© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

ARTICLES

Serotonin modulates the response of embryonicthalamocortical axons to netrin-1
Alexandre Bonnin1,2,5, Masaaki Torii1,2,3,5, Lilly Wang4, Pasko Rakic3 & Pat Levitt1,2
Modifying serotonin (5-HT) abundance in the embryonic mouse brain disrupts the precision of sensory maps formed bythalamocortical axons (TCAs), suggesting that 5-HT influences their growth. We investigated the mechanism by which 5-HT
influences TCAs during development. 5-HT1B and 5-HT1D receptor expression in the fetal forebrain overlaps with that of the axon
guidance receptors DCC and Unc5c. In coculture assays, axons originating from anterior and posterior halves of the embryonicday 14.5 dorsal thalamus responded differently to netrin-1, reflecting the patterns of DCC and Unc5c expression. 5-HT converts
the attraction exerted by netrin-1 on posterior TCAs to repulsion. Pharmacological manipulation of 5-HT1B/1D receptors andintracellular cAMP showed the signaling cascade through which this modulation occurs. An in vivo correlate of altered TCA
pathfinding was obtained by transient manipulation of 5-HT1B/1D receptor expression abundance in the dorsal thalamus by
in utero electroporation. These data demonstrate that serotonergic signaling has a previously unrecognized role in the modulationof axonal responsiveness to a classic guidance cue.

Specific neurotransmitter systems may be the targets of disrupted
development in neuropsychiatric disorders1,2. There are at least two
potential outcomes of such disturbances, which relate to the pleiotropic
nature of neurotransmitters: (i) direct disruption of neuronal signaling
in the adult and (ii) perturbations of brain histogenetic processes thatare mediated in part by biogenic amines before they act as classicalneurotransmitters. For example, temporally restricted genetic deletion of
the 5-HT1A receptor3 during development is sufficient to produce
altered behavior in the adult. Similarly, transient pharmacologicalinhibition of the 5-HT transporter (SERT) during early development
produces abnormal emotional behaviors in adult mice4. These findings
suggest that atypical 5-HT function during a sensitive developmental
period alters the formation and subsequent function of behaviorally
relevant brain circuitry. There have been long-standing hypotheses
regarding such nonclassical roles for 5-HT because of its very early
expression in embryogenesis and the developing CNS. Some early
actions of 5-HT include effects on the morphogenesis of CNS andcraniofacial structures, as well as subtle effects on neuronal growth, cell
migration and survival5–9. The reported effects vary widely, probably
reflecting the fact that, as in the adult, 5-HT actions during development
differ according to the receptor subtypes and downstream transduction
mechanisms. Perhaps the most notable example of a prominent developmental disruption following altered 5-HT levels is the aberrant
formation of the somatosensory map in the mouse barrel cortex ingenetic mutants of monoamine oxidase A (MAOA)10 and of SERT11.
We recently documented the dynamic expression patterns of 5-HT1
receptor subfamily members in the telencephalon of the prenatal

mouse12. There were unexpected spatial and temporal expression
patterns of 5-HT1B and 5-HT1D (Htr1b and Htr1d) receptor transcripts, particularly in the dorsal thalamus, where there is overlap withexpression of the axon guidance receptors DCC, Unc5c (Unc5h3), andRobo1 and Robo2 (refs. 13,14). In transfected cell lines and in the adultbrain, both 5-HT1B and 5-HT1D receptors are coupled to the inhibitionof adenylate cyclase via Gi/o proteins, which leads to an intracellulardecrease of cAMP15,16. Because altering intracellular cAMP and cyclic
guanine monophosphate can influence the axon guidance of isolated
spinal and peripheral neurons by modulating their responsiveness to
attractive or repulsive cues17–20, we hypothesized that 5-HT could
influence aspects of TCA development by modulating intracellularsecond messenger systems through Gi/o coupled 5-HT1 receptors. We
tested this hypothesis using explant cultures and found that 5-HT
converts the attraction exerted by netrin-1 on developing posterior
dorsal thalamus axons to repulsion through a 5-HT1B/1D receptor–
dependent mechanism that controls intracellular cAMP. We also report
the disruption of TCA development in vivo by using in utero electroporation of siRNAs and expression plasmids, targeting specificallythe developing dorsal thalamus, to transiently decrease or increase
5-HT1B/1D receptor expression, respectively.
RESULTS
Previously, we showed that 5-HT1A, 5-HT1B, 5-HT1D and 5-HT1F
receptors are expressed transiently and in distinct patterns in the fetal
dorsal thalamus12. Htr1b and Htr1d transcripts are expressed in thedorsal thalamus by embryonic day 14.5 (E14.5), the age used to harvest

1Department of Pharmacology, Vanderbilt University, 465 21st Ave. South, Nashville, Tennessee 37232, USA. 2Vanderbilt Kennedy Center for Research on Human Development,
Vanderbilt University, Box 40 Peabody, 230 Appleton Place, Nashville, Tennessee 37203, USA. 3Department of Neurobiology and Kavli Institute of Neuroscience, Yale University
Medical School, 333 Cedar St SHM C316, New Haven, Connecticut 06510, USA. 4Department of Biostatistics, Vanderbilt University School of Medicine, S-2323 Medical CenterNorth, Nashville, Tennessee 37232, USA. 5These authors contributed equally to this work. Correspondence should be addressed to P.L. (pat.levitt@vanderbilt.edu).

Received 18 January; accepted 19 March; published online 22 April 2007; doi:10.1038/nn1896

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Htr1b

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Str

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Figure 1 Bright-field micrographs illustrate the distribution of Htr1b, Htr1d, Dcc and Unc5c receptor
transcripts and Gbx2 transcription factor transcripts in embryonic dorsal thalamus (DT). (a–j) In situ
hybridization performed with DIG-labeled riboprobes in the horizontal plane (at two different ventrodorsal
levels, right) showed that Htr1b (a,f), Htr1d (b,g) and Dcc (d,i) receptor transcripts were expressed
throughout the DT at E14.5, with a slight anterior-low to posterior-high gradient. Unc5c (c,h) expression
was restricted to anterior and mediolateral regions of the DT at this age. The Gbx2 (e,j) expression
pattern delineates the boundaries (red dotted line) of the DT. (k–s) Sections in the coronal plane showed
Htr1b (k,m) and Htr1d (l,n) expression in the DT at E14.5 and E16.5; expression patterns appeared
more regionalized by E16.5 as DT nuclei were forming and present, like Gbx2 (o), a medial to lateral
gradient in the posterior dorsal thalamus. (p,q) Unc5c and Dcc expression in the DT at E16.5 onhorizontal sections; both receptors were expressed in the anterior DT, but compared with Unc5c (p),Dcc expression also included the posterior DT (q, red arrows); netrin-1 was expressed in a anterior-high
to posterior-low gradient in the striatum (r; see also Supplementary Fig. 4). At E16.5, expression of
Gbx2 was restricted ventrally to the medial DT and dorsally to the lateral DT (s). Cc, cingulate cortex;
H, hippocampus; M/LHb, medial/lateral habenula; Str, striatum; Vlg, ventral lateral geniculate nucleus;Mg, medial geniculate nucleus; D, dorsal; R, rostral. Scale bars, 150 mm.

