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An integrated microprobe for the brain
Carolina Gutierrez Herrera & Antoine R Adamantidis
How do neurons and glial cells acting in
concert give rise to the high-level functions
of the brain, from coordinated information
processing to adaptive behaviors? Our ability
to answer this question has been limited by a
lack of integrated technologies for measuring the complexity of neural and non-neural
circuits. In this issue, Canales et al.1 describe
a tour-de-force approach for overcoming
this hurdle—an implantable fiber that combines several functions in a single device,
allowing monitoring of electrical activity,
optogenetic manipulation of cell function
and drug delivery for long-term experiments in freely moving animals (Fig. 1).
The new fiber represents a major leap toward
a probe that allows physiologically relevant
study of brain functions in a truly integrative fashion.
Neuroscientists possess a range of techniques for studying the brain, including
in vitro and in vivo electrophysiology, genetic
engineering, microscopy and brain imaging. But there are few devices that combine
multiple techniques—a necessity if we are
to develop systems-level descriptions of
brain structures and of the functional units
(neurons and glia) that produce, release and
recycle metabolites, membrane receptors,
neurotransmitters and neuromodulators.
The communications among cells in the brain
are extremely complex and depend on the
cells’ molecular equipment (e.g., membrane
receptors), on their input/ouput connectivity
maps and on their local environments (e.g.,
cells, fluid, metabolites). Multimodal devices
Carolina Gutierrez Herrera and Antoine R.
Adamantidis are at the University of Bern,
Department of Neurology, Inselspital University
Hospital, Bern, Switzerland. Antoine R.
Adamantidis is also at McGill University,
Department of Psychiatry, Douglas Institute,
Montreal, Quebec, Canada.
e-mail: [email protected]
would offer the possibility to both modulate
and measure these different determinants
of brain function with high spatiotemporal
resolution.
The different components of the probe
developed by Canales et al.1 are assembled
into a macroscopic preform that is heated
and pulled into a 200-fold smaller optical
fiber. The resulting diameter of the probe is
extremely small (~10–50 µm), allowing targeting of single cells in the brain. With such
a miniature format, extracellular spikes from
single or multiple neurons can be recorded
in freely moving animals, cells can be optogenetically manipulated by delivering light
through the optical fiber, and drugs can
be infused through the hollow channel—
either serially or simultaneously (Fig. 1).
By combining polymer and polymer-metal
materials having high mechanical flexibility, the authors succeed in minimizing tissue
damage due to the fiber’s motion, enhancing
its signal-to-noise ratio and enabling longterm use (up to months) in freely moving
animals.
As one example, the probe could potentially be applied to first monitor neuronal
activity during a specific behavior and then,
in a second step, to replay the recorded
neuronal firing pattern by optogenetic control of cells (made light sensitive by opsin
expression). Ultimately, such approaches
will help unravel the importance of specific
cells or circuits in higher brain functions
and behavior.
In addition, the fiber’s hollow core allows
local drug delivery. As extracellular molecules
are important modulators of neural activity,
simultaneous drug delivery and electrical
recording should facilitate identification of
the molecules and pathways responsible for
local modulation of circuits over longer time
scales. For instance, one could control receptor activation by changing the local concentration of agonists, antagonists, hormones,
peptides or metabolites and then evaluate the
effects of such perturbations on the activity
and plasticity of brain circuits. This would be
an interesting approach for studying hypothalamic circuits and their orchestration of
feeding behaviors upon modulation by circulating leptin, insulin and glucose2.
One limitation of the fiber developed
by Canales et al.1 is the lack of an optical
imaging capability. Imaging of brain cell
activity using calcium or voltage indicators has been accomplished with in vivo
two-photon microscopy at unprecedented
cellular and subcellular spatial resolution3.
This approach is restricted to a head-fixed
configuration and surface imaging of cortical circuits (to a maximum of 700 µm deep),
a limitation that has been overcome by the
development of small epi-fluorescence
Katie Vicari/Nature Publishing Group
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© 2015 Nature America, Inc. All rights reserved.
A multimodal fiber can both record and manipulate neural activity in mice.
Electrical activity
recording
Optogenetic control
H
O N
O N
H
N
+
N
O
O–
Drug delivery
Figure 1 The optical fiber–based microprobe can be implanted over long time periods on the skull of
freely moving rodents. It allows simultaneous recording of neural activity, optogenetic control and local
drug delivery in long-term experiments.
nature biotechnology volume 33 number 3 March 2015
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© 2015 Nature America, Inc. All rights reserved.
n e w s and vi e w s
microscopes (or micro-endoscopes) 4,5 and
fiber photometry 6,7. These implantable
technologies allow imaging of cellular or
synaptic activity, respectively, in deep brain
structures, including the hippocampus, cerebellum and hypothalamus of freely moving
animals. A recently described fiber optic–
based glass microprobe combines both electrical and optical imaging capabilities 8,9.
Although this probe is not suitable for longterm implantation in freely moving mice, it
has been successfully used for recording of
local field potentials and neuronal activity,
for optical imaging of fluorescent signals
deep in the brain and for photo-labeling
recorded cells.
