The neuropeptide PACAP38 induces dendritic spineremodeling through ADAM10–N-cadherinsignaling pathway

Fabrizio Gardoni1,*, Claudia Saraceno1, Matteo Malinverno1, Elena Marcello1, Chiara Verpelli2,3, Carlo Sala2,3

and Monica Di Luca1

1Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy2Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Pharmacology, University ofMilan, via Vanvitelli 32, 20129 Milan, Italy3Neuromuscular Diseases and Neuroimmunology, Neurological Institute Foundation Carlo Besta, Via Celoria 11, 20133 Milan, Italy

*Author for correspondence (fabrizio.gardoni@unimi.it)

Accepted 14 November 2011Journal of Cell Science 125, 1401–1406� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.097576

SummaryThe neuropeptide pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) has been implicated in the induction of synapticplasticity at the excitatory glutamatergic synapse. In particular, recent studies have shown that it is involved in the regulation of N-methyl-D-aspartate (NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation. Here we

demonstrate the effect of PACAP38 on the modulation of dendritic spine morphology through a disintegrin and metalloproteinase 10(ADAM10)–N-cadherin–AMPA receptor signaling pathway. Treatment of primary hippocampal neurons with PACAP38 induced anaccumulation of ADAM10 at the postsynaptic membrane. This event led to a significant decrease of dendritic spine head width and to a

concomitant reduction of GluR1 colocalization with postsynaptic markers. The PACAP38-induced effect on dendritic spine head widthwas prevented by either treatment with the ADAM10-specific inhibitor or transfection of a cleavage-defective N-cadherin constructmutated in the ADAM10 cleavage site. Overall, our findings reveal that PACAP38 is involved in the modulation of dendritic spinemorphology in hippocampal neurons, and assign to the ADAM10–N-cadherin signaling pathway a crucial role in this modification of the

excitatory glutamatergic synapse.

Key words: PACAP38, ADAM10, Spine morphology

IntroductionThe neuropeptide pituitary adenylate cyclase-activating

polypeptide 38 (PACAP38) is expressed in the different regions

of the hippocampus starting during embryogenesis, diminishing

only slowly towards adulthood (Jaworski and Proctor, 2000). It has

been shown that PACAP38 modulates a variety of signaling

pathways within the excitatory glutamatergic synapse (MacDonald

et al., 2005; Yang et al., 2009; Yang et al., 2010), ranging

from activation of different protein kinases, i.e. protein kinase A

(PKA) and the mitogen-activated protein (MAP) kinase to the

mobilization of calcium (Harmar et al., 1998; Vaudry et al., 2000;

Kojro et al., 2006; Ohnishi et al., 2008; Yang et al., 2010).

Interestingly, induction of endogenous PACAP38 responded to

different signals, i.e. cAMP signals and KCl-dependent membrane

depolarization (Fukuchi et al., 2004).

A recent study showed that treatment with PACAP38 at

a very low concentration reduced a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA) receptor excitatory postsynaptic

currents (EPSCs) through PAC1-receptor independent signaling

(Costa et al. 2009). By contrast, another study showed that the

application of PACAP38 enhanced AMPA-evoked currents (Michel

et al., 2006). PACAP38 has been also shown to induce a form of

long-term depression (LTD) in hippocampal neurons that requires

interaction of AMPA receptor subunits with scaffolding proteins

(Kondo et al. 1997; Roberto et al. 2001; Ster et al., 2009), thus

validating its role in learning and memory. This has been further

confirmed by the use of transgenic animals (Yang et al., 2010).

Nonetheless, the mechanism by which PACAP38-dependent

signaling can modify AMPA receptors remains almost unknown

and no information is available on the effect of PACAP38 on

AMPA receptor subunit localization at the synapse.

