Presynaptic Partners of Dorsal Raphe
Serotonergic and GABAergic Neurons
Brandon Weissbourd,1Jing Ren,1Katherine E. DeLoach,1Casey J. Guenthner,1,2Kazunari Miyamichi,1,3,*
and Liqun Luo1,2,*
1Department of Biology and Howard Hughes Medical Institute
2Neurosciences Graduate Program
Stanford University, Stanford, CA 94305, USA
3Present Address: Department of Applied Biological Chemistry and JST ERATO Touhara Chemosensory Signal Project, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
*Correspondence: email@example.com (K.M.), firstname.lastname@example.org (L.L.)
The serotonin system powerfully modulates physi-
ology and behavior in health and disease, yet the
circuit mechanisms underlying serotonin neuron
activity are poorly understood. The major source of
forebrain serotonergic innervation is from the dorsal
raphe nucleus (DR), which contains both serotonin
and GABA neurons. Using viral tracing combined
with electrophysiology, we found that GABA and se-
rotonin neurons in the DR receive excitatory, inhibi-
tory, and peptidergic inputs from the same specific
brain regions. Embedded in this overall similarity
are important differences. Serotonin neurons are
more likely to receive synaptic inputs from anterior
neocortex while GABA neurons receive dispropor-
tionallyhigher input from the centralamygdala. Local
input mapping revealed extensive serotonin-sero-
tonin as well as GABA-serotonin connectivity with
a distinct spatial organization. Covariance analysis
suggests heterogeneity of both serotonin and GABA
neurons with respect to the inputs they receive.
These analyses provide a foundation for further func-
tional dissection of the serotonin system.
Understanding modulatory neurotransmitter and neuropeptide
signaling will be indispensable for understanding information
flow through neural circuits (Bargmann and Marder, 2013). There
isa particularly urgent need for advances in thisfield, as the most
widely prescribed drugs for neurological disorders target whole-
brain modulatory signaling, yet often suffer from low efficacy
of current brain-wide treatments suggest that it is essential to
rons, which directthe spatiotemporal patterns of their transmitter
release, and in the interpretation of their output by the circuits
being modulated. The need for such understanding is perhaps
best exemplified by the monoamine modulatorytransmittersero-
tonin, famous as the target system of the most widely prescribed
class of antidepressants (Walker, 2013). Serotonin (5-hydroxy-
tryptamine) is an ancient molecule that is instrumental in circuit
function and behavior in diverse organisms, from Aplysia and C.
elegans to mammals (e.g., Brunelli et al., 1976; Liu et al., 2011;
Sawin et al., 2000). It has been implicated in various functions
and dysfunctions of the mammalian brain: from feeding, aggres-
sion, sexual behaviors, and pain modulation to autism, schizo-
phrenia, depression, and anxiety (reviewed in Mu ¨ller and Jacobs,
The serotonin system exerts its widespread effects from a
group of relatively small brainstem nuclei. Serotonin-producing
brain as well as descending projections to the spinal cord
(Dahlstro ¨m and Fuxe, 1964; reviewed in Hornung, 2010). These
projections form classical synaptic connections as well as vari-
cosities with no associated postsynaptic structure (Descarries
et al., 2010). Upon release, serotonin acts primarily on G protein
coupled receptors (and a single ionotropic receptor) encoded by
more than a dozen distinct genes, and many more isoforms, that
are differentially expressed in the brain (Bockaert et al., 2010).
The dorsal raphe (DR) is the largest serotonergic nucleus,
containing more than half of the estimated 20,000 total seroto-
nin-producing neurons in the rat (Descarries et al., 1982). It
has been an area of intensive study due to its innervation of
the forebrain and direct links to behavior, particularly related to
stress, mood, and anxiety (Hale et al., 2012; Maier and Watkins,
2005). However, a number of other cell types are also present
both within the DR and in closely apposed nuclei, including large
and overlapping populations of GABAergic, glutamatergic, and
ropeptides. In addition to heterogeneity with respect to trans-
mitter synthesis, there is also considerable heterogeneity within
serotonergic neurons (and these other cell types) with respect to
connectivity, physiological properties, and receptor expression
(e.g., Calizo et al., 2011; Kirby et al., 2003; Urbain et al., 2006;
reviewed in Gaspar et al., 2003; Hale and Lowry, 2011).
