REVIEWS

Improved tools to study astrocytes

Xinzhu Yu1,3, Jun Nagai1,3 and Baljit S. Khakh   1,2 ✉
Abstract | Astrocytes are a type of glial cell that tile the CNS. They interact with multiple cell
types, including neurons, glial cells and blood vessels, and are involved or implicated in brain
disorders. Progress has been made in understanding astrocytes, but the field lacks detailed
information concerning how they perform their multifarious functions, and how and when
they influence the operations of the neural circuits with which they interact. One recognized
bottleneck to progress has been the paucity of reliable tools with which to explore astrocytes
within the adult vertebrate CNS in vivo. However, improved tools for molecular, genetic,
morphological and physiological assessments have been developed recently or have been
adapted from their original purposes to study neurons and are now being used to systematically
document and interrogate astrocyte biology in vivo. These tools, their uses and limitations, and
the insights that they afford are summarized in this Review.

Gliotransmission
The release of transmitters
from astrocytes owing to
intracellular Ca2+ elevations,
through vesicular exocytosis or
by another regulated
mechanism.
1
Department of Physiology,
David Geffen School of
Medicine, University of
California Los Angeles,
Los Angeles, CA, USA.
2
Department of
Neurobiology, David Geffen
School of Medicine, University
of California Los Angeles,
Los Angeles, CA, USA.
3
These authors contributed
equally: Xinzhu Yu,
Jun Nagai.
✉e-​mail: bkhakh@
mednet.ucla.edu
https://doi.org/10.1038/
s41583-020-0264-8
Astrocytes tile the CNS and interact with multiple cell
types, including neurons, other glial cells and blood
vessels1. Important strides have been made in under-
standing astrocytes, especially their crucial roles in syn-
apse formation and removal2,3. However, the field lacks
detailed understanding of how these versatile cells per-
form their many functions, which vary depending on the
cells they contact, and how they influence the specialized
operations of the neural circuits in which they exist4–7.
A major bottleneck has been the lack of reliable and
selective tools with which to explore astrocyte functions
within the vertebrate adult CNS in vivo.
Given their now-​recognized potential contributions
to brain disorders8, interest has grown in studying fun-
damental astrocyte biology and evaluating the causa-
tive and correlative roles of astrocytes in the operation
of neural circuits, as well as astrocytic contributions to
behaviour and disease or disease-​related phenotypes. In
the astrocyte field, as in other areas of biology, global
and conditional gene-​deletion mouse models have
provided powerful means to explore the functions of
these cells. Furthermore, tools to monitor and manipu-
late cells precisely in vivo that are now broadly advanc-
ing neuroscience9–11 have also found applications in
studying astrocytes. Major long-​standing questions
concerning whether, how and when astrocytes contri­
bute to the functions of the nervous system12,13 are now
being addressed with methods that more directly permit
manipulation and interrogation of astrocytes.
A saying attributed to Sydney Brenner, one of the
giants of biology, is that, “Progress in science depends on
new techniques, new discoveries and new ideas, prob-
ably in that order”14. This saying is apropos for astro-
cytes: the use of new techniques and tools has resulted in
unexpected discoveries and testable hypotheses regard-
ing these fascinating cells, which, for far too long, have
been the neglected, less glamorous, siblings of neurons.
Here, we focus on recent studies with newer types of
tools that have been deployed in vivo. We explore four
astrocyte-​relevant topics spanning the pertinent gamut
of biology — ranging from molecules, cells and circuits
to behaviour. We start with a brief introduction to the
key challenges in each topic and then describe recently
developed tools used to address them, finishing each
section with some notes of caution and further con-
sideration where necessary. One overriding emergent
theme is the need to choose the tool after carefully con-
sidering the specifics of the question or questions being
explored; these types of decision will need to be made
on a case-​by-case basis.
Although we do not discuss the evidence for or
against gliotransmission in depth (reviewed elsewhere15–17),
we do discuss some recent tools that have been used in
studies of the reported phenomenon. We do not consider
astrogliosis, because at this juncture it is hard to establish
a clear definition for the process, and also because it has
been excellently reviewed18. In the interests of space, we
have concentrated mainly on work performed in the past
5 years in adult mice.
Unmasking molecules and mechanisms
The challenge. To perform hypothesis-​driven exper-
iments with astrocytes, it is valuable to have compre-
hensive profiles of their gene and/or protein expression.
Such unbiased data are also useful to shed light on com-
plex phenomena and to give initial direction to explora-
tory experimental work. Several methods have been used
to uncover astrocyte transcriptional and translational

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Immunopanning
A method of cell purification
that uses cell-​type-specific
antibodies immobilized
to a solid surface (such as
a cell culture plate) to
immunoprecipitate
specific cell populations.
profiles, including earlier evaluations using cultured
astrocytes and more recent in vivo assessments using
designer tools and astrocyte-​selective gene targeting
in adult mice (Tables 1,2).
Molecular profiling of isolated astrocytes. The trans­
crip­tomes of purified astrocytes can be assessed
using fluorescence-​a ctivated cell sorting (FACS),
immunopanning19 and magnetic-​activated cell sorting in
combination with reporter mouse lines and antibodies
against astrocyte-​specific molecules. Astrocytes from
various brain regions, developmental stages and disease
conditions have been studied in this way.
Soon after landmark explorations with astrocyte
cultures20, an antibody against the astrocytic glutamate
transporter GLT1 and transgenic mice overexpressing
green fluorescent protein (GFP) under the control of
the human glial fibrillary acidic protein (GFAP) pro-
moter (GFAP-​GFP reporter mice) were used to docu-
ment the transcriptional profiles of cortical astrocytes
from 10–12-week-​old mice21, representing the first
analyses of adult astrocyte gene expression from a
defined brain area. By comparing the transcriptional
profiles of GLT1+;GFAP-​GFP+ astrocytes with those of
GLT1+;GFAP-​GFP– astrocytes, the authors found sub-
tle differences in gene expression associated with GFAP
expression, suggesting that GFAP may be of limited utility
as a universal astrocyte marker.
Not long thereafter, forebrain astrocytes were iso-
lated using FACS from mice expressing enhanced GFP
(EGFP) under the control of the human S100 calcium-​
binding protein-​β (S100β) gene promoter (S100B-​EGFP
reporter mice), from postnatal day 1 (P1) to P30 and
for more than 20,000 genes analysed with microarrays22.
From this study, Aldh1l1, which encodes aldehyde dehy-
drogenase family 1 member L1 (ALDH1L1), emerged as
a pan astrocyte marker and was confirmed with Aldh1l1-
EGFP reporter mice that fortuitously already existed23.
The RNA of cortical astrocytes from the Aldh1l1-EGFP
mice was sequenced, providing a platform for analys-
ing and comparing transcriptomes in the brain24. Using
such methods, alterations in the gene expression pro-
files of purified astrocytes with ageing, ischaemic stroke
and neuro­inflammation have been documented25,26, and
these data have been used to glean deeper insights into
possible underlying mechanisms and implications27.
SOX9 is a more recently identified astrocyte-​specific
nuclear marker that identifies a population of astro-
cytes that overlaps highly with those identified by GLT1
expression28. This merits specific mention, because
SOX9 could be useful to isolate nuclei from post-​
mortem human brain tissue to assess astrocytes in
pathophysiology. An innovative intersectional approach
in which astrocytes isolated from Aldh1l1-EGFP mice
were grouped depending on their expression of cell-​
surface markers was used to document astrocyte

Table 1 | online database resources of astrocyte transcriptomes and proteomes

Name (website) Technique species (line) Ages Brain regions refs
Brain RNA-​seq RNA-​seq Mouse (Aldh1l1-EGFP) P7 Cortex 24

Human a
17–20 GW (fetal), Cortex 30

8–18 years (juvenile),

21–63 years (adult)
Adult Astrocyte RNA-​seq Mouse (RiboTag × Aldh1l1- P63–P82 Cortex, striatum and 39,40

RNA-Seq Explorer Cre/ERT2) hippocampus

LC–MS/MS Mouse (Aldh1l1-EGFP) P30 Striatum and 40

hippocampus
Astrocyte Aging RNA-​seq Mouse (RiboTag × Gfap-​Cre) 4 months, 2 years Visual cortex, motor 43

Transcription cortex, hypothalamus

and cerebellum
DropViz scRNA-​seq Mouse P60–P70 9 brain regions 53

Mouse Brain Atlas scRNA-​seq Mouse P12–P30, 6 weeks 19 regions of central, 52

and 8 weeks peripheral and enteric

nervous system
SCope scRNA-​seq Drosophila melanogaster 0, 1, 3, 6, 9, 15, 30 Brain 194

and 50 days
Mouse P21–P31 Somatosensory cortex 50

and hippocampus
P56–P80 Hypothalamus 195

snRNA-​seq Human 20–51 years Visual cortex, frontal 196

cortex and cerebellum

Allen Brain Atlas, scRNA-​seq Mouse P53–P59 Cortex and thalamus –
RNA-​seq data
snRNA-​seq Human 24–66 years Cortex and thalamus –
Macaque 2.3–17.9 years Thalamus –
Aldh1l1, encodes aldehyde dehydrogenase family 1 member L1; Cre, Cre recombinase; EGFP, enhanced green fluorescent protein;
ERT2, oestrogen receptor 2; GFAP, glial fibrillary acidic protein; GW, gestational weeks; LC–MS/MS, liquid chromatography-​tandem
mass spectrometry ; P, postnatal day; RiboTag, haemagglutinin-​tagged ribosomal protein; RNA-​seq, RNA sequencing; scRNA-​seq,
single-​cell RNA sequencing; snRNA-​seq, single-​nucleus RNA sequencing. aImmunopanning was used to isolate human astrocytes.

