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: firstname.lastname@example.org (K.M.), email@example.com (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
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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.
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(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).
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(legend on next page)
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Serotonin and GABA Neurons Receive Specific
To investigate input tracing in more detail, we first compared the
distribution of inputs to DR serotonin and GABA neurons from
subregions of interest in subcortical areas (Figure 3; Table S1).
To test whether these subregions send excitatory or inhibitory
inputs, we combined TCB-based rabies tracing with ISH using
vGlut1/2 or Gad1/2 probes, respectively.
Across subregions, the distribution of inputs from Sert-cre
versus Gad2-cre tracing experiments was significantly different
(p < 0.0001, two-way ANOVA), and projections from each of the
large regions above (Figure 2D) were mostly from a specific
subset of subregions. For example, thalamic inputs to both
DR cell types were predominantly from the LHb, which ac-
counted for 87% ± 2% of thalamic inputs (Sert and Gad pooled,
Figure 3A1). LHb and PVT inputs were predominantly vGlut2
positive (Figure 3A2), though we also observed sparse Gad1/
2-positive projection neurons in the LHb (Figure 3A3). From
within the cerebellum, greater than 95% of inputs to both sero-
tonin and GABA neurons came from the DCN (Figure 3B1),
which sent glutamatergic (Figure 3B2) and not GABAergic (Fig-
ure 3B3) projections.
The hypothalamus contributes similar proportions of inputs
to both DR serotonin and GABA neurons, with 34% of hypotha-
lamic inputs coming from the lateral hypothalamus (Sert and
Gad pooled, Figure 3C1). While no differences in hypothalamic
input to serotonin and GABA neurons reach statistical signifi-
cance, there are notable trends. These include the particularly
large proportions of input to serotonin neurons from the lateral
hypothalamus and to GABA neurons from the parasubthalamic
nucleus (Figure 3C1). The lateral hypothalamus contains both
vGlut- and Gad-positive inputs to both serotonin and GABA
neurons (estimated 2-fold more Gad-positive overall) with
considerable variation between subregions of the LH (Figures
We next subdivided the amygdala complex into four regions:
the CeA, EAM (see Experimental Procedures for definition),
BNST, and the remaining amygdalar nuclei combined. We
found that three subdivisions account for nearly all amygdalar
projections to the DR: the EAM, CeA, and BNST (Figure 3D1).
While the EAM makes up a similar proportion of the total inputs
to serotonin and GABA neurons, the CeA makes up a signifi-
cantly larger proportion of inputs to GABA neurons (2.8-fold
enrichment) and sends GABAergic projections (Figures 3D2–
3D4). The BNST has a notable trend toward projecting to DR
GABA neurons and sends GABAergic projections from the dor-
sal BNST (Figures 3D1and 3D5–3D7), though sparse vGlut2-
positive inputs to the DR exist in other BNST subregions
specific subcortical structures. Embedded in the overall similar-
ity between Sert-cre and Gad2-cre tracing experiments are
considerable differences in input distribution, including striking
differences in the central amygdala.
DR Neurons Receive Inputs from Diverse Cell Types in
Much like the DR, each anatomically defined subregion that we
havefocusedonthusfarcontains acomplex andheterogeneous
group of neurons. We next combined TCB-based transsynaptic
tracing from Sert-cre or Gad2-cre mice with ISH to identify the
cell types from within the biased CeA and unbiased PVH that
send inputs to DR serotonin and GABA neurons.
The central amygdala is composed of many populations of
cells, including subsets that produce neuropeptides. From the
Allen Brain Atlas (Lein et al., 2007), we identified four neuropep-
tide-encoding genes Tac1, Tac2, Preproenkephalin, and Crh
(encoding corticotropin-releasing hormone) that are expressed
in subsets of CeA neurons; we also included PKCv, a marker
of a functional subset of CeA neurons (Haubensak et al.,
2010). We found that Tac2-positive neurons account for
?40% of the CeA inputs to both serotonin and GABA neurons
(Figures 4A1–4A4), whereas Pkcv+ projections account for
?8% (Sert-cre and Gad2-cre experiments pooled; Figures
4B1–4B4). There were no significant differences in the proportion
of CeA inputs from either cell type projecting to DR serotonin
versus GABA neurons. We also observed many Crh-positive in-
puts from the CeA to both serotonin and GABA neurons in the
DR (21% pooled average), as well as sparse inputs from
Preproenkephalin- (to both serotonin and GABA neurons) and
Tac1-expressing neurons (only serotonin neurons tested)
Thus, inputs to serotonin and GABA neurons from the central
amygdala are from a mixture of cell types, this mixture is of
similar proportions, and a Tac2-expressing population accounts
Figure 2. Overview of Whole Brain Input to DR Serotonin and GABA Neurons
(A and B) Coronal sections of a Sert-cre (A) and a Gad2-cre (B) tracing brain showing the distribution of presynaptic partners. Approximate section planes are
shown in the top right on a sagittal section of a schematic brain, with approximate distance (anterior-posterior) from the bregma. Images at the bottom are higher
magnification views of the rectangular regions on the top images.