Interestingly, PACAP38-dependent activation of PAC1

receptor can also activate key enzymes, i.e. a disintegrin and

metalloproteinase 10 (ADAM10), localized within the excitatory

postsynaptic density (Kojro et al., 2006; Marcello et al., 2007;

Marcello et al., 2010; Malinverno et al., 2010). In particular,

Kojro and coworkers showed that PACAP38 leads to a strong

increase of ADAM10 activity through the intervention of three

signaling cascades (PKC, MAP kinase and PI 3-kinase) (Kojro

et al., 2006). Notably, we have recently described that inhibition

of ADAM10-dependent cleavage of the cell-adhesion molecule

N-cadherin induces a modification of the number and the current

of synaptic AMPA receptors in hippocampal neurons, and a

significant increase in the size of dendritic spines (Malinverno

et al., 2010). Accordingly, several reports demonstrated the

existence of a close coordination between AMPA receptor

content at synapses, N-cadherin activation and spine size (Kasai

et al., 2003; Kopec et al., 2007; Xie et al., 2008).

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Here we show that in hippocampal neurons PACAP38 induces

dendritic spine shrinkage correlated with a decrease of synaptic

AMPA receptors through modulation of ADAM10-dependent

cleavage of N-cadherin.

Results and DiscussionTreatment with PACAP38 leads to a modification ofADAM10 and GluR1 subunit levels at synapses in primaryhippocampal neurons

A previous report described the ability of PACAP38 to stimulate

ADAM10 activity in neuroblastoma cells (Kojro et al., 2006). We

performed immunocytochemical labeling to verify whether

treatment of hippocampal neurons with PACAP38 also induces

a modification of ADAM10 localization at synaptic sites, where

most of the ADAM10 neuronal substrates are located (Uemura

et al., 2006; Malinverno et al., 2010). As shown in Fig. 1A,B,

treatment with PACAP38 (300 nM, 30 minutes) led to an

increase of ADAM10 colocalization with the postsynaptic marker

Shank (PACAP38 300 nM vs control, 61.866.7%, **P,0.001).

Treatment with PACAP38 at lower concentrations (1–10 nM)

(MacDonald et al., 2005) did not induce any effect on ADAM10

localization in the postsynaptic compartment (PACAP38 1 nM vs

control, –1.9610.9%, P50.902; PACAP38 10 nM vs control,

10.264.4%, P50.344).

PACAP38 can modulate different intracellular pathways

within the excitatory synapse (Macdonald et al., 2005; Yang

et al., 2009, 2010). In particular, PACAP38 induces PKA, and

several cellular effects of PACAP38 are mediated by MAP kinase

activation (Kojro et al., 2006). Treatment of hippocampal

neurons with PACAP38 in the presence of a p38-specific

inhibitor (SB203580, 5 mM) did not abolish the effect of

PACAP38 on ADAM10 localization (PACAP38 + SB203580

vs control, 64.864.4%, P,0.0001; PACAP38 + SB203580 vs

PACAP38, 1.862.8%, P50.788). Conversely, co-treatment with

a PKA inhibitor (H89, 5 mM) blocked the PACAP38-induced

increased in the colocalization of ADAM10 with Shank

(Fig. 1A,B; PACAP38 + H89 vs control, 6.267.2%, P50.431;

PACAP38 +H89 vs PACAP 38, P,0.001).

PACAP38 has a high affinity for its specific receptor (PAC1),

but it also has an affinity for the VIP-specific receptors

Fig. 1. PACAP38 leads to a modification of ADAM10 and GluR1 localization at synapses. (A) Ten days in vitro (DIV10) primary hippocampal neurons were

immunolabeled for ADAM10 (green) and Shank (red) as a postsynaptic marker. (B) ADAM10 and Shank colocalization expressed as a percentage of the control,

in neurons treated with PACAP38 (300 nM, 30 minutes) in the absence or the presence of a PKA inhibitor (H89, 5 mM) or a VIP antagonist (VIP(6-28), 1 mM;

