Please cite this article in press as: Horn et al.

, DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Cell Reports

Article

DCC Expression by Neurons Regulates

Synaptic Plasticity in the Adult Brain
Katherine E. Horn,1 Stephen D. Glasgow,2 Delphine Gobert,1 Sarah-Jane Bull,1 Tamarah Luk,1 Jacklyn Girgis,1
Marie-Eve Tremblay,3 Danielle McEachern,1 Jean-François Bouchard,1,6 Michael Haber,4 Edith Hamel,1
Paul Krimpenfort,5 Keith K. Murai,4 Anton Berns,5 Guy Doucet,3 C. Andrew Chapman,2 Edward S. Ruthazer,1
and Timothy E. Kennedy1,*
1Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4, Canada
2Center for Studies in Behavioural Neurobiology, Department of Psychology, Concordia University, Montreal, QC, H4B 1R6, Canada
3Groupe de Recherche sur le Système Nerveux Central, Département de Pathologie et Biologie Cellulaire, Université de Montréal,

Montréal, QC, H3C 3J7, Canada

4Centre for Research in Neuroscience, Montreal General Hospital, McGill University, Montreal, QC, H3G 1A4, Canada
5Department of Molecular Genetics, Cancer Genomics Centre, Centre for Biomedical Genetics, Netherlands Cancer Institute, Amsterdam,

1066 CX, The Netherlands

6Current address: School of Optometry, Université de Montréal, Montreal, Quebec, Canada H3T 1P1

*Correspondence: timothy.kennedy@mcgill.ca
http://dx.doi.org/10.1016/j.celrep.2012.12.005

SUMMARY to development. Although both netrin-1 and DCC are essential

for normal development, their function in the adult nervous
The transmembrane protein deleted in colorectal system is not known. Studies in several species support a role
cancer (DCC) and its ligand, netrin-1, regulate synap- for netrins in influencing synaptogenesis during development.
togenesis during development, but their function Genetic analyses have identified a role for netrin in nerve-muscle
in the mature central nervous system is unknown. synaptogenesis in Drosophila. When the amount of netrin ex-
Given that DCC promotes cell-cell adhesion, is pressed by muscle cells is increased, more synaptic connec-
tions are made by motoneurons (Mitchell et al., 1996; Winberg
expressed by neurons, and activates proteins that
et al., 1998), whereas in the absence of DCC, fewer synapses
signal at synapses, we hypothesized that DCC form (Kolodziej et al., 1996). In Caenorhabditis elegans, the
expression by neurons regulates synaptic func- netrin-1 homolog Unc-6 regulates synaptogenesis by organizing
tion and plasticity in the adult brain. We report that the subcellular distribution of presynaptic proteins (Colón-
DCC is enriched in dendritic spines of pyramidal Ramos et al., 2007; Poon et al., 2008; Stavoe and Colón-Ramos,
neurons in wild-type mice, and we demonstrate 2012). In Xenopus, application of netrin-1 protein to the optic
that selective deletion of DCC from neurons in the tectum increases the number of axon branches and synapses
adult forebrain results in the loss of long-term poten- made by retinal ganglion cells through a DCC-dependent
tiation (LTP), intact long-term depression, shorter mechanism (Manitt et al., 2009). The contribution of netrins to
dendritic spines, and impaired spatial and recogni- synapse formation suggests that DCC expressed by neurons
tion memory. LTP induction requires Src activation in the mature mammalian brain may influence synapse func-
tion and plasticity. Notably, DCC activates the cytoplasmic
of NMDA receptor (NMDAR) function. DCC deletion
tyrosine kinase Src in neurons (Li et al., 2004). Activation of Src
severely reduced Src activation. We demonstrate
regulates NMDA receptor (NMDAR) function and is essential
that enhancing NMDAR function or activating Src for long-term potentiation (LTP), a form of activity-dependent
rescues LTP in the absence of DCC. We conclude synaptic plasticity (Lu et al., 1998). Here, we tested the hypoth-
that DCC activation of Src is required for NMDAR- esis that DCC expressed by neurons regulates synaptic plas-
dependent LTP and certain forms of learning and ticity in the adult brain.
memory.
RESULTS
INTRODUCTION
DCC Enrichment at Synapses
Axon guidance cues are emerging as regulators of synaptogen- To establish whether netrin-1 and DCC are present at synapses
esis during development; however, their potential contribution to in the mature mammalian brain, we fractionated subcellular
synaptic plasticity in the mature central nervous system (CNS) is components of adult rat hippocampus (Huttner et al., 1983).
not clear (Shen and Cowan, 2010). Here, we asked whether the We found that both netrin-1 and DCC are present in synapto-
netrin receptor, deleted in colorectal cancer (DCC), plays a role in somes (fraction P2, Figure 1A). Following synaptosome lysis
synaptic function and plasticity in the adult brain. Many types of and further fractionation, netrin-1 and DCC were present in
neurons express netrin-1 and DCC, and expression is not limited fraction LP1, which is composed of pre- and postsynaptic

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 1

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

plasma membranes, consistent with enrichment of GluA2/3 in

this fraction. DCC and netrin-1 were also enriched in fraction
LP2, which contains synaptotagmin- and synaptophysin-posi-
tive transmitter vesicles and various cargo transport vesicles.
Fractionation of adult rat brain to enrich for the postsynaptic
density (PSD) (Fallon et al., 2002) revealed DCC cofractionating
with the PSD protein PSD-95 (Figure 1A). These results provide
evidence that netrin-1 and DCC are enriched at mature synapses
associated with synaptic plasma membranes and intracellular
vesicles, and that DCC cofractionates with the PSD.
Dendritic spines are postsynaptic specializations that undergo
activity-dependent changes in shape and number that are
thought to be important for learning and memory (Nimchinsky
et al., 2002). To examine the distribution of DCC in CA1 pyra-
midal cell dendrites and spines, we imaged hippocampal
organotypic slices infected with a Semliki Forest virus encoding
membrane-targeted farnesylated red fluorescent protein (fRFP)
(Haber et al., 2006). The results demonstrated a striking enrich-
ment of DCC immunoreactivity in the head of dendritic spines
(Figure 1B). Consistent with this distribution, immunoelectron
microscopy revealed enrichment of DCC in PSDs in the CA1
stratum radiatum of adult rat brain (Figure 1C). These findings
demonstrate that DCC is enriched at mature synapses in
dendritic spines.