The future development of multimodal
fibers in systems neuroscience largely
depends on advances in related fields, such
as materials science, electronics and wireless technologies. For example, the utility of
such probes could be further enhanced by
analytical devices that detect rapid changes
in the concentrations of neurotransmitters,
metabolites, glucose, hormones, neuropeptides, enzymes or pH in the vicinity of
the probe. This would further support our
understanding of nonsynaptic transmission or
peptidergic modulation of neural circuits10,
where classical detection technologies such as
microdialysis are limited by their low temporal resolution (i.e., sampling rates). As probes
for multimodal recording and control of the
spontaneous activity of neural and non-neural
brain cells continue to be improved, they will
undoubtedly open up a wide array of new
approaches to understanding the molecular
and cellular mechanisms that underlie brain
activity with exceptional spatial and temporal
resolution.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
1. Canales, A. et al. Nat. Biotechnol. 33, 277–284
(2015).
2. Kim, J.D., Leyva, S. & Diano, S. Front. Physiol. 5, 480
(2014).
3. Grienberger, C. & Konnerth, A. Neuron 73, 862–885
(2012).
4. Ziv, Y. et al. Nat. Neurosci. 16, 264–266 (2013).
5. Jennings, J.H. et al. Cell 160, 516–527 (2015).
6. Cui, G. et al. Nature 494, 238–242 (2013).
7. Gunaydin, L.A. et al. Cell 157, 1535–1551 (2014).
8. LeChasseur, Y. et al. Nat. Methods 8, 319–325
(2011).
9. Dufour, S. et al. PLoS ONE 8, e57703 (2013).
10.van den Pol, A.N. Neuron 76, 98–115 (2012).
Singling out blood development
Eva M Fast & Len I Zon
Analysis of gene expression in thousands of single cells generates a model of
the blood regulatory network.
The more we learn about the intricacies of
embryonic development, the more it seems
that populations of apparently similar cells
are in fact heterogeneous, with individual
cells developing at different rates than their
neighbors. Efforts to measure these differences have been hampered by technological
limitations, and most studies of transcription
in embryos have been carried out on pools
of cells, making it difficult to tease apart the
gene regulatory networks that control developmental processes. In this issue, Moignard
et al.1 describe an approach for inferring the
regulatory interactions of blood development
by assessing gene expression in thousands of
single cells and computationally reducing
these multidimensional datasets to direct
Eva M. Fast and Len I. Zon are in the
Department of Stem Cell and Regenerative
Biology, Harvard University, Cambridge,
Massachusetts, USA.
e-mail: [email protected]
260
interactions between genes. This powerful
analysis not only provides a global glimpse
of blood development but may serve as a
blueprint for future modeling studies based
on single-cell data.
Moignard et al.1 are among the first to assay
cellular differentiation using single-cell expression analysis over an in vivo time course2,3.
They begin by capturing 3,934 blood precursor cells from mouse embryos at four successive developmental stages, making this the
most comprehensive single-cell expression
study of organ development to date (Fig. 1a).
The cells are isolated by fluorescence-activated
cell sorting using the mesodermal marker Flk1
(Kdr) and the blood-specific marker Runx1.
Capturing the entire population with bloodforming potential is a pre­requisite for building a
comprehensive model of blood development.
Next, the expression levels of 42 transcription factors and marker genes related to blood
and endothelium are measured in each of the
3,934 cells by qRT-PCR. It is not surprising
that the resulting multidimensional data
set of >150,000 expression scores requires
extensive and sophisticated computational
analysis. This analysis yields a network that
models genetic interactions during blood
development using the Boolean rules AND,
OR and NOT, known as an asynchronous
Boolean network.
The computational analysis involves two
main steps. The first step aims to developmentally link the four Flk1+ cell populations
despite their being collected at different
time points and from different embryos.
Specifically, the authors expect that cells destined to become blood or endothelium will
follow different developmental trajectories.
These two populations of Flk1 + cells cannot
be resolved by conventional methods, such
as hierarchical clustering and principal component analysis. But application of a newly
developed computational approach based
on diffusion metrics, called diffusion maps,
succeeds in ordering all the sorted cells
more closely according to developmental
time (Fig. 1b).
One possible reason why standard methods fail to cluster the data is that Flk1 is a
very general marker. Flk1+ cells in the early
embryo have the potential to form both blood
and endothelial cells but also other mesodermal lineages, such as cardiac tissue. Moignard
et al.1 address this possibility by performing
RNA-seq analysis on pools of 50 cells from
each of the four time points, which allows
them to analyze genes that were not included
in the predefined qRT-PCR set used for the
single-cell analysis. The authors conclude
that most of the sorted single cells are indeed
destined to become blood, a precondition for
the subsequent analysis.
The second step, and the central part
of the paper, involves distilling the gene
expression states of the putative blood lineage into a regulatory network that models
blood development (Fig. 1c). Single-cell
expression states are transformed into regulatory interactions between genes through
a pioneering analytic method called the
single-cell network synthesis (SCNS) toolkit
(http://scns.stemcells.cam.ac.uk/) (Fig. 1c).
Briefly, gene expression is discretized in an
on-and-off pattern, and all existing expression
states are ordered in a state diagram based
on similarity. Incorporating the time variable
into these expression states allows the authors
to model potential genetic interactions as an
asynchronous Boolean network.
It could be argued that Boolean logic cannot capture the full complexity of biological
systems. Nevertheless, constructing a whole
gene network based on single-cell expression
volume 33 number 3 March 2015 nature biotechnology