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Table 2 | Transgenic mouse lines and viral vectors for targeting astrocytes
Line or virus Promoter or gene locus Proportion expression in refs
of astrocytes astrocytes relative
targeteda to other cellsb
Mouse lines
Gfap-​Cre Mouse Gfap High Low 39,197,198

GFAP-​Cre Human GFAP Very low Very low 60

GFAP-​Cre/ERT2 Human GFAP High Low 59,199

GFAP-​tTA Human GFAP Moderate Moderate 200,201

Aldh1l1-Cre/ERT2 Aldh1l1 Very high Very high 39,66

Slc1a3-Cre/ERT2 Slc1a3 (which encodes GL AST) Moderate Low 39,62,63

S100B-​Cre Mouse S100B Moderate Moderate 202

Slc6a11-Cre/ERT2 Slc6a11 (which encodes GAT3) Moderate Moderate 39

Gjb6-Cre/ERT2 Gjb6 (which encodes CX30) Very high Moderate 39,63

Fgfr3-Cre/ERT2 Fgfr3 High Low 203

Viral vectors
AAV5, AAV8 and AAVrh43 Gfa2 Very high High 204,205

AAV5, AAV9 and AAV-​PHP.eB GfaABC1D Very high Very high 41,45,46,
71–74,79,206

AAV5, AAV8 and AAV9 Rat Aldh1l1 Low Very low 78

AAV-​DJ Human ALDH1L1 Low Very low 77

Please note that this table is a summary: readers are strongly encouraged to consult the original articles cited herein and examine
the relevant data sets carefully before making a choice about which reagent to use. AAV, adeno-​associated virus; Aldh1l1, encodes
aldehyde dehydrogenase family 1 member L1; Cre, Cre recombinase; ERT2, oestrogen receptor 2; GFAP, glial fibrillary acidic
protein; S100β, S100 calcium-​binding protein-​β; tTA , tetracycline-​controlled transactivator protein. aUsed as a convenient
description to convey the proportion of targeted astrocytes in relation to the total number of astrocytes in the region of interest.
b
Used as a convenient description to convey the proportion of astrocytes relative to other cells such as neurons.

Bacterial artificial
chromosome (BAC)
transgenic
Describing a transgenic
organism generated by
random but stable integration
of large segments of DNA
(up to 300 kb) from BAC
vectors into the genome.
Tamoxifen
A selective oestrogen receptor
modulator that specifically
activates tamoxifen-​inducible
Cre recombinase (for example,
Cre/ERT2) and results
in the shuttling of the
Cre recombinase into
the nucleus, where the
recombination occurs.
diversity between brain areas and to identify analogous
cell subtypes in gliomas29.
Immunopanning19 was recently adapted to isolate
astrocytes30. The method is useful when fluorescent
reporter mouse lines are not available and/or in model
species for which genetic modifications are not routine.
Astrocytes from fetal, juvenile and adult human brain
tissue were immunopanned with antibodies against
hepatocyte cell adhesion molecule (HEPACAM), a cell-​
surface protein expressed by human astrocytes. RNA
sequencing (RNA-​seq) identified >2,000 genes differen-
tially expressed in fetal versus mature human astro­cytes
and revealed functionally relevant molecular diff­erences
between human and mouse astrocytes 30 that were
anticipated from careful neuroanatomical work31.
A similar approach called magnetic-​activated cell
sorting, which conjugates cell-​type-specific antibodies
to magnetic beads, also enables isolation of different
cell types from postnatal mice32,33, is relatively straight­
forward to implement and has been used for RNA-​seq34.
Magnetic-​activated cell sorting32–34 preserves astrocyte
morphology better than does FACS, does not involve cell
culture and overcomes some of the key limitations of
other methods that use single cells.
Molecular profiling of astrocytes in vivo. Newer strat-
egies have assessed translated mRNAs in specific cell
populations of the brain without physical cell dissociation.
One approach, termed translating ribosome affinity
purification (TRAP)35,36, genetically targeted expression
of an EGFP-​tagged ribosomal protein L10a, which can
be harvested using EGFP-​specific antibodies to enable
sequencing of ribosome-​associated mRNAs. Multiple
bacterial artificial chromosome (BAC) transgenic mouse
lines exist to target TRAP to neurons, oligodendro-
cytes and astrocytes (bacTRAP lines). An Aldh1l1 bac-
TRAP line was used to identify brain-​region-dependent
transcriptional differences in astrocytes across the
mouse lifespan37 (see following section for discussion
of mouse lines used).
RiboTag is a related method to isolate ribosome-​
associated mRNAs from any cell type with an available
Cre driver38. A BAC transgenic mouse line was generated
to enable cell-​type-specific expression of haemaggluti-
nin (HA)-tagged RPL22 (a ribosomal protein) upon Cre
recombinase-​mediated deletion of an upstream floxed
STOP cassette38. RPL22HA incorporates into polyribo-
somes and can be immunoprecipitated with reliable
HA-​targeting antibodies to yield actively translating
mRNAs from required cell populations. This RiboTag
mouse line has been crossed with a tamoxifen-​inducible
Aldh1l1-Cre/ERT2 mouse line for RNA-​seq of mature
astrocytes from the cortex, striatum and hippocam-
pus39,40 (Fig. 1), and with Gfap-​Cre mice to enable analy-
sis of molecular changes in astrocytes after spinal cord
injury41 and in a mouse model of multiple sclerosis42.
Adult and aged astrocyte transcriptomes from dif-
ferent brain regions were also obtained using RiboTag
mice crossed with Gfap-​Cre mice43. With ageing, genes
related to synapse elimination and inflammatory path-
ways were upregulated, whereas genes related to choles-
terol synthesis were downregulated. Recently, RiboTag

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a Aldh1l1-Cre/ERT2 × RiboTag mice b Top 500 astrocyte-enriched genes in each area

Cortex
128

40 49

283

78 69
mRNA from
all cells
Striatum 99 Hippocampus
Bulk RNA-seq

Anti-HA antibodies
Common genes
Magnetic beads Core functions:
• Potassium homeostasis
Magnetic IP • Neurotransmitter uptake
• Neurotransmitter degradation
• Gap junction signalling
mRNA from • Fatty acid oxidation
astrocytes • Serine biosynthesis
• Neuroinflammation signalling
Astrocyte RNA-seq

Fig. 1 | Transcriptomic profiling of astrocytes from cell-​specific rNA sequencing in adult mice. a | RiboTag mice
expressing the ribosomal subunit RPL22 with a haemagglutinin (HA) tag (RPL22HA) in a Cre-​dependent manner from the
Rosa26 locus were crossed with astrocyte-​specific Aldh1l1-Cre/ERT2 mice. The adult offspring that were positive for both
alleles were treated with tamoxifen to drive the expression of RPL22HA selectively in astrocytes. Subsequently , different
brain areas were dissected and the total mRNA from a ‘soup’ of all the included cells was sequenced (bulk RNA sequencing
(RNA-​seq)). Astrocyte-​specific RNA was also extracted using a well-​established immunoprecipitation (IP) technique, in
which magnetic beads were bound to HA epitopes of RPL22HA and its associated mRNA , which was then subjected to
sequencing (astrocyte RNA-​seq). b | In the example shown, the overlap between the top 500 genes showing astrocyte
enrichment in the cortex, hippocampus and striatum is shown, as are the numbers of genes specifically expressed in each
area. Functional analysis using Gene Ontology and Ingenuity Pathway Analysis of the 283 shared genes suggests the core
functions of astrocytes in these three brain areas. Original data are published in refs39,40 and are available at Adult
Astrocyte RNA-​Seq Explorer. Aldh1l1, encodes aldehyde dehydrogenase family 1 member L1; ERT2, oestrogen receptor 2.

Adeno-associated virus
(AAV) vector
A vector based on an AAV,
composed of exogenous DNA
(up to 5 kb) flanked by two
145-nucleotide-​long inverted
terminal repeats.
was incorporated into viral vectors, including a floxed
RiboTag adeno-​a ssociated virus (AAV) vector and an
astrocyte-​specific RiboTag AAV that can be deployed
relatively easily in control and disease-​model mice to
investigate astrocyte function44–46.
Molecular profiles of astrocytes at a single-​cell level.
Analyses of single-​cell transcriptional profiles have
unmasked cellular diversity within and between organs,
species, developmental stages, and health and disease,
with unparalleled clarity47–49. Briefly, in single-​cell RNA-​
seq or single-​nucleus RNA-​seq, individual cells or cell
nuclei, respectively, are isolated from a given tissue (such
as a particular brain area) and sequenced. Subsequent
analysis can yield information on complex and/or rare
cell populations, the developmental trajectories of cell
lineages and/or the functions of single cells in their
environment.
Single-​cell transcriptomic analyses have advanced
our understanding of astrocyte diversity. For example,
mouse cortical astrocytes form two subclasses distin-
guished by differential expression of Gfap and Mfge8
with different laminar locations and morphologies50.
Similarly, using a form of single-​cell RNA-​seq that lim-
its transcriptional alterations typically caused by con-
ventional cell preparations, amygdalar astrocytes were
classified into three subpopulations with distinct spatial
distribution and signalling pathways: one subpopulation
was enriched for genes involved in cell adhesion and
RNA processing, and another was enriched for genes
involved in apoptosis regulation and cell proliferation51.
Two projects together used RNA-​seq of more than 1
million single cells to generate a detailed taxonomy of
the cellular architecture of the entire mouse nervous sys-
tem52,53. Seven molecularly distinct astrocyte types were
identified, with discrete spatial distributions that corre-
lated with those of glutamate and glycine signalling52.
Improved methods for single-​cell sequencing appear
frequently and their use will probably accelerate, ena-
bling the field to map, in detail, the molecular profiles of
astrocytes within different brain areas, and to compare
them with astrocytes in other brain areas and with other
cell types of the body54.
Considerations. Studies with purified astrocytes have
reported valuable information, but there are several
aspects to consider.
First, a necessary step in assessing astrocytic profiles
is to purify these cells without introducing molecular
alterations in the process. It seems implausible that iso-
lated astrocytes are unaltered relative to their in vivo
counterparts, given the stress of cell dissociation and

