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Developmental Cell

Article

Local CRH Signaling Promotes Synaptogenesisand Circuit Integration of Adult-Born NeuronsIsabella Garcia,1,2 Kathleen B. Quast,3 Longwen Huang,4 Alexander M. Herman,1 Jennifer Selever,3 Jan M. Deussing,5

Nicholas J. Justice,6 and Benjamin R. Arenkiel1,3,4,7,*1Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA2Medical Scientist Training Program, Baylor College of Medicine, Houston, TX 77030, USA3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA4Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA5Max Planck Institute of Psychiatry, 80804 Munich, Germany6Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, USA7Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, TX 77030, USA

*Correspondence: [email protected]

http://dx.doi.org/10.1016/j.devcel.2014.07.001

SUMMARY

Neural activity either enhances or impairs de novosynaptogenesis and circuit integration of neurons,but how this activity is mechanistically relayed inthe adult brain is largely unknown. Neuropeptide-ex-pressing interneurons arewidespread throughout thebrain and are key candidates for conveying neuralactivity downstream via neuromodulatory pathwaysthat are distinct from classical neurotransmission.With the goal of identifying signaling mechanismsthat underlie neuronal circuit integration in the adultbrain, we have virally traced local corticotropin-releasing hormone (CRH)-expressing inhibitory inter-neurons with extensive presynaptic inputs onto newneurons that are continuously integrated into theadult rodent olfactory bulb. Local CRH signalingonto adult-born neurons promotes and/or stabilizeschemical synapses in the olfactory bulb, revealing aneuromodulatory mechanism for continued circuitplasticity, synapse formation, and integration of newneurons in the adult brain.

INTRODUCTION

Synaptogenesis and circuit integration of neurons in the brain is

governed by diverse repertoires of synaptic and extrasynaptic

inputs. Excitatory input by principal neurons has profound

effects on sculpting and pruning synaptic connectivity (Favero

and Castro-Alamancos, 2013; Le Roux et al., 2013). However,

recent evidence suggests that interneurons also play significant

roles in modulating synapse formation (De Marco Garcıa et al.,

2011; Hensch et al., 1998; Le Magueresse and Monyer, 2013).

Inhibitory interneurons are highly heterogeneous and, depending

on the brain region, can vastly outnumber principal neurons

(Chen and Greer, 2004; Isaacson and Strowbridge, 1998; Lledo

et al., 2008). Neurochemical classification schemes have shown

that interneurons not only express GABA and calcium-binding

Developmen

proteins such as parvalbumin (PV), calretinin (CR), and calbindin

(Barinka et al., 2012; Kosaka and Kosaka, 2008; Rudy et al.,

2011), but also a cast of neuromodulatory peptides, including so-

matostatin (SST), cholecystokinin, oxytocin, and corticotropin-

releasing hormone (CRH) (Huang et al., 2013; Le Magueresse

and Monyer, 2013; Ma et al., 2006; Rudy et al., 2011; Xu et al.,

2013). Neuropeptidergic interneurons are promising candidates

for modulating changes in local synapse and circuit structure,

and are pervasive throughout the rodent olfactory bulb (OB).

Endowed with the feature of ongoing neurogenesis (Alvarez-

Buylla and Temple, 1998), the mouse olfactory system offers

an excellent in vivomodel to investigate mechanisms that under-

lie synaptogenesis, circuit plasticity, and the integration of new

neurons into existing networks (Abrous et al., 2005; Ming and

Song, 2005). Adult-born neurons are continuously generated in

the subventricular zone (SVZ), migrate via the rostral migratory

stream (RMS), and populate the OB where the vast majority

become inhibitory granule cells that form connections with OB

principal mitral and tufted cells (Mori et al., 1983; Price and Po-

well, 1970a, 1970b; Carleton et al., 2003; Shepherd and Greer,

2004). This interaction influences olfactory behaviors and odor-

related memories (Abraham et al., 2010; Breton-Provencher

et al., 2009; Mouret et al., 2009; Rochefort et al., 2002).

Studies have found that the survival and integration of adult-

born granule cells is activity-dependent during a developmental

critical period between 2 and 4 weeks after their birth (Kelsch

et al., 2009; Yamaguchi and Mori, 2005), when they receive in-

puts from local interneuron subtypes, including deep and super-

ficial short axon cells and Blanes cells (Arenkiel et al., 2011; Eyre

et al., 2008; Pressler and Strowbridge, 2006), as well as centrif-

ugal fibers from deeper regions of the brain (Arenkiel et al., 2011;

Balu et al., 2007; Panzanelli et al., 2009; Whitman and Greer,

2007). Maturing granule cells extend their dendrites into the

external plexiform layer (EPL), where they connect with principal

mitral cells. Interestingly, the EPL also harbors amore dispersed,

and heterogeneous population of neuropeptidergic interneurons

(Kosaka and Kosaka, 2008; Lepousez et al., 2010a, 2010b) that

also form reciprocal synaptic connectivity with mitral cells

(Huang et al., 2013; Kato et al., 2013; Miyamichi et al., 2013). Un-

like granule cells, EPL interneurons are generated in the early

postnatal period and remain stable throughout life (Batista-Brito

tal Cell 30, 645–659, September 29, 2014 ª2014 Elsevier Inc. 645

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Neuropeptide Signaling and Circuit Integration

646 Developmental Cell 30, 645–659, September 29, 2014 ª2014 Elsevier Inc.

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et al., 2008), but their potential neuromodulatory role in shaping

the integration of adult-born neurons is unknown.

