as it
happens, through the lenses of a mi-
croscope. In this way, GECIs report the
strength and frequency of a neurons
output from a synapse, but tells us
nothing about the source that drives
the neuron to communicate with its
neighbours in the first place. To obtain
a complete picture of how neurons
process incoming synaptic inputs and
respond (or don’t respond), a sensor
of the electrical component is needed,
a genetically encoded voltage indica-
tor or GEVI, and they’ve been a long
time coming.
Since the 1940’s the gold standard
procedure for recording the electri-
cal activity of neurons has been to
jam electrodes into brains or attach
them to single neurons. These two
approaches measure either the com-
bined electrical activity of groups of
neurons that are close to the elec-
trode, or the extremely precise electri-
cal changes of single neurons. Neither
case is optimal as you either gen-
eralize clusters of neurons to single
“units” or you attempt to understand
the entire brain one neuron at a time,
and there’s 86 billion of those suck-
ers. The potential of GEVIs therefore
lies in their ability to report the electric
activity of neurons visually allowing
researchers to discern the responses
of individual neurons within a network
of active neurons and thus visualize
their electrical conversations, if you
will. GEVIs are great indicators of the
inputs fed into a neuron due to their
ability to fluoresce even during small
changes in membrane potential and
thus report small synaptic voltage
changes.
So that’s two halves of one very cool
possibility, you see where this is go-
ing?
That’s right, if a neuron could geneti-
cally express both a GECI and a GEVI
simultaneously researchers would be
able to see which synapses stimulat-
ing a neuron caused it to fire, or, how
many synaptic inputs are integrated
Purkinje cells have big planar dendritic
trees
July 2018 | NEUROMAG |
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