Conditional Deletion of DCC from Forebrain Neurons

DCC is expressed by dentate gyrus granule cell neurons,
hippocampal CA1 and CA3 pyramidal neurons, and neurons
throughout the neocortex during postnatal development and
in adults (Livesey and Hunt, 1997; Volenec et al., 1997). Conven-
tional DCC null mice die within hours of birth, which makes it
impossible to examine synaptic plasticity in the adult CNS in
these animals (Fazeli et al., 1997; Serafini et al., 1996). To delete
DCC selectively from neurons in the mature brain, we adopted
a cre/loxP gene-targeting strategy (Sauer, 1998). Floxed DCC
(DCCf/f) mice were crossed to a line expressing cre regulated
by the promoter of the a-subunit of the Ca2+/calmodulin-depen-
dent kinase II gene (T29-1 CaMKIIa-cre), which drives expres-
sion exclusively by neurons in the adult forebrain (Benson
et al., 1992; Burgin et al., 1990; Jacobs et al., 1993). Although
endogenous CaMKIIa is not expressed during embryogenesis,
upregulation occurs postnatally (Bayer et al., 1999). Critically,
cre is expressed by neurons after axon guidance is complete,
which gives us the opportunity to selectively address DCC
function in mature neural circuits in vivo.
Cre is first expressed in T29-1 CaMKIIa-cre mice at
2.5 weeks of age and is initially restricted to CA1 hippocampal
pyramidal neurons (Tsien et al., 1996). By 1 month of age (Sonner
Figure 1. DCC Is Enriched in Mature Dendritic Spines
(A) Subcellular fractionation of adult rat brain. The diagram illustrates fractions et al., 2005), cre is expressed by CA1 and CA3 pyramidal
of interest. DCC and netrin-1 are present in synaptic fractions, and DCC neurons, dentate gyrus granule cells, and neurons throughout
enriches with the PSD. H, whole-brain homogenate; P1, pellet with nuclear and
cellular debris; P2, mitochondria and synaptosome-enriched pellet; S2 and
S3, soluble fractions; P2*, synaptosome-enriched pellet; LP1, synaptic plasma arrow). Scale bars, left: 0.5 mm; right: 0.25 mm. at, axon terminal; ds, dendritic
membrane enriched; LP2, synaptic vesicle enriched. spine.
(B) DCC immunoreactivity (white) on dendritic spines along an fRFP-labeled (D) Cre is broadly expressed in the forebrain in progeny of T29-1 CaMKIIa-Cre
dendrite. Scale bar, 1 mm. Enlarged images of DCC-immunoreactive spines crossed with ROSA26-lacZ mice. Brain sections were obtained from b-gal-
are shown below. negative (left) and -positive (right) 2- and 12-month-old mice (b-gal stain).
(C) Immunoelectron microscopy detects DCC immunostaining at PSDs Neuronal expression of cre was observed in CA1, CA3, the dentate gyrus, and
of a subset of dendritic spines (wide arrows) but not at others (narrow the neocortex in coronal (top) and sagittal (bottom) sections. Scale bar, 1 mm.

2 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

the neocortex, but not by glia. We confirmed this pattern of we examined the brains of young (2–4 months) and older
expression by crossing T29-1 mice with ROSA26-lacZ reporter (>5 months) DCCf/f,cre+ and age-matched controls using the
mice (Soriano, 1999), which express b-galactosidase (b-gal) Golgi-Cox staining technique (Figure 3C). Spines were quantified
following cre-induced recombination (Figure 1D). Crossing the along segments of dendrites of CA1 hippocampal neurons
DCCf/f mice with T29-1 mice generated conditional DCC knock- (Figure 3A) by an investigator blind to genotype. No significant
outs (DCCf/f,cre+) that are homozygous DCCf/f and carry at least difference was found in spine density or head width, but a signif-
one copy of CaMKIIa-cre. The crosses also yield DCCf/f,wt/wt icant decrease in spine length was detected along dendritic
and DCCf/wt,wt/wt littermates that were used as wild-type (WT) branches in older DCCf/f,cre+ mice compared with control mice
controls. All analyses used male mice only. (Figure 3D). Importantly, no significant difference was detected
Levels of DCC protein in the hippocampus of DCCf/f,cre+ mice in the younger mice, indicating that the loss of DCC expression
were substantially reduced at 3, 8, and 18 months of age (Figures by mature neurons in the DCCf/f,cre+ mice results in a decrease
2A and 2B) but were unchanged at postnatal day 14 (P14) (Fig- in spine size as the mice age.
ure 2A), indicating that normal levels of DCC are present
throughout embryogenesis and initial maturation of the nervous DCC Loss Impairs Memory
system. Conventional DCC knockouts lack a corpus callosum To test the hypothesis that DCC contributes to memory, we used
(Fazeli et al., 1997). Cresyl violet staining revealed no obvious the Morris water maze, a hippocampus-dependent spatial
gross morphological changes in the brains of adult DCCf/f,cre+ memory task (Clark and Martin, 2005). For 3 days, mice were
mice (Figure 2C), which is consistent with normal expression of trained to swim to a visible platform. All of the mice performed
DCC in young mice and demonstrates the feasibility of removing comparably and reached the platform within the same time on
DCC from neurons after axon guidance is complete. the third day, indicating intact sensory and motor function. After
We previously reported defects in axo-oligodendroglial para- training, the spatial visual cues in the surroundings were
nodal junctions in conventional DCC knockout mice that result switched and the mice learned anew to swim to a submerged
from the absence of DCC function in oligodendrocytes (Jarjour platform located in a different quadrant of the maze. On the
et al., 2008). No such deficit was found in the hippocampi of adult eighth day, 2 hr after the last test, the platform was removed,
DCCf/f,cre+ mice, in which DCC is deleted only from neurons (Fig- and a probe trial was run in which the time and distance spent
ure 2D). Consistent with selective deletion of DCC from neurons, in the appropriate quadrant were measured. Testing young
immunohistochemical staining for cre and the astrocyte marker DCCf/f,cre+ and littermate control mice (2–4 months old) revealed
glial fibrillary acidic protein (GFAP) did not label the same cells no significant difference between groups (Figures 4A–4C).
(Figure 2E, top panels). We also assessed the distribution of cells However, when the same mice were tested using this 8 day
expressing cre using b-gal expression in the progeny of T29-1 protocol at >5 months of age, the probe trial revealed that
mice crossed with ROSA26-lacZ reporter mice. b-gal, indicating the control mice traveled significantly farther and spent more
cre expression, did not overlap with tyrosine hydroxylase (TH)- time in the appropriate quadrant than their DCCf/f,cre+ counter-
immunopositive neurons, consistent with cre not being ex- parts, and made more passes over the former location of
pressed by ventral midbrain dopaminergic neurons (Figure 2E, the submerged platform (Figures 4D–4F). Swimming speed did
bottom panels). not vary between genotypes at any age (Figures 4A and 4D).
In 2- to 4-month-old mice (hereafter referred to as young These findings identify a spatial memory impairment in older
adults) and 5-month-old and older mice (hereafter referred to DCCf/f,cre+ mice.
as older adults), although the levels of DCC in hippocampal We also applied the novel-object-recognition test, which is
homogenates were significantly decreased, we did not detect based on the tendency of normal mice to interact more with novel
significant changes in the expression of a variety of synaptic objects than with familiar objects (Bevins and Besheer, 2006). In
proteins (Figure 2F). this test, each mouse is first habituated to an empty field, and
the next day the mouse is returned to the same open field, now
DCC-Deficient Dendritic Spines Are Smaller containing two identical, biologically neutral objects, which it is
To determine whether DCC influences dendritic spine morpho- allowed to explore for 5 min. After a 4 hr rest, the mouse is re-
logy, we first established hippocampal organotypic slice cultures turned to the open field, where one familiar object has been re-
derived from conventional DCC knockout mice (Fazeli et al., placed by a novel object (Figure 4G). The relative amount of
1997). Adult DCC heterozygotes were crossed to generate time spent attending to the novel object can be used as a
litters composed of newborn DCC null pups (Fazeli et al., measure of the memory for the familiar object (Bevins and Besh-
1997), heterozygotes, and WT littermate controls. To visualize eer, 2006). We then calculated cognition and difference scores
dendrites, hippocampal cultures derived from null and WT for 24 DCCf/f,cre+ mice and 24 controls, with the experimenter
pups were infected with Semliki Forest virus encoding being blind to genotype. The total time spent exploring the
membrane-targeted green fluorescent protein (GFP) (Haber two objects did not differ between genotypes (Figure 4H);
et al., 2006). Analysis of dendritic spine morphology in pyramidal however, novel-object recognition was significantly impaired in
neurons that had never expressed DCC revealed significantly DCCf/f,cre+ mice (Figures 4I and 4J). When performance was
smaller spines (Figure 3A), with reduced spine head size and binned based on age, young (2–4 months) DCCf/f,cre+ mice were
neck width, compared with controls (Figure 3B). not different from controls, whereas older (>5 months) DCCf/f,cre+
To determine whether selective postnatal deletion of DCC mice were significantly impaired in recognition memory com-
from neurons would alter dendritic spine morphology in vivo, pared with age-matched controls (Figures 4I and 4J).