PACAP38 300 nM vs control, **P,0.001, n512; PACAP38 + H89 vs control, P50.431, n512; PACAP38 +H89 vs PACAP 38, §P,0.001, n512; PACAP38 +

VIP(6-28) vs control, #P,0.005, n512; PACAP38 + VIP(6-28) vs PACAP38, #P,0.005, n512). (C) DIV10 primary hippocampal neurons were immunolabeled

for GluR1 (green) and Shank (red) as a postsynaptic marker. (D) GluR1 and Shank colocalization expressed as a percentage of the control, in neurons treated with

PACAP38 (300 nM, 30 minutes) in the absence or the presence of a PKA inhibitor (H89, 5 mM) or VIP antagonist [VIP(6-28), 1 mM; PACAP38 vs control,

**P,0.001, n518; PACAP38 + H89 vs control, *P50.016, n518; PACAP38 + H89 vs PACAP38, **P,0.001, n518; PACAP38 + VIP(6-28) vs control,#P,0.005, n518; PACAP38 + VIP(6-28) vs PACAP38, *P50.013, n518].

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(VPACRs) VPAC1 and VPAC2, similar to VIP (Yang et al.,2010). As shown in Fig. 1A,B co-treatment of hippocampal

neurons with PACAP38 and VIP antagonist [VIP(6-28), 1 mM]partially blocked the increased colocalization of ADAM10 withShank induced by PACAP38 [PACAP38 + VIP(6-28) vs control,41.463.6%, P,0.005; PACAP38 + VIP(6-28) vs PACAP38,

P,0.005].

Modulation of ADAM10 levels at synaptic sites is a crucialway to regulate AMPA receptors at hippocampal excitatory

synapses, leading to a modification of AMPA receptor currentsand subunits composition (Malinverno et al., 2010). Confocalanalysis revealed that treatment with PACAP38 (300 nM,

30 minutes) induces a reduction of GluR1 localization inShank-positive postsynaptic clusters (Fig. 1C,D; PACAP38 vscontrol, –37.363.5%, **P,0.001), thus indicating a decrease inAMPA receptor availability at synaptic sites following activation

of PACAP38-dependent signaling.

Treatment of hippocampal neurons with PACAP38 in thepresence of a PKA inhibitor (H89, 5 mM) partially blocked the

reduction of GluR1 colocalization with Shank induced byPACAP38 (Fig. 1C,D; PACAP38 + H89 vs control, –14.56

2.5%, P50.016; PACAP38 + H89 vs PACAP38, P,0.001),

suggesting a role for PKA in the complex mechanism by whichPACAP38-dependent signaling can modify AMPA receptors(Yang et al., 2010). Interestingly, co-treatment with a VIP

antagonist [VIP(6-28), 1 mM] also partially blocked thereduction of GluR1 localization at synaptic sites induced byPACAP38 [Fig. 1C,D; PACAP38 + VIP(6-28) vs control,–21.763.5%, P,0.005; PACAP38 + VIP(6-28) vs PACAP38,

P50.013].

Treatment with PACAP38 leads to a significant decrease ofspine head width in primary hippocampal neurons

ADAM10 plays a crucial role in the complex sequence of events

that regulates dendritic spine maturation and/or stabilization

and in the modulation of the structural organization of the

glutamatergic synapse (Malinverno et al., 2010).

Accordingly, we investigated the onset of a possible effect of

PACAP38 on dendritic spine morphology by time-lapse confocal

imaging. In control neurons, no significant modification of average

spine head width was observed over 30 minutes (data not shown). A

decrease of spine head width (Fig. 2A,B), that became statistically

significant 21 minutes after treatment, was observed in PACAP38-

treated neurons compared with control ones (Fig. 2A,B; P,0.0001

PACAP38 vs control). Furthermore, a significant increase in the

percentage of shrinking spines (control 24.761.2%, PACAP38

46.367.0%, P50.038) and a corresponding decrease in the

percentage of growing ones (control 28.960.2%, PACAP38

7.563.4%, P50.049) was detected 30 minutes after treatment

with PACAP38 (Fig. 2C).