We have previously shown that odor enrichment increases the

number of inputs onto adult-born neurons in the OB (Arenkiel

et al., 2011), causing enhanced cell survival and integration.

However, the precise signaling mechanisms between these

inputs and granule cells remain in question. In this study, we

have mapped local neuropeptidergic EPL interneurons with

anatomical and functional connectivity onto granule cells during

peak periods of synaptogenesis and circuit integration. Using

loss- and gain-of-function analyses, conditional viral-genetic

technologies, optical imaging, electrophysiological recordings,

and optogenetic stimulation, we have uncovered a neuro-

peptidergic signaling mechanism between local CRH+ EPL

interneurons and adult-born granule cells that plays an important

role in synapse formation, circuit plasticity, and the integration of

new neurons into the existing networks, revealing a dual func-

tional role for neuropeptidergic inhibitory interneurons in the

mouse OB.

RESULTS

Adult-Born Neurons Receive Inputs from Local EPLInterneuronsTo reveal the identities of the presynaptic inputs made onto

adult-born granule cells, we performed targeted monosynaptic

tracing using genetically engineered rabies virus (RV), SADG-

EGFP RV (Arenkiel et al., 2011; Wickersham et al., 2007a,

2007b). RV is a neurotropic virus that travels retrogradely be-

tween connected neurons. Endowing it with the avian coat pro-

tein EnvA provides selectivity of infection to source cells that ex-

press the TVA receptor, and replacing the glycoprotein G with

an EGFP reporter in the viral genome provides a fluorescently

labeled map of connectivity. Using a conditional knockin mouse

(ROSALSL-Rabies G-IRES-TVA, referred to herein asROSARITVA/RITVA)

that expresses the elements for conditional monosynaptic

tracing (rabies-G and TVA) (Takatoh et al., 2013), we selectively

targeted adult-born neurons in the OB for transsynaptic tracing.

ROSARITVA/RITVA mice were stereotaxically injected with a lenti-

virus that expressed Cre recombinase and a tdTomato reporter

into the RMS (Figure 1A). TdTomato marked adult-born neurons

red, and Cre recombinase allowed for conditional expression of

TVA and G. 28 days postlentiviral injection, we introduced RV

into the core of the OB to target newly integrated granule cells

for connectivity mapping. One week post-RV infection, the tar-

geted OB showed high-level EGFP expression (Figure 1B)

compared to negative controls in which no Cre was present (Fig-

ure S1A available online), suggesting that (1) adult-born neurons

Figure 1. Adult-Born Granule Cells Receive Presynaptic Inputs from E

(A) Genetic strategy for targeting adult-born neurons for transsynaptic tracing.

(B) Dorsal view of mouse brain showing a labeled OB 7 days post infection with

(C) Cross-section of (B). RMS, rostral migratory stream; GCL, granule cell layer; EP

(D) High-magnification view of (C). tdTomato+/EGFP+ cells (arrows) are newborn n

EPL presynaptic inputs; asterisks mark mitral cell inputs). MCL, mitral cell layer.

15 mm.

(E and F) EPL presynaptic inputs are PV+ (E, arrows; scale bar represents10

represents10 mm).

See also Figure S1.

Developmen

were successfully targeted for tracing, and (2) that retrograde

jumping of RV had occurred. Tissue sections revealed strong

expression of tdTomato in adult-born ‘‘source’’ cells, and high-

efficiency RV labeling in presynaptic inputs (Figures 1C and

1D). As positive controls, we identified presynaptic EGFP la-

beling in mitral cells (Figure 1D), validating RV jumping to known

synaptic partners. Interestingly, we found that adult-born

granule cells also received extensive inputs from both deep

and superficially located interneurons, with highest enrichment

from cells located in the EPL (Figure 1D).

Local CRH+ EPL Interneurons Make Connections ontoAdult-Born Granule CellsImmunohistochemical characterization of the presynaptic EGFP+

EPL cells revealed that granule cell inputs constituted a subset of

nondopaminergic interneurons (GFAP, 0%; tyrosine hydroxylase,

0%; CR+, 92% ± 5%; Figures S1B–S1D), with strong colabeling

of PV (95% ± 3%; Figure 1E), and SST (45% ± 5%; Figure S1E).