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 3

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 2. Characterization of DCCf/f,cre+ Mice

(A) Western blots of hippocampal homogenates from P14, 3-month-old, and 8-month-old controls and DCCf/f,cre+ littermates show decreased levels of DCC
in adult DCCf/f,cre+ mice.
(legend continued on next page)

4 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 3. DCC Deficiency Decreases Spine

Size
(A) Illustration of CA1 pyramidal neuron dendritic
branching and spine morphology of DCC-deficient
mice.
(B) Hippocampal organotypic slice cultures from
P0 conventional DCC knockout or WT pups were
infected with a virus encoding farnesylated GFP
(fGFP). Analysis revealed decreased spine head
length and width and neck width in DCC null
neurons (***p < 0.005; error bars depict SEM).
(C) Representative images of Golgi-Cox stained
spines from control and DCCf/f,cre+ mice. Scale
bar, 10 mm.
(D) Analysis of Golgi-Cox stained spines of young
(2–4 months) and older (>5 months) mice. Spines
from proximal CA1 pyramidal dendritic branches
in older DCCf/f,cre+ mice exhibit significantly
reduced spine length (***p < 0.005; error bars
depict SEM).

collaterals. Analysis of the input/output

relationship at CA3-CA1 synapses across
a range of stimulus intensities did not
detect significant differences between
hippocampal slices derived from older
DCCf/f,cre+ and control mice (Figure 5A).
Critically, this indicates that CA3-CA1
synaptic contacts are intact in animals
lacking DCC, and that basal levels of
synaptic transmission in DCCf/f,cre+ mice
are not altered by the deletion of DCC.
To determine whether DCC deletion influ-
Importantly, the behavioral tests revealed no significant differ- ences synaptic plasticity, we next assessed LTP and long-term
ence between the young DCCf/f,cre+ and control mice. We depression (LTD) at CA3-CA1 Schaffer collateral synapses
conclude that deficits develop during aging as a result of the (Figure 5).
absence of DCC function in neurons, and that DCC expression LTP is an experimental model of activity-dependent synaptic
by neurons in the mature brain contributes to spatial memory strengthening that may function as a neural substrate underlying
and the recognition of novelty. learning and memory (Bliss and Collingridge, 1993). To assess
the role of DCC in LTP, we used high-frequency stimulation
Impaired LTP but not Long-Term Depression (HFS; 1 s, 100 Hz) to induce LTP in hippocampal slices derived
in DCC-Deficient Mice from DCCf/f,cre+ mice and age-matched controls (Figures 5B
To determine whether DCC loss leads to changes in synaptic and 5C). Whereas slices from control animals showed robust
efficacy, we used acute hippocampal slices from both young LTP, slices from older (>5 months) DCCf/f,cre+ mice exhibited
(2–4 months) and older (>5 months) adult DCCf/f,cre+ or age- a striking absence of potentiation 1 hr after induction (Figure 5B).
matched control mice to record field excitatory postsynaptic To determine whether this impairment was due to a develop-
potentials (fEPSPs) in CA1 evoked by stimulation of the Schaffer mental deficit in DCCf/f,cre+ animals, we tested hippocampal

(B) Immunostained CA1 in hippocampal sections from 18-month-old control and DCCf/f,cre+ mice (red, b-tubulin III; green, DCC; blue, Hoechst; scale bar, 10 mm).
(C) Cresyl-violet-stained coronal sections of 18-month-old control and DCCf/f,cre+ mice. Scale bar, 1 mm. CC, corpus callosum.
(D) Axo-oligodendroglial paranodes of 8-month-old control and DCCf/f,cre+ mice exhibit no significant differences in width of Caspr immunoreactivity or distance
between Kv1.2 juxtaparanodes. Error bars depict SEM.
(E) Immunohistochemical staining of cre-positive and -negative coronal brain sections. A section from cre-positive progeny of T29-1 CaMKIIa-Cre crossed with
ROSA26-lacZ mice (far left) shows the location of immunostained regions (scale bar, 1 mm). Top panels show hippocampal CA1 from cre-positive and -negative
mice (red, GFAP; green, Cre; blue, Hoechst; scale bar, 10 mm). Bottom panels show adjacent brain sections containing substantial nigra (TH-positive) of
cre-positive progeny. An overlay of stained sections is shown in the far-right bottom panel (red, TH; green, Hoechst; blue, b-gal; scale bar, 1 mm).
(F) Western blots of hippocampal homogenates of young (3 months) and older (>5 months) mice. Histograms plot the average intensity from control and
DCCf/f,cre+ mice (n/genotype indicated under histogram) normalized using b-tubulin III as a loading control (Student’s two-tailed t test, *p < 0.05, **p < 0.01;
error bars depict SEM).

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 5

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 4. Impaired Spatial and Recognition Memory in Aged DCCf/f,cre+ Mice

(A–F) Morris water maze. Young (A) and older (D) controls and DCCf/f,cre+ mice swim at similar speeds. Genotypes show no difference between young (B) and
older (E) mice during training to learn the location of a submerged platform. In a probe trial to test spatial memory of the location of the submerged platform, 2 hr
after the last day of training, passes over the former location of the platform, distance, and time in the appropriate quadrant were analyzed (C and F). Young
(2–4 months) control and DCCf/f,cre+ mice perform similarly (n = 8/group) (C). (F) Older (>5 months) DCCf/f,cre+ mice score lower than controls (control: n = 7,
DCCf/f,cre+: n = 8). Statistical analysis was performed using a two-tailed t test (*p < 0.05, **p < 0.01; error bars depict SEM).
(G) Diagram of the novel-object-recognition test.
(H) Young (2–4 months) and older (>5 months) control and DCCf/f,cre+ mice explore objects for similar durations (young control: n = 13; young DCCf/f,cre+: n = 11,
older control: n = 11; older DCCf/f,cre+: n = 13).
(I and J) Performance was worse for older DCCf/f,cre+ mice in cognition (I) and difference (J) scores (two-tailed t test; *p < 0.05; error bars depict SEM).

slices from 2- to 4-month-old DCCf/f,cre+ mice (Figure 5C). In transmitter release. PPF ratios were not significantly different in
contrast to their aged counterparts, fEPSPs in slices from slices from older (>5 months) control and DCCf/f,cre+ mice at
young (2–4 months) DCCf/f,cre+ mice and their age-matched intervals ranging from 20 to 100 ms (Figure 5E), or in slices
controls demonstrated significant potentiation 1 hr post-tetanus from younger control and DCCf/f,cre+ mice (data not shown).
(DCCf/f,cre+ versus age-matched controls, p > 0.05). We The absence of a difference in PPF supports the conclusion
conclude that the impairment exhibited by older animals is not that deletion of DCC does not result in a significant alteration in-
due to a deficit in the early development of DCCf/f,cre+ mice. presynaptic transmitter release, and is consistent with DCC
We also assessed fEPSP amplitudes during the HFS train deletion resulting in a postsynaptic deficit.
and found no significant differences between genotypes (Fig- Repeated low-frequency stimulation (LFS) induces LTD of
ure 5D), suggesting that the lack of LTP did not resulting from evoked responses at CA3-CA1 synapses. To determine whether
an inability to follow the HFS train. DCC contributes to LTD, we used a paired-pulse LFS (PPLFS)
To determine whether LTP impairment may be due to altered paradigm to induce LTD (15 min, 1 Hz paired-pulse stimulation,
presynaptic function, we examined paired-pulse facilitation 1,800 pulses, 25 ms interpulse interval; Kourrich et al., 2008).
(PPF) across a range of stimulus intervals. Changes in PPF are Hippocampal slices from >5 months old DCCf/f,cre+ mice and
generally attributed to changes in the probability of presynaptic their age-matched controls demonstrated significant depression