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Tetracycline
sorting procedures. In addition, the complete transcrip- areas from which astrocytes are assessed are not known
An antibiotic used to either tional landscape of astrocytes in vivo may be impossible or accounted for experimentally or computationally.
repress (Tet-​Off) or activate to capture with purified astrocytes, because the latter do Nonetheless, early FACS and cell-​culture studies mark-
(Tet-​On) gene expression in not retain the archetypal bushy morphology of astro- edly advanced the field and promoted molecular and
a Tet system. Its derivative,
doxycycline, is also widely
cytes in vivo (Fig. 2). For the most highly expressed genes, mechanistic investigations of astrocyte biology, hand in
used. the effects of astrocyte dissociation and purification may hand with physiology.
be inconsequential; however, for functionally important Complementary tools will be needed to exploit the
genes with low expression, even subtle changes could be available single-​cell sequencing data to test specific
relevant for downstream analyses. hypotheses in silico, in vitro or in vivo. Furthermore,
Second, it is necessary to be confident that the pro­ more research is necessary to establish where and how
cedures are astrocyte-​s elective. Depending on the astrocyte populations are distributed in the brain and
procedures employed, certain subpopulations of astro- whether they have specific locations in relation to anato­
cytes may be preferentially selected, because many mically defined pathways, local microcircuits and areas.
reporter lines and antibodies are not completely specific As an example, chordin-​like protein 1-expressing astro-
and do not target all astrocytes ubiquitously (see the next cytes are enriched in layer II/III of the visual cortex,
section). where they regulate the plasticity of thalamocortical
Third, a further consideration when interpreting synapses58. Such avenues are probably best explored by
some past studies is that multiple brain areas seem combining gene expression analyses in anatomically
to have been pooled during isolation steps. As astro- intact preparations.
cyte transcriptomes vary depending on the brain Mass spectrometry-​b ased proteomics of FACS-​
region29,40,42,43,55–57, such pooling could potentially influ- isolated astrocytes from the striatum and the hippocam-
ence the interpretation of the resulting data, if the brain pus of Aldh1l1-EGFP mice has revealed similarities and
differences between brain areas that were consistent with
data gathered using RNA-​seq. However, mass spectrom-
Scale Methods etry detected fewer proteins than were predicted to be
Tissue • Multicolour intracellular expressed by RNA-​seq. To comprehensively address
~1 mm iontophoresis (dye-filling) these inconsistencies without bias, advances in pro-
• Transgenic mouse reporter lines
• Viral vector-mediated reporter teomics will be necessary to examine protein expression
expression in astrocytes that retain their complex morphology. As
• Plasmid vector-mediated an example of the mismatch between gene expression
reporter expression
and function, nearly all astrocytes show intracellular
increases in Ca2+ in response to extracellular ATP, but
RNA-​seq data suggest that expression of transcripts
Major encoding ATP P2Y G protein-​c oupled receptors
Cellular • Immunohistochemistry
~50 μm branch (GPCRs) is very low39,40,44–46. Detailed proteomics of the
• Intracellular iontophoresis
(dye-filling) various compartments that comprise complex astrocyte
Soma • Transgenic mouse reporter lines morphology (Fig. 2) in different brain regions will bring
• Viral vector-mediated reporter
expression us closer to uncovering physiological functions.
Territory
• Plasmid vector-mediated
reporter expression Genetic targeting
The challenge. Mouse lines for targeting and manip-
ulating astrocytes in vivo would be useful. However,
Subcellular Branchlets and leaflets • Intracellular iontophoresis
~0.1–1 μm (dye-filling) there is no ‘perfect’ mouse line for targeting astrocytes,
• Transgenic mouse reporter lines and every available line has its own limitations — some
• Viral vector-mediated reporter minor, others considerable. The properties of key astro-
expression
• Plasmid vector-mediated cyte mouse lines are summarized in Table 2 with a focus
reporter expression on those expressing Cre recombinase, because such lines
can be used in conjunction with other Cre-​dependent
Fine processes in territory mice or viruses to express designer reporters, sensors
and actuators.
Synaptic • Electron microscopy
~10–100 nm • Super-resolution microscopy
• NAPA Cre lines and drivers. When choosing a genetic strategy
to express or delete genes in astrocytes, two factors must
be considered: the proportion of astrocytes in the region
of interest that are targeted (which we term efficiency),
and the extent to which other cell types are excluded
from targeting (which we term specificity).
Much effort has been devoted to optimize both
Fig. 2 | Methods to study astrocyte morphology. The cartoon illustrates the scales at parameters with the use of GFAP, ALDH1L1, S100β,
which astrocyte morphology can be studied, with the methods currently in use listed to GLAST and GLT1 reporter and Cre lines. To date, sev-
the right. Please refer to the main text for a discussion of the strengths and weaknesses of eral Cre-​dependent or tetracycline-​controllable trans-
each individual approach. NAPA , neuron–astrocyte proximity assay. genic mouse lines have been applied to manipulate gene

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Microglial reactivity
A microglial activation
response to tissue damage or
pathological insults, to mediate
inflammatory responses.
expression through promoters of astrocyte-​marker genes
(Table 2). However, because the expression of astrocyte
genes depends on the brain region, developmental stage
and physiological condition, and most astrocyte-​marker
genes are often active in neural progenitor cells and
detected in non-​astrocytic cell types, one has to choose
and characterize each system based on experimental
need. For example, transgene expression driven by the
human GFAP promoter does not occur for all astrocytes
and is often detected in large numbers of neurons59,60.
Mouse Gfap-​Cre lines are better than those that use
the human GFAP promoter, but neuronal expression is
expected and reported for both lines61. Slc1a3-Cre/ERT2
(also known as GLAST) lines label almost 100% of
Bergmann glia in the cerebellum, but only about
20–30% of astrocytes in the cortex, hippocampus and
striatum39,62–65.
The best available transgenic model to target astro-
cytes across the entire brain is the tamoxifen-​inducible
Aldh1l1-Cre/ERT2 mouse line, which permits temporal
control of gene expression and has demonstrated high
astrocyte specificity and efficiency when induced in
adulthood39,66. However, Aldh1l1, like the other marker
genes (including Gfap and Slc1a3), is expressed in
postnatal neurogenic zones67 and thus early postnatal
induction of this line will affect other cells.
Viral methods. Although powerful, gene targeting with
transgenic approaches affects multiple brain regions
as well as some peripheral organs, making it difficult
to investigate local circuit modulation and to interpret
behavioural outcomes. One alternative approach to
overcome this limitation is to use viral vectors for gene
delivery to astrocytes in specific brain regions.
Lentiviruses and AAVs are the most commonly used
viral vectors in astrocyte biology and have several attrac-
tive properties compared with mouse lines, including
their region specificity and flexibility of application in
different species and disease models. Lentiviruses can
carry larger cargoes (up to about 8 kb) than can AAVs
(up to about 5 kb), but AAVs do not integrate into the
genome and can be concentrated to higher titres for
improved efficiency. Some viruses display natural selec-
tivity for astrocytes in vivo68,69 and astrocyte specificity
can be enhanced by combining with astrocyte-​specific
promoters (Table 2).
The GfaABC1D promoter (681 bp) was derived from
the human GFAP promoter gfa2 (2.2 kb). It is about twice
as active as gfa2, and its smaller size is better suited to
viral vectors70. The expression of cargo genes under the
GfaABC1D promoter in astrocytes is highly efficient
and specific in many studies41,45,46,71–76. However, leaky
expression in other cell types has been reported with
Cre-​mediated recombination39,45.
Notably, the Aldh1l1 promoter preferentially drives
neuronal expression in many regions of the rodent
brain when used in viral vectors 77,78. The recently
evolved AAV variant PHP.eB with the GfaABC 1D
promoter enables widespread and efficient transduc-
tion in astrocytes of the entire brain through intra-
venous injection 79, offering an alternative strategy
to the use of transgenic mouse lines that avoids any
microinjections into the brain, but, of course, it is not
brain-​region-specific.
Considerations. It is worth noting that viral tropism may
vary depending on development, brain region, mouse
genetic background, cargo and administration routes.
Until the precise mechanisms by which viruses target
specific cell types are better elucidated, careful character-
ization and control experiments will be needed for each
study to empirically assess efficiency and specificity on
a case-​by-case basis.
At this time, the most reliable mouse line seems to
be Aldh1l1-Cre/ERT2 (refs39,66), and the most reliable
viral approach is the use of AAV2/5 with the GfaABC1D
promoter41,45,46,68,69,71–76, but better tools will probably be
developed before too long.
As with all gene-​targeting strategies, one needs to
assess astrocyte and microglial reactivity. This has not so
far proved a major problem, but parallel controls will
still be necessary — especially in brain areas not yet
studied with such approaches. Furthermore, depending
on the question, even targeting strategies that are not
astrocyte-​selective can be useful — for example, when
mosaic expression of a reporter such as GFP is needed
for morphological studies and neuron expression can
simply be ignored in downstream analyses, because neu-
rons and astrocytes can easily be discriminated based on
the expression of GFP.
Exploring morphology
The challenge. A key task is to reliably measure, and
track changes in, astrocyte morphology. A stereotypical
mature astrocyte in the mammalian brain is remarkably
complex, with an oval soma and several major branches
that each split into secondary and tertiary branchlets,
which in turn ramify into fine processes called leaflets
(Fig. 2). The elaboration of this intricate morphology
emerges postnatally and establishes boundaries between
neighbouring mature astrocytes, a phenomenon called
‘tiling’.
Astrocyte morphology has long been recognized to
be non-​uniform. For example, protoplasmic astrocytes,
which reside in the grey matter, are highly morpholog-
ically complex, whereas fibrous astrocytes in the white
matter are less complex. Numerous studies have con-
firmed that astrocyte morphology is heterogeneous
within and across brain regions, and dynamic in both
physiological and pathological states. We review the
approaches available to document the complexity and
track the plasticity of astrocyte morphology (Fig. 2).
We do not consider immunohistochemistry of
GFAP as this underestimates astrocyte complexity80,81
and is not detectable in many astrocytes. Furthermore,
although several astrocyte markers, such as S100β,
glutamate transporters (GLAST and GLT1) and glu-
tamine synthetase (GS), can reveal details of astro-
cytes, these markers essentially show the distribution
of the cognate protein and not the cellular architecture.
Furthermore, some marker proteins are not specific for
astrocytes in some brain regions; for example, S100β
also identifies oligodendrocytes and oligodendrocyte
progenitor cells, and GLT1 is detectable in some neurons.
www.nature.com/nrn