Highest neuropeptide expression was observed with CRH

(68% ± 4%; Figure 1F), suggesting that adult-born neurons

received direct input from resident CRH+ interneurons in the

OB. Although immunohistochemistry identified enrichment of

CRH protein in EPL interneurons, we also detected substantial

extracellular CRH in the EPL (Figure 1F), suggesting that CRH is

locally secreted. To more precisely identify CRH+ interneurons

as bona fide inputs, we used genetic labeling techniques.

To this end, we crossed CRH-Cre mice to conditional

tdTomato reporter mice (CRH-Cre+/�; ROSALSL-tdTom+/�), andobserved strong tdTomato signal in the paraventricular nucleus

of the hypothalamus, a known hub for CRH synthesis and secre-

tion (Figure S2), and high levels of expression in EPL interneurons

of theOB (Figure 2A),whichwepreviously characterized asmulti-

polar and anaxonic fast-spiking PV+ interneurons (Huang et al.,

2013). To determine if adult-born granule cells received inputs

from CRH+ EPL interneurons, we performed transsynaptic

tracing in CRH-Cre+/�; ROSALSL-tdTom+/� mice and electropo-

rated the avian TVA receptor and rabies-G glycoprotein into the

SVZ of CRH-Cre+/�; ROSALSL-tdTom+/� pups (Figure 2B), tran-

siently targeting neural progenitors that give rise to adult-born

neurons. 28 days later, mice were injected with RV into the core

of the OB, and killed 7 days later. OB slices revealed strong

EGFP expression, with high efficiency infection of targeted

granule source cells and presynaptic inputs (Figure 2C). Both

source cells and all inputs were labeled EGFP+, whereas CRH+

inputs were EGFP+/tdTomato+. Through this differential label-

ing, we verified that new granule cells indeed received extensive

input from local CRH+ EPL interneurons with 86% (±5% SEM) of

EPL presynaptic inputs expressing CRH via lineage tracing.

xternal Plexiform Layer Interneurons

SADG-EGFP RV. Scale bar represents 500 mm.

L, external plexiform layer; GL, glomerular layer. Scale bar represents 300 mm.

euron source cells. EGFP+ cells are presynaptic inputs (open arrowheadsmark

Scale bar represents 80 mm. Inset shows a source cell. Scale bar represents

mm) and CRH+ (F, arrows; arrowheads mark extracellular CRH; scale bar


Figure 2. Local CRH+ EPL Interneurons

Make Connections onto Adult-Born Granule

Cells

(A) Genetic lineage of CRH+ neurons in the OB of

CRH-Cre+/�; ROSALSL-tdTom+/� mice. GL, glomer-

ular layer; EPL, external plexiform layer; MCL,

mitral cell layer; GCL, granule cell layer; scale bars

represent 500, 150, and 25 mm).

(B) Experimental scheme to identify presynaptic

inputs in CRH lineage-traced mice.

(C) SADDG-EGFP RV transsynaptic tracing in

CRH-Cre+/�; ROSALSL-tdTom+/� mice (arrows

identify newborn neuron source cells; open

arrowheads mark CRH+ presynaptic EPL in-

terneurons; scale bars represent 150 and 80 mm).

See also Figure S2.

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Adult-Born Granule Cells Dynamically Express CRHR1CRH is best known as a hypothalamic regulatory hormone

that mediates systemic stress responses (Vale et al., 1981;

Vale et al., 1983). In addition, CRH has been implicated both

as a neurotransmitter and neuromodulator in the hippocam-

pus, amygdala, and cerebellum (Maras and Baram, 2012; Roo-

zendaal et al., 2008; Schmolesky et al., 2007). CRH can bind to

two G protein coupled receptors, CRHR1 and CRHR2 (Perrin

et al., 1993, 1995; Perrin and Vale, 1999), but in the brain

shows higher affinity to CRHR1, which mediates many of its

physiological effects (Bale and Vale, 2004). When bound to

CRHR1, Gs-coupled signaling is activated (Berger et al.,

2006; Blank et al., 2003; Perrin et al., 1993; Thiel and Cibelli,

1999).