(C) Six horizontal sections of one hemisphere of a Sert-cre tracing brain showing the location of presynaptic partners. Approximate section planes and distances
from bregma are shown in the top right on a schematic sagittal brain section.
four individual Sert-cre (blue) and Gad2-cre (red) tracing experiments. Serotonin neurons receive a greater proportion of their input from the cortex. BNST, bed
Thal, thalamus; Cereb, cerebellum; Striat, striatum; HP, hippocampus.
Scale, 250 mm. Statistical analysis here, and in Figures 3 and 5, used two-way ANOVA on normalized cell counts with Bonferroni corrections (see Experimental
Procedures). Hypothalamus values are underestimates, as not all subregions were counted (see Table S1). In this and subsequent figures, error bars indicate
SEM. Significance notation: *p < 0.05; **p < 0.01; ***p < 0.001. Figure S2 shows horizontal and sagittal views of inputs to DR serotonin and GABA neurons from
3D-reconstructed coronal sections. Figures S3 and S4 show starter cell distributions for each of the eight experiments used.
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Figure 3. Quantitative Analysis of Subcortical Input Distribution
cre (red) tracing experiments and are shown as the proportion of the total cells counted in an experimental brain located in a given subregion. Accompanying
photomicrographs show glutamatergic or GABAergic projections from subregions of interest. Green, rabies-GFP; red, ISH using a vGlut1 and/or vGlut2 probe or
a Gad1+2 probe mix. All arrowheads point to double-labeled cells. Values in the upper right indicate approximate distance from bregma for each set of images.
(A) (A1) Proportion of total inputs from thalamic subregions. Inputs from the thalamus are almost entirely from the lateral habenula (LHb) and glutamatergic (A2),
though we observe sparse LHb GABAergic presynaptic neurons as well (A3).
(B) (B1) Proportion of total inputs from the cerebellum. Inputs from the cerebellum are almost entirely from the deep cerebellar nuclei (DCN) and are glutamatergic
(B2) and not GABAergic (B3).
(legend continued on next page)
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neurokinin B, which is involved in the regulation of sexual matu-
ration and function in the hypothalamus (Lasaga and Debeljuk,
2011), but whose role in the CeA remains unknown. As we
have shown that the CeA makes up a larger proportion of inputs
to DR GABA neurons (Figure 3D1), these cell types are likely to
disproportionately affect DR GABA neuron function.
We next used the same strategy to identify subsets of PVH
neurons that project to DR serotonin and GABA neurons. Cells
in the PVH express vGlut2 and rarely Gad1/2; consistent with
this observation, we see many vGlut2-positive but no Gad1/2-
positive inputs from the PVH to the DR (Figures 4E1and 4E2).
We next labeled oxytocin- or vasopressin (AVP)-producing neu-
rons and observed that both serotonin and GABA neurons
receive inputs from both cell types (Figures 4C and 4D) and
that numerous oxytocin (Figure 4E3) and AVP (Figure 4E4) neu-
rons in the PVH also express vGlut2.
AVP and oxytocin inputs were consistent across Sert-cre
tracing experiments, with a trend toward preferential targeting
of serotonin neurons by AVP (Figure 4C4). However, there
was high variability between Gad2-cre samples (Figure 4C4),
similar to the Gad2-cre-specific high variation in the PVH
seen in the subregion mapping above (Figure 3C1). This sug-
gests that PVH populations may target specific subsets of
DR GABA neurons, making tracing results more susceptible
to differences in the starter cells sampled (see Figure 8;
Anterior Cortical Neurons Send Biased Input to DR
Serotonin Neurons Compared to GABA Neurons
The connectivity between the anterior cortex and the DR has
attracted great interest as a circuit involved in modulating
stress and depressive behaviors (Amat et al., 2005; Warden
et al., 2012). Interestingly, our analysis of large brain structures
suggests that the cortex as a whole preferentially targets sero-
tonin neurons (Figure 2D). We therefore investigated cortical
input patterns in more detail by first counting the number of
labeled cells in seven subregions of the neocortex (Figures 5A
inputs to DR serotonin neurons compared to DR GABA neurons
(Figures 5B, 5D, and 5E). We also observed smaller but signifi-
cant biases in the orbital and prelimbic/cingulate (PrL/Cg)
cortices toward DR serotonin neurons (Figures 5B, 5D, and
5E). These input neurons from the cortex to the DR are glutama-
tergic (Figures 5F1and 5F2). The anterior-posterior distributions
of cortical inputs were similar for Sert-cre and Gad2-cre brains,
with strong bias toward anterior cortical regions (Figures 5C–
5E). Thus, the anterior cortex in general, and the insular cortex
in particular, sends biased input to DR serotonin neurons
compared to GABA neurons.