To understand the circuits that control serotonergic modula-
tion of animal behavior and physiology, it is essential to deter-
mine the direct synaptic inputs that control the activity of
Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc. 645
serotonin neurons. Previous studies using anterograde and
retrograde tracers have identified numerous brain areas that
send projections to the DR (reviewed in Hornung, 2010; Jacobs
and Azmitia, 1992). While providing a valuable outline of possible
inputs to DR cell types, most of these studies are limited by the
inability to distinguish axons that pass by the DR from those that
onto which they synapse. The development of monosynaptic
retrograde transsynaptic tracing based on modified rabies virus
(Wickersham et al., 2007) has provided a means to systemati-
cally map the inputs to genetically defined populations of neu-
rons in specific areas of the brain. Here we applied recently
improved strategies for mapping both long-distance and local
synaptic inputs (Miyamichi et al., 2013) to identify and compare
neurons that send direct input to serotonin- and GABA-produc-
ing neurons in the DR.
Figure 1A shows the schematic organization of serotonin (blue)
and GABA (red) neurons in the vicinity of the DR in a series of
coronal sections. This schematic was based on immunostaining
against tryptophan hydroxylase 2 (Tph2) to label serotonin-pro-
ducing neurons (hereafter called serotonin neurons) and in situ
hybridization (ISH) for Gad1 and Gad2, encoding glutamate
decarboxylases 1 and 2, to label GABA-producing neurons
Figure 1. DR Serotonin and GABA Neurons as Starter Cells for Rabies-Based Transsynaptic Tracing
(A) Schematic representation of serotonin (blue) and GABA (red) neurons on coronal sections through the DR and surrounding regions, including the central and
rostral linear nucleus raphe (CLi and RLi, respecitively), midbrain reticular nucleus (MRtN), and ventrolateral PAG (vlPAG). The approximate location targeted for
viral injections and spread of infection is indicated with tan circles. Only serotonin and GABA neurons within these regions are drawn. Aqueduct (Aq).
(B) Schematic of rabies-based transsynaptic tracing. Sert-cre or Gad2-cre mice were transduced with two AAVs in the DR followed by EnvA-pseudotyped,
glycoprotein (RG)-deleted, and GFP-expressing rabies virus. Serotonin or GABA starter cells are labeled in yellow, and presynaptic partners throughout the brain
are labeled in green, as shown on a schematic sagittal section of the mouse brain. TCB, wild-type TVA-mCherry fusion used in Figures 2–5; TC66T, TVA-mCherry
with a point mutation (66T) in the TVA receptor used in Figure 7; CAG, a ubiquitous promoter; triangles: loxP and Lox2272 sites that cause the transgene
expression to be Cre dependent (FLEx).
(C) Left, 60 mm coronal section through the DR of a Sert-cre tracing brain showing the location of starter cells (yellow). Right, z projected confocal stacks of a
different Sert-cre tracing brain in approximately the same position, triple labeled in green for GFP from rabies virus, in red for mCherry from TCB, and in magenta
with anti-Tph2 staining. All starter cells are Tph2 positive (arrowheads).
(D) Same as in (C), except from Gad2-cre tracing. Right panels show that none of the starter cells (arrowheads) are Tph2 positive.
Scale, 100mm.Inthis and all other figures, abbreviations areas follows: A, anterior;P,posterior;D, dorsal; V,ventral; M, medial; L,lateral. Anatomical schematics
and coordinates here and throughout are modified from Paxinos and Franklin (2001). Figure S1 describes further characterization of starter cell populations and
the rabies tracing technique.
Inputs to the Dorsal Raphe
646 Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc.
(GABA neurons, hereafter) (Figures S1A, available online, and
7A). These clusters of serotonin neurons are distributed in
continuous populations across multiple anatomical regions.