Having identified that CRH+ EPL interneurons provide inputs

onto granule cells, we next sought to investigate the expression

of CRH receptors in theOB. RT-PCR revealed that bothCRH and

CRHRmRNAswere present in the bulb, and that CRHR1was ex-

pressed at much higher levels than CRHR2 (Figure 3A). Because

available antibodies for CRHR are not useful for detecting

endogenous CRHR1 (Refojo et al., 2011) and the expression


pattern is not conclusive in the OB (Fig-

ure S3A), we used genetic strategies

to determine the CRHR1 cell type-spe-

cific expression pattern. Consistent with

mRNA transcript detection, tissue sec-

tions from CRHR1-EGFP BAC transgenic

mice, whose expression pattern was pre-

viously validated to match endogenous

CRHR1 (Justice et al., 2008), revealed

high levels of spatially restricted CRHR1

in granule cells (Figure 3B). Finally, to

determine the precise spatial localiza-

tion of CRH and CRHR1 neurons within

the OB,CRHR1-EGFPmicewere crossed

to CRH-Cre+/��; ROSALSL-tdTom+/� mice

to generate CRHR1-EGFP+/�; CRH-

Cre+/�; ROSALSL-tdTom+/� double reporter

mice. OB sections showed a cell type-

specific juxtaposition between EGFP-

labeled CRHR1+ granule cells, and

tdTomato-labeled CRH+ EPL interneu-

rons (Figure 3C). Whereas CRHR1+ granule cell bodies were

located throughout the GCL with superficial enrichment, CRH+

EPL interneurons almost exclusively resided in the EPL, were

absent from the GCL (Figure S3B), and directly juxtaposed

CRHR1+ dendrites. These data support the idea that CRH ligand

is locally released by EPL interneurons and directly acts on

CRHR1+ granule cell dendrites in the EPL.

EGFP expression was nearly absent from the RMS and

became gradually enriched in the outer GCL, with strong enrich-

ment in superficial regions (Figures 3B and 3C). These data sug-

gested that CRHR1 might exhibit a dynamic expression pattern

with granule cell maturation, and that CRHR1 might be ex-

pressed both in early postnatal-born granule cells which pre-

dominantly localize to the superficial GCL (Lemasson et al.,

2005), as well as adult-born granule cells. To test if CRHR1 is

dynamically regulated during periods of newborn granule cell

synaptogenesis and to better determine its spatiotemporal

expression profile, CRHR1-EGFP mice were pulsed with the

proliferation marker EdU, and killed between 7 and 60 days,

spanning early, intermediate, and late phases of synaptogenesis

and circuit integration (Figure 3D; Carleton et al., 2003). We

Figure 3. Adult-Born Granule Cells Dynami-

cally Express CRHR1

(A) Semiquantitative RT-PCR for CRH and CRHR1/

2 of whole OB RNA.

(B) OB cross-section of CRHR1-EGFP BAC

transgenic mice. RMS, rostral migratory stream;

GCL,granule cell layer; EPL, external plexiform

layer; GL, glomerular layer; scale bar represents

200 mm.

(C) Reporter expression of CRHR1-EGFP; CRH-

Cre+/�; ROSALSL-tdTom+/� mice (arrows point to

CRHR1+ granule cells; open arrowheads mark

CRH+ EPL interneurons; scale bars represent 60

and 20 mm).

(D) Experimental scheme to determine the devel-

opmental expression profile of CRHR1 expression

in granule cells.

(E) CRHR1 expression in newborn neurons 28 days

post EdU injection (scale bar represents 60 mm).

(F) Quantification of CRHR1-expression in granule

cells (data points represent averages ± SEM, n = 3

animals per time point).

(G) CRHR1::EGFP expression in adult-born

granule cells (scale bars represent 100 and 20 mm).

See also Figure S3.

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Developmental Cell 30, 645–659, September 29, 2014 ª2014 Elsevier Inc. 649


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found that the ratio of EdU-labeled granule cells that expressed

CRHR1 was very low at 7 days of age (5.8% ± 3.9%; Figure 3F),

and substantially increased between 14 (34.5% ± 4.1%), 21

(59.2% ± 4.2%), and 28 days of age (81.3% ± 2.2%). This value

slightly increased further at 40 days post-EdU pulsing (87.8% ±

4.5%), and the number of colabeled neurons plateaued at

60 days (87.5% ± 3.2%). Intriguingly, dynamic enrichment of

CRHR1 coincided with critical periods of activity-dependent

survival, synaptogenesis, and circuit integration between 14

and 28 days of granule cell age (Kelsch et al., 2009; Mouret

et al., 2008; Yamaguchi and Mori, 2005).

Finally, to determine the subcellular localization of CRHR1, we

expressed a CRHR1::EGFP fusion construct with a tdTomato

cell fill in new granule cells. We observed CRHR1::EGFP in den-

drites, with enriched subcellular localization in a subset of den-

dritic spines in the EPL (Figure 3G). Interestingly, CRHR1::EGFP

was also present in extrasynaptic dendritic regions (Figures

S3C–S3E), suggesting that CRH-mediated local neuropeptide

signaling might not occur exclusively at synapses, but also via

extrasynaptic mechanisms not restricted to dendritic spine

structures.

Together, these data support the idea that CRH is synthesized

locally by EPL interneurons and can signal to granule cells via

time-dependent expression of CRHR1, suggesting a possible

role for secreted CRH in the long-term survival and circuit inte-

gration of adult-born neurons.