tissue for our in vitro coculture experiments, and at E16.5, when TCAs
actively grow in the basal forebrain toward the dorsal pallium21. Thereis Htr1b expression in the dorsal thalamus at E14.5, which increases at
E16.5, particularly in the most lateral regions, where the ventrolateral,
ventral posterolateral and posteromedial thalamic nuclei are forming(Fig. 1). Similarly, there is abundant expression of Htr1d mRNA in thedorsal thalamus, peaking in intensity at E16.5 (Fig. 1b,g,i,n).
The expression patterns of 5-HT receptor transcripts in the dorsal
thalamus overlap with those of receptors for several guidance cues,
including DCC, Unc5c (ref. 13) and Robo1 and 2 (ref. 22). To compare
directly Htr1b and Htr1d expression with netrin-1 receptor expression
in the dorsal thalamus, we carried out in situ hybridization using
riboprobes complementary to the 3¢ untranslated region (UTR) of the
Dcc and Unc5c transcripts at E14.5 (Fig. 1c,d,h,i) and E16.5 (Fig. 1p,q).
The transcription factor gastrulation brain homeobox 2 (Gbx2)23 wasused to delineate the boundaries of the developing dorsal thalamus at
E14.5 and E16.5 (Fig. 1e,j,o,s). Analysis at different dorsoventral levels
showed that Gbx2 transcript was not uniformly expressed throughout

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 5 [ MAY 2007

the dorsal thalamus, but in combination theanalyses provided an accurate demarcation.
In situ hybridization carried out in the horizontal plane at two different ventrodorsal levels
showed that at E14.5, Dcc and Unc5 transcripts
overlap broadly in the anterior dorsal thalamus, although Unc5c transcripts are much
more abundant (Fig. 1 c,h). In contrast with
Gbx2, which was expressed over the whole
rostrocaudal extent of the dorsal thalamus at
E14.5 (Fig. 1e,j), Unc5c expression formed a
distinct rostral-high to caudal-low gradient(Fig. 1c,h) throughout the ventrodorsal axis.
At both ventrodorsal levels of the dorsal thalamus shown in Figure 1, Dcc was expressed in avery slight anterior-low to posterior-high gradient (Fig. 1d,i). At E16.5, both Dcc and Unc5c
were intensely expressed in the most anterior
aspect of the dorsal thalamus, whereas Dcc
expression appeared to be stronger than thatof Unc5c in the posterior dorsal thalamus(Fig. 1p,q). These mapping data indicatethat the expression patterns of the Htr1b/
Htr1d, Dcc and Unc5c transcripts broadly
overlap at E14.5 and E16.5 in the posterior
dorsal thalamus. These transcript patterns
for DCC and Unc5c are consistent with previously published studies (refs. 13,24,25 andthe Brain Gene Expression Map database,
http://www.stjudebgem.org).

Effect of netrin-1 on dorsal thalamus axons
To examine the influence of specific guidancecues on directed dorsal thalamus axonal outgrowth, we employed an in vitro assay using
E14.5 dorsal thalamus explants, which we
cocultured with aggregates of HEK-293 cells
that stably expressed soluble forms of netrin-1
or slit-2 (ref. 26,27) in a matrix that allowed
for three-dimensional outgrowth of axons (seeMethods and ref. 13). Given the distinct
patterns of receptor expression that we identified, the dorsal thalamus was subdivided into
the anterior and posterior regions to assess potential differences in
responsiveness to guidance cues. We used quadrant-based quantitative
analysis of the orientation of dorsal thalamus axon outgrowth (Fig. 2
and Methods). Not every axonal response in this assay was identical,
which was expected given that the tissue contains different developing
thalamic nuclei that show distinct projection patterns in vivo. The
initial analysis showed that fibers emerging from explants of theposterior half (Fig. 2a) of the dorsal thalamus were attracted toward
the source of netrin-1 (Fig. 2d), but were repelled away from a source ofslit-2 (Fig. 2f). Parental HEK-293 cells do not influence the directionality of dorsal thalamus axon outgrowth (Supplementary Fig. 1
online). In contrast, axons growing out of explants isolated from theanterior half of the dorsal thalamus are repelled from sources of either
netrin-1 or slit-2 (Fig. 2c,e). Statistical analysis of the data obtained
from over four independent experiments showed that the attractive
effect of netrin-1 on axons from the posterior dorsal thalamus was
significantly different from the mainly repulsive effect that netrin-1
exerts on axons from the anterior dorsal thalamus (Fig. 2g).

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Posterior DT

Anterior DT

c
DT

DT

netrin-1

E14.5

b

T

D

f Slit-2

Slit-2ARhnetrin-1

Slit-2*

100
Percentage explants

100
Percentage explants© 2007 Nature Publishing Group http://www.nature.com/natureneurosciencemmetric
SyaDT

Anterior DT

e

HEK

Ctx

Slit-2

Posterior DTgd netrin-1

netrin–1

a Ctx

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60
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TowardSymmetrical
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5-HT (Supplementary Fig. 1). In the concentration range tested (3 nM–30 mM), 5-HT did
not significantly influence the direction oroutgrowth rate of axons when dorsal thalamus
explants were cocultured with parental HEK293 cells (Supplementary Fig. 1), nor did it
modulate the repulsive effect of either netrin-1
on anterior dorsal thalamus axons or slit-2 onanterior and posterior dorsal thalamus axons(data not shown). However, when explantsfrom posterior dorsal thalamus, grown in thepresence of netrin-1–expressing HEK-293
cells, were treated with 30 mM 5-HT, axons
were repelled from, instead of being attracted
to, the source of netrin-1 (Fig. 3). Doseresponse analysis showed that nanomolar
concentrations of 5-HT were sufficient toalter the responsiveness of axons to netrin-1,
with the optimal response at 30 mM (datanot shown).