6 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 5. Impaired LTP but Intact LTD in DCCf/f,cre+ Mice

(A) No significant difference is detected between control and DCCf/f,cre+ in CA3-CA1-evoked fEPSP amplitudes in older animals.
(B) Following HFS in older animals (>5 months), DCCf/f,cre+ does not display LTP. The mean amplitude of fEPSPs is potentiated in control (137.6% ± 8.0%,
p < 0.001) but not in DCCf/f,cre+ slices at 1 hr (116.1% ± 8.2%; p > 0.05). Representative fEPSPs from control (left) and DCCf/f,cre+ (right) before (gray) and after
(black) HFS (arrow) are shown.
(C) Slices from young (2–4 months) DCCf/f,cre+ mice and age-matched controls remain significantly potentiated 1 hr after HFS (113.9% ± 5.2% in DCCf/f,cre+ versus
121.3% ± 12.5% in controls, p > 0.05).
(D) No significant differences in fEPSP amplitude during the HFS train between older (>5 months) control and DCCf/f,cre+ mice are observed.
(E) PPF ratios in slices from older control and DCCf/f,cre+ mice do not differ significantly (p > 0.05).
(F) PPLFS (bar, 15 min) induced LTD in older control (n = 7) and DCCf/f,cre+ mice (n = 8; p < 0.01 versus baseline).

of synaptic responses following PPLFS (p < 0.01; Figure 5F), indi- ifenprodil to selectively block the function of GluN2B-contaning
cating that the LTP deficit is the not the result of a general loss NMDARs. In the presence of ifenprodil, we observed no differ-
of synaptic plasticity. ence in the evoked NMDAR-mediated responses between
DCCf/f,cre+ mice and their age-matched controls (Figure 6D).
DCC Regulates NMDAR Subunit GluN2B Expression This result indicates that the increased expression of GluN2B
We then investigated the mechanism that underlies the deficit in in DCCf/f,cre+ mice does not alter the electrophysiological
LTP induction in DCCf/f,cre+ mice. Western blot analyses revealed response, which suggests that the increased GluN2B protein is
no significant change in the expression of the synaptic proteins not located at synapses.
synaptophysin, N-ethylmaleimide-sensitive factor (NSF), AMPA
receptor (AMPAR) subunits GluA1 and GluA2/3, or NMDAR DCC Regulates Src, Phospholipase C g, and
subunits GluN1 and GluN2A in hippocampal homogenates of Phosphorylated Src Family Kinase Expression
adult DCCf/f,cre+ mice compared with controls (Figures 2F and DCC activates both phospholipase C (PLC) and Src family
6A). In contrast, a significant increase in the amount of NMDAR kinases (SFKs) in neurons (Liu et al., 2004; Xie et al., 2006),
subunit GluN2B was found in young (3 months) and older and activation of the tyrosine kinase Src is necessary and suffi-
(>5 months) DCCf/f,cre+ mice (Figure 6A) that persisted in cient for the induction of LTP (Lu et al., 1998). Src regulates
20-month-old DCCf/f,cre+ mice (Figure 6B). Increased GluN2B NMDAR function by phosphorylating the GluN2A subunit (Salter
protein was detected in whole hippocampal homogenates and and Kalia, 2004), and PLC activation of Src through protein
in the synaptosome LP1 plasma membrane fraction (Figure 6B), kinase C (PKC) increases the opening probability and open
which includes the synaptic apposition and extrasynaptic time of NMDAR without changing the channel conductance or
plasma membranes. reversal potential (MacDonald et al., 2007; Yu et al., 1997; Yu
To determine whether increased levels of GluN2B contribute and Salter, 1998). We therefore investigated how loss of DCC
to electrophysiological differences in slices from control and might alter the expression and function of these proteins.
DCCf/f,cre+ mice, we examined the various currents that con- In hippocampal homogenates of older (>5 months), but not
tribute to the fEPSP response in slices from older (>5 months) young (3 months) DCCf/f,cre+ mice, we detected a significant
control and DCCf/f,cre+ mice by applying appropriate pharmaco- decrease in the amount of phosphorylated PLCg1, but not total
logical inhibitors (Figure 6C). Following application of picrotoxin PLCg1 (Figure 7A), consistent with reduced PLCg1 activation in
(PTX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the absence of DCC. We also identified a significant decrease in
block GABA receptors and AMPARs, respectively, we added the amount of total Src protein in the DCCf/f,cre+ animals, but no

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 7

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 6. DCC Regulates NMDAR Function

(A) Protein expression levels of NMDAR subunits in
hippocampal homogenates from young (3 months)
and older (>5 months) mice (n = 3/genotype; two-
tailed t test, *p < 0.05; error bars depict SEM).
(B) Subcellular fractionation of hippocampi from
control and DCCf/f,cre+ mice (20 months old). In
DCCf/f,cre+ mice, GluN2B is enriched in the
LP1 synaptic membrane fraction. No significant
changes were detected between genotypes in
levels of GluA1, PSD-95, or synaptophysin.
(C) Pharmacological inhibitors were applied to
slices of older (>5 months) control and DCCf/f,cre+
mice in low-Mg2+ (0.1 mM) ACSF. fEPSP ampli-
tude was measured during this treatment.
(D) No significant difference was detected in
fEPSP amplitudes measured for control and
DCCf/f,cre+ mice. Normalized fEPSP amplitude in
the presence of PTX and CNQX is shown; error
bars depict SEM.

We then directly tested the hypothesis

that the LTP deficit in older (>5 months)
DCCf/f,cre+ mice is specifically due the
lack of activated Src. We thus aimed to
selectively activate Src in the DCCf/f,cre+
mice and determine whether this was
sufficient to rescue LTP in the absence
of DCC. To do this, we applied pituitary
adenylate cyclase activating peptide-38
(PACAP-38), a 38 amino acid peptide
that activates PAC1R, a G-protein-
coupled receptor expressed by hippo-
change in the SFK Fyn. Furthermore, we detected decreased campal neurons (Miyata et al., 1990; Zhou et al., 2000).
pan-SFK Y416 phosphorylation, revealing substantially reduced PACAP-38 binding of PAC1R signals through PLC and PKC to
SFK activity in neurons lacking DCC (Figure 7A). Activation of activate Src and enhance NMDAR function (Macdonald et al.,
DCC by addition of netrin-1 to synaptosomes purified from 2005). Application of PACAP-38 during the HFS train (1 s,
the cortex of WT adult mice increased phosphorylated SFK 100 Hz) rescued LTP in hippocampal slices derived from older
(pSFK) compared with unstimulated control synaptosomes (>5 months) DCCf/f,cre+ mice in ACSF containing 2.0 mM of
(Figure 7B), providing evidence that netrin-1 activates SFKs at Mg2+. Critically, this rescue was blocked by addition of the
synapses. The emergence of these deficits with age supports SFK inhibitor PP2 but not its inactive analog, PP3 (Figure 7D),
the conclusion that loss of DCC results in a deficit in key synaptic indicating that SFK activation was essential for PACAP-38 to
signaling mechanisms in older adults. rescue LTP. We conclude that in the absence of DCC, sub-
The influx of calcium (Ca2+) through NMDARs is critical for stantially reduced SFK signaling underlies deficient NMDAR
the induction of LTP, but is blocked by magnesium (Mg2+) function that results in a severe deficit in the capacity to induce
at resting membrane potentials. SFK activation promotes the LTP, with coincident defects in hippocampal-dependent
influx of Ca2+ through the NMDAR by increasing both the prob- memory (Figure 7E).
ability of NMDAR opening and the open time (Lu et al., 1998;
Salter and Kalia, 2004; Yu and Salter, 1998). To determine DISCUSSION
whether the LTP deficit in older (>5 months) DCCf/f,cre+ mice is
a consequence of reduced Ca2+ influx through NMDARs, we Many proteins that are essential for normal neural development
facilitated NMDAR function by reducing the concentration of are also expressed in the adult brain, raising the intriguing
Mg2+ from 2.0 mM to 1.3 mM in the artificial cerebrospinal fluid possibility that they may in some way influence plasticity. Here,
(ACSF) perfusing the slice. This resulted in a striking rescue of we report that DCC-dependent activation of Src in mature
LTP following HFS (1 s, 100 Hz) in hippocampal slices derived hippocampal neurons is required for the induction of NMDAR-
from older (>5 months) DCCf/f,cre+ mice, resulting in LTP that dependent LTP, and that DCC expression by forebrain neurons
was indistinguishable from that evoked in age-matched controls contributes to spatial and recognition forms of memory. Further-
in the same ACSF (Figure 7C). more, DCC deletion from mature neurons resulted in shorter