For a more detailed morphological analysis, dendritic spines

were categorized according to their shape (mushroom, thin and

stubby) using a highly validated classification method (see

Materials and Methods section). As shown in Fig. 2D, treatment

with PACAP38 induced a significant reduction in the proportion

of mushroom spines and a concomitant significant increase in the

proportion of thin and stubby ones (mushroom: P50.00009, GFP

+ PACAP38 vs GFP; stubby: P50.0002, GFP + PACAP38 vs

GFP; thin: P50.002, GFP + PACAP38 vs GFP). No significant

change in the mean spine density was found (GFP 4.10860.329

Fig. 2. Modulation of dendritic spine morphology by PACAP38. (A) Spine head width was measured at all set times and expressed as a percentage of initial

values (*P,0.0001). (B) Representative time-lapse images of hippocampal neurons transfected with GFP (DIV7) and incubated or not (DIV10) with PACAP38.

(C) Spine growth and shrinkage in neurons treated or not with PACAP38 for 30 minutes, expressed as a percentage of the total number of spines (PACAP38 vs

control, percentage of growing spines (*P50.049; PACAP38 vs control, percentage of shrinking spines, §P50.038). (D) Dendritic spines were divided in

three different categories depending on their morphology: stubby, thin and mushroom, as indicated in the line drawing on the right. The diagram shows the

percentage of total spines belonging to each category in GFP-transfected neurons treated or not with PACAP38 for 30 minutes (GFP + PACAP38 vs GFP control,

percentage of mushroom spines, **P50.00009; GFP + PACAP38 vs GFP control, percentage of stubby spines, #P50.0002; GFP + PACAP38 vs GFP control,

percentage of thin spines, §P50.002).

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spines/10 mm; GFP + PACAP38 4.14160.498 spines/10 mm;

P50.89).

Inhibition of ADAM10 activity by treatment with TIMP-1

blocks the effects of PACAP38 on the ADAM10–GluR1

pathway and dendritic spine morphology

The results described above suggest that PACAP38 induces

a profound modification of dendritic spine morphology in

hippocampal neurons correlated with an alteration of the

synaptic localization of ADAM10 and GluR1 subunits of the

AMPA receptor. Interestingly, we have recently demonstrated

that treatment of hippocampal neurons with the ADAM10-

specific inhibitor TIMP-1, which was previously shown to block

the ADAM10-dependent pathway downstream of PAC1 receptor

activation (Kojro et al., 2006), induced a substantial increase in

GluR1 localization in Shank-positive clusters (Malinverno et al.,

2010).

Based on these considerations, we treated hippocampal

neurons with PACAP38 in the presence or absence of TIMP-1.

As expected, incubation with TIMP-1 did not affect ADAM10

localization at the postsynaptic compartment in PACAP38-

treated hippocampal neurons (TIMP-1 + PACAP38 vs

PACAP38, 6.3611.9%, P50.68), indicating that TIMP-1

inhibits ADAM10 activity without affecting its subcellular

localization. However, co-treatment of hippocampal neurons

with PACAP38 + TIMP-1 led to a significant rescue of

GluR1 colocalization with Shank-positive postsynaptic clusters

(Fig. 3A,B; TIMP-1 + PACAP38 vs PACAP38, 30.7610.3%,

P50.02; TIMP-1 + PACAP38 vs control, P50.896).

We then performed morphological analysis of primary

hippocampal neurons after treatment with PACAP38 in the

presence or absence of TIMP-1. Statistical analysis revealed a

significant increase of spine head width in neurons treated with

PACAP38 + TIMP-1 compared with PACAP38-treated ones

(Fig. 3C,D; P50.0002, PACAP38 vs control; P50.03, TIMP-1 +

PACAP38 vs PACAP38; P50.059, TIMP-1 + PACAP38 vs

control).