To test whether the biased input from anterior cortex to DRse-
rotonin neurons over GABA neurons reflects biased functional
connections, we employed channelrhodopsin-assisted circuit
mapping (CRACM) (Petreanu et al., 2007) to examine connectiv-
fill a large proportion of anterior cortex. Four weeks later, we
transduced the DR with an AAV expressing Cre-dependent
mCherry to label either serotonin or GABA neurons. Two weeks
later, acute coronal DR slices were used for whole-cell patch
recording in response to photostimulation of cortical axon termi-
nals (Figure 6A).
All animals with expression of ChR2 in the anterior cortex
(n = 8 Sert-cre mice, n = 8 Gad2-cre mice) had bright EYFP+
axon fibers in DR slices (Figure 6B), indicating direct projections
from anterior cortical neurons. In voltage-clamp mode, brief
blue light illumination (5 ms) evoked immediate excitatory post-
synaptic potentials (EPSCs) in a subset of mCherry+ serotonin
and GABA neurons when cells were held at?65 mV (Figure 6C1).
These responses were eliminated by application of 6,7-dinitro-
quinoxaline-2,3-dione (DNQX; 10 mM), a selective antagonist
of AMPA-type glutamate receptors (Figures 6C and 6D), indi-
cating that anterior cortical axons release glutamate (n = 7).
The short latency as well as pharmacological inhibition and
reinstatement (Figure S6) indicate that these connections are
Of a total of 43 serotonin neurons from four Sert-cre mice, 15
cells (35%) exhibited EPSCs in response to photostimulation. Of
the 57 GABA neurons recorded from five Gad2-cre mice, only
nine cells (16%) produced EPSCs in response to photostimula-
tion (Figure 6E). Post hoc staining of neurobiotin-filled cells that
received direct input from anterior cortex confirmed their cell
type identity and their location within the DR (Figures 6B). These
results demonstrate monosynaptic, glutamatergic inputs from
anterior cortical neurons onto a subset of DR serotonin and
GABA neurons, with a higher connectivity rate with serotonin
neurons. This is consistent with our transsynaptic tracing results
showing that serotonin neurons receive a greater fraction of their
inputs from the anterior cortex (Figure 5B). Interestingly, DR
GABA neurons that received direct anterior cortical input were
preferentially concentrated in the ventral wing (Figure 6F).
Anterior Cortical Inputs to DR Serotonin and GABA
Neurons Exhibit Different Postsynaptic Properties
In addition to anatomical connectivity, functional differences
may be present in the synaptic properties of these connections.
Wetherefore examinedthepostsynapticproperties ofcortex-to-
DR connections by characterizing the voltage dependence of
photostimulation-induced EPSCs.Both cell typesexhibited rela-
tively large EPSCs at hyperpolarized holding potentials, while
(C) (C1) Proportion of total inputs from subregions of the hypothalamus. The lateral hypothalamus (LH) makes up the majority of hypothalamic input, though many
regions send considerable projections to the DR. The LH sends both glutamatergic and GABAergic projections ([C2]–[C5]).
(D)(D1)Proportion of totalinputs from subregionsof theamygdala.The central amygdala (CeA) makes upalarger proportion of totalinputto DRGABAcompared
to serotonin neurons (Bonferroni correction against the 33 subregion comparisons). The central amygdala sends GABAergic and not glutamatergic input ([D2]–
[D4]). The dorsal BNST sends GABAergic input ([D6] and [D7]), and we have seen sparse vGlut2+ projections from other subregions (D5).
Scale, 100 mm. Abbreviations color code are as follows: subregions of the thalamus (orange), cerebellum (magenta), hypothalamus (blue), and amygdala (red).
Table S1 contains cell counts for each subregion, including those not shown here, and qualitative information on subregions not counted.