0
Anterior DT

Posterior DT

5-HT1B/1D and cAMP modulate axon
response to netrin-1
The expression of Htr1b and Htr1d transcripts
in the dorsal thalamus at the time the explants
are generated suggests that these receptors
are likely to mediate the 5-HT effect on directed axon outgrowth that we observedin vitro. Consistent with this, immunohistochemical staining of thalamic explants after
3 days in vitro (DIV) showed that 5-HT1D
receptor protein was distributed along
dorsal thalamus axons and growth cones(Fig. 3c,d), similar to 5-HT1B immunoreactivity shown previously29.
To determine whether 5-HT1B/1D receptors
mediate the modulation of netrin-1 responsiveness by 5-HT, we used a potent 5-HT1B/1D
agonist, L694,247 (ref. 30), and antagonist, BRL-15572 (ref. 31).
Cotreating posterior dorsal thalamus explants with 5-HT and
BRL-15572 at 30 mM completely blocked 5-HT modulation of
netrin-1 responsiveness of dorsal thalamus axons (Fig. 3b,e). A doseresponse study showed that concentrations as low as 300 nM in theculture medium potently blocked the 5-HTmodulation of the responseto netrin-1 (data not shown). BRL-15572 alone had no significant effecton the response of dorsal thalamus axons to netrin-1 (data not shown).
L694,247 mimicked the effect of 5-HT in changing the response ofposterior dorsal thalamus axons to netrin-1 from attraction to repulsion (Fig. 3f).
Our results demonstrate that activation of 5-HT1B/1D receptors on
posterior dorsal thalamus neurons switches the response of TCAs to
netrin-1 from attraction to repulsion. 5-HT1B/1D receptors are coupled
to the inhibition of adenylate cyclase via Gi/o proteins, and theiractivation leads to an intracellular decrease of cAMP15,16. On thebasis of a previous analysis of isolated spinal neurons18, we hypothesized that 5-HT1B/1D receptors modulate the response of TCAs to
netrin-1 by changing the intracellular abundance of cyclic nucleotides
in dorsal thalamus neurons. To test this directly, we modulated theabundance of cAMP using a well-characterized pharmacological strategy18,20. Bath application of 20 mM Rp-cAMPS (a nonhydrolyzableanalog of cAMP and antagonist of protein kinase A, PKA32) converted

Figure 2 Analysis of attractive and repulsive effects of netrin-1 and slit-2 on DT axons in vitro.
(a) Sagittal view of the prosencephalon at E14.5 illustrates the DT region dissected for the explant
assays (dotted circle), which includes the mediolateral extent of the DT and most medially, part of theepithalamus (the pretectal area is avoided). The anterior and posterior halves of the tissue were obtainedby a single bisecting cut (dotted line). Ctx, cortex; D, dorsal; R, rostral. (b) Method used to quantify axon
outgrowth from DT explants, as either toward (T) or away (A) from the source of soluble guidance cues
(HEK cells) or ‘symmetrical’ around the DT explant (no preferential direction; dotted circle). (c–f) DT
explants at E14.5 + 3 DIV immunostained for Tuj1 show that axons from the anterior DT are repelledaway (c), whereas axons from the posterior DT are attracted toward (d) a source of netrin-1. (e, f) Slit-2
repels axons originating from either anterior (e) or posterior (f) DT. Scale bar, 100 mm. (g) Quantification
over four independent experiments shows that the response of anterior DT axons to netrin-1 wassignificantly different than the response of posterior DT axons (*, w2 ¼ 16.57, P o 0.0001, d.f. ¼ 2,
w2; n ¼ 20 and 19 explants, respectively). (h) In contrast, the response to slit-2 was not significantly
affected by the origin of explants (anterior versus posterior, w2 ¼ 2.80, P ¼ 0.2779, d.f. ¼ 2, w2;
n ¼ 16 and 26 explants, respectively; values ± s.e.m.).

Consistent with this difference between anterior and posterior
segments, in experiments in which we isolated more finely dissected
anterior, middle and posterior dorsal thalamus, we obtained differentially repelled, nonpreferential and attractive responses of dorsal
thalamus axons to netrin-1 cues, respectively (Supplementary
Fig. 1). Conversely, there was no significant difference between the
responses of anterior and posterior dorsal thalamus axons to slit-2(Fig. 2h). The distinct outgrowth responses to netrin-1 by the anterior
and posterior halves of the dorsal thalamus are consistent and predictable in the context of our mapping data and with previously
published reports showing that DCC alone mediates netrin-1 attraction, whereas DCC-Unc5c complexes mediate netrin-1 repulsion28.
Moreover, the data are consistent with the intense DCC immunoreactivity observed on axons and growth cones originating fromposterior dorsal thalamus explants (Supplementary Fig. 1).
5-HT modulation of posterior axon response to netrin-1
On the basis of the Htr1b and Htr1d transcript expression we observedin the dorsal thalamus, we next tested the possibility that 5-HT
modulates the response of dorsal thalamus axons to netrin-1 and/or
slit-2. Dorsal thalamus explants were exposed to increasing concentrations of 5-HT. High-performance liquid chromatography analysis
showed that the basal serum-free culture medium did not contain