Iontophoresis
An approach to deliver charged
molecules (such as fluorescent
dye) into cells by applying
electric current.
Ezrin
A protein that functions
as a linker between the
plasma membrane and
the actin cytoskeleton.
Lck-GFP
A green fluorescent protein
(GFP) tagged with a membrane-​
targeting sequence of Lck (from
Src tyrosine kinase).
Spaghetti monster
fluorescent proteins
Modified fluorescent proteins
with multiple epitope tags that
can be targeted using highly
specific antibodies.
Nature Reviews | Neuroscience
Reviews
Visualizing astrocyte morphology with intracellular observed to be dynamic and regulated by intracellular
dye-​filling. Astrocytes in brain sections fixed with short Ca2+ and metabotropic glutamate receptors89.
exposure to fixative have been visualized by delivering Postnatal electroporation of plasmids encoding
fluorescent dyes into them through a sharp electrode, cytosolic and membrane-​tagged fluorescent proteins
in an iontophoresis-​based protocol80; the dye diffuses was used to label astrocytes in mouse visual cortex,
into the volume of the cell, revealing total astrocyte to measure and track the volume and complexity of
morphology. The method can be readily combined astrocytes and their processes. This approach revealed
with immunohistochemistry to confirm cell identity experience-​dependent increases in astrocytic complexity
and characterize protein expression in different cellular between P7 and P21, mediated by neuroligin–neurexin
compartments. The dye most frequently delivered in this interactions90.
way is lucifer yellow, a bright fluorophore that is easily Lck-​GFP has been used to examine the effects of
imaged with confocal microscopy. Using this method, brain-​derived neurotrophic factor (BDNF) on astro-
subtle differences were identified in the morphology of cyte morphology34. In another study, its expression was
striatal and hippocampal astrocytes40. Remarkably, when induced through intracerebroventricular injections of
two adjacent hippocampal astrocytes were filled with virus at P0, to sparsely label astrocytes for morphological
differently coloured dyes, they formed discrete, non-​ and anatomical examination at different postnatal time
overlapping territories during postnatal development82 points91. The use of spaghetti monster fluorescent proteins
that remained intact following injury 83. could be powerful and improve signal-​to-noise ratios
Although intracellular dye-​filling is time consuming further for sparse labelling92, although they would
and can sample only limited numbers of cells at a time, require antibody labelling and may not be appropriate
it nevertheless provides detailed, high signal-​to-noise for experiments such as those requiring live imaging.
images with, by definition, single-​cell resolution. It is
also relatively straightforward to implement84, is reliableVisualizing finer astrocyte morphology. Astrocytes
and, owing to the use of lightly fixed tissue (short expo- have extensive, ramified fine processes that measure
sure to fixative), preserves in vivo morphology. Lucifer on a scale of tens of nanometres in thickness; together,
yellow intracellular iontophoresis has elegantly shown these processes constitute up to 95% of total cell vol-
how astrocyte morphogenesis is compromised by neo- ume93. Therefore, to resolve the complete structure of
natal exposure to general anaesthesia via a mechanism astrocytes, evaluations with submicrometre resolution
involving ezrin and intracellular Ca2+ signalling85. are crucial.
Astrocytes can be identified by the presence of glyco­
Visualizing astrocyte morphology with genetic gen granules and intermediate filaments in the cyto-
approaches. Reporter expression — through viruses plasm using electron microscopy94,95. Three-​dimensional
or transgenic mouse lines — has been used for sparse reconstructions with serial-​section electron microscopy
labelling in vivo for single-​cell morphological stud- images have demonstrated that astrocyte processes con-
ies. Cytosolic fluorescent proteins (such as GFP and tact most, but not all, synapses in the rat hippocampal
tdTomato) and membrane-​tethered fluorescent pro- CA1 region: approximately 57% of the synapses were
teins (including Lck-​GFP) have been expressed using contacted by astrocyte processes and, in these cases,
astrocyte-​specific promoters. about 43% of the synaptic interface was surrounded
One study labelled astrocytes with EGFP through by astrocyte processes95,96. A similar phenomenon was
use of GFAP-​tTA mice, which express tetracycline-​ found in the cortex, where the extent of synapse cov-
controlled transactivator protein (tTA) under the control erage by astrocytes was greater in layers II/III than in
of the human GFAP promoter86. Single cortical astro- layer VI87.
cytes were seen to enwrap an average of four neuronal Super-​resolution microscopy refers to various appro­
somata and to contact 300–600 neuronal dendrites, aches that achieve spatial resolution below the diff­raction
forming synaptic ‘islands’ through which astrocytes limit of light microscopy. Combined with mathemat-
may synchronize and coordinate neuronal networks. ical reconstructions, super-​resolution microscopy has
Cortical astrocytes that were sparsely labelled using the enabled imaging of fine astrocytic processes and certain
Slc1a3-Cre/ERT2 mouse line were classified into four proteins within them, including those near synapses97.
distinct clusters on the basis of 24 morphological para­ Such approaches have great potential to visualize astro-
meters, including the territory volume, cell orientation cyte structures at a scale of tens of nanometres, includ-
and extent of ramification87. The distribution of these ing when combined with expansion microscopy 98,
subpopulations was also layer-​specific and depended on which physically expands fixed tissues. Stimulated
the laminar organization of cortical neurons87. emission depletion (STED) microscopy creates super-​
Another recently developed sparse labelling method, resolution images by quenching fluorophores in a
called mosaicism with repeat frameshift (MORF), takes doughnut-​shaped area around the excitation spot, to
advantage of frameshift mutations in a long mononucleo­ minimize the area of illumination at the focal point99.
tide repeat to stochastically label individual genetically Although super-​resolution methods have not yet been
defined neurons88, and could be used to label astrocytes. extensively used to study astrocyte fine structure, this
In a study in organotypic slices, membrane-​targeted is likely to change as the methods become increasingly
EGFP and mCherry were expressed by cell-​type-specific routine.
viruses in CA1 neurons and hippocampal astrocytes, Fluorescent proteins and a genetically targeted
respectively; putative astrocyte–spine contacts were neuron–astrocyte proximity assay (NAPA) have been
Reviews
Förster resonance energy
transfer
(FRET). Energy transfer
between two appropriate light-​
sensitive molecules, such as
fluorophores, via dipole–dipole
coupling. The transfer typically
reports distances on the tens
of nanometres scale or lower
and so can be used to assess
the proximity between cognate
fluorophores that undergo
Förster resonance energy
transfer.
Microdomains
Highly localized, small (several
micrometres wide) territories of
a cell.
Photoisomerable
Able to undergo reversible
structural conversion between
isomers in response to
photoexcitation.
used73,89 to measure the spatio-​temporal interactions
between astrocyte processes and synapses. The NAPA
depends on Förster resonance energy transfer (FRET)
between GFP expressed in astrocytes and mCherry
expressed in presynaptic neurons. Thus, detecta-
ble FRET reflects interactions of astrocyte processes
and presynaptic compartments within approximately
10 nm of each other. The NAPA could potentially be
developed for imaging astrocyte–neuron interactions
in awake behaving animals — for example, with two-​
photon fluorescence lifetime imaging microscopy100. In
addition, the NAPA could also be expanded to assess
spatial relationships between multiple cell types, such as
oligodendrocytes and microglia.
Considerations. Since the pioneering work that first doc-
umented the shapes of astrocytes, advances in imaging
and labelling techniques have expanded our knowledge
of astrocyte biology. However, a single perfect solution
for imaging and tracking astrocyte morphology does not
exist, and many questions remain open. For instance,
do astrocytes with similar but separable morphologies
in a given brain area perform distinct functions? What
are the molecular mechanisms regulating astrocyte
morphology within and between regions? Is structural
rearrangement of astrocytes a cause or a consequence of
neuronal plasticity and/or pathophysiology?
A major challenge is to develop methods that are
innocuous and that can be used for live imaging and,
subsequently, for three-​dimensional reconstructions
by electron microscopy. Related to this point, the tissue
fixation conditions used for electron microscopy affect
ultrastructural preservation of astrocytes and other cells101.
Furthermore, advances in automated tracing of cellu-
lar elements are needed for fine-​scale reconstructions
of astrocytes, because existing methods that work for
neurons do not work reproducibly for astrocytes102.
Machine-​learning approaches may solve this problem
before too long.
Although the NAPA appears useful, a formal possi-
bility remains that, by detecting only the most proximal
astrocyte–neuron interactions, the method does not
permit assessment of the dynamics of astrocyte–neuron
spatial interactions at a scale of tens of nanometres73.
Again, methods must be chosen after due considera-
tion of the specifics of the question or questions being
investigated.
Exploring calcium signalling
The challenge. Firing no action potentials and displaying
no known propagated electrical signals, astrocytes are
largely electrically silent in comparison with neurons13,
and were thus long viewed as lacking dynamic signals
for bidirectional communication with other astrocytes,
microglia and neurons. This view is beginning to change
with the use of improved tools. About 30 years ago, intra-
cellular Ca2+ signalling was discovered in astrocyte cell
cultures103,104, and was subsequently reported in brain
slices105, in anaesthetized106 and behaving animals107,108,
and in human cells30,31. Since these landmark studies,
dynamic and diverse astrocyte Ca2+ signals have been
widely reported, including those in highly localized
microdomains of astrocyte processes93,109,110. These Ca2+
signals are proposed to underlie important physiologi-
cal functions of astrocytes in multiple species, including
worms, flies, zebrafish, mice and, potentially, humans.
The cellular sources of astrocyte Ca2+ signals include
multiple pathways, including those involving various
receptors, channels, exchangers and pumps on the plasma
membrane, as well as within intracellular organelles such
as mitochondria, endoplasmic reticulum, Golgi and acidic
organelles111. The relative contributions of these sources
to overall astrocyte Ca2+ dynamics are not clear and may
require a combination of experimental and modelling
approaches to delineate. Nevertheless, the available data
clearly imply that Ca2+ signalling in astrocytes is as rich,
fascinating and complex as that in other cells of diverse
organisms111–114.
The larger question of whether astrocyte Ca2+ signals
cause changes in neural circuit function and behaviour
has now been explored. These experiments have become
possible because of methodological advances (mostly
from the Genetically-​Encoded Neuronal Indicator and
Effector (GENIE) project at Janelia) in monitoring astro-
cyte Ca2+ with genetically encoded calcium indicators
in vivo and in adult brain slices115–118 (Table 3). These
advances have in turn necessitated the use of methods
to increase and decrease Ca2+ signals within astrocytes
in vivo (Fig. 3), and to evaluate the molecular and cellular
consequences of such manipulations (Fig. 4). Here, we
consider tools for selectively stimulating and attenuating
astrocyte Ca2+ signalling in vivo and the insights they
have revealed. Studies using conventional pharmacolog-
ical approaches are not considered here but are reviewed
elsewhere119,120.
Increasing astrocyte Ca2+ with LiGluR. The light-​gated
ionotropic glutamate receptor (LiGluR) is an ionotropic
glutamate receptor type 6 isoform with a mutation
that enables the reversible, covalent attachment of a
photoisomerable molecule called maleimide–azoben-
zene–glutamate (MAG)121,122. Photostimulation of MAG-​
bound LiGluRs on astrocytes induced channel-​pore
opening and increases in intracellular Ca2+. Interestingly,
light-​stimulated astrocytes released glutamate through
anion channels in their membranes, resulting in prop-
agation of Ca2+ signalling to neighbouring astrocytes
lacking the LiGluR123. LiGluRs have not yet been used
to stimulate astrocytes in vivo, perhaps owing to the
difficulty of innocuously delivering MAG to the brain.
Increasing astrocyte Ca2+ with DREADDs. Astrocyte
GPCRs have been studied for more than two deca­
des124. Pharmacological and gene expression studies
have suggested that astrocytes express a wide range of
GPCRs24,39,124, and much attention has focused on astro-
cyte GPCRs that promote inositol 1,4,5-trisphosphate
receptor (IP3R)-dependent Ca2+ release from intra­
cellular endoplasmic reticulum stores. However, as many
endogenously expressed GPCRs are also found on neu-
rons and other cells, pharmacologically targeting such
mechanisms to explore causal roles within astrocytes,
although valuable in cultures of purified astrocytes30,31,125,
has proved challenging in brain slices and in vivo120.
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 Reviews