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Figure 4. Characterizing DR Input Cell Types in the Central Amygdala and Paraventricular Hypothalamic Nucleus
(A and B) Central amygdala sections with Tac2 (A) or Pkcd (B) ISH (red), alone with anatomical boundaries ([A1] and [B1]) or in a Sert-Cre ([A2] and [B2]) or a Gad2-
Cre ([A3] and [B3]) brain; quantification of GFP+ cells that are Tac2+ or Pkcd+ is also shown ([A4] and B4]). High magnification images to the right of each image
correspond to boxes in the low magnification images to the left. BLA, basolateral amygdala.
(C and D) PVH sections with vasopressin (C) or oxytocin (D) ISH (red) alone with anatomical boundaries ([C1] and [D1]) or in a Sert-cre ([C2] and D2]) or a Gad2-Cre
([C3] and [D3]) brain; quantification of GFP+ cells that express vasopressin (C4) or oxytocin (D4) is also shown.
(E) PVH projections to the DR are vGlut2 positive (E1) and not Gad1/2 positive (E2). Populations of both oxytocin (E3) and vasopressin (E4) neurons coexpress
Scale, 100 mm. All arrowheads (except in [E3] and [E4]) indicate double-labeled cells for GFP from rabies virus and ISH probes. Analysis by two-tailed, unpaired
t tests followed by Bonferroni correction. Each data point represents one experimental animal. Figure S5 shows additional cell types that send input to DR
serotonin and GABA neurons from the CeA.
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GABA neurons showed much smaller outward current at depo-
larized potential (Figures 6G and 6H1). Thus, in contrast to
serotonin neurons whose I–V curve was generally linear, GABA
neurons showed a rectified I–V curve (Figure 6I) with (green
line) or without (red line) the NMDA receptor antagonist APV in
the recording solution. This suggests that DR GABA neurons
express GluA2-lacking AMPA receptors, which confer distinct
synaptic properties compared to GluA2-containing AMPA re-
ceptors, including increased synaptic conductance and perme-
ability to Ca2+(Isaac et al., 2007). In addition, EPSCs in GABA
neurons did not have a marked NMDA component (Figure 6H1).
For five GABA neurons tested, APV (50 mM) had a relatively small
effect on the EPSC when clamped at +60mV (Figures 6H2and
6J, lower panel). In contrast, EPSCs in serotonin neurons ex-
hibited an increased slow component when the holding potential
went up from ?40mV to +60mV (Figures 6G and 6J). In serotonin
nent was eliminated, indicating significant NMDA receptor con-
tributions to postsynaptic currents of serotonin neurons (Figures
6G and 6J).
Serotonin Neurons Receive Diverse Local Input with a
Specific Spatial Pattern
In order to control serotonin neuron activity, the complex, long-
range inputs that we have described thus far interact with a
largely uncharacterized local DR circuit containing diverse cell
types (see Introduction). Interestingly, the specific location of
cortical inputs to DR GABA neurons in the CRACM experiments
(Figure 6) further implies that there are spatially distinct subsets
of DR serotonin and GABA neurons. To gain insight into how
Figure 5. Quantitative Analysis of Cortical Input Distribution
(A) Schematic showing the location of cortical subregions quantified in (B) at one of the many coronal planes quantified.
(B) Inputs to DR serotonin (blue) or GABA (red) neurons from subregions of the anterior cortex. Serotonin neurons receive a higher proportion of their total input
from insular, orbital (orb), and prelimbic/cingulate (PrL/Cg) cortices. ‘‘Other’’ includes all other neocortical regions.
corpus callosum and the anterior border of the BNST indicated on graph.
(D and E) Coronal sections of a Sert-cre (D) and a Gad2-cre (E) brain with the locations of cortical subregions indicated.
(F) Cortical projections are all glutamatergic (F1) and not GABAergic (F2).
Scale, 200 mm. Som, somatosensory cortex; Infra, infralimbic cortex.
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Figure 6. Connectivity and Synaptic Properties of Cortical Inputs to DR Serotonin and GABA Neurons
(A) Schematic drawing of virus injection and slice recording procedures. Top, sagittal view showing AAVDJ-CaMKII-ChR2(H134R)-EYFP injection into anterior
cortex (AC) of either Sert-cre (n = 6) or Gad2-cre (n = 5) mice. AAVDJ-EF1a-FLEx-mCherry was injected into the DR to label serotonin or GABA neurons. Bottom,
(legend continued on next page)
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654 Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc.
serotonin neurons integrate long-range and local inputs, partic-
ularly in their interactions with DR GABA neurons, we next char-
acterized local synaptic input to serotonin neurons utilizing the
TC66T-based tracing strategy with minimal local background
(Figure S1E) (Miyamichi et al., 2013).