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VOLUME 10 [ NUMBER 5 [ MAY 2007 NATURE NEUROSCIENCEARTICLES
5-HT1B/1D expression influences TCA
pathfinding in vivo
Our in vitro studies indicated that 5-HT is apotent modulator of posterior dorsal thalanetrin-1
mus axon responsiveness to netrin-1, and+5-HT 30 µM+5-HT+BRL-15572
suggested that 5-HT1B/1D activation in thedorsal thalamus may influence TCA pathfindnetrin-1
Toward100efSymmetricaling in vivo. To test this possibility, we used*
netrin-1
100
Away
targeted in utero electroporation to the dorsal
80
thalamus to selectively alter the expression of80
both 5-HT1B/1D receptors. 5-HT1B/1D siRNAs
60
60
or control siRNA were injected into the thirdventricle at E12.5 (Fig. 5) with the pCAG4040
EYFP plasmid (encoding enhanced yellow
fluorescent protein), which labels all trans20
20
fected cells (see ref. 33). The EYFP label
facilitates visualization of the complete axonal
00Untreated
Untreated
100 nM
1 µM
10 µM+5-HT (30 µM) +5-HT (30 µM)
projection of transfected dorsal thalamus neu+BRL15572 (30 µM)
L694,247
rons. The technical difficulty of targeting thedorsal thalamus in utero at E12.5 creates
Figure 3 5-HT1B/1D receptors mediate 5-HT modulation of DT axons response to netrin-1. (a,b) Tuj1
variability between brains in terms of theimmunostaining of posterior DT explants at E14.5 + 3DIV. When 5-HT (30 mM) was added to the culture
precise location of the electroporated plasmedium, axons were repelled from the source of netrin-1 (a); this effect was inhibited by a 5-HT1D
mids. We therefore identified, while blindedantagonist (BRL-15572, b). Scale bar, 100 mm. (c) Confocal image showing 5-HT1D immunostaining
on axons and growth cones of DT neurons in vitro at E14.5 + 3 DIV. Scale bar, 20 mm. (d) Higher
to treatment and before any analysis, thosemagnification of the growth cone area boxed in c. Scale bar, 5 mm. (e) Quantification over fourbrains in which only the dorsal thalamus was
independent experiments showed that 5-HT (30 mM) had a significant effect in switching thelabeled (n ¼ 25). Potential alterations of the2
response of posterior DT axons to netrin-1 from attraction to repulsion (*, w ¼ 44.00, P o 0.0001,
TCA pathway were investigated using netrind.f. ¼ 2, w2). In cultures treated with both 5-HT and the 5-HT1D antagonist BRL-15572 (30 mM),
G1 immunostaining, which specifically labels
the response of posterior DT axons to netrin-1 was not significantly different from untreated cultures
TCAs at E16.5 and E18.5 (ref. 34; and see(n ¼ 4 independent experiments; w2 ¼ 3.57, P ¼ 0.2083, d.f. ¼ 2, w2; untreated explants: n ¼ 35;
5-HT–treated explants: n ¼ 34, 5-HT + BRL-15572–treated explants: n ¼ 30; values ± s.e.m.).
Methods for details). When control siRNAs(f) Dose-response study of the effect of the 5-HT1B/1D agonist L694,247 showed that concentrations
and EYFP reporter plasmids are electropoas low as 100 nM mimic the effect of 5-HT in changing the response of posterior DT axons to netrin-1
rated at E12.5, EYFP+ cells are present
from attraction to repulsion.
throughout the dorsal thalamus at E18.5(Fig. 5b). Labeled axons (EYFP+) that originate from electroporated dorsal thalamus
the response of posterior dorsal thalamus axons to netrin-1 from neurons projected normally through the internal capsule and toward
attraction to repulsion (Fig. 4). In contrast, Sp-cAMPS (20 mM), an the cortex (Fig. 5d), as previously described35,36.
analog of cAMP that activates PKA32, blocked the effect of 5-HTon the
Further immunohistochemical staining for the CAM-like adhesion
posterior dorsal thalamus axon response to netrin-1 (Fig. 4e). molecule L1, which labels subpopulations of both thalamocortical and
Sp-cAMPS alone had no direct effect on the response of dorsal corticofugal axons at this stage of brain development37, showed thatthalamus axons to netrin-1 (Fig. 4c). These observations are consistent L1+ corticothalamic axons followed a normal trajectory on thewith our hypothesis that the ability of 5-HT to modulate the response electroporated side of the brain (data not shown). After control
of posterior dorsal thalamus axons to netrin-1 is mediated by siRNA electroporation in the dorsal thalamus at E12.5, the pathsa 5-HT1B/1D–induced intracellular decrease of cAMP in dorsal followed by netrin-G1+ axons around the cortico-striatal boundary
thalamus neurons.
and in the cortex at E18.5 were identical on the control (Fig. 6) andelectroporated (Fig. 6b,c) sides. The overlap of netrin-G1+ axon pathways on the control and electroporated sides is shown (Fig. 6d).
netrin-1
Toward*Quantification of netrin-G1 immunofluorescent intensities along theSymmetrical100c 5-HT / Tuj1b netrin-1

d 5-HT / Tuj1
1D

Percentage explants

1D

Percentage explants

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NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 5 [ MAY 2007

Figure 4 Modulation of the posterior DT axon response to netrin-1 ismediated by a 5-HT1B/1D–mediated change of intracellular cAMP. (a–e) The
DT axon response to netrin-1 was measured under the following conditions:
untreated (a), 20 mM Rp-cAMPs (b), 20 mM Sp-cAMPs (c), 30 mM 5-HT (d)and 30 mM 5-HT + 20 mM Sp-cAMPs (e). Rp-cAMPs, like 5-HT, switched the
DT axon response to netrin-1 from attraction to repulsion (*, w2 ¼ 24.07,
P o 0.0001, d.f. ¼ 2, w2; untreated explants: n ¼ 24; Rp-cAMPS–treatedexplants: n ¼ 19). Sp-cAMPs alone had no significant effect on the DT axon
response to netrin-1 (w2 ¼ 4.23, P ¼ 0.1256, d.f. ¼ 2; Sp-cAMPS–treatedexplants: n ¼ 13), but significantly blocked the effect of 5-HT (responses
from untreated and [5-HT + Sp-cAMPS]-treated explants were notsignificantly different; w2 ¼ 5.53, P ¼ 0.0747, d.f. ¼ 2; values ± s.e.m.).