Cumulative frequency plots of spine head width confirmed a

significant shift towards smaller spine size in the presence of

PACAP38 that was rescued in the presence of TIMP-1 (Fig. 3E).

No significant changes in the mean spine density were found

(control 3.08760.299 spines/10 mm, PACAP38 2.82060.118

spines/10 mm, PACAP38 + TIMP-1 3.079260.140 spines/

10 mm; P50.366, PACAP38 vs control; P50.132, PACAP38 +

TIMP-1 vs PACAP38; P50.97, PACAP38 + TIMP-1 vs control).

Transfection of the ADAM10 cleavage-defective N-

cadherin construct blocks any effect of PACAP38 on

spine morphology

Interfering with ADAM10 activity at synapses is sufficient to induce

a significant decrease in ADAM10-mediated N-cadherin cleavage,

leading to accumulation of N-cadherin full length (FL) and to a

significant enlargement of dendritic spine head width (Malinverno

et al., 2010). Accordingly, we checked whether the morphological

Fig. 3. ADAM10 inhibitor prevents the effects of PACAP38 on GluR1 and dendritic spine morphology. (A) DIV10 primary hippocampal neurons were

immunolabeled for GluR1 (green) and Shank (red) as a postsynaptic marker. (B) GluR1 and Shank colocalization expressed as a percentage of control neurons.

Treatment with PACAP38 (300 nM, 30 minutes) decreased colocalization (TIMP-1 + PACAP38 vs PACAP38, 30.7610.3%, *P50.0256; TIMP-1 + PACAP38 vs

control, P50.896; n56 neurons for each group). (C) Representative images showing dendrites from neurons treated or not with PACAP38 and TIMP-1 + PACAP38.

(D) Average spine head width. PACAP38 vs control –14.962.9%, **P50.0002; PACAP38 + TIMP-1 vs control –6.663.1%, P50.0595; TIMP-1 + PACAP38 vs

PACAP38, P50.0392; n.780 spines from 10 different neurons for each group, from four different experiments. (E) Cumulative frequency plot of spine head width.

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effects induced by PACAP38 could be correlated to an alteration of

ADAM10-dependent N-cadherin cleavage. By using an antibody

raised against N-cadherin C-terminal intracellular domain (C-32),

we detected two main immunoreactive bands corresponding to

the N-cadherin FL and to its C-terminal fragment (CTF), which

derives from ADAM10-mediated cleavage (Uemura et al., 2006;