8 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Figure 7. Netrin-1 and DCC Regulate Src Activation

(A) Phosphorylated and total SFKs and PLCg1 proteins in hippocampal homogenates in control and DCCf/f,cre+ mice, normalized to b-tubulin III (n/genotype under
histogram; two-tailed t test, *p < 0.05). Error bars depict SEM.
(B) Netrin-1 stimulation of P2* purified synaptosomal fraction isolated from adult WT brain significantly increases levels of pSFKs, normalized to synaptophysin as
a loading control (n = 6/condition; two-tailed t test, *p < 0.05; error bars depict SEM).
(C) Slices from older (>5 months) control and DCCf/f,cre+ mice in reduced 1.3 mM Mg2+ ACSF remain significantly potentiated 1 hr after HFS (1 s, 100 Hz, 148.6% ±
10.9% in DCCf/f,cre+; 139.4% ± 6.6% in age-matched controls; p < 0.01).
(D) Slices from older DCCf/f,cre+ mice treated with PACAP-38 during HFS and perfused with ACSF containing 2.0 mM Mg2+ and the inactive compound PP3 remain
significantly potentiated after 1 hr. The SFK inhibitor PP2 blocks potentiation (124.7% ± 4.5% in PP3; 108.2% ± 7.7% in PP2; p < 0.05).
(E) Model.

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 9

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

dendritic spines and increased levels of NMDAR subunit DCC Regulation of GluN2B Expression
GluN2B, indicating that DCC is required to maintain mature Following DCC loss, we detected increased levels of the NMDAR
synaptic morphology and an appropriate balance of NMDAR subunit GluN2B. During early development, high levels of
subunit expression. These findings identify a role for DCC as GluN2B relative to GluN2A are normally present at synapses,
an essential upstream activator of Src signaling at mature CNS with a switch to more GluN2A and less GluN2B occurring during
synapses, and of synaptic plasticity and memory formation in maturation (Barria and Malinow, 2002; Sheng et al., 1994; Wil-
the mature mammalian brain. liams et al., 1993). NMDARs present at immature hippocampal
A key aspect of these findings is the relatively minor differ- synapses are largely GluN1-2B complexes (Tovar and West-
ences detected between young DCCf/f,cre+ and control mice, brook, 1999) that are thought to inhibit the recruitment of AMPAR
and the increased severity of the deficits with age, which support GluA1 subunits to the plasma membrane and compromise
the conclusion that impairments develop during aging due to synapse maturation (Kim et al., 2005). Interestingly, transgenic
loss of DCC function in neurons. At P14, the level of DCC protein mice that selectively increase GluN2B expression in forebrain
in the hippocampus of DCCf/f,cre+ mice did not differ from that neurons exhibit enhanced hippocampal LTP and improved
in controls. Between P14 and 3 months of age, levels of DCC learning and memory (Tang et al., 1999). In contrast, we found
protein were substantially reduced. Although memory was intact that increased GluN2B expression due to DCC deletion is asso-
and significant changes in most synapse-associated proteins ciated with a deficit in LTP induction and compromised spatial
were not detected in young (2–4 months) DCCf/f,cre+ mice, and recognition memory. Importantly, although we detected
increased levels of GluN2B were present in the hippocampal increased GluN2B protein in hippocampal homogenates and in
homogenates of these mice. This indicates that although DCC the LP1 synaptosomal plasma membrane fraction isolated
loss has not yet resulted in dramatic defects, the initial con- from mature brain, electrophysiological analyses revealed no
sequences of deleting DCC can be detected at this age. In difference in the contribution of GluN2B to fEPSP responses
contrast, levels of Src, pSFK, PLCg1, and pPLCg1 in young between genotypes, suggesting that the increased GluN2B
DCCf/f,cre+ mice were not significantly different compared with detected in DCCf/f,cre+ mice may be chiefly extrasynaptic. This
controls. Mean levels of Src and pSFK showed a tendency to suggests that defects induced by loss of DCC may occlude
be slightly reduced, however, which may represent the onset the expected enhancement of synapse function induced by
of deficits that become more severe in older animals. We increasing levels of GluN2B.
conclude that DCC expression by these neurons is essential to
maintain the function of synapses that contribute to memory, Netrin-1 and DCC Function at Synapses
but that in young adult mice (2–4 months), the deficits are not The requirement for DCC in activity-dependent plasticity raises
yet sufficiently severe to disrupt memory formation. These questions about how DCC and netrins might be regulated by
findings support the hypothesis that DCC loss results in a activity. Both netrin-1 and DCC are enriched in the LP2 fraction
progressive deficit in synapse function as the mice age. of adult brain synaptosomes, consistent with trafficking in
cargo vesicles at synapses. Whether netrin-1 is secreted from
Beyond Axon Guidance: A Postsynaptic Function neurons by constitutive or regulated pathways is unknown,
for DCC in Mature Neurons and it remains to be determined whether exocytosis of netrin-1
Subcellular fractionation and immunohistochemical analyses may be regulated in an activity-dependent manner. In contrast,
indicate that DCC is enriched in dendritic spines and associated we previously reported that membrane depolarization recruits
with the PSD. Previous studies of DCC function in neurons DCC to the plasma membrane of embryonic cortical neurons,
focused on axonal growth cones, where DCC directs the and that this promotes axon outgrowth in response to netrin-1
organization of F-actin to regulate motility and adhesion (Lai (Bouchard et al., 2008). This finding raises the tantalizing possi-
Wing Sun et al., 2011). Actin is also the major cytoskeletal bility that DCC trafficking may be similarly regulated by activity
element that regulates the structure of dendritic filopodia and at synapses, and that activity-induced recruitment of DCC to
spines. Notably, the actin regulatory proteins Nck1 (Dock), the synaptic plasma membrane may enhance NMDAR function.
Pak1, and Rho GTPases (Cdc42, Rac1, and RhoA) all regulate
dendritic spine morphology (Tada and Sheng, 2006), and all Src Is Essential for LTP Induction
are downstream effectors of DCC in axons (Lai Wing Sun et al., Netrin-1 signaling through DCC activates PLCg (Xie et al., 2006)
2011). Our findings raise the possibility that DCC functions and Src in neurons (Li et al., 2004), and activation of Src by PLC
in dendrites upstream of Rho GTPases to maintain mature (MacDonald et al., 2007) is required for Schaffer collateral CA1
dendritic spine morphology. NMDAR-dependent LTP (Lu et al., 1998). The NMDAR GluN2A
Little, if any, DCC immunoreactivity was detected in pre- subunit is phosphorylated by Src (Salter and Kalia, 2004), and
synaptic terminals in mature neurons. This is in contrast to the NMDAR function is enhanced by signaling from PLC to PKC to
role of DCC in directing extending axons, which upon reaching activate Src (MacDonald et al., 2007). DCC-deficient mice
an appropriate target form presynaptic terminals. Our findings exhibit reduced levels of Src protein, reduced activation of
highlight a postsynaptic role for DCC; however, it remains to SFK and PLCg, and a severe deficit in the induction of LTP.
be determined how DCC is distributed within dendrites during We therefore tested the hypothesis that reduced activation of
maturation, when DCC becomes predominantly localized to Src results in a deficit in NMDAR function that underlies the
postsynaptic spines, and whether DCC function in mature absence of LTP. We found that enhancing NMDAR func-
neurons is restricted to a postsynaptic role. tion either by decreasing extracellular levels of Mg2+ or by