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compared with the axons of nonelectroporated neurons (netrin-G1+ only; Fig. 6m).
a
b
c
H
H
Such a ventrolateral reduction (after overexCtx
Dlg
Vlg
pression) or expansion (after siRNA treatDT
EYFP
DTment) of TCA pathways was never observed
Vlg
siRNAsDT
when control siRNA or control expression~30°
plasmids were electroporated in identical
V3
E18.5
EYFP / DAPI
DAPI
dorsal thalamus regions (n ¼ 8 out of 8;Fig.
6a–e); therefore, disruption of TCA
Control siRNAdpathfinding can be specifically attributed to
Ctx
TCAs
Ctx
Ctx
Ctx
alteration of 5-HT1B/1D–mediated signaling.
Ctx
To understand the possible basis for theseH
H
shiftsin dorsal thalamus axon trajectory, we
IC
IC
DT
DT
carried out a more detailed analysis of theStrStrpatterns of netrin-1 expression in the ventral
TCAs
forebrain, where dorsal thalamus axon sortingTCAs
occurs. Netrin-1 expression in the internal
D
E18.5 EYFP / DAPI
capsule at E14.5 and E16.5 (Fig. 7 and SupRostral
CaudalL
plementary Fig. 4 online) showed a very
complex tridimensional gradient (Fig. 7e)
Figure 5 In utero electroporation of E12.5 DT neurons to examine TCA pathway formation in vivo.
that is compatible with a potential role in(a) Schematic representation of the electroporation protocol. Solutions containing 5-HT1B/1D
the ‘sorting’, or proper ventrodorsal and medoverexpression plasmids or siRNAs and pCAG-EYFP reporter plasmids were injected into the thirdventricle at E12.5 and an electric pulse was applied through the DT with the cathode apposed on theiolateral positioning, of TCAs before they
dorsomedial side of the brain, forming a 301 angle with the horizontal plane. (b) Brain electroporated
reach the cortex (see ref. 13). In experimentsin the DT at E12.5, harvested at E18.5 and stained for EYFP; neurons throughout the DT expressed thein which TCAs from the anterior and posterEYFP reporter protein (green). Some cells can be seen populating the Vlg at E18.5 (arrow). (c) DAPI
ior dorsal thalamus at E14.5 were labeled withstaining reveals that in utero electroporation by itself does not alter the normal development of the DT,
the fluorescent lipophilic probes DiI and DiA,
as assessed by cell density and cytoarchitecture. Scale bar, 200 mm. (d) Coelectroporation of a control
respectively, we observed that in all of thesiRNA and EYFP reporter plasmid at E12.5 in the DT allowed the subsequent tracking of TCAs at E18.5(EYFP+, green) throughout the rostrocaudal extent of the brain; control siRNA or electroporation by itself
brains analyzed (n ¼ 10), anterior dorsal
did not alter the normal projection patterns of TCAs. Scale bar, 200 mm. L, lateral. Ctx, cortex; IC,thalamus axons grew preferentially followinginternal capsule; V3, third ventricle; Dlg, dorsal lateral geniculate nucleus.
a dorsomedial course in the internal capsule,along the lower–netrin-1 part of the gradient.
Conversely, posterior dorsal thalamus axons
mediolateral axis of the cortex (see Methods) confirmed that netrin- followed a more ventrolateral course, along the higher–netrin-1 part of
G1+ axons distribute identically on both sides of control siRNA– the gradient (Fig. 7d). The growth trajectory was also highly dependent
on the medial or lateral placement in either the anterior or posterior
electroporated brains (Fig. 6e).
In contrast to control siRNA, electroporation of siRNAs that dorsal thalamus, as medial axons in either region tended to project
specifically reduce Htr1b/Htr1d expression in the dorsal thalamus more anteriorly than those originating from lateral regions (Fig. 7d and(see Supplementary Fig. 2 online) produced an expansion of the Supplementary Figs. 4 and 5 online). Thus, the changes in the TCA
netrin-G1+ axon pathway into more lateral-ventral regions of the pathway that we observed at E18.5 after manipulation of 5-HT1B/1D
dorsal pallium at E18.5 (n ¼ 5 out of 5 brains; Fig. 6f–h). Furthermore, expression levels could be due to an altered response of dorsal thalamus
the subpopulation of shifted netrin-G1+ axons on the electroporated axons to netrin-1 in the internal capsule, consistent with our observaside were also EYFP+ (Fig. 6h), indicating that these axons originated tion in vitro. We therefore examined, after in utero dorsal thalamus
from dorsal thalamus neurons that had reduced expression of electroporations at E12.5, TCA pathways in the internal capsule at
Htr1b/Htr1d. Measures of netrin-G1 immunofluorescent intensity E16.5, when axons are still actively growing through the region. Afteralong the TCA pathway (at the level shown in Supplementary Fig. 3 Htr1b/Htr1d siRNA electroporation (Fig. 8), a subpopulation of TCAs
online) confirmed that axons originating from dorsal thalamus neu- (EYFP+ axons; Fig. 8b,c) spread ventrally in the internal capsule androns electroporated with Htr1b/Htr1d siRNA expand ventrolaterally in cerebral wall, compared with brains electroporated with control siRNAthe cerebral wall as compared with the control side (Fig. 6j). We (Fig. 8a). In contrast, when 5-HT1B/1D receptors were overexpressed
obtained the exact opposite growth pattern of TCAs after overexpres- (EYFP+ axons; Fig. 8d,e), TCAs followed a more dorsally restrictedsion of 5-HT1B/1D receptors by electroporation of expression plasmids path in the internal capsule and coursed more medially in the cerebral
into the dorsal thalamus at E12.5. In these brains, the pathway of wall compared with normal (not shown). Therefore, increasing andnetrin-G1+ axons was more restricted dorsomedially in the dorsal reducing expression of 5-HT1B/1D receptors in the dorsal thalamus
pallium (Fig. 6k,l; n ¼ 5 out of 6 brains), with similar patterns observed (compare Fig. 8b,d and 8c,e) produce opposite effects on TCA pathat multiple rostrocaudal levels. Measures of immunofluorescent inten- ways in the internal capsule at E16.5 (Fig. 8e–g). Note that in bothsity on each side of the brain (Fig. 6o) confirmed that netrin-G1 situations, EYFP+ axons were still distributed in the internal capsule,immunoreactivity was decreased laterally in the cerebral wall. A albeit abnormally, within the normal boundaries of the TCA pathway.
detailed examination of the corticostriatal boundary showed that
EYFP+ axons (which originated from dorsal thalamus neurons over- DISCUSSION
expressing 5-HT1B/1D receptors) made a sharp turn in the internal The present study demonstrates that TCA responsiveness tocapsule and followed a somewhat more medial course in the cortex netrin-1 is modulated by 5-HT through Gi/o-coupled 5-HT1B/1D© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

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Figure 6 Modifying 5-HT1B/1D receptor expression in vivo alters the topography of a subpopulation of TCAs. (a–e) After in utero DT electroporation ofcontrol siRNA at E12.5, netrin-G1+ axons at E18.5 extended ventrolaterally in the cortex along similar pathways on the nonelectroporated (control, a)and electroporated (EP, b) sides. (c) Netrin-G1+ and EYFP+ axons overlapped on the electroporated side (d). Netrin-G1 immunofluorescent quantification (e)showed that TCA pathways on the control side (gray in d, blue curve in e) and control siRNA-electroporated side (red in d, red curve in e) overlapped in
the cortex. (f–h) 5-HT1B/1D siRNA electroporation at E12.5 in the DT induced a ventrolateral shift/expansion of netrin-G1+/EYFP+ axons on the electroporatedside of the cortex (g,h) at E18.5 compared with the control side (f). (i,j) Schematic representation (i) and quantification (j) of netrin-G1 immunofluorescentdistribution illustrating the ventrolateral shift/expansion on the electroporated side (red curve, j) of the cortex. (k–m) pCAG–5-HT1B/1D electroporation at E12.5
reduced the number of netrin-G1+ axons projecting ventrolaterally in the cortex. Netrin-G1+/EYFP+ axons in the cortex at E18.5 (l,m) appeared to be shifteddorsally on the electroporated side compared with the control side (k) of the same brain. (n) Schematic representation of netrin-G1+ axon pathways shown ink (gray) and l (red). (o) Quantification illustrating the ventrolateral decrease of netrin-G1+ fibers on the electroporated side (red curve) of the cortex. Scalebars, 100 mm.

receptor subtypes through the alteration of intracellular cyclic
nucleotide levels. In vivo, we observed consistent, though modest,
disruptions of TCA pathway topography following in utero alterationof 5-HT1B/1D receptor expression. The lack of 5-HT modulation ofeither netrin-1 (on anterior dorsal thalamus axons) or slit-2 repulsive
cues in general suggests a highly selective mechanism throughwhich specific G protein–coupled receptors affect intracellulartranslation of axon guidance receptor activity on defined subsets
of axons. These data provide a potential mechanism for altered
TCA patterning after prenatal increases in 5-HT10,11. The results
also suggest that long-term changes in behavior following
developmental disruption of 5-HT receptor signaling could occur
as a result of altered circuit formation, rather than through alterations
later in life in the 5-HT modulation of fast synaptic transmission.