Malinverno et al., 2010). As shown in Fig. 4A, treatment with

PACAP38 induced a significant decrease in the N-cadherin FL/CTF

ratio (PACAP38 control, –34.769.7%, P50.02) that was

completely rescued by co-treatment with TIMP-1 (TIMP-1 +

PACAP38 vs PACAP38, P50.001; TIMP-1 + PACAP38 vs control

4.5610.6%, P50.68). These results suggest an involvement of

ADAM10-dependent N-cadherin cleavage in the morphological

outcome of the treatment with PACAP38. Accordingly, treatment

with PACAP38 and subsequent morphological analysis were

repeated in neurons transfected with GFP, GFP–N-cadherin wild-

type (wt) or with the cleavage-defective GFP–N-cadherin GD

construct, mutated in the ADAM10 cleavage site (Uemura et al.,

2006; Malinverno et al., 2010). Both N-cadherin constructs had the

same expression pattern at the postsynaptic site in hippocampal

neurons as demonstrated by colocalization analysis with Shank-

positive clusters (data not shown). Statistical analysis revealed a

significant increase of spine head width in neurons treated with

GFP–N-cadherin GD (Fig. 4B,C; P50.002, N-cadherin GD control

vs GFP control) but not in those treated with GFP–N-cadherin wt,

thus confirming that blocking ADAM10-dependent cleavage of

N-cadherin leads per se to an increase of spine size (see also

Malinverno et al., 2010). Treatment with PACAP38 induces a

significant decrease in spine head width in neurons transfected with

GFP (Fig. 4B,C; P50.02 GFP + PACAP38 vs GFP control) or with

N-cadherin wt (Fig. 4B,C; P50.0003 N-cadherin wt + PACAP38 vs

N-cadherin wt control) but not in those transfected with GFP N-

cadherin GD (Fig. 4B,C; P.0.05 N-cadherin GD + PACAP38 vs

N-cadherin GD control), thus indicating that blocking ADAM10-

dependent cleavage of N-cadherin prevents any effect of PACAP38

on dendritic spine size.

Morphological regulation of dendritic spines at excitatory

synapses represents one of the main relevant and efficient

cellular mechanisms involved in the induction of different forms

of plasticity within the central nervous system (Bourne

and Harris, 2011). Accordingly, formation, stabilization and

elimination of dendritic spines are highly organized and complex

mechanisms based on a variety of cellular and molecular

regulatory steps. Here we demonstrate that treatment of

hippocampal neurons with PACAP38 leads to a significant

modulation of dendritic spine morphology dependent on

activation of the ADAM10–N-cadherin signaling pathway. In

fact, co-treatment with the ADAM10-specific inhibitor TIMP-1

or transfection of N-cadherin GD construct, mutated in the

ADAM10 cleavage site, are both sufficient to block any

morphological effect observed after incubation with PACAP38.

Notably, PACAP38-dependent shrinkage of dendritic spines is

strictly paralleled by a reduction of AMPA receptor GluR1

subunit localization at synaptic sites.

PACAP38 induces a form of LTD in hippocampal neurons that

depends on Rap-1 and p38-MAPK activation and on synaptic

levels of AMPA receptors, and that was mutually occluded by

the Rap guanine nucleotide exchange factor (Epac) LTD (Ster

et al., 2009). However, we did not observe any effect of the

Epac activator (8pCPT-2Me-cAMP, 200 mM) on ADAM10

colocalization with the postsynaptic marker Shank compared

with controls (21.9166.78%, P50.8464), further indicating that

the Epac–p38 pathway is not involved in PACAP38-dependent

modulation of ADAM10 function.

In conclusion, our findings demonstrate that the neuropeptide

PACAP38 plays a key role in the modulation of dendritic spine

morphology through activation of the ADAM10–N-cadherin–

AMPA receptor signaling pathway in hippocampal neurons.

Fig. 4. ADAM10-dependent cleavage of N-cadherin modulates the effects

of PACAP38 on spine morphology. (A) Western blot analysis performed in

a Triton-insoluble postsynaptic fraction with an antibody against the C-

terminal domain of N-cadherin. Treatment with PACAP38 induced a

statistically significant decrease in the N-cadherin FL/CTF ratio (*P50.0256

PACAP38 vs control) that was rescued by co-treatment with TIMP-1

(§P50.0011 TIMP-1 + PACAP38 vs PACAP38). (B) Representative images

show dendrites from neurons transfected with GFP and co-transfected with

either GFP–N-cadherin wt or GFP-N-cadherin GD. For each condition,

neurons were treated with PACAP38 or left untreated (control).

(C) Average spine head width (percentage GFP: GFP + PACAP38

212.663.1%, *P50.02271 GFP + PACAP38 vs GFP; N-cadherin wt

0.962.5%; N-cadherin wt + PACAP38 –19.261.5, **P50.00039 N-cadherin

wt + PACAP38 vs N-cadherin wt; N-cadherin GD 16.563.1%, §P50.00236

N-cadherin GD vs GFP; N-cadherin GD + PACAP38 12.662.5%).