CRH Signaling Is Required for Normal Levels of Adult-Born Granule Cell SurvivalTo determine how CRH signaling affects adult-born neurons,

mice lacking CRH or its receptor (CRH�/� or CRHR1�/�) were

pulsed with bromodeoxyuridine (BrdU) and killed either 24 hr

later to assay proliferation in the SVZ (BrdU+/Ki67+ cells), or

30 days later to assay survival in the GCL (BrdU+ cells; Fig-

ure 4A), focusing on deeper regions where adult-born granule

cells reside (Lemasson et al., 2005). Compared to wild-type lit-

termates, CRH�/� mutants showed increased SVZ proliferation

(p < 0.05, 4,942 �/� 302 cells in CRH�/�, compared to 3,906 ±

354 cells in CRH+/+ mice), but decreased cell survival in the OB

(p < 0.005, 25 ± 1 cell in CRH�/�, compared to 36 ± 2 cells in

CRH+/+ mice; Figures 4B and C). This increased proliferation in

CRH�/� mice is consistent with previous reports that stress

impairs neurogenesis both in the SVZ and in the hippocampus

(de Andrade et al., 2013; Hitoshi et al., 2007, Schoenfeld and

Gould, 2013), and that CRH�/� mice show decreased stress

levels (Jacobson et al., 2000). Cleaved caspase-3 and TUNEL

staining revealed increased apoptosis in the GCL of CRH�/�

Figure 4. CRH Signaling Is Required for Normal Levels of Adult-Born G

(A) Experimental scheme to determine cellular proliferation and granule cell surv

(B) Quantification of proliferating cells (BrdU and Ki67 double-positive cells) in th

(C) Quantification of adult-born granule cell survival in control and CRH�/� mice.

(D) Quantification of proliferating cells in the SVZ of CRHR1�/� mice (p > 0.05 St

(E) Quantification of adult-born granule cell survival in control and CRHR1�/� mi

(F) Representative images of the GCL of CRHR1+/+ and CRHR1f/f mice that expr

(G) Quantification of the ratio of Cre-EGFP+/tdTom+ granule cells (*p < 0.01 Stu

(H–P)Western blots of the synaptic proteins Synapsin, PSD95, andNR2B of OBs o

(*p < 0.05 Student’s t test). All data points represent averages ± SEM, n = 4 anim

See also Figure S4.

Developmen

mice (Figure S4A). To investigate whether granule cell apoptosis

was secondary to loss of CRH+ interneurons, we examined the

integrity of the EPL and performed cell counts using DAPI and

CR, which overlaps with EPL interneurons (Huang et al., 2013;

Figures S1D, S4B, and S4C) and found no difference. Moreover,

because CRH has important systemic effects as a regulatory

hormone, many of which are mediated by corticosteroids, we

questioned whether the attrition of granule cells in CRH�/�

mice was corticosteroid-dependent and supplemented adult

CRH�/� mice with corticosterone. Corticosteroid supplementa-

tion at a concentration that readily crosses the blood-brain bar-

rier and is capable of rescuing embryonic phenotypes in utero

(Muglia et al., 1995), did not change granule cell survival in

CRH�/� mice (Figure S4F), suggesting that adult-born neuron

survival was not mediated by systemic CRH signaling, but likely

through local CRH.

As loss of secreted CRH systemically in CRH�/� mice could

have secondary effects in the OB, we next assayed for prolifera-

tion and cell survival in CRHR1�/� mice, and saw no change in

SVZ-based proliferation compared to control littermates (p >

0.05, 4,172 ± 113 cells in CRHR�/� versus 4,337 ± 303 cells in

CRHR+/+ mice, Figure 4D). These data were consistent with

the observation that the SVZ lacked CRHR1 expression in

CRHR1-EGFP mice (data not shown), and that SVZ proliferation

might be mediated by systemic signaling rather than through

central CRH receptor activation. However, decreased numbers

of granule cells were noted in the OB 30 days post-BrdU pulsing

(p < 0.001, 24 ± 1 cells inCRHR�/� versus 32 ± 2 cells inCRHR+/+

mice, Figure 4E), as well as increased numbers of apoptotic cells

(Figure S4D). Moreover, this phenotype was also corticosteroid-

independent (Figure S4G).

To bypass any potential systemic effects of using germline

CRH loss-of-function alleles, we next conditionally removed

CRHR1 specifically from adult-born granule cells by injecting a

mixture of equal titers of AAV particles that expressed either

Cre-P2A-EGFP or tdTomato (control) into the RMS of

CRHR1+/+ or CRHR1flox/flox mice (Kuhne et al., 2012), and re-

vealed a 24.8% ± 2.8% decrease in the ratio of Cre-EGFP+/

tdTom+ granule cells between CRHR1+/+ and CRHR1flox/flox

mice (Figures 4F and 4G). Interestingly, morphological analysis

on the proportion of surviving granule cells showed no difference

between Cre and tdTomato+ neurons (data not shown).