However, they are mostly concentrated in the DR near the
midline ventral to the aqueduct and in ‘‘wings’’ that extend into
the ventrolateral periaqueductal gray (vlPAG). GABA neurons
scale, serotonin and GABA neurons are intermingled, including a
small number of cells coexpressing Gad1/2 and Tph2 (Fig-
ure S1A), consistent withprevious reports (Belin et al., 1983; Shi-
kanai et al., 2012). We chose a tracing protocol that would allow
surrounding structures as a whole, despite losing subregion res-
olution. We will use ‘‘DR’’ to refer to these groups shown in Fig-
ure 1A for the remainder of this study.
Strategies for Tracing Inputs to DR Serotonin and GABA
Rabies-based, retrograde, transsynaptic tracing (Wickersham
et al., 2007) relies on two modifications to the rabies virus that
allow for (1) cell-type-specific initial infection with rabies and (2)
monosynaptic spread from these cells. The first aim is achieved
byusing EnvA-pseudotyped rabies virusin combination withtar-
geted expression of the cognate receptor (TVA) in specific cell
types. The second aim is achieved using rabies glycoprotein
(RG)-deleted rabies virus, allowing for rabies spread only when
RG is provided in trans. To generate targeted rabies tracing,
we used two Cre-dependent AAVs—expressing either TVA
receptor-mCherry fusion or RG—in combination with mice that
express Cre in specific cell types (Miyamichi et al., 2013; Wa-
tabe-Uchida et al., 2012). Starter cells are both mCherry+ (from
the TVA-mCherry fusion) and GFP+ (from rabies virus), whereas
their presynaptic partners are only GFP+.
We utilized two complementary strategies that differed in the
TVA receptor used (Miyamichi et al., 2013). The first strategy
utilizes an optimized construct expressing the wild-type TVA
receptor-mCherry (TCB), which allows for high-efficiency, long-
range tracing, but exhibits considerable local background.
The second strategy utilizes a mutant TVA receptor-mCherry
(TC66T), which lowers overall transsynaptic tracing efficiency
compared to TCB, but reduces background to ?0 (Miyamichi
et al., 2013) (Figure S1). We used TCBfor whole-brain input map-
ping, excluding regions near the DR, and TC66Tfor local input
To restrict starter cells to serotonin or GABA neurons, weused
Sert-cre (Gong et al., 2007) and Gad2-cre (Taniguchi et al., 2011)
mice, respectively. Figures 1C and 1D show examples of starter
cells fromSert-cre (C) and Gad2-cre (D) experimental mice. Anti-
Tph2 staining indicated that nearly all starter cells from Sert-cre
tracing were Tph2 positive, while starter cells from Gad2-cre
tracing were predominantly Tph2 negative (Figures 1C and 1D,
inset; Figure S1B). Consistent with our previous result (Fig-
ure S1A), ?5% of starter cells from Gad2-cre tracing were
Tph2 positive (Figure S1B; see Figure S1 and Supplemental
Experimental Procedures for discussion of the rabies tracing
technique as applied to the DR). Together, these experiments
validated our strategy of tracing input to largely distinct popula-
tions of DR serotonin and GABA neurons.
Long-Range Inputs to DR Serotonin and GABA Neurons
To determine the presynaptic partners of DR serotonin and
GABA neurons, we analyzed serial coronal (Figures 2A and 2B)
naptic tracing. Sections from representative Sert-cre (Figures
2A1–2A5and 2C1–2C6) and Gad2-cre (Figures 2B1–2B5) brains
revealed rabies-GFP+ presynaptic input neurons located only
in specific brain nuclei in a bilaterally symmetrical manner.
Figure S2 provides horizontal and sagittal projections from
3D-reconstructed coronal sections. Overall, DR serotonin and
GABA neurons receive input from the same brain regions. Im-
ages from a Sert-cre and a Gad2-cre tracing experiment are
available at http://web.stanford.edu/group/luolab/DR.shtml.