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Materials and MethodsAntibodies and reagentsThe following monoclonal antibodies were used: anti-GFP and anti-pan Shank,purchased from NeuroMab (Davis, CA). Anti-ADAM10 N-terminal polyclonalantibody was purchased from Abcam (Cambridge, MA) and anti-GluR1 polyclonalantibody from Millipore (Billerica, MA); Alexa Fluor secondary antibodies werepurchased from Invitrogen (Carlsbad, CA). TIMP-1 and PACAP38 were purchasedfrom Calbiochem (Darmstadt, Germany). p38 mitogen-activated protein kinaseinhibitor (SB203580), VIP antagonist [VIP(6-28)], EPAC activator (8CPT-2Me-cAMP) and protein kinase A inhibitor (H89) were purchased from Tocris (Bristol,UK).

DNA constructsThe plasmid encoding for GFP-tagged N-cadherin was kindly provided by MariaPassafaro (Milano, Italy); the cleavage-defective (N-cadherin GD) construct wascreated by site-direct mutagenesis of R714G and I715D. Transfection assay inCOS7 cells was performed to verify that the N-cadherin GD mutant form leads to adramatic decrease in the formation of the C-terminal fragment (CTF) products,thus indicating a significant decrease in N-cadherin cleavage (data not shown).

Neuronal cultures preparation and transfectionHippocampal neuronal primary cultures were prepared from embryonic day 18–19(E18–E19) rat hippocampi as previously described (Piccoli et al., 2007). Neuronswere transfected between DIV7 using the calcium-phosphate method.

ImmunocytochemistryFor morphological studies, transfected neurons were fixed in 4%paraformaldehyde with 4% sucrose at room temperature and immunostained forGFP; primary and secondary antibodies were applied in GDB buffer (Sala et al.,2001) (30 mM phosphate buffer, pH 7.4, containing 0.2% gelatin, 0.5% Triton X-100 and 0.8 M NaCl). Cells were chosen randomly for quantification.Fluorescence images were acquired using a Zeiss confocal LSM510 system witha 636 objective and a sequential acquisition setting at 102461024 pixelsresolution; for each image two to four 0.5 mm sections were acquired and a z-projection was obtained.

For colocalization studies, hippocampal neurons were fixed in methanol at220 C and immunostained for GluR1, ADAM10 and Pan-Shank; primary andsecondary antibodies were applied in GDB buffer. Cells were chosen randomly forquantification.

Live imagingTime-lapse images were obtained in an environmentally controlled chamber with5% CO2 at 37 C using a Zeiss Confocal LSM510 (a gift from FondazioneMonzino, Milano, Italy) system with 636 objective and zoom function set on 4;for each image, three to four 1 mm sections were acquired and a z-projection wasobtained. Images of non-treated cells used as control were acquired every3 minutes for 30 minutes; after injection of PACAP38 images were acquired every3 minutes for 30 minutes. Morphological analysis was conducted at each timepoint.

Quantification and statistical analysisQuantification of western blotting analysis was performed with ImageJ softwareand values were expressed as means 6 s.e.m. Colocalization analysis of confocalexperiments was performed using Zeiss AIM 4.2 software. Analysis of dendriticspine morphology was performed with ImageJ software; for each dendritic spinelength, the head and neck width were measured, which was used to classifydendritic spines into three categories (thin, stubby and mushroom) (see also Harriset al., 1992), as shown in Fig. 2D. In particular, the length and the ratio betweenthe width of head and the width of neck (Wh/Wn) were used as parameters for theclassification as follows: protrusions having a length of more than 3 mm wereconsidered as filopodia, the others as spines; spines with a Wh/Wn ratio biggerthan 1.7 were considered mushrooms; spines with a Wh/Wn ratio smaller than 1.7were divided in stubby, if shorter than 1 mm, and thin if longer than 1 mm.Statistical evaluation of all confocal experiments was performed by using one-wayANOVA, followed by Bonferroni’s post-hoc test. An operator who was ‘blind’ tothe experimental conditions performed both image acquisition and quantification.

FundingThis work was supported by Fondazione Cariplo [grant numbers2319-2008 to M.D.L. and 264-2009 to C.S.].

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