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 5 [ MAY 2007

Anterior and posterior thalamic axon response to netrin-1
Detailed expression mapping revealed some unexpected data regarding
the differential expression of netrin-1 and 5-HTreceptors in subregionsof the fetal dorsal thalamus (see also ref. 12). Individually, thesepatterns are consistent with previous studies that show a widespread
expression of Dcc throughout the dorsal thalamus13,24 and a more
restricted Unc5c expression to anterior dorsal thalamus domains(ref. 25, Supplementary Fig. 6 online and http://www.stjudebgem.org).
We interpreted the distinct outgrowth responses of anterior dorsal
thalamus axons to netrin-1 (compared with posterior dorsal thalamus
axons) as being related to the repellant activity mediated through thecoexpressed Unc5c and DCC receptors, as has been shown previously28.
These differential responses may be an important mechanism for the
initial sorting of dorsal thalamus axons. For example, anterior and593

Figure 7 Ntn1 (netrin-1) transcript expression in
the striatum/IC area at E14.5 and E16.5. (a) At
E14.5, Ntn1 showed a graded expression
throughout the forebrain; Ntn1 was strongly
expressed in the rostroventral domain of thestriatum and, more caudally, showed aventrolateral (pial side) high to dorsomedial(ventricular side) low gradient in the IC (indicatedby red arrows). This three-dimensional gradient is
schematized in (e). (b) A similar distribution wasobserved at E16.5. (c) Higher magnification ofareas boxed in a and b show that netrin-1
expression was graded (high ventrolateral to
low mediodorsal) in the IC proper. (d) TCAs fromthe anterior and posterior DT at E14.5 (sites of
injection are indicated by asterisks) were labeledwith DiI (red) and DiA (green), respectively. In allbrains analyzed (n ¼ 10), anterior DT axons(red) grew preferentially following a dorsomedialcourse in the IC, along the low netrin-1 part of
the gradient. Conversely, posterior DT axons(green) followed a more ventrolateral course,
along the higher-Ntn1 part of the gradient. Scalebar, 100 mm. CP, caudate putamen; Spt, septum;
M, medial.

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Anterior(Fig. 7 and Supplementary Fig. 4). Conversely, the attractive influenceof netrin-1 on posterior dorsal thalamus axons, which was consistent
with previous reports (see ref. 13), could account in part for their more
ventrolateral and more widespread outgrowth throughout the striatum(Fig. 7 and Supplementary Fig. 4, and see ref. 38).
Two recent reports claim that during fetal development, TCAs fromanterior and posterior dorsal thalamus preferentially grow anteriorly
and posteriorly in the internal capsule, respectively38,39. This simplified
model does not agree with previously described projection patterns40–42,
which illustrate a more complex organization. Our own DiI and DiA
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posterior dorsal thalamus axons follow different routes through theinternal capsule to reach distinct cortical areas. The graded expressionof netrin-1 throughout the rostrocaudal extent of the striatum andinternal capsule (ventrolateral (pial side) high to dorsomedial(ventricular side) low) seen with detailed transcript mapping is
consistent with the finding that Dcc/Unc5c–expressing axons fromthe anterior dorsal thalamus grew preferentially along the lower
netrin-1 part of the gradient; these axons followed a more dorsomedialcourse in the internal capsule that may have been due partly to a
repulsive influence of the more dense ventrolateral source of netrin-1

Electroporation site

© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

ARTICLES

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Figure 8 Modifying 5-HT1B/1D receptor expression in vivo alters TCA distribution in the internal capsule. Embryos were electroporated at E12.5 in the dorsal
thalamus and harvested at E16.5. (a) When control siRNAs (or pCAG plasmid) were electroporated in the DT (lower), EYFP+ TCAs were distributed in astereotypical manner in the IC where they spread out (dotted circle) before turning sharply into the cortex. (b,c) In 5-HT1B/1D siRNA–electroporated brains,
EYFP+ TCAs spread across a larger domain ventrolaterally in the IC (dotted circle) and took a slightly more ventrolateral course when turning in the cortex
compared with similarly electroporated controls. (d,e) After 5-HT1B/1D overexpression in the DT, EYFP+ TCAs followed a more dorsally restricted course in the
IC (dotted circle), and a more medial course in the cortex, than similarly electroporated controls. Differences in TCA pathfinding after 5-HT1B/1D siRNA andoverexpression were observed across embryos with similar electroporation patterns in the DT (compare a,c,e lower with b,d lower). Scale bars, 100 mm.

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ARTICLES
double-labeling also showed a complex pattern, in which the anteriorposterior organization of TCAs in the internal capsule depended on theanterior-posterior and the medial-lateral origin of the axons in thedorsal thalamus. Thus, labeled axons in the antero- or posteromedialaspect of the dorsal thalamus project anteriorly, whereas those in theantero- or posterolateral aspects of the dorsal thalamus project to moreposterior cortical areas. This suggests that complex guidance mechanisms occur in the internal capsule to regulate the pathway of TCAs fromvarious sites in the dorsal thalamus. It is important to note that in ourin vitro assays, anterior and posterior dorsal thalamus explants werecomposed of mixed populations of neurons of medial and lateral origin
in the dorsal thalamus. This is consistent with the observation thatin vitro the overall response of anterior and posterior dorsal thalamus
axons to netrin-1 was not uniform, but rather included subpopulationsof explants whose axons are attracted, repelled and unresponsive tonetrin-1. These were all included in the statistical analyses of responses.
We did not expect major disruption of the TCA pathway to result
from altering 5-HT signaling in vivo, as there are other guidance cues
expressed in the basal forebrain that also influence the pathways
followed by TCAs, which are suggested by the modest disruption of
TCA pathfinding in the striatum of netrin-1 (Ntn1) knockout mice13.
However, it is difficult to interpret the phenotype in these knockout
mice, as our data showed that netrin-1 also is expressed in the medial
dorsal thalamus prenatally.
5-HT, pleiotropic signal changing attraction to repulsion
We hypothesized that 5-HT, acting through 5-HT1B/1D receptors, may
promote a regional modulation of TCA guidance. This was supported
by several observations. First, 5-HT axons are present in the vicinity ofgrowing TCAs, reaching the basal telencephalon by E14–15, in close
apposition with TCAs along the internal capsule7. Moreover, 5-HT and
its metabolites accumulate at nanomolar concentrations by E16.5 in theforebrain (Supplementary Fig. 1). The physiological concentration of
5-HT in the synaptic cleft is in the millimolar range (1–5 mM)43. Afterrelease, the extrasynaptic concentration of 5-HT can still reach
10 mM44. Therefore, physiologically relevant concentrations of 5-HT
in and around the synaptic cleft are much higher than those wedetermined by HPLC in the whole forebrain, where the amine isdiluted. The effective concentration of 5-HT that modulated netrin-1
was well within this range.
What is the molecular basis for this modulation? A recent study has
shown that PKA stimulation increased DCC translocation in commissural neurons in vitro45. We colocalized 5-HT1D and DCC receptors
along dorsal thalamus axons and at growth cones. Therefore, it ispossible that in growing dorsal thalamus axons, 5-HT1B/1D–mediatedcAMP-dependant inhibition of PKA might modulate DCC receptor
translocation at the membrane. As noted above, DCC alone mediates
attraction, whereas a DCC-Unc5c complex mediate repulsion28. We
showed that Unc5c is expressed in a marked anterior-high to posteriorlow gradient in the dorsal thalamus (see Supplementary Fig. 6),
whereas Dcc is expressed throughout the dorsal thalamus. Thus, if
signaling through 5-HT1B/1D receptors inhibits DCC translocation, it
could increase the proportion of DCC-Unc5c receptor complexes
present at the surface of posterior dorsal thalamus axons and switch
responses to netrin-1 from attraction to repulsion. This hypothesis also
is consistent with anterior dorsal thalamus explants showing a basal
repulsive response to netrin-1 that is not modulated by 5-HT.
5-HT1B/1D receptors modulate TCA topography in vivo
Because of the widespread distribution of 5-HT receptor family
members in forebrain regions outside of the dorsal thalamus,