The densest long-range labeling, from anterior to posterior,
was observed in anterior neocortex (Figures 2A1 and 2C);
extended amygdala (EAM), including the bed nucleus of the stria
terminalis (BNST) (Figures 2A2and 2C3–2C6); lateral habenula
(LHb), central amygdala (CeA), and subregions of the hypothala-
area (Figures 2A4, 2C4, and 2C5); as well as deep cerebellar
nuclei (DCN) and the medulla (Figures 2A5, 2C2, and 2C4–2C6).
Despite very dense labeling of these input sites, large regions
of the brain were either blank or sporadically labeled. These re-
striatum, hippocampus, and the majority of the thalamus. While
the central and EAM were densely labeled, there was little label-
ing in the medial, basolateral, and cortical amygdala.
aptic partners of DR serotonin and GABA neurons, we divided
each brain into 33 regions of interest and counted the number
of cells in each (see Experimental Procedures). These regions
accounted for nearly all long-range inputs, omitting the densely
background from TCB-based tracing. Data from four Sert-cre
brains and four Gad2-cre brains representing those with high-ef-
ficiency tracing and starter cells most restricted to the DR were
used in the quantitative analysis described below (Figures S3
On average, tracing from serotonin neurons yielded higher
numbers of long-range GFP+ cells (3,919, 27,582, 35,778, and
50,862 cells per mouse) than tracing from GABA neurons
(2,697, 6,291, 11,862, and 12,665 cells per mouse). This differ-
ence cannot be accounted for by differences in starter cell
numbers (2,147 ± 556.9, Sert-cre and 3,402 ± 1,940, Gad2-
cre; mean ±SEM). As each brain had a different total number
of input cells, in order to directly compare between experiments,
for each mouse we plotted these counts as the fraction of input
neurons counted within a given region over the total number of
input neurons (Figures 2, 3, and 5).
Grouping the 33 subregions that we quantified into eight large
regions, and considering the serotonin and GABA tracing brains
together, the hypothalamus contributed most of the long-range
inputs to the DR, followed by the amygdala, medulla, cortex,
thalamus, cerebellum, striatum, and hippocampus (Figure 2D).
The hippocampus was excluded from further analysis due
to lack of labeling. Even at this coarse resolution, DR serotonin
neurons received a higher proportion of their inputsfrom the cor-
tex (2-fold enrichment, Figure 2D).
Inputs to the Dorsal Raphe
Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc. 647
enriched. Conditional to the caveats of rabies tracing, this sug-
gests that many local GABA neurons may not directly synapse
onto DR serotonin neurons. Several possibilities may account
for this. First, some GABA neurons may act mainly on the pre-
synaptic terminals of neurons that synapse onto serotonin or
other DR neurons (Soiza-Reilly et al., 2013). Second, some
GABA neurons may inhibit other GABA neurons or other local
neurons such as glutamate and dopamine neurons. Third,
many DR GABA neurons are known to send long-range
projections (Bang and Commons, 2012). Given the abundant
long-range GABAergic projections from the DR, it is intriguing
to consider the DR as two parallel but interacting subsystems
that integrate similar inputs and send either serotonergic or
GABAergic outputs. Data presented here suggest that DR
GABA neurons are particularly heterogeneous and may
therefore be ideal first targets for further dissection of DR
We hope that this map of synaptic input to serotonin and
GABA neurons with respect to brain areas, neurotransmitter
phenotypes, and synaptic properties will serve as a foundation
for future functional interrogation of specific DR pathways.
All animal procedures followed animal care guidelines approved by Stanford
University’s Administrative Panel on Laboratory Animal Care (APLAC). All
handling of rabies virus followed procedures approved by Stanford Univer-
sity’s Administrative Panel on Biosafety (APB) for Biosafety Level 2.
Mice and Anatomical Regions
Four Sert-cre and four Gad2-cre brains were chosen based on high tracing
efficiency and starter cells largely restricted to the DR. The DR clusters of se-
rotonin and GABA neurons are in close apposition to those of the rostral- and
central-linear raphe nucleus (RLi, CLi), which is directly ventral to the DR at
certain planes, as well as the midbrain reticular nucleus (MRtN). These exper-
iments included starter cells in these regions, but we excluded brains with sig-
nificant starter cells in other regions, particularly the VTA and median raphe
(Figures S3 and S4). These brains were chosen from seven Sert-cre and 11
Gad2-cre tracing experiments, not including TC66Texperiments and brains
for ISH, which were processed differently (see Supplemental Experimental
Procedures). For the replication cohort, three Sert-cre and four Gad2-cre
experiments were selected from five additional injections. Each included one
brain from the original seven Sert-cre and 11 Gad2-cre that had not been cho-
sen as one of the original eight but was still restricted in starter cells and effi-
cient in transsynaptic spread.