Figures 7A1–7A4show coronal sections through the DR with
rabies-GFP tracing from serotonin neurons combined with
Gad1/2 ISH. The DR and vlPAG contain large, distinct clusters
of GABA neurons, and inputs to serotonin neurons are enriched
in particular subregions. Interestingly, large regions of these
neighboring DR GABAergic neurons were essentially unlabeled,
particularly when compared to the very dense inputs from a
nearby subregion of the midbrain reticular nucleus (MRtN)
(e.g., Figure 7A2). In contrast, we observed many Tph2-positive
local inputs (Figures 7B1and 7B2).
of Gad1/2 ISH: dorsal wings (DWs), ventral wings (VWs), and the
midbrain reticular nucleus (Figures 7C1–7C3) and grouped these
subregions into four bins across the anterior-posterior axis
(similar to Bang and Commons, 2012). We then quantified the
number of local inputs to serotonin neurons that were coming
from each of these subregions. Beginning with all local inputs
regardless of their cell type, we found that the three subdivisions
the total input, the VWs for 27% ± 4%, and the MRtN for 38% ±
7%. When the spatial distribution of local inputs was analyzed
using heatmaps to show relative enrichment of a subregion
along the A-P axis, it was evident that specific subregions
were responsible for the majority of local inputs (Figure 7E).
We next examined the distribution of cell-type-specific inputs
to DR serotonin neurons. Surprisingly, local inputs were as likely
to be from other serotonin neurons (41% ± 6%) as from neigh-
boring GABA neurons overall (39% ± 0.7%, Figure 7D). This
is likely an underestimate of serotonin-serotonin connectivity,
as the subregions we used to analyze local input (Figure 7C)
excluded serotonin neurons on the midline, where local seroto-
nin inputs were densely intermingled with starter cells. The
roughly 20% that is unaccounted for is composed of multiple
cell types, including vGlut1-3+ input, notably from the Gad-
negative, anterior PAG clusters in Figure 7A1(data not shown).
We next looked at the spatial distributions of Gad1/2- and
Tph2-positive inputs across the subregions described above
(Figures 7F and 7G). Local GABAergic input primarily came
from the MRtN (Figure 7F1), while serotonergic inputs were
more evenly distributed across the MRtN, DWs, and VWs (Fig-
ure 7G1). Further, GABAergic and serotonergic local inputs to
DR serotonin neurons came from specific subregions along the
A-P axis: serotonergic local inputs were enriched in the ventral
and DWs in the central DR, while GABAergic local inputs were
mostly from posterior DWs and the anterior midbrain reticular
nucleus (Figures 7F2and 7G2).
In summary, these results demonstrate the diversity of
local input and the presence of a specific spatial organiza-
tion—including the finding that an MRtN subregion sends dense
inputs to DRserotonin neurons. Theseresults also suggest, con-
ditional to the caveats of rabies-based tracing, that many local
GABA neurons may not play a major role in directly inhibiting
DR serotonin neurons.
Covariance between Input Regions Suggests DR Circuit
Our analysisof localinputssuggests that theDRishighly hetero-
geneous with spatially distinct populations. In our analysis of
long-range tracing, we see considerable variability between an-
variability may indicate that starter cells in each experiment
sample from subpopulations of DR neurons that have different
underlying connectivity. We therefore explored the variability
between experiments in more detail. To increase the number
of independent samples, and to validate our previous findings,
we quantified the number of cells in the anterior cortex (anterior
to the corpus callosum crossing), central amygdala, medulla,
LHb, PVH, and BNST for a replication cohort of three Sert-cre
whole-cell recording from mCherry+ cells in coronal sections containing the DR coupled with the laser stimulation from anoptical fiber placedimmediately above
(B) Confocal z projections showing recorded DR serotonin (upper panels) and GABA (lower panels) neurons filled with neurobiotin during recording (cyan)
the area covered by ChR2-EYFP+ axonal terminals. All are mCherry+. Gad2-cre slices were stained with Tph2 antibody (pink); all neurobiotin-labeled neurons
were negative for Tph2. Scale, 50 mm.
(C) EPSCs evoked by photostimulation (blue bar, 5 ms) are mediated by AMPA receptors. Stimulation-induced EPSCs (C1) were abolished by application of
DNQX (C2), an AMPA receptor antagonist. Top traces are the average of six trials from the same serotonin neuron, with 20 s intertrial intervals. Bottom graph
shows the change in EPSC amplitude over time. Each dot represents an EPSC generated by optical stimulation at fixed 20 s intervals.
Red dots, GABA neurons. Paired t test, n = 7 cells.
(E) Connectivity rate of anterior cortical input to DR serotonin (blue) and GABA neurons (red).