NATURE NEUROSCIENCE VOLUME 10 [ NUMBER 5 [ MAY 2007

conditional gene targeting would be necessary to manipulate gene
expression in the dorsal thalamus without altering expression elsewherein the forebrain; however, such mouse lines are not available for
5-HT1B/1D receptors. We developed an alternative strategy of in utero
electroporation selectively into the dorsal thalamus, using siRNA andoverexpression plasmids to manipulate gene expression. Because
5-HT1B and 5-HT1D receptors have similar spatiotemporal expression
patterns in the dorsal thalamus and signal through the same pathway,
the impact of altered 5-HT signaling was maximized by reducing theexpression of both receptors simultaneously. A technical challenge ofthis method is the inherent difficulty of reproducibly targeting theidentical areas of the dorsal thalamus in different embryos. Thisrequired the electroporation of more embryos, and before any analysisof axonal patterning, identification (blind to the manipulation) of
those cases in which the electroporated region included only the dorsal
thalamus. Qualitative analysis revealed that changing 5-HT1B/1D receptor expression caused altered distribution of TCAs ventrodorsally in theinternal capsule and subcortical white matter. Unlike gene-targetingstudies, in which altered patterning of several forebrain structures
through which TCAs normally navigate confounds the analysis, ourresults showed that a selective alteration of 5-HT signaling in dorsal
thalamus neurons led to shifted axonal topography through an otherwise normal terrain.
Why would altering 5-HTsignaling induce shifts in TCA trajectories?
The in vitro results demonstrate that 5-HT signaling, through
5-HT1B/1D–mediated changes of intracellular cAMP levels, switcheddorsal thalamus axon responses to netrin-1 from attraction to repulsion. The analysis of TCA pathways performed in netrin-1 knockout
mice13 suggested that netrin-1 is important for proper ventrodorsal
sorting of growing TCAs, and netrin-1 also is likely to influence
anteroposterior organization. We show that TCA response to netrin1 is modulated by cAMP, suggesting that the sorting of TCAs in theinternal capsule likely depends on the control, through 5-HT modulation, of intracellular cAMP levels in dorsal thalamus neurons. The
experimentally induced shifts that we observed were consistent with theorganization of dorsal thalamus axons in the internal capsule justbefore birth (Supplementary Fig. 7 online; at midrostral and middle
levels of the internal capsule, the axons arising from posterior dorsal
thalamus are situated ventrolateral to the more dorsomedially locatedanterior dorsal thalamus axons). In the present study, decreased
intracellular levels of cAMP in dorsal thalamus neurons as a result of
5-HT1B/1D overexpression resulted in an increased repulsive response of
a subpopulation of TCAs that normally are attracted by netrin-1 in theinternal capsule. In the coronal plane, these axons adopted a more
dorsal trajectory to avoid the ventrolateral netrin-1–rich region. We
consistently obtained this shift by 5-HT1B/1D overexpression in thedorsal thalamus. Conversely, in dorsal thalamus neurons in which
5-HT1B/1D expression is decreased by siRNA electroporation, a likely
loss of negative regulation of cAMP levels in a subpopulation of TCAs
would favor an attractive response to netrin-1, thus positioning themmore ventrally.
We suggest that changes in netrin-1 responsiveness by 5-HT are due
to an altered DCC/Unc5c ratio in individual axons. We cannot exclude,
however, the possibility that altered 5-HT signaling disrupts the
responsiveness of dorsal thalamus axons to other guidance cues,
because changes in cAMP levels influence axon responsiveness tocues such as slit-2 and semaphorin 3A/C45,46. We note that in our
explant assays, 5-HT was ineffective in modulating the responsivenessof dorsal thalamus axons to slit-2. There also is a possibility that altered
5-HT signaling could change TCA fasciculation, leading to the
expanded and restricted pathways observed in the internal capsule.