For quantifications of subregions, boundaries were based on the Allen Insti-
tute’s reference atlas (Lein et al., 2007) with consultation of Paxinos and
Franklin (2001). The EAM is treated particularly differently in these two atlases
(Heimer et al., 1997). According to the Allen atlas, our definition includes the
substantia innominata, magnocellular nucleus, anterior amygdalar area, and
the fundus of striatum, though we often used Paxinos and Franklin (2001)
to adjust borders around subregions not annotated in the Allen atlas, such
as the interstitial nucleus of the posterior limb of the anterior commissure
(IPAC). The infralimbic cortex and medulla are as defined in the Allen atlas,
though for medulla, sections anterior to the appearance of the DR were
omitted due to possible local background (Figure S1). For counts of thalamic
subregions, we were conservative while counting sections that border
midbrain nuclei, so our counts may underestimate posterior thalamic subre-
gions. For all regions except the BNST, arcuate nucleus, DMH, and VMH,
every third section was counted, and the final number is adjusted by a factor
of three. These four exceptions are relatively small and rapidly changing re-
gions, so every second section was counted to get a more accurate estimate,
and the final number was adjusted by a factor of two. Note that we did not
adjust for the possibility of double counting cells, which likely results in over-
estimates, with the extent depending on the size of the cells in the regions
For long-range tracing data, cell counts for each experiment were first normal-
ized to the lowest efficiency tracing experiment (2,697 total cells) so that the
total number of cells in each brain was equal. As most of the variance could
be accounted for by the number of cells in a region (R2= 0.85 for Sert-cre
and R2= 0.73 for Gad2-cre), we took the logarithm of the number of cells in
each region, which allowed us to perform two-way ANOVA as the variances
were equal across regions (Brown-Forsythe test). Normality was confirmed
with the D’Agostino and Pearson test. All p values for subregion post hoc tests
contained less than 1% of total labeling were omitted).
Analysis oflocalsubregioninputsinFigure7usedone-wayANOVA followed
by Bonferronni corrections (equal SD, Brown-Forsythe test). All graphing and
analysis described above was done using Prism software (GraphPad). For
analysis of clustering in Figure 8, we created a vector for each experimental
brain containing the proportions of GFP+ cells in each subregion. We then
generated pairwise correlations in Matlab (Mathworks) and graphed relation-
ships using Prism (GraphPad). Heatmaps and dendrograms were generated
in R (http://www.r-project.org/).
Supplemental Experimental Procedures contain detailed descriptions of
rabies-mediated transsynaptic tracing, rabies tracing combined with in situ
hybridization (ISH), histology and imaging, PCR primers used to prepare tem-
plates for ISH probes, and CRACM.
Supplemental Information includes seven figures, one table, and Supple-
mental Experimental Procedures and can be found with this article online at
We thank R. Malenka for advice on electrophysiology; M. Lochrie and The
Stanford Viral Core for AAV production; T. Mosca for help with image analysis;
X.Gao, C. Lowe, and W.Allenfor advice on data analysis; C.Manalac for tech-
members of the Luo Lab for discussions and critiques on the manuscript. We
thank N. Uchida and K. Meletis for coordinating submission. B.C.W. is sup-
ported by a Stanford Graduate Fellowship and an NSF Graduate Research
Fellowship (grant number DGE-114747). C.J.G. was supported by a National
Defense Science and Engineering Graduate Fellowship. K.M. was a Research
Specialist, and L.L. is an Investigator, of the Howard Hughes Medical Institute.
Supported by an HHMI Collaborative Innovation Award (HCIA).
Accepted: June 19, 2014
Published: August 6, 2014
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