(F) Summary diagram showing the locations of recorded DR serotonin (blue, left) and GABA neurons (red, right). Purple, nonresponding cells. Aq, aqueduct.
(G) EPSCs of a DR serotonin neuron at different membrane potentials. APV application abolished the slow (NMDA receptor) component at +60mV (inset, green
trace). Each trace is an average of six repeats.
(H) (H1) EPSCs of a DR GABA neuron at different membrane potentials. (H2) APV application had minimal effect on the EPSC at +60mV (inset, green trace). Each
trace is an average of six repeats.
(I) I/V curves of serotonin (blue, n = 7) and GABA neurons with (green, n = 5) or without (red, n = 5) APV in the recording solution.
(J) Decay time of photostimulation-evoked EPSCs. For serotonin neurons (blue), decay time of EPSCs significantly increased at +60mV compared to ?65mV
and was dramatically reduced by APV application. Paired t test, n = 7 cells. For GABA neurons (red), little difference was seen between EPSCs recorded
at ?65mV, +60mV, or with APV application.
Figure S6 provides evidence that connections between anterior cortical axons and DR serotonin and GABA neurons are monosynaptic.
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(legend on next page)
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656 Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc.
and four Gad2-cre tracing experiments. These new experiments
support our previous findings, with the cortex and medulla
making up a larger proportion of inputs to serotonin neurons
and the CeA and BNST making up a larger proportion of inputs
to GABA neurons (Figure S7).
When combining these replicate experiments with our eight
previously analyzed brains, we observed that more sparsely
labeled brains tended to be more variable, while densely labeled
brains often trended toward the mean. This suggests that our
tracing results may represent a sample drawn from subsets of
DR neurons with differential connectivity, with high-efficiency
brains more widely sampling from, and effectively averaging,
these subsets. We therefore asked whether inputs from subre-
gions were covarying, which might indicate that subpopulations
brain regions and provide candidate pairs of regions that inner-
vate the same subsets of cells.
Using the seven Sert-cre and the eight Gad2-cre experiments
included above, we performed pairwise correlations followed by
hierarchical clustering analysis between the six regions quanti-
fied. Interestingly, we observed considerable clustering in both
Sert-cre and Gad2-cre cohorts (Figures 8A and 8B). For
Sert-cre tracing, the LHb, PVH, and cortex formed a cluster
separate from the medulla, CeA, and BNST. We observed a
particularly strong negative correlation between the cortex and
medulla (Figures 8A and 8C). Generally, clustering and correla-
tions were more striking in Gad2-cre tracing experiments, with
the CeA and BNST forming a particularly strong cluster distinct
from the cortex, LHb, and medulla (Figure 8B). The medulla
was negatively correlated with the CeA (Figure 8D) and BNST
(r = ?0.84), while the CeA was positively correlated with the
BNST (Figure 8E). Additionally, the CeA and BNST were both
negatively correlated with the cortex (r = ?0.72 and ?0.82,
neurons receive inputs from different combinations of regions.
For example, serotonin neurons receiving cortical input may be
largely distinct from those innervated by the medulla, and
GABA neurons receiving CeA inputs may be the same as those
receiving BNST input, yet distinct from those receiving cortical
and medullary input. This could occur at the level of individual
cells, small clusters, or large spatial regions, as our sampling
of the DR is concentrated around a spatially defined injection
site. Interestingly, Gad2-cre tracing experiments appeared
more clustered, suggesting that DR GABA neurons are
composed of distinct subsets more easily distinguishable with
DR serotonin and GABA neurons receive direct excitatory, inhib-
itory, and peptidergic input from diverse yet specific regions
(Figure 8F). Glutamatergic neurons in the anterior cortex prefer-
entially synapse onto DR serotonin neurons, whereas DR GABA
neurons receive a higher proportion of their inputs from the
GABAergic central amygdala. CRACM confirmed biased input
of cortical projections to serotonin neurons and identified
different postsynaptic properties of DR cell types. Analysis of
local connectivity within the DR demonstrated that a large
proportion of inputs to serotonin neurons are from other seroto-
nin-producing cells and that local input comes from distinct
spatial locations (Figure 8G). Analysis of long-range inputs also
provides evidence for DR subcircuits and predicts which brain
regions may preferentially coinnervate DR subpopulations.
Below we discuss the limitations, advances, and implications
of our study.