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However, if this was the case, we would expect a more dramatic
phenotype: for example, as reported in the L1-knockout mice37
where TCAs appear disorganized in the internal capsule. Notably, thechanges in TCA topography reported here occurred within the normalboundaries of the TCA pathway, suggesting that disruption of 5-HT
signaling does not grossly alter the initial trajectories of these axons(for example, their course through the ‘corridor region’ lying upstreamof the internal capsule). In vivo, the site of 5-HT action in themodulation of TCA guidance is likely to reside in the ventral telencephalon, because this is the earliest region of overlap between 5-HT
axons and netrin-1 (ref. 47). There is little innervation of the dorsal
thalamus at this age, indicating that 5-HT released by serotonergic
afferents signals via the 5-HT1B/1D receptors expressed on the growthcones of TCAs as they navigate through the internal capsule.
The data reported here have important implications in understanding the long-term consequences of 5-HT disruption in development.
Previous studies show that dramatic increases in 5-HT levels during
development10,11 alter cortical barrel field organization. What could be
the outcome of a subtle topographical shift of the TCA pathway as
described here? We hypothesize that there will be subtle topographicalmismatches after both decreased 5-HT1B/1D signaling (siRNAs), in
which posterior dorsal thalamus axons would innervate more lateraland posterior domains of the somatosensory areas (S1), and increased
5-HT1B/1D signaling (overexpression), in which posterior dorsal thalamus axons would innervate more dorsal and anterior areas of S1 in thecortex. Although it will be technically challenging, this question may be
addressed using markers of barrel fields (for example, SERT) in early
postnatal pups with retro- and anterograde tracing of TCAs in the adultbrain after in utero electroporation of 5-HT1B/1D receptors siRNAs andoverexpression plasmids. There is a possibility that genetic and/or
environmental modifications resulting in specific developmental
alterations of 5-HT signaling are likely to generate subtle topographicaldefects of TCA circuit formation. If such defects have consequences forthe function of adult TCA circuits, then the regulation of 5-HT
signaling during development, via highly selective spatiotemporalcontrol of differential 5-HT receptor expression, forms a potential
basis for the neurodevelopmental etiology of mental health disorders inwhich 5-HT signaling is implicated.
METHODS
Animals. Timed-pregnant C57BL/6J and CD-1 mice were purchased from theJackson Laboratory. All research procedures using mice were approved by theInstitutional Animal Care and Use Committee at Vanderbilt University andconformed to US National Institutes of Health guidelines. Unless otherwise
noted, all reagents were purchased from Sigma.
In situ hybridization. In situ hybridization for Htr1b and Htr1d transcripts were performed as described12. We obtained Dcc and Unc5c (Unc5h3)
receptor gene sequences from the project ‘Ensembl’ mouse genome
database (www.ensembl.org) using the following accession numbers: Dcc(ENSMUST00000073379) and Unc5c (ENSMUSG00000059921). We designed
probes (510 base pairs (bp) for Dcc and 465 bp for Unc5c) to target uniquesequences (verified using the National Center for Biotechnology Information
BLAST server) in the 3¢ untranslated regions of each receptor. We generated
the Ntn1 riboprobe from a 376-bp cDNA fragment (clone ID: 1139227;
Invitrogen). The Gbx2 probe was kindly provided by J. Rubenstein. Probes
were synthesized and in situ hybridizations performed as described12.
Explant assays. We used a coculture assay to monitor axonal growth fromdorsal thalamus explants toward or away from a source of soluble guidancecues (HEK-293 cells stably expressing netrin-1 or slit-2 were a gift from
J. Wu26,27). The dorsal thalamus48 was dissected at E14.5 (Fig. 2a). We
generated explants by cutting posterior and anterior halves of the dorsal

596

thalamus into 16 pieces of approximately 300 mm in diameter. Hanging drops49
of HEK-293 cells were embedded in Matrigel (35 ml, BD Bioscience) and four
dorsal thalamus explants were carefully positioned approximately equidistant(200–400 mm) from the HEK-293 blocks. Cocultures were grown serum-free in
Neurobasal medium supplemented with N2 and B27 (Invitrogen). After 72 h,
cultures were fixed in 4% paraformaldehyde (PFA) and processed for immunohistochemistry. The attractive or repulsive effects of guidance cues were
quantified by blind scoring using a virtual quadrant strategy (Fig. 2b andref. 13). Explants from three or four independent experiments were scored.
Data were analyzed using SAS (Windows version 9, SAS Institute). Pearson
w2 statistics were used to compare the distribution of the orientations of axons.
All statistical comparisons were performed using two-sided tests at the 5%
significance level. When sample sizes are small (any of the expected cell counts
is less than 5), exact P values were calculated.
Explants immunohistochemistry. PFA-fixed explants were incubated overnight in primary antibody (2% BSA, 0.2% Tween-20 in PBS) using thefollowing antibody dilutions: anti-DCC (Pharmingen, 1:500; mouse IgG
directed against the intracellular domain of DCC), anti-Tuj1 (Covance,
1:500) and anti-5HT1D (Abcam, 1:2,000, and a previously characterized
rabbit polyclonal antibody, 1:10,000; the latter was a kind gift from
A. Basbaum; both antibodies yielded similar staining patterns). Explants were
washed, incubated overnight with Cy2,3-conjugated secondary antibodies(1:1,000), washed and imaged using an Axiocam CCD camera coupled to a
Leica MZFLIII stereoscope.
In utero electroporation, localization of EYFP+ and netrin-G1+ axons. We
carried out in utero electroporation at E12.5, as described33. Briefly, the uterine
horns of anesthetized (50 mg per kg of body weight, Nembutal, intraperitoneal)
pregnant C57BL/6 mice (E12.5) were exposed. We injected solutions ofexpression plasmids or siRNAs (plasmid sources and details are listed in
Supplementary Methods online) for each gene (2–3 mg ml–1 each forexpression plasmids; 0.5 mg ml–1each for siRNAs; 1 ml total), control plasmid(4 mg ml–1) or siRNA (1 mg ml–1) with pCAG-EYFP or PCAG-DsRed2 (1 mg
ml–1). DNA solutions were injected with a glass micropipette into the thirdventricle. Tweezer-type electrodes were placed on each side of the fetal head
forming a B301 angle from the horizontal plane (Fig. 5a) and electronic pulses
were charged with an electroporator (BTX). Natural reabsorption of embryos
occurred with a frequency B10–20%. We harvested electroporated embryos at
E16.5 or E18.5. Brains were fixed with 4% PFA overnight, sectioned at 70 mm
on a vibratome and processed for immunohistochemistry (Supplementary
Methods). For each animal, we delimited a region for quantification from themedial cortex to the claustrum at each level and on each side of the brain(Supplementary Fig. 3).
Axonal tracing. After fixation in 4% PFA, E14.5 and E16.5 brains were bisectedin the sagittal plane. We inserted small crystals of DiI and DiA (Invitrogen)with a fine needle into the anterior and posterior parts of the dorsal thalamus
in either lateral or medial regions of the exposed surface. After storage infixative about 2 weeks in the dark at 37 1C, we collected 100-mm slices andcounterstained using DAPI (Molecular Probes).
Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTS
We thank J. Wu and H.H. Wu for technical help and comments, and
K.L. Eagleson and G. Stanwood for comments on the manuscript. The work was
supported in part by US National Institute of Mental Health grant MH65299
and US National Institute of Child Health and Human Development
P30HD15052 to P.L.
AUTHOR CONTRIBUTIONS
A.B. participated in the design of the project, the writing of the manuscript andconducted and analyzed the experiments. M.T. conducted and participated in
the analyses of crucial in utero electroporation experiments and helped to write
the manuscript. L.W. performed the statistical analyses. P.R. contributed tothe in vivo experiments and helped to write the manuscript. P.L. contributed
to the design of the experiments, interpretation of results and writing of
the manuscript.

VOLUME 10 [ NUMBER 5 [ MAY 2007 NATURE NEUROSCIENCEARTICLESCOMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.

© 2007 Nature Publishing Group http://www.nature.com/natureneuroscience

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Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions

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