Having found decreased numbers of surviving granule cells in

CRH loss-of-function mutants, we wondered if synaptic protein

expression was affected in these models and performed west-

ern blot analysis, which showed significantly decreased levels

of Synapsin, PSD95, and NR2B in the OBs of CRH�/�,

ranule Cell Survival and Synaptic Protein Expression

ival in CRH mutant alleles (scale bars represent 100 and 15 mm).

e SVZ of control and CRH�/� mice (*p < 0.05 Student’s t test).

udent’s t test).

ce (*p < 0.001 Student’s t test).

essed Cre-EGFP or tdTomato in granule cells (scale bar represents 50 mm).

dent’s t test).

fCRHR1�/�,CRHR1�/�, andCRHR1f/fmice injectedwith Cre or control viruses

als each.


Figure 5. Constitutive CRHR Signaling in Granule Cells Promotes

Synaptogenic Changes in the OB

(A) Representative images of granule cells expressing tdTomato or tdTomato

and a constitutively active (CA) CRHR1-EGFP fusion construct (CA)

CRHR::EGFP. Scale bar represents 50 mm.

(B–D) Quantification of average granule cell length (B, p > 0.05 Student’s t test),

(C) mean total dendrite length (*p < 0.05), and (D) average dendritic branch

number in tdTomato and (CA)CRHR+ granule cells (*p < 0.01 Student’s t test).

(E) Scholl analysis of the number of intersections in tdTomato or (CA)CRHR+

granule cells (*p < 0.05 ANOVA).

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652 Developmental Cell 30, 645–659, September 29, 2014 ª2014 Els

CRHR�/�, and CRHR1flox/flox mice compared to controls (Fig-

ures 4H–4P). Together, these data imply an important role for

CRH signaling in the maintenance and/or generation of synap-

ses in the OB.

We next asked if CRHR1 expression correlates with cell sur-

vival, and stained CRHR1-EGFP olfactory bulb tissue with acti-

vated caspase-3 (Figure S4H). Interestingly, we did not observe

any CRHR1-EGFP+/caspase-3+ cells. Together, these loss-of-

function and molecular marker data suggest that granule cells

require local CRH signaling for normal development andmatura-

tion, and that CRHR1 expression correlates with synapse for-

mation and/or survival, allowing granule cells to integrate into

existing brain circuits.

Gain-of-Function CRH Signaling Promotes IncreasedSynaptic Protein Expression in the OBHaving found that lack of CRH signaling leads to impaired

granule cell survival (Figure 4), we next queried the consequence

of enhanced CRH signaling. To determine a role for CRHR1 in

shaping synapse development and neuronal maturation, we ex-

pressed a constitutively active version of CRHR1 fused to EGFP,

(CA)CRHR1::EGFP (Nielsen et al., 2000) in newborn granule cells

(Figure 5A). Morphological characterization of granule cells

expressing (CA)CRHR1::EGFP revealed normal average neuron

length comparable to tdTomato controls (p > 0.05, 284 ± 21 mm

in (CA)CRHR1:EGFP versus 316 ± 19 mm in controls, Figure 5B),

but increased total dendrite length (p < 0.05, 894 ± 30 mm versus

672 ± 25 mm, Figure 5C), dendritic branch number (p < 0.01,

10.5 ± 0.65 mm versus 7 ± 0.7 mm, Figure 5D), and branch

intersections at both proximal and distal radii from the soma

(p < 0.05; Figure 5E). Next, we quantified dendritic spines

and found that total spine number was increased in (CA)

CRHR1:EGFP neurons (Figure 5F). Neurolucida reconstructions

revealed increased complexity in (CA)CRHR1::EGFP granule

cells compared to control neurons (Figure 5G).

Then, we targeted granule cells in the OB for conditional

gain-of-function studies with spatiotemporal specificity during

periods of endogenous CRHR1 expression to determine if

CRHR1 activation is sufficient for modulating synaptogenic

changes. For this, we first generated a BAC transgenic allele

to drive Cre recombinase from the CRHR1 promoter. To vali-

date cell type specificity of Cre expression, we generated

CRHR1-EGFP;CRHR1-Cre+/;ROSALSL-tdTom+/� mice, and saw

Cre activity that matched the expression pattern of CRHR1-

EGFP transgenic mice in the OB (Figure S5A). We next gener-

ated a conditional adeno-associated virus that carries (CA)

(F) Quantification of the number of dendritic spines between tdTomato and

(CA)CRHR+ granule cells (*p < 0.05 ANOVA). N = 10 cells each from three

animals.

(G) Representative granule cell morphology reconstructions.

(H) Experimental scheme for targeting CRHR1+ OB granule cells for consti-

tutive CRHR1-activation.

(I) Expression pattern of AAV-flex-(CA)CRHR1::GFP in granule cells (arrows

point to (CA)CRHR1::GFP+ neurons; scale bars represent 1,000, 100, and

20 mm).