Table 3 | Genetically encoded single-​wavelength sensors useful for astrocyte work

Analyte sensor Kd Wavelengtha (nm) ref.
excitation emission
Intracellular analytes
Ca2+ GCaMP6f 375 nM 485 510 117

GCaMP6m 167 nM 485 510 117

GCaMP6s 144 nM 485 510 117

Lck-​GCaMP6f 375 nMb 488 510 39

jGCaMP7s 68 nM 485 510 116

jGCaMP7f 174 nM 485 510 116

jGCaMP7b 82 nM 485 510 116

jGCaMP7c 298 nM 485 510 116

jRCaMP1a 214 nM 575 595 207

jRCaMP1b 712 nM 570 595 207

jRGECO1a 148 nM 565 590 207

XCaMP-​B 71 nM 405 446 208

XCaMP-​G 200 nM 488 514c 208

XCaMP-​Gf 128 nM 488 514c 208

XCaMP-​Gf0 115 nM 488 514c 208

XCaMP-​Y 81 nM 488 527c 208

XCaMP-​R 97 nM 561 593 208

cAMP Flamindo2 2.1 µM 505 522 209

Pink Flamindo 7.2 µM 567 590 210

K+ GINKO1 0.42 mM 488 520 211

Extracellular analytes
Glutamate iGluSnFR 4–5 μM 470 550 212

SF-​iGluSnFR .A184S 0.6 μM 470 525 213

SF-​iGluSnFR .A184V 2.1 μM 470 525 213

SF-​iGluSnFR .S72A 34 μM 470 525 213

SF-​Azurite-iGluSnFR 9 μM 383 450 213

SF-​mTurquoise2-iGluSnFR ND 434 474 213

SF-​Venus-iGluSnFR 2 μM 515 530 213

ATP iATPSnFR1.0 350 μM 473 525 214

iATPSnFR1.1 138 μM 473 525 214

GABA iGABASnFR 30 μM 485 510 215

iGABASnFR .F102G 42 μM 485 510 215

iGABASnFR .F012Y.Y137L 106 μM 485 510 215

Dopamine dLight1.1 330 nM 490 516 216

dLight1.2 770 nM 490 516 216

dLight1.3a 2.3 μM 490 516 216

dLight1.3b 1.7 μM 490 516 216

dLight1.4 4.1 nM 490 516 216

dLight1.5 110 nM 490 516 216

GRABDA1m 130 nM 470 510 217

GRABDA1h 10 nM 470 510 217

Acetylcholine GACh 0.7–2 μM 480 520 218

Noradrenaline GRABNE1m 930 nM 488 525 219

GRABNE1h 83 nM 488 525 219

Not all these sensors have been used to explore astrocytes, but their availability means such experiments can now commence. Readers
are strongly encouraged to read the original articles cited here regarding the specific features, including the kinetic properties, of each
sensor. Kd, equilibrium dissociation constant; ND, not determined. aValues for peak excitation and emission wavelengths are approximate
and are from the cited papers based on the information provided (in some cases, the values have been estimated from the spectra in
the original papers — for example, for jRCaMP1a, jRCaMP1b, jRGECO1a, Flamindo2, GRABDA1m and GRABDA1h). All values are for single-​
photon imaging, because not all the sensors have been tested with two-​photon microscopy. Several sensors, such as Ca2+ and glutamate
sensors, have been iteratively improved over several years; given space constraints, reference is made to the most recent versions.
b
Kd of Lck-​GCaMP6f is assumed by that of GCaMP6f, based on comparisons between cytosolic-​targeted and membrane-​targeted
GCaMP2 (ref.110). cXCaMP-​G and XCaMP-​Y can be optically separated with linear unmixing, as suggested in the cited paper.