10 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

pharmacologically activating Src completely rescued LTP cut with a cryostat. CA1 dendrite segments were traced and analyzed
in DCCf/f,cre+ mice. We conclude that DCC expression by (Neurolucida 9 and NeuroExplorer 9; MBF Bioscience, Williston, VT, USA;
spines in young mice: WT: n = 204, DCCf/f,cre+: n = 208; spines in aged mice:
hippocampal pyramidal neurons is essential to maintain the
WT: n = 192, DCCf/f,cre+: n = 267; dendritic segments young: WT: n = 11,
morphology of mature dendritic spines, and that DCC activation DCCf/f,cre+: n = 11; dendritic segments aged: WT: n = 13, DCCf/f,cre+: n = 15).
of Src is required for the induction of NMDAR-dependent LTP,
with consequences critical for memory in adult animals. Morris Water Maze
Mice were trained to find a submerged platform in a water maze in an 8-day
EXPERIMENTAL PROCEDURES training protocol as previously described (Nicolakakis et al., 2011). Swimming
was tracked with the use of an overhead video tracking system (2020 Plus
Animals tracking system, Ganz FC62D video camera; HVS Image, Mountain View,
All procedures involving animals were performed in accordance with the CA, USA) and tracking software (Water 2020 software; HVS Image). The
Canadian Council on Animal Care’s guidelines for the use of animals in time and distance traveled in each quadrant were calculated using Water
research. T29-1 CaMKIIa-cre mice were obtained from The Jackson Labora- 2020 software.
tory (Bar Harbor, ME, USA). Floxed DCC mice were generated as previously
described (Krimpenfort et al., 2012). Novel-Object-Recognition Test
Recognition memory was assessed by means of the novel-object-recognition
Immunostaining test (Bevins and Besheer, 2006). Exploration time (duration % body length
Western blot analyses utilized the following antibodies: mouse a-b-tubulin III away from the object, head pointed toward object) was recorded by overhead
(1:500, T4026; Sigma-Aldrich, St. Louis, MO, USA), mouse a-DCCin video (VideoTrack; ViewPoint Life Sciences, Otterburn Park, QC, Canada).
(1:1,000, 554223; BD PharMingen, San Diego, CA, USA), rabbit a-Fyn (gift of Recordings (24 mice/genotype) were assessed by an investigator who was
Dr. Andre Veillete; Davidson et al., 1992), rabbit a-GluR1 (1:1,000, AB1504; blind to genotype. Difference score = (time with novel object – time with familiar
Chemicon, Temecula, CA, USA), rabbit a-GluR2/3 (1:1,000, AB1506; Chemi- object). Cognition score = (time with novel object/total exploration time).
con), mouse a-NR2B (1:3000, N59/60; NeuroMab, Davis, CA, USA), rabbit
a-NSF (1:5,000, AB1764; Chemicon), mouse a-phospho-PLCg1 (1:200, Electrophysiology
pY783.27; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit a-phos- Acute brain slices (350–400 mm) were obtained from mice as previously
pho-Src family (Tyr416; 1:1,000, 2101; Cell Signaling Technology, Beverly, described (Glasgow and Chapman, 2007, 2008). During recording, slices
MA, USA), mouse a-PLCg1 (1:1,000, 05-163; Millipore, Billerica, MA, USA), were continuously perfused with oxygenated ACSF (95% O2, 5% CO2, 1.5–
mouse a-PSD95 (1:500; BD PharMingen), mouse a-Src family (WTAPE), clone 2 ml/min).
2E8.2 (1:1,000, 05-1461; Millipore), and mouse a-synaptophysin (1:10,000, LTP and LTD tests began after 20–30 min baseline (intensity adjusted to
S-5768; Sigma-Aldrich). evoke responses 40%–70% of maximum). LTD was assessed with prolonged
Immunohistochemical analyses of 16 mm cryostat sections utilized PPLFS (900 paired pulses, 25 ms interval, 1 Hz for 15 min). LTP was induced
a-b-tubulin III, goat polyclonal a-DCCex (1:500, A20: sc-6535; Santa Cruz by HFS (1 s, 100 Hz train). In the LTP rescue experiment with reduced Mg2+, the
Biotechnology), Hoechst stain (1:10,000, 33258; Sigma-Aldrich), and ACSF contained 1.3 mM Mg2+ and 2.5 mM Ca2+ (versus 2 mM Mg2+ and 2 mM
secondary donkey a-goat Alexa-488 (1:500, A11055; Invitrogen, Eugene, Ca2+). In the LTP rescue experiment with PACAP-38, either PP2 or PP3 (1 mM in
OR, USA) or donkey a-mouse Alexa-555 (1:500, A31570; Invitrogen). DMSO) was present in the 2.0 mM Mg2+ ACSF for the duration of recording,
and PACAP-38 (1 nM) was administered from 10 min before HFS to 20 min
Confocal and Electron Microscopy after HFS. For analysis of fEPSPs in the presence of pharmacological
Axonal-oligodendroglial paranodes labeled with mouse a-Caspr (#75-001; inhibitors, slices were recorded in low-Mg2+ ACSF (0.1 mM Mg2+) to which
NeuroMab) and rabbit a-Kv1.2 (#APC-010; Alomone Labs, Jerusalem, Israel) 100 mM PTX, 5 mM CNQX, 3 mM ifenprodil, and 100 mM APV (Tocris, Minneap-
were imaged with a confocal microscope (510 LSM; Carl Zeiss, Toronto, olis, MN, USA) were added sequentially.
Canada). Immunoelectron microscopy on adult rat CA1 followed a pre-
embedding immunoperoxidase protocol as previously described (Tremblay Data Analysis
et al., 2009) using mouse a-DCCin (1:200, G97-449; BD PharMingen), Statistical significance was tested at the 95% confidence level (p < 0.05). In the
and sections were examined at 60 kV (CM100 electron microscope; Philips, graphs, error bars indicate SEM. Student’s two-tailed t test was used to
Eindhoven, The Netherlands). compare differences between two means.
For additional details, see Extended Experimental Procedures.
Subcellular Fractionation
Subcellular fractionation utilized adult rat brain and mouse hippocampi or
SUPPLEMENTAL INFORMATION
cortex as previously described (Huttner et al., 1983). PSD fractionation of adult
rat brain (unstripped, 7–8 weeks old, ID 56004-2; Pel-Freez Biologicals,
Supplemental Information includes Extended Experimental Procedures and
Rogers, AR, USA) was carried out as previously described (Fallon et al.,
can be found with this article online at http://dx.doi.org/10.1016/j.celrep.
2002). Western blots were probed with antibodies against DCCin, GluR1,
2012.12.005.
netrin-1 (rabbit polyclonal PN2, 11760; Manitt et al., 2001), NR2B, PSD-95,
synaptophysin, and synaptotagmin (rabbit polyclonal 8907, provided by
P. De Camilli, Yale University, New Haven, CT, USA). LICENSING INFORMATION