(J–O) Western blot analysis of synaptic protein expression of CRHR-Cre+/�

OBs injected with either flexed GFP or flexed-(CA)CRHR-GFP AAV (*p < 0.05

Student’s t test, n = 4 animals each). All data points are averages ± SEM.

See also Figure S5.

evier Inc.

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CRHR1::EGFP in an inverted ‘‘flexed’’ configuration (Atasoy

et al., 2008) and stereotaxically targeted the RMS for infection

of granule cells in CRHR1-Cre+/� mice (Figures 5H and 5I). To

confirm enhanced signaling through CRHR1, we isolated whole

OBs for western blot analysis. Control lysate was obtained from

CRHR1-Cre+/� mice injected with serotype-matched flexed

GFP virus. Since CRHR1 is Gs-coupled and leads to activation

of CREB via phosphorylation (Blank et al., 2003; Thiel and Ci-

belli, 1999), we first assayed pCREB levels in (CA)CRHR1-ex-

pressing OBs (Figure 5J), which were significantly increased.

Interestingly, we also observed significant changes in levels of

synaptic protein expression, including a large increase in the

presynaptic protein Synapsin (Figure 5K), suggesting the forma-

tion of de novo synapses or the strengthening of existing synap-

ses with enhanced CRHR1 signaling. Intriguingly, however,

although the quantity of PSD95 showed an increased trend of

expression, these changes were not statistically significant (Fig-

ure 5L). Because granule cells are GABAergic and form recip-

rocal dendrodendritic synapses with mitral cells (Mori et al.,

1983; Panzanelli et al., 2009; Shepherd and Greer, 2004), we

also assayed for changes in both inhibitory and excitatory re-

ceptor expression. The mitral cell-specific GABA-Aa1 (Whitman

and Greer, 2007), as well as NR1, and NR2B receptor subunits

showed increased expression (Figures 5M, 5N, and S5B),

whereas levels of AMPA receptor subtypes were decreased

compared to GFP controls (Figures 5O and S5C). Immunohisto-

chemistry for the upregulated proteins revealed increased

levels of NMDA and GABA receptor expression in the EPL of

gain-of-function experiments compared to controls (Figures

S5D–S5F). Intriguingly, increased NR1 and NR2B expression

was localized to both (CA)CRHR1::EGFP+ as well as EGFP

lacking dendritic structures, which could be due to the

nature of the membrane-bound overexpression construct. For

example, the fusion protein is not exclusively targeted to all

synaptic structures within newborn granule cells, and its syn-

aptogenic properties may in fact influence the formation of syn-

aptic structures in a more widespread manner. Consistent with

this, we also noted significant upregulation of GABA-Aa1,

which is expressed by mitral/tufted cells in the OB, and is

not expected to colocalize with (CA)CRHR::EGFP structures.

Together, these data suggest an overall increase in synap-

tic connectivity within the OB circuitry with constitutive

CRHR1 signaling, which then also may lead to non-cell-auton-

omous and more widespread increases in synaptic protein

expression.

Together, increased spine numbers, upregulated Synapsin

and NMDA receptor expression, combined with decreased

AMPA receptors suggests that CRH signaling in the OB pro-

motes the formation of new immature synapses, and/or potential

synaptic scaling of existing excitatory synapses (Turrigiano et al.,

1998; Turrigiano and Nelson, 2004).

Gain-of-Function CRH Signaling Promotes FunctionalSynaptogenesis in the OBA hallmark of bona fide circuit integration is functional syn-

aptic connectivity. Having determined that constitutively active

CRHR1 signaling leads to synaptogenic changes in the OB (Fig-

ure 5), we next queried the functional consequence of gain-of-

function CRH signaling using electrophysiology.

Developmen

To determine if soluble CRH ligand influenced granule cell

electrophysiological properties, we made whole cell recordings

from CRHR1-EGFP+ granule cells while bath-applying CRH,

which showed no change in the frequency of miniature excit-

atory postsynaptic currents (mEPSCs), but induced a significant

decrease in amplitude (p < 0.05, 11.55 ± 0.72 pA before, and

8.8 ± 0.35 pA after CRH; Figures 6A–6D). Whole cell recordings

from granule cells that expressed (CA)CRHR1 compared to

EGFP controls showed no change in mEPSC frequency, but

significantly decreased mEPSC amplitudes (p < 0.05, 7.73 ±

1.38 pA in (CA)CRHR1 versus 11.69 ± 1.27 pA in EGFP controls;

Figures 6E–6H). These data were consistent with the observa-

tion that CRH gain-of-function signaling through activated

CRHR1 led to decreased AMPA receptor levels (Figures 5O

and S5C) and further suggested that upregulation of glutamater-

gic synapses via NMDA receptor expression likely reflects func-

tionally silent or immature synapses. Worth noting, we did not

observe any rapid changes in firing, passive membrane proper-

ties, or membrane potential following CRH application (data not

shown), suggesting that in the OB CRH does not act directly as a

neurotransmitter, but likely functions as a true neuromodulator.