Nature Reviews | Neuroscience

Reviews

a Increasing astrocyte Ca2+ signalling via GPCR pathways

DREADDs

hM3Dq rM3Ds hM4Di Melanopsin Opto-XRs

Synthetic
b ligands Light
a Opsin

Intracellular
Major c Soma loop of GPCR
Gαq Gαs Gαi Gα Gα
branches

IP3
ER Ca2+

Territory IP3R
d comprising
branchlets Cytosol
and leaflets

b Increasing astrocyte Ca2+ signalling via channels c Decreasing astrocyte Ca2+ signalling d Decreasing astrocyte Ca2+
signalling via pumps
Light-gated Light-gated
channel channel
380 nm light Human Ca2+
K+ K+ Endogenous ligand PMCA2w/b
Tethered ligand (CalEx)

ChR2 LiGluR GPCR

Gα
Na+ Na+ PLC IP3 sponge
H+ Ca2+
(Ca2+) IP3 ADP ATP

IP3R2
deletion

Fig. 3 | cartoon of the tools used to manipulate astrocyte intracellular
ca2+ signalling. Several genetically encoded tools and approaches exist to
selectively increase and decrease astrocyte intracellular Ca2+ levels. a | Five
tools for stimulating astrocyte Ca2+ signalling through G protein-​coupled
receptor (GPCR) pathways are shown. All downstream pathways of the tools
involve inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release
from the endoplasmic reticulum (ER). DREADDs (designer receptors
exclusively activated by designer drugs) are engineered GPCRs that can be
activated by inert chemicals and not by endogenous ligands. Each DREADD
interacts with distinct Gα proteins upon synthetic ligand binding: Gq-​coupled
hM3Dq, Gs-​coupled rM3Ds and Gi-​coupled hM4Di. Melanopsin, a type of
photopigment, can elicit intracellular signal cascades when stimulated with
blue light (about 470–480 nm). Opto-​XRs are engineered chimaeras
containing a photo-​activatable extracellular part from the pigment rhodopsin
(blue) and the intracellular part of endogenous GPCRs (red) that trigger
G protein-​mediated signalling. b | Two tools for increasing astrocyte Ca2+ levels
through channels are shown. Photostimulation of channelrhodopsin 2 (ChR2)
causes non-​selective cation flow through the plasma membrane, including
Na+, H+, K+ and Ca2+. However, the Ca2+ permeability of ChR2 is reported to be
low220 and its mechanisms and physiological relevance are under debate
(also see Considerations in the section Exploring calcium signalling).
Light-​gated ionotropic glutamate receptor (LiGluR) is a mutated ionotropic
glutamate receptor 6 that is Ca2+ permeable. Upon photostimulation, the
photoisomerable tethered ligand maleimide–azobenzene–glutamate (MAG)
opens the LiGluR channel pore to induce Ca2+ influx. Notably , both tools
induce K+ efflux. Increases in extracellular K+ resulting from ChR2 activation
have been shown to be consequential for neuronal firing. c | Two strategies
for blocking GPCR–IP3R pathways to attenuate Ca2+ signalling are shown that
include genetic deletion of IP3R2, which is thought to be the most abundant
IP3R isoform in astrocytes, and a recombinant IP3 sponge that competes the
endogenous IP3Rs for IP3 binding. d | Human plasma membrane Ca2+ ATPase
splice variant w/b (PMCA2w/b) is a Ca2+ pump that has no endogenous
expression in astrocytes and acts as a constitutive Ca2+ extruder (known as
the CalEx approach).

In these regards, the development and use of DREADDs
(designer receptors exclusively activated by designer
drugs) has been extremely useful to explore astrocyte
biology in brain preparations, such as slices, and in vivo.
DREADDs are engineered GPCRs that have strongly
attenuated responses to their endogenous ligands and
have been engineered to respond to synthetic, biolog-
ically inert chemical actuators126,127. Because the syn-
thetic ligands are delivered in the animal’s water or food
supply, or by systemic injection, DREADDs enable
relatively non-​invasive stimulation of GPCR pathways in a
genetically targeted manner in vivo.
Several types of DREADD have been developed to tar-
get major Gα-​protein signalling pathways: Gq-​coupled
hM3D126, Gi-​coupled hM4D126,128, Gs-​coupled rM3D129
and G12-coupled DREADD130. These engineered recep-
tors are chimaeras between a human or rat muscarinic
receptor and the turkey β1-adrenoceptor to confer speci­
fic G protein pathway activation, and/or carry point
mutations to kill sensitivity to endogenous ligands.

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 Reviews

That such receptors are so frequently used as biological
tools is testimony to the deep value of the careful biochemi­
cal and biophysical studies of GPCRs that permitted
their design127,131–133.
AAVs for expressing DREADDs selectively in stri-
atal and hippocampal astrocytes were used to make
three observations40. First, application of the ligand,
CNO, evoked astrocyte Ca2+ elevations similar to the
Ca2+ signals mediated by endogenous astrocyte GPCRs.
Second, Gq, Gi and Gs DREADDs all evoked astrocyte
Ca2+ signalling40,134, despite the fact that they usually
have separable effects on neuronal membrane potential.
However, astrocytes and neurons are distinct cells, and
so it is simplistic to think they would respond similarly

Astrocyte-specific
manipulation Effect on astrocytes Consequences Cellular mechanism

Circuits Behaviour
a
Gq-DREADD hM3Dq Increased Ca2+ signalling Increased FOS+ neurons Enhanced conditioned
recruited during fear fear memory acquisition
CNO conditioning

Enhanced LTP
Gαq via D-serine
FOS+

Ca2+ neuron
IP3R
Hippocampal CA1
ER

b
Gi-DREADD hM4Di Increased Ca2+ signalling Increased MSN firing Hyperactivity and
disrupted attention

Gαi
Synapse
formation
Ca2+ via TSP1
MSN

Dorsal striatum

c
Human PMCA2w/b (CalEx) Attenuated Ca2+ signalling Reduced MSN activity with Excessive self-grooming
increased correlation
Ca2+ Ca 2+

export

Regulation of
GABA-mediated
tonic inhibition
ADP ATP
Dorsal striatum

Fig. 4 | causative roles of astrocyte intracellular ca2+ signalling revealed
by new tools. a | Activation of the DREADD (designer receptor exclusively
activated by designer drug) hM3Dq in astrocytes of the hippocampal CA1
region causes increases in astrocyte Ca2+ signalling, as well as regulation of
synaptic transmission by d-​serine, and FOS expression in neurons recruited
during fear conditioning. Chemogenetic and optogenetic activation of the
astrocyte Gq pathway was used to delineate the time-​locked changes
associated with the behavioural effect: enhanced memory acquisition71.
b | hM4Di activation in dorsal striatal astrocytes increased astrocyte Ca2+
signalling, excitatory synaptic transmission and firing of medium spiny
neurons (MSNs) in vivo. The behavioural consequences were hyperactivity
with disrupted attention, mimicking certain symptoms of attention deficit
hyperactivity disorder in humans. The mechanisms involved astrocytic
release of the synaptogenic cue thrombospondin 1 (TSP1). Importantly ,
gabapentin, an anticonvulsant drug, antagonized the TSP1 synaptogenic
effects and rescued the synaptic, circuit and behavioural changes
observed following astrocyte Gi pathway activation45. c | Strong astrocyte
Ca2+ signalling attenuation by calcium extruder (CalEx; expression of a
Ca2+-exporting pump) in the dorsal striatum caused excessive self-​grooming
behaviour in mice reminiscent of behaviours in obsessive–compulsive
disorder in humans. This was due to altered MSN activity in vivo and reduced
tonic GABA inhibition resulting from increases in the functional expression
of astrocyte GABA transporter, GAT3. SNAP-5114, a pharmacological
inhibitor of GAT3, rescued the excessive self-​grooming behaviour caused
by CalEx46. ER , endoplasmic reticulum; IP3R , inositol 1,4,5-trisphosphate
receptor ; PMCA2w/b, plasma membrane Ca2+ ATPase splice variant w/b.