Organotypic Slice Culture and Spine Morphology This is an open-access article distributed under the terms of the Creative
Slices (250 mm thick) from the hippocampi of P0 DCC knockout pups and Commons Attribution License, which permits unrestricted use, distribution,
WT littermates were cultured 60 DIV, infected with a virus encoding farnesy- and reproduction in any medium, provided the original author and source
lated fluorescent protein, and fixed 24 hr later. Analysis of spines (WT: n = are credited.
275 spines; DCC / : n = 392 spines) used Reconstruct software (John Fiala,
Boston University, Boston, MA, USA). ACKNOWLEDGMENTS

Golgi Staining and Spine Morphology We thank W. Sossin, A. Di Polo, and J. Goldman for critical discussions, and M.
Mouse brains (n = 3/condition) were processed (FD Rapid GolgiStain Kit; Cayouette and D. Bowie for reagents. The project was supported by CIHR
FD Neurotechnologies, Catonsville, MD, USA) and R50 mm sections were operating grant #247564 to T.E.K. K.E.H. was funded by a CIHR Frederick

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 11

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

Banting and Charles Best Canada Graduate Scholarship Doctoral Award. Jacobs, K.M., Neve, R.L., and Donoghue, J.P. (1993). Neocortex and hippo-
S.D.G. received grants from NSERC, and C.A.C. received grants from NSERC campus contain distinct distributions of calcium-calmodulin protein kinase II
and FRSQ. D.G. holds an FRSQ postdoctoral fellowship. M.E.T. held an FRSQ and GAP43 mRNA. J. Comp. Neurol. 336, 151–160.
doctoral training award. E.S.R. holds a tier II Canada Research Chair. T.E.K. Jarjour, A.A., Bull, S.J., Almasieh, M., Rajasekharan, S., Baker, K.A., Mui, J.,
holds an FRSQ Chercheur Nationaux award and is a Killam Foundation Antel, J.P., Di Polo, A., and Kennedy, T.E. (2008). Maintenance of axo-oligo-
Scholar. dendroglial paranodal junctions requires DCC and netrin-1. J. Neurosci. 28,
11003–11014.
Received: May 21, 2012
Kim, M.J., Dunah, A.W., Wang, Y.T., and Sheng, M. (2005). Differential roles of
Revised: October 1, 2012
NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and
Accepted: December 13, 2012
AMPA receptor trafficking. Neuron 46, 745–760.
Published: January 3, 2013
Kolodziej, P.A., Timpe, L.C., Mitchell, K.J., Fried, S.R., Goodman, C.S., Jan,
REFERENCES L.Y., and Jan, Y.N. (1996). frazzled encodes a Drosophila member of the
DCC immunoglobulin subfamily and is required for CNS and motor axon
Barria, A., and Malinow, R. (2002). Subunit-specific NMDA receptor trafficking guidance. Cell 87, 197–204.
to synapses. Neuron 35, 345–353. Kourrich, S., Glasgow, S.D., Caruana, D.A., and Chapman, C.A. (2008).
Bayer, K.U., Löhler, J., Schulman, H., and Harbers, K. (1999). Developmental Postsynaptic signals mediating induction of long-term synaptic depression
expression of the CaM kinase II isoforms: ubiquitous gamma- and delta-CaM in the entorhinal cortex. Neural Plast. 2008, 840374.
kinase II are the early isoforms and most abundant in the developing nervous Krimpenfort, P., Song, J.Y., Proost, N., Zevenhoven, J., Jonkers, J., and Berns,
system. Brain Res. Mol. Brain Res. 70, 147–154. A. (2012). Deleted in colorectal carcinoma suppresses metastasis in p53-
Benson, D.L., Isackson, P.J., Gall, C.M., and Jones, E.G. (1992). Contrasting deficient mammary tumours. Nature 482, 538–541.
patterns in the localization of glutamic acid decarboxylase and Ca2+/calmod- Lai Wing Sun, K., Correia, J.P., and Kennedy, T.E. (2011). Netrins: versatile
ulin protein kinase gene expression in the rat central nervous system. Neuro- extracellular cues with diverse functions. Development 138, 2153–2169.
science 46, 825–849. Li, W., Lee, J., Vikis, H.G., Lee, S.H., Liu, G., Aurandt, J., Shen, T.L., Fearon,
Bevins, R.A., and Besheer, J. (2006). Object recognition in rats and mice: a E.R., Guan, J.L., Han, M., et al. (2004). Activation of FAK and Src are
one-trial non-matching-to-sample learning task to study ‘recognition memory’. receptor-proximal events required for netrin signaling. Nat. Neurosci. 7,
Nat. Protoc. 1, 1306–1311. 1213–1221.
Bliss, T.V., and Collingridge, G.L. (1993). A synaptic model of memory: long- Liu, G., Beggs, H., Jürgensen, C., Park, H.T., Tang, H., Gorski, J., Jones, K.R.,
term potentiation in the hippocampus. Nature 361, 31–39. Reichardt, L.F., Wu, J., and Rao, Y. (2004). Netrin requires focal adhesion
Bouchard, J.F., Horn, K.E., Stroh, T., and Kennedy, T.E. (2008). Depolarization kinase and Src family kinases for axon outgrowth and attraction. Nat. Neuro-
recruits DCC to the plasma membrane of embryonic cortical neurons sci. 7, 1222–1232.
and enhances axon extension in response to netrin-1. J. Neurochem. 107, Livesey, F.J., and Hunt, S.P. (1997). Netrin and netrin receptor expression in
398–417. the embryonic mammalian nervous system suggests roles in retinal, striatal,
Burgin, K.E., Waxham, M.N., Rickling, S., Westgate, S.A., Mobley, W.C., and nigral, and cerebellar development. Mol. Cell. Neurosci. 8, 417–429.
Kelly, P.T. (1990). In situ hybridization histochemistry of Ca2+/calmodulin- Lu, Y.M., Roder, J.C., Davidow, J., and Salter, M.W. (1998). Src activation in
dependent protein kinase in developing rat brain. J. Neurosci. 10, 1788–1798. the induction of long-term potentiation in CA1 hippocampal neurons. Science
Clark, R.E., and Martin, S.J. (2005). Interrogating rodents regarding their object 279, 1363–1367.
and spatial memory. Curr. Opin. Neurobiol. 15, 593–598. Macdonald, D.S., Weerapura, M., Beazely, M.A., Martin, L., Czerwinski, W.,
Colón-Ramos, D.A., Margeta, M.A., and Shen, K. (2007). Glia promote local Roder, J.C., Orser, B.A., and MacDonald, J.F. (2005). Modulation of NMDA
synaptogenesis through UNC-6 (netrin) signaling in C. elegans. Science 318, receptors by pituitary adenylate cyclase activating peptide in CA1 neurons
103–106. requires G alpha q, protein kinase C, and activation of Src. J. Neurosci. 25,
Davidson, D., Chow, L.M., Fournel, M., and Veillette, A. (1992). Differential 11374–11384.
regulation of T cell antigen responsiveness by isoforms of the src-related MacDonald, J.F., Jackson, M.F., and Beazely, M.A. (2007). G protein-coupled
tyrosine protein kinase p59fyn. J. Exp. Med. 175, 1483–1492. receptors control NMDARs and metaplasticity in the hippocampus. Biochim.
Fallon, L., Moreau, F., Croft, B.G., Labib, N., Gu, W.J., and Fon, E.A. (2002). Biophys. Acta 1768, 941–951.
Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are Manitt, C., Colicos, M.A., Thompson, K.M., Rousselle, E., Peterson, A.C., and
co-localized in lipid rafts and postsynaptic densities in brain. J. Biol. Chem. Kennedy, T.E. (2001). Widespread expression of netrin-1 by neurons and
277, 486–491. oligodendrocytes in the adult mammalian spinal cord. J. Neurosci. 21, 3911–
Fazeli, A., Dickinson, S.L., Hermiston, M.L., Tighe, R.V., Steen, R.G., Small, 3922.
C.G., Stoeckli, E.T., Keino-Masu, K., Masu, M., Rayburn, H., et al. (1997). Manitt, C., Nikolakopoulou, A.M., Almario, D.R., Nguyen, S.A., and Cohen-
Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) Cory, S. (2009). Netrin participates in the development of retinotectal synaptic
gene. Nature 386, 796–804. connectivity by modulating axon arborization and synapse formation in the
Glasgow, S.D., and Chapman, C.A. (2007). Local generation of theta- developing brain. J. Neurosci. 29, 11065–11077.
frequency EEG activity in the parasubiculum. J. Neurophysiol. 97, 3868–3879. Mitchell, K.J., Doyle, J.L., Serafini, T., Kennedy, T.E., Tessier-Lavigne, M.,
Glasgow, S.D., and Chapman, C.A. (2008). Conductances mediating intrinsic Goodman, C.S., and Dickson, B.J. (1996). Genetic analysis of Netrin genes
theta-frequency membrane potential oscillations in layer II parasubicular in Drosophila: Netrins guide CNS commissural axons and peripheral motor
neurons. J. Neurophysiol. 100, 2746–2756. axons. Neuron 17, 203–215.
Haber, M., Zhou, L., and Murai, K.K. (2006). Cooperative astrocyte and Miyata, A., Jiang, L., Dahl, R.D., Kitada, C., Kubo, K., Fujino, M., Minamino, N.,
dendritic spine dynamics at hippocampal excitatory synapses. J. Neurosci. and Arimura, A. (1990). Isolation of a neuropeptide corresponding to the
26, 8881–8891. N-terminal 27 residues of the pituitary adenylate cyclase activating polypep-
Huttner, W.B., Schiebler, W., Greengard, P., and De Camilli, P. (1983). Synap- tide with 38 residues (PACAP38). Biochem. Biophys. Res. Commun. 170,
sin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association 643–648.
with synaptic vesicles studied in a highly purified synaptic vesicle preparation. Nicolakakis, N., Aboulkassim, T., Aliaga, A., Tong, X.K., Rosa-Neto, P., and
J. Cell Biol. 96, 1374–1388. Hamel, E. (2011). Intact memory in TGF-beta1 transgenic mice featuring