Caveats and Limitations
It is important to note that the precise mechanisms and possible
caveats of rabies-based tracing are not entirely known, particu-
larly as they apply to previously untested cell types and connec-
tions. It has been established in many systems that rabies virus
spreads effectively across known synaptic connections (Hau-
bensak et al., 2010; Miyamichi et al., 2011, 2013; Stepien
et al., 2010; Takatoh et al., 2013; Ugolini, 1995; Watabe-Uchida
et al., 2012; Wickersham et al., 2007) and does not infect axons
in passage (e.g., Miyamichi et al., 2011). However, it is unknown
whether thereare biasesin which synapsesare crossed, and itis
difficult to prove complete synaptic specificity in complex CNS
circuits in vivo; for example, whether rabies virus will cross an
axo-axonal synapse that is onto the presynaptic terminal of an
input to a starter cell. Lastly, it is not known whether the
Figure 7. Distribution of Local Inputs to DR Serotonin Neurons
(A) Coronal sections (anterior to posterior) from a Sert-cre brain showing rabies-GFP+ local inputs (green) to serotonin neurons (TVA-mCherry, magenta) with
Gad1+2 ISH (red). Aq, aqueduct; MRtN, midbrain reticular nucleus.
with white boxes. Arrowheads indicate Tph2+ local input neurons.
(C) Coronal sections with outlines indicating regions for quantification. DW, dorsal wing; VW, ventral wing.
(D) Proportion of local inputs to DR serotonin neurons coming from other serotonin neurons (blue) or GABA neurons (red); n = 3 tracing experiments.
(E) Spatial distribution of local input to DR serotonin neurons across DR subregions, regardless of their cell type. For each subregion, the proportion of total local
n = 5 tracing experiments.
(F) The proportion of GABAergic local inputs to serotonin neurons located in each of the three subregions (F1) and their spatial distribution (F2). GABAergic inputs
are largely from the MRtN compared to the dorsal (2.4-fold) and ventral (5.6-fold) wings (F1). Inputs are mostly from anterior MRtN and posterior DWs (F2). Spatial
distribution shown as a heatmap, as in (E2). Analysis of n = 3 tracing experiments by one-way ANOVA with Bonferonni correction.
(G) The proportion of serotonergic (Tph2-positive) local inputs to serotonin neurons located in each of the three subregions (G1) and their spatial distribution (G2).
While serotonergic local input neurons are evenly distributed across the three subregions, their detailed spatial distribution shows dense inputs from the dorsal
and ventral wings in the central DR. Analysis of n = 3 tracing experiments, as above.
Scale: 200 mm.
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Figure 8. Covariance between Input Regions and Summary of Findings
(A and B) Pairwise correlations and cluster analysis of six regions counted in seven Sert-cre (A) and eight Gad2-cre (B) experiments. Heatmaps represent high
correlation (red) or anticorrelation (blue) between regions. Cortex includes all neocortical regions anterior to the corpus callosum crossing.
(C–E) Example graphs showing a strong negative correlation between cortical and medullary inputs in Sert-cre tracing experiments (C), a strong negative cor-
(F) Summary of inputs to the DR on a schematic sagittal section showing regions that make up greater than 1% of total inputs. Percentage of total input is coded
by gray scales (inset). Stars indicate biased input to DR serotonin (blue) or GABA (red) neurons. The primary neurotransmitters expressed by input regions are
shown as small circles as indicated in the inset.
(G) Schematic of local inputs to DR serotonin neurons. Blue, serotonergic inputs; red, GABAergic inputs. The number of circles in each region reflects the
quantitative distribution of local inputs of each cell type.
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658 Neuron 83, 645–662, August 6, 2014 ª2014 Elsevier Inc.
differences in tracing efficiency observed between serotonergic
and GABAergic neurons reflect real, underlying connectivity
rates or are a result of the tracing technique and how this might
affect input distribution.
A second limitation is that some DR neurons express both
Tph2 and Gad2, so that serotonin and GABA input tracing
experiments contain an overlapping population of starter cells.
However, these Tph2+Gad2+ cells are a small fraction of the
population, making it unlikely that they would alter our conclu-
sions. A third limitation is that our study samples large popula-
tions of starter cells across the DR, which likely combine many
distinct subpopulations of DR neurons (discussed below).
From these caveats and limitations, we suggest that this study
of inputs to populations of DR neurons has reliably revealed
trends and biases that likely underestimate the true specificity
within DR circuits.
DR Neurons Receive Diverse Inputs
Our results are consistent with previous DR input studies using
classic tracers (reviewed in Hornung, 2010; Jacobs and Azmitia,
1992). As a specific example, a comprehensive retrograde
tracing study in the rat (Peyron et al., 1998) labeled cells in all
of the forebrain areas that we identified as sending input to the
DR. Our study has extended these previous studies by enabling
us to identify the presynaptic partners of specific DR cell types.