Nature Reviews | Neuroscience

Reviews
Remote memory
The enduring memory of
events that happened or were
learnt in the distant past.
Retinal ganglion cells
Output neurons in the ganglion
cell layer of the retina,
receiving visual input from
photoreceptors and
interneurons.
to GPCRs or any other receptor. Indeed, multiple GPCR ChR2-mediated astrocyte excitation has been used to
pathways converge on astrocyte Ca2+ signalling, includ- ‘excite’ astrocytes (but see Considerations) and explore
ing the Gi pathway124,133, and changes in Ca2+ signalling their roles in regulating circuits and behaviour, implicat-
are not analogous to changes in membrane potential or ing them in breathing143, response selectivity in the visual
excitability and rely on different molecular machinery. cortex144, regulation of blood flow145, sleep146,147 and cer-
Third, although Gq-​DREADD-mediated Ca2+ signals ebellar motor function148 through various mechanisms.
were similar for striatal and hippocampal astrocytes, A recent study demonstrated that ChR2 stimulation of
Gi-​DREADD-mediated signals were substantially greater astrocytes caused transient increases in extracellular
in striatal astrocytes than in hippocampal astrocytes. K+ levels that affected neuronal firing in slices, in silico
Therefore, GPCR-​mediated astrocyte Ca2+ signalling and and in vivo149. In addition, changes in extracellular
its downstream effects may vary between brain areas. K+ levels affect not only neurons but also astrocytes,
Studies using DREADDs suggest that astrocyte Ca2+ microglia and blood vessels150. In another study, the
signalling is sufficient to modulate behaviour. Activation Arch opsin, which is inhibitory in neurons, was used
of Gq DREADDs in medial basal hypothalamic astro- to increase cortical astrocyte Ca2+ signalling76, again
cytes altered food intake by modulating the activity of highlighting the possibility that certain tools may have
neurons expressing agouti-​related protein (AGRP)135,136. different effects on neurons versus astrocytes.
Activating Gq DREADDs on astrocytes in the central
amygdala induced ATP or adenosine release, which Increasing astrocyte Ca2+ with melanopsin. Light-​
reduced neuronal firing, suppressing the gating of con- activated GPCRs have been used to manipulate astrocyte
ditioned fear expression137. Gq-​DREADD stimu­lation Ca2+ signalling. Melanopsin is a photoactivated GPCR
in hippocampal CA1 astrocytes induced astrocyte endogenously expressed in the plasma membrane of
d-​serine release and increased neuronal recruitment mammalian retinal ganglion cells but not in the brain.
during memory acquisition, enhancing recent memory71 Melanopsin can be activated by blue light (~470–480 nm
(Fig. 4a). In another study, activation of the Gq pathway wavelength) and deactivated by yellow light. Although it
in hippocampal astrocytes caused astrocytic release can couple to Gi/o, when examined in neurons melanopsin
of glutamate that directly activated neuronal NMDA almost exclusively induces Gq signalling151,152.
receptors134. Recently, AAV-​expressed melanopsin was deployed
A few studies have explored the consequences of acti- to selectively stimulate astrocyte Ca2+ signalling153.
vating the Gi pathway in astrocytes. In the dorsolateral Continuous photostimulation of melanopsin-​expressing
striatum of mice, activation of astrocyte Gi DREADDs hippocampal astrocytes for 1–20 s increased the fre-
induced the expression of thrombospondin 1 (TSP1), an quency of astrocyte Ca2+ signals in fine processes (micro-
astrocyte synaptogenic cue45. This, in turn, boosted fast domains), whereas stimulation for longer than 10 s was
excitatory synaptic transmission onto striatal medium required to evoke Ca2+ signals in astrocyte somata. These
spiny neurons, increased medium spiny neuron firing evoked Ca2+ signals were abolished in IP3R2-lacking
in vivo and resulted in behavioural hyperactivity with mice, confirming IP3-dependent signalling. When stim-
disrupted attention phenotypes45 (Fig. 4b). In the hippo­ ulated with a brief train of light pulses, stronger astro-
campus, activation of Gi DREADDs enhanced long-​ cyte Ca2+ signalling was associated with potentiation of
term potentiation of Schaffer-​collateral–CA1 pyramidal evoked excitatory postsynaptic currents in CA1 pyra­
neuron synapses and contextual memory acquisition138. midal neurons lasting about 10 minutes — that is,
Similar responses that are mediated by endogenous mimick­ing short-​term potentiation. By contrast, low-​
μ-​opioid receptors (MORs) on astrocytes may be rele- frequency optical stimulation of melanopsin-​expressing
vant to the acquisition of reward memories138. Activation astrocytes triggered long-​term potentiation and enhanced
of astrocytic MORs in the nucleus accumbens caused episodic memory, consistent with DREADD stimulation
the release of astrocyte glutamate that activated NMDA of the same population of astrocytes in vivo71. Melanopsin
receptors on neurons139, similar to that following acti- seems a very useful tool to optically stimulate astrocytes
vation of the Gi pathway in hippocampal astrocytes134. in a time-​controlled manner and to potentially mimic the
A preliminary report suggests that Gi-​DREADD acti- endogenous oscillatory patterns of GPCR pathways.
vation of hippocampal CA1 astrocytes does not affect
recent memory (unlike activation of the Gq pathway)71, Increasing astrocyte Ca2+ with opto-​XRs. Optical control
but impairs remote memory recall140, although the under- of GPCR pathways provides more rapid temporal con-
lying molecular and cellular mechanisms remain to be trol than do conventional pharmacological approaches,
explored. and is thus useful for assessing time-​locked changes in
syna­ptic function and behaviour71. In addition to naturally
Increasing astrocyte Ca with channelrhodopsin 2. occurring opsins, such as melanopsin (which is typically
2+
Channelrhodopsin 2 (ChR2) is a light-​gated, non-​ coupled to Gq), conopsin (Gi/o) and the non-​mammalian
selective cation channel originally found in algae; when rhodopsin–guanylyl cyclase (Gs)154, several engineered
stimulated with blue light (~470 nm), ChR2 mediates light-​sensitive GPCRs, called opto-​XRs, have been used.
the flow of cations such as protons and sodium into the In these opto-​XRs, the intracellular loops of vertebrate
cell. As ChR2 can cause membrane potential depolar- rhodopsins are replaced by those of α-​adrenoceptors and
ization and action potentials when expressed in neu- β-​adrenoceptors to create light-​sensitive mutant proteins
rons, the channel and its derivatives have immeasurably that recruit Gq-​mediated and Gs-​mediated pathways,
advanced systems neuroscience141,142. respectively155. Several opto-​XRs, including opto-​α1-AR
www.nature.com/nrn