12 Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors

 Please cite this article in press as: Horn et al., DCC Expression by Neurons Regulates Synaptic Plasticity in the Adult Brain, Cell Reports (2013), http://
dx.doi.org/10.1016/j.celrep.2012.12.005

chronic cerebrovascular deficit: recovery with pioglitazone. J. Cereb. Blood Tang, Y.P., Shimizu, E., Dube, G.R., Rampon, C., Kerchner, G.A., Zhuo, M.,
Flow Metab. 31, 200–211. Liu, G., and Tsien, J.Z. (1999). Genetic enhancement of learning and memory
Nimchinsky, E.A., Sabatini, B.L., and Svoboda, K. (2002). Structure and in mice. Nature 401, 63–69.
function of dendritic spines. Annu. Rev. Physiol. 64, 313–353. Tovar, K.R., and Westbrook, G.L. (1999). The incorporation of NMDA receptors
with a distinct subunit composition at nascent hippocampal synapses in vitro.
Poon, V.Y., Klassen, M.P., and Shen, K. (2008). UNC-6/netrin and its receptor
J. Neurosci. 19, 4180–4188.
UNC-5 locally exclude presynaptic components from dendrites. Nature 455,
669–673. Tremblay, M.E., Riad, M., Chierzi, S., Murai, K.K., Pasquale, E.B., and Doucet,
G. (2009). Developmental course of EphA4 cellular and subcellular localization
Salter, M.W., and Kalia, L.V. (2004). Src kinases: a hub for NMDA receptor
in the postnatal rat hippocampus. J. Comp. Neurol. 512, 798–813.
regulation. Nat. Rev. Neurosci. 5, 317–328.
Tsien, J.Z., Chen, D.F., Gerber, D., Tom, C., Mercer, E.H., Anderson, D.J.,
Sauer, B. (1998). Inducible gene targeting in mice using the Cre/lox system.
Mayford, M., Kandel, E.R., and Tonegawa, S. (1996). Subregion- and cell
Methods 14, 381–392.
type-restricted gene knockout in mouse brain. Cell 87, 1317–1326.
Serafini, T., Colamarino, S.A., Leonardo, E.D., Wang, H., Beddington, R.,
Volenec, A., Bhogal, R.K., Moorman, J.M., Leslie, R.A., and Flanigan, T.P.
Skarnes, W.C., and Tessier-Lavigne, M. (1996). Netrin-1 is required for
(1997). Differential expression of DCC mRNA in adult rat forebrain. Neuroreport
commissural axon guidance in the developing vertebrate nervous system.
8, 2913–2917.
Cell 87, 1001–1014.
Williams, K., Russell, S.L., Shen, Y.M., and Molinoff, P.B. (1993). Develop-
Shen, K., and Cowan, C.W. (2010). Guidance molecules in synapse formation mental switch in the expression of NMDA receptors occurs in vivo and
and plasticity. Cold Spring Harb. Perspect. Biol. 2, a001842. in vitro. Neuron 10, 267–278.
Sheng, M., Cummings, J., Roldan, L.A., Jan, Y.N., and Jan, L.Y. (1994). Winberg, M.L., Mitchell, K.J., and Goodman, C.S. (1998). Genetic analysis of
Changing subunit composition of heteromeric NMDA receptors during devel- the mechanisms controlling target selection: complementary and combinato-
opment of rat cortex. Nature 368, 144–147. rial functions of netrins, semaphorins, and IgCAMs. Cell 93, 581–591.
Sonner, J.M., Cascio, M., Xing, Y., Fanselow, M.S., Kralic, J.E., Morrow, A.L., Xie, Y., Hong, Y., Ma, X.Y., Ren, X.R., Ackerman, S., Mei, L., and Xiong, W.C.
Korpi, E.R., Hardy, S., Sloat, B., Eger, E.I., 2nd, and Homanics, G.E. (2005). (2006). DCC-dependent phospholipase C signaling in netrin-1-induced neurite
Alpha 1 subunit-containing GABA type A receptors in forebrain contribute to elongation. J. Biol. Chem. 281, 2605–2611.
the effect of inhaled anesthetics on conditioned fear. Mol. Pharmacol. 68,
Yu, X.M., and Salter, M.W. (1998). Gain control of NMDA-receptor currents by
61–68.
intracellular sodium. Nature 396, 469–474.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter
Yu, X.M., Askalan, R., Keil, G.J., 2nd, and Salter, M.W. (1997). NMDA channel
strain. Nat. Genet. 21, 70–71.
regulation by channel-associated protein tyrosine kinase Src. Science 275,
Stavoe, A.K., and Colón-Ramos, D.A. (2012). Netrin instructs synaptic vesicle 674–678.
clustering through Rac GTPase, MIG-10, and the actin cytoskeleton. J. Cell Zhou, C.J., Kikuyama, S., Shibanuma, M., Hirabayashi, T., Nakajo, S., Arimura,
Biol. 197, 75–88. A., and Shioda, S. (2000). Cellular distribution of the splice variants of the
Tada, T., and Sheng, M. (2006). Molecular mechanisms of dendritic spine receptor for pituitary adenylate cyclase-activating polypeptide (PAC(1)-R) in
morphogenesis. Curr. Opin. Neurobiol. 16, 95–101. the rat brain by in situ RT-PCR. Brain Res. Mol. Brain Res. 75, 150–158.

Cell Reports 3, 1–13, January 31, 2013 ª2013 The Authors 13