We found that the major input regions are often associated
with processing autonomic and emotional information, such as
the amygdala, hypothalamic subregions, and LHb (Swanson,
2011; Lammel et al., 2012; Matsumoto and Hikosaka, 2009).
Additional input regions include the anterior cortex and cere-
bellar nuclei that play diverse roles in coordinating sensation
and action, motor control, and cognitive function (Swanson,
This study treated the DR as a homogenous unit for the pur-
pose of characterizing its overall input. Previous studies (see
Introduction) and data presented here (Figures 6F, 7, and 8) sug-
gest that these results likely represent input to starter cells that
consist of multiple subtypes that receive unique combinations
of inputs. We also observed that input regions were differentially
variable (Figures 3, 5, and 8). This differential variability sug-
gests that highly variable input regions innervate specific cell
while less variable regions may more widely innervate the DR or
target less clustered populations. These highly variable input
regions are exciting candidates for the dissection of functional
Cortical Inputs to the DR
Our map of long-range inputs has identified interesting differ-
ences between DR serotonin and GABA neurons (Figure 8F).
Among inputs to the DR, medial prefrontal cortex (mPFC) has
received particular attention (mPFC is mostly composed of our
PrL/Cg and infralimbic subdivisions) (Figure 5). Stimulation of
the rat mPFC causes a reduction in the firing rates of DR seroto-
nin neurons in vivo (Celada et al., 2001; Hajo ´s et al., 1998),
suggesting that mPFC axons mainly synapse onto DR GABA
neurons, which in turn inhibit serotonin neurons. In support of
this hypothesis, an electron microscopic (EM) study using dual
labeling of mPFC afferents and Tph2 (serotonergic neurons) or
GABA found more frequent mPFC terminals synapsing onto
GABA-labeled dendrites than Tph2-labeled dendrites (Jankow-
ski and Sesack, 2004). Our study found that cortical subregions
(including the PrL/Cg) were preferentially labeled when starter
cells were DR serotonin neurons rather than GABA neurons,
and our CRACM analysis found that serotonin neurons had a
2-fold higher chance of receiving input from the anterior cortex
as a whole when compared to GABA neurons.
Differences in the techniques used (and the subsequent con-
clusions) in our study and these previous ones may provide
insight into DR circuit structure. It is clear from this combination
of studies that mPFC projections synapse onto both serotonin
and GABA neurons. The bias found in the EM study (Jankowski
cortical input to DR cell types vary across DR and/or cortical
subregions. This is consistent with our observation that specific
cortical subregions make up a larger fraction of input to DR
serotonin neurons compared to GABA neurons (notably not the
infralimbic cortex) (Figure 5B). Further, GABA neurons receiving
cortical input are clustered in a specific portion of the DR, within
which the connectivity rate is comparable to that observed for
serotonin neurons overall (Figure 6F). Interestingly, the inhibition
of DR serotonin neurons by mPFC stimulation observed in
Celada et al. (2001) was in part due to activation of inhibitory
serotonin autoreceptors, consistent with local tracing that iden-
tified extensive interconnectivity of DR serotonin neurons (Fig-
ure 7). We therefore suggest the following: (1) cortical inputs to
the DR, taken overall, preferentially synapse onto serotonin neu-
rons; (2) connectivity rates are highly variable over subregions of
complex local circuit that may feature substantial serotonergic
On the Relationship between DR Serotonin and GABA
Several possibilities could account for the finding that DR sero-
tonin and GABA neurons receive inputs from the same regions.
It is possible that specificity is diluted due to the technical ca-
veats described above. However, the coinnervation of serotonin
and GABA neurons may reflect complex computations by the
local DR circuit and a need to control serotonin activity in a
spatiotemporally precise manner. For example, excitation of a
population of serotonin neurons may be accompanied by inhibi-
the same serotonin neurons to limit the duration of excitation,
akin to lateral inhibition and feedforward inhibition in many other
systems (Isaacson and Scanziani, 2011). Feedforward inhibition
may contribute to the low firing rates of serotonin neurons
(Jacobs and Azmitia, 1992; Urbain et al., 2006).
Whereas GABA neurons in the DR are generally thought to
locally inhibit serotonin neurons, our local tracing studies re-
vealed spatial selectivity of the direct GABAergic input to
serotonin neurons. We identified a subregion of the midbrain
reticular nucleus, ventral to the periaqueductal gray, as a
particularly strong source of GABAergic input to DR serotonin
neurons. By comparison, the GABA neurons in the dorsal
and ventral wings within the periaqueductal gray are not as
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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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