Tet system
An inducible gene expression
system that reversibly activates
(Tet-​On) or represses (Tet-​Off)
transcription in the presence of
a tetracycline transactivator
(tTA) protein, a DNA sequence
called the tetracycline
response element (TRE) and
treatment with tetracycline or
its derivatives.
Nature Reviews | Neuroscience
(Gq), opto-​β2-AR (Gs), opto-A2A-​R (Gs) and opto-​MOR
(Gi/o), have been validated in neurons154.
Stimulation of opto-​α1-ARs in astrocytes recapitu-
lated the enhanced fear memory effect that was triggered
by Gq-​DREADD stimulation of hippocampal astro-
cytes71 — presumably through the same mechanism. As
far as we know, other opto-​XRs have not yet been tested
in astrocytes.
Although opto-​XRs allow temporally precise opti-
cal stimulation, light from the tip of an optical fibre is
expected to spread into a restricted volume of brain
tissue and to scatter back, thus complicating anatom-
ically precise stimulation based on fibre position156.
Light-​dependent tissue heating can also cause neuronal
firing156,157 and, in most cases, fewer astrocytes are stim-
ulated with light than using chemogenetics where chem-
ical actuators spread in the tissue, possibly complicating
direct comparisons between these methods. From these
perspectives, it is interesting to consider that chemo­
genetic and opto-​α1-AR Gq pathway activation in hippo­
campal astrocytes produced almost identical effects on
fear memory71.
Decreasing astrocyte Ca2+ by targeting IP3. Astrocyte
Ca2+ signalling is complex and mediated by multiple
mechanisms111,113. In one major pathway leading to ele-
vated intracellular Ca2+ in astrocytes, IP3 generated by
activated cell-​surface GPCRs stimulates endoplasmic
reticulum IP3Rs, resulting in Ca2+ release. IP3 recep-
tor type 2 (IP3R2) is the predominant IP3R isoform in
astrocytes in the rodent brain158–160. Mice genetically
lacking IP3R2 display strongly attenuated spontaneous
and Gq-​GPCR-mediated Ca2+ signals in the somata of
astrocytes from multiple brain regions. These mice and
the interpretations of these data have been extensively
reviewed16,17.
Another approach to disrupt IP3-dependent Ca2+
signall­ing is to use molecules that buffer cytosolic IP3, to
prevent IP3-mediated Ca2+ release from the endoplasmic
reticulum. An ‘IP3 sponge’ based on the IP3 ligand-​
binding site of IP3R1 was constructed to compete with
endogenous IP3Rs for IP3 binding161. In a transgenic
mouse line using the Tet system with a GLT1 promoter,
IP3 sponges were expressed in astrocytes in most brain
regions, except the cerebellum, resulting in an approxi-
mately 30% reduction in the integrated area of metabo-
tropic glutamate receptor (mGluR)-mediated Ca 2+
responses in hippocampal astrocytes and reduced astro-
cytic coverage of synapses. These changes were associated
with deficits in hippocampus-​dependent behaviours162.
However, leaky expression of the GLT1-promoter-​driven
transgene was also detected in hippocampal neurons162,163,
possibly complicating the interpretation of these data.
The pleckstrin homology domain of PLC-​like pro-
tein p130 (p130PH) acts as a mobile cytosolic IP3 buffer
and, when expressed by a recombinant AAV with the
astrocyte-​specific promoter GfaABC1D, suppresses
ATP-​induced Ca2+ elevation in cortical astrocytes by
about 70% in vitro and in vivo164. Selective expression
of p130PH in hypothalamic astrocytes considerably
reduced the food intake of mice. By contrast, feeding
behaviour was increased by selectively activating the
Reviews
Gq-​coupled hM3D DREADD in the same cells136. How
p130PH affects basal and spontaneous Ca2+ signals in
astrocytes is not clear, because their mechanisms may be
independent of IP3 signals. Notably, p130PH also binds
other phospholipids, including PtdIns(4,5)P2, possibly
complicating the interpretation of such studies165.
Decreasing astrocyte Ca2+ with calcium extruder. Given
the diversity of astrocytic Ca2+ signalling, a genetic
approach was developed to attenuate such signalling
regardless of the Ca2+ source46. An efficient Ca2+ pump,
called plasma membrane Ca2+ ATPase isoform 2 splice
variant w/b (PMCA2w/b), constitutively extrudes Ca2+
out of cells and is not endogenously expressed in astro-
cytes. AAVs overexpressing human PMCA2w/b under
the control of the GfaABC1D promoter were used to
induce expression of the pump on the surface of stri-
atal astrocytes. This calcium extruder (CalEx) approach
reduced the amplitude and duration of spontaneous
and GPCR-​mediated Ca2+ signals by approximately 80%
without causing overt changes in their frequency.
Attenuating astrocyte Ca2+ signalling in the dorsolateral
striatum of adult mice resulted in repetitive behaviour,
reminiscent of certain symptoms of obsessive–compulsive
disorder in humans (Fig. 4c). Available data suggest that
PMCA2w/b does not detectably alter extracellular Ca2+
concentrations sufficiently to alter neurotransmitter
release probability46, which has a fourth power depend-
ency on extracellular Ca2+. The pronounced behavioural
phenotype was mediated by reductions in tonic GABA
inhibition of striatal neurons through upregulation of
astrocyte GABA transporter GAT3. Further examina-
tion using astrocyte-​specific RNA-​seq revealed that
reducing astrocyte Ca2+ levels leads to a decrease in
Rab11a expression, which in turn results in increased
GAT3 function166.
Instrumentation and software for assessing astrocyte
Ca2+. Several software packages have become available
to analyse astrocyte Ca2+ signals167,168. Of these, Astrocyte
Quantitative Analysis (AQuA) software seems poten-
tially most useful168 as it allows quantification of the
spatio-​temporal patterns of Ca2+ signalling.
Furthermore, there have been advances with instru-
mentation to complement single-​photon and multi-
photon microscopy. For example, fluorescence lifetime
imaging can measure the absolute concentration of
Ca2+ in different astrocyte compartments169, and three-​
dimensional imaging can monitor Ca2+ signals in parts
of astrocytes that are larger than those that can be
imaged in a single plane170. Wearable miniature micro-
scopes have been used to image astrocyte Ca2+ in freely
behaving mice171, and an optical fibre imaging system
was developed to monitor astrocyte Ca2+ signals from
two different brain areas simultaneously172.
Various computational methods and models for
analy­sing data and making predictions for new exper-
iments are also emerging173–178, although these will need
modifying for astrocytes in specific brain areas. Overall,
developments in software, modelling and instrumenta-
tion are allowing finer-​scale investigation of astrocytes at
multiple spatial and temporal scales in vivo and in silico.
Reviews
Acidification
The act of something such
as a cellular compartment
becoming more acidic
(that is, becoming protonated).
Considerations. The studies above demonstrate that
activation of astrocytic GPCR pathways results in gene
expression changes that affect synaptic function, as well
as more rapid changes from the release of glutamate,
ATP or adenosine, leading to slow and fast modulation
of neural circuits with discernible behavioural conse-
quences. Differences between the effects of Gq and Gi
modulation suggest that these pathways may serve sep-
arable functions. However, several considerations are
worth noting.
Whether glutamate gliotransmission exists under
physiological conditions is debated owing to contradic-
tory findings16,17, and further demonstration of this pheno­
menon with DREADDs (see earlier sections), although
supportive, does not address this point. That is, DREADD
activation allows one to trigger a specific pathway in
astrocytes but, without additional evidence, this does
not necessarily mean that those pathways are employed.
Furthermore, although activation of GPCRs in astro-
cytes increases intracellular Ca2+ levels, caution is needed
before concluding that Ca2+ is the effector responsible for
the observed synaptic, circuit and behavioural effects.
Activation of the Gi pathway is expected to cause
multiple intracellular signalling events, including
changes in cyclic AMP levels and gene expression. Some
or all of these changes may contribute to the reported
neural-​circuit and behavioural changes, in a brain-​
area-dependent, context-​dependent and experimental-​
approach-dependent manner. Furthermore, to interpret
the results usefully, several parallel controls for off-​target
effects of CNO and its breakdown products must be
performed when using DREADDs179. Studies that do
not report ‘CNO-​only’ controls should be interpreted
cautiously179.
Future studies may benefit from the DREADD
agonist compound 21 (ref.180), which is not degraded.
Another inhibitory κ-​opioid receptor-​based Gi DREADD
(KORD) is activated by the inert ligand salvinorin B128.
To our knowledge, KORD has not been tested in astro-
cytes, but could be useful in combination with other
DREADDs in a multiplexed approach, to enable Gi and
Gq GPCR pathways to be stimulated concomitantly in
the same animal.
A final concern with the use of DREADDs, especially
in vivo, is that the effects require at least tens of min-
utes to occur and can last hours — largely depending
on the pharmacodynamics of the DREADD ligands127,181.
Thus, DREADDs are not appropriate for every astro-
cyte experiment, and due consideration is needed about
the specific question being addressed and the kinetics
of DREADD-​mediated responses in vivo relative to the
metrics used to evaluate them in brain slices.
In regard to ChR2, how ion flux and depolarization
of photostimulated ChR2-expressing astrocytes affect
their physiology is not fully understood, because astro-
cytes express few voltage-​gated ion channels and do
not display propagated voltage signals. Furthermore,
the effects of ion fluxes on the multiple ion-​coupled
transporters that astrocytes express have not been
addressed experimentally and will not be straightfor-
ward to deduce empirically. These concerns are great-
est for long-​duration optical stimuli lasting seconds or
longer. It may be that ChR2 stimulation of astrocytes
may not mimic physiological astrocytic processes in
astrocytes — at least, not those that have so far been
readily identifiable— but might reflect pathophysi-
ology when  acidification occurs182. Further points to
consider when using optogenetics for astrocyte stud-
ies include the non-​specific effects of light157,183, the
effects of tissue warming157 and, in the case of silencing
approaches, off-​target circuit-​related effects184,185. Some
of these concerns will undoubtedly be addressed with
future variants of ChR2 and its derivatives.
The findings with IP3 sponges and with CalEx are
interesting, as they begin to tackle questions related to
causation. However, until we recognize the molecular
identity of astrocyte Ca2+ sources, our understanding
about the nature of the attenuated signals and their
endogenous engagement will be limited. As more gene
and protein expression data become available, the field
could move to more specific approaches to delete or
modify certain molecules. Reversible optical activa-
tion and silencing of endogenous GPCRs186 seems to
be a promising and emerging approach for future use;
there are some obvious candidates to explore, such as
α-​adrenoceptors167,172,187.
Perhaps the best way forward is to capitalize on
insights about astrocyte Ca2+ signalling in flies and
zebra­f ish166,188–191 that were revealed by performing
genetic screens to identify molecular pathways agnos-
tically. As it is not restricted to a specific Ca2+ pathway,
CalEx enables circuit-​specific suppression of astrocyte
Ca2+ signalling in the mature brain. However, CalEx
could be further developed to become more useful — for
example, by making it ligand-​activated through designer
engineering, or by modifying it to enable fine temporal
control that could enable it to reveal rapid Ca2+-triggered
physiological changes. Of course, intracellular dialysis
with the Ca2+ buffer BAPTA can attenuate astrocyte
Ca 2+ signalling. However, this approach is hard to
deploy in vivo, and recent work suggests that BAPTA
has off-​target effects, including effects on ion homeo-
stasis regulated by Na,K-​ATPase192, that may complicate
interpretation of synaptic and circuit responses.
Many genetically encoded sensors for Ca2+, neuro-
transmitters and neuromodulators have been developed
in the past few years. Genetically encoded single-​
wavelength sensors are summarized in Table 3, with
appropriate references to their development and/or
use in astrocytes. Additional sensors will probably be
developed in the next few years for most neurotrans-
mitters and neuromodulators, and will allow research-
ers to directly monitor the downstream consequences of
astrocyte Ca2+ manipulations.
Finishing remarks
The past few years have witnessed inspiring advances in
several complementary approaches and tools to explore
astrocyte molecular mechanisms, signalling and func-
tions in adult mice. Although important progress has
occurred, further technical advances are necessary.
With currently available tools, it is possible to design
experiments to explore astrocyte biology from mole-
cules to systems. Additional tools are needed to explore
www.nature.com/nrn
 Reviews

how various cell types of the nervous system, including
astrocytes, neurons and microglia, interact structurally
and functionally in health and in neuroimmune and
brain–periphery interactions. Such tools may crack open
completely new areas of biology related to how neurons
and glia work synergistically in the brain and how the
brain responds to sensory input and controls the body.
From this perspective, emerging work with zebrafish
and mice may be portentous. There, the use of improved
tools is starting to provide evidence that astrocytes
contribute to complex behavioural and cognitive pro-
cesses45,46,71,72,191,193. Building on fruitful partnerships
between tool generators, modellers and experimental-
ists will be crucial for the growth of glial biology and
could catalyse major breakthroughs in cell biology
and medicine.
Published online xx xx xxxx

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Publisher’s note
Acknowledgements Springer Nature remains neutral with regard to jurisdictional
The authors are supported by the US National Institutes of claims in published maps and institutional affiliations.
Health (NS111583, DA047444, NS060677 and MH104069),
a Paul G. Allen Distinguished Investigator Award and the CHDI
Foundation (to B.S.K.). B.S.K. was also partly supported by the Related links
Ressler Family Foundation. X.Y. was supported partly by an Adult Astrocyte RNA-​seq explorer: http://astrocyternaseq.org/
American Heart Association Postdoctoral Fellowship Allen Brain Atlas, RNA-​seq data: https://celltypes.brain-​map.
(16POST27260256). J.N. was partly supported by a Japan org/rnaseq
Society for the Promotion of Science (JSPS) Overseas Research Astrocyte Ageing Transcription: http://igc1.salk.edu:3838/
Fellowship (H28-729) and the Uehara Memorial Foundation astrocyte_aging_transcriptome/
Overseas Postdoctoral Research Fellowship (201730082). The Brain RNA-​seq: http://www.brainrnaseq.org/
authors regret that many papers could not be cited (especially Dropviz: http://dropviz.org/
early studies), because of space limits and the requirement to Mouse Brain Atlas: http://mousebrain.org
focus primarily on the past 5 years. The authors thank mem- sCope: http://scope.aertslab.org
bers of the Khakh laboratory for useful discussions, and the
anonymous reviewers for their comments. © Springer Nature Limited 2020
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