With the observation that GABA-Aa1 receptor subunit expres-

sion increased with CRH signaling (Figure 5M), we next hypoth-

esized that functional GABAergic synapses from granule cells

onto mitral cells might be upregulated. To address this, we

recorded miniature inhibitory postsynaptic currents (mIPSCs)

from mitral cells in OBs where CRHR+ granule cells expressed

(CA)CRHR1::EGFP, and found a significant increase in fre-

quency of mIPSCs (p < 0.05, 2.21 ± 0.39 Hz in (CA)CRHR1

versus 1.17 ± 0.18 pA in EGFP controls, Figures 6I–6L), sug-

gesting either increased formation or stabilization of granule

cell-mitral cell synapses or changes in presynaptic release prop-

erties. Hence, biochemical and electrophysiological evidence

suggests that enhanced CRHR signaling in granule cells leads

to increased functional synaptogenesis and circuit plasticity in

the OB.

Acute Optogenetic Activation of CRH+ EPL InterneuronsPromotes Release of CRH in the OBCRH+ EPL interneurons make connections onto granule cells,

which in concert dynamically express CRHR1 (Figures 1, 2,

and 3). Lack of CRHR1 expression during a critical time window

in granule cell maturation and circuit integration leads to

decreased cell survival (Figure 4), and constitutively active

CRHR1 enhances synapse formation and circuit integration of

adult-born neurons (Figures 5 and 6). Together these data

support a mechanism by which activity-induced release of

CRH from EPL interneurons may influence granule cell synapto-

genesis. We next questioned if manipulating the activity of CRH+

EPL interneurons directly and acutely could dynamically recapit-

ulate the physiological effects observedwith constitutive CRHR1

activation.

To activate CRH+ EPL interneurons with spatiotemporal spec-

ificity, CRH-Cre+/� mice were crossed to ROSALSL-ChR2 mice

to obtain CRH-Cre+/�; ROSALSL-ChR2 mice that selectively ex-

pressed the light gated cation channel channelrhodopsin 2

(ChR2; Boyden et al., 2005; Nagel et al., 2003) in CRH+ EPL in-

terneurons in theOB. From thesemice, wemade slices of theOB

(Figure 7A) and hypothalamus (Figure S6), which were acutely



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Developmental Cell


photostimulated ex vivo in small volumes of artificial cerebral spi-

nal fluid (ACSF). Light-stimulated depolarization of CRH+ neu-

rons led to CRH release in both hypothalamic control (Figure 7B)

and OB slices (Figure 7C), whereas soluble CRH levels in light-

stimulated controls were unchanged. Together, these data sug-

gest that the targeted depolarization of CRH+ neurons elicited

the release of stored CRH neuropeptide from EPL interneurons.

Finally, to determine the effects of CRH+ EPL interneuron

activation on granule cell synapses in vivo, CRH-Cre+/�;ROSALSL-ChR2+/� mice were chronically implanted with fiberop-

tics directly over the olfactory bulb (Ung and Arenkiel, 2012)

and photostimulated with blue laser light (Figure 7D). Acute

in vivo photostimulation in awake and behaving mice led to

enhanced olfactory bulb expression of pCREB (Figure 7E), reca-

pitulating the effect seen in constitutive CRHR1 activation (Fig-

ure 5J). Western blots of stimulated animals showed significant

upregulation of the synaptic proteins Synapsin, PSD95, and

NR2B (Figures 7F–7H), suggesting a conserved synaptogenic

effect of photostimulated release of CRH from EPL interneurons

in vivo, which mirrored constitutively active CRHR1 signaling

(Figure 5).

DISCUSSION

Neuropeptides, including CRH, have been implicated in a variety

of neuronal processes, ranging from neuromodulation and den-

dritic outgrowth, to neuroprotection (Chen et al., 2004; Hanstein

et al., 2008; Sheng et al., 2012). However, the precise role of CRH

in the maturation and integration of granule cells in the OB has

not been investigated. Here, we describe a neuropeptidergic

function for inhibitory interneurons in shaping cell survival, syn-

aptogenesis, and circuit integration of new neurons in the adult

brain that is distinct from classical neurotransmitter signaling.

Together, our data support a potential mechanism for a tripar-

tite-like interaction between mitral cells, EPL interneurons, and

granule cells (Figure 7I). We and others have recently shown

that mitral cells and EPL interneurons exhibit reciprocal connec-

tivity (Huang et al., 2013, Kato et al., 2013; Miyamichi et al.,

2013). It is conceivable that neuronal activity via olfactory sen-

sation is conveyed to mitral cells, which in turn stimulate EPL

interneurons to both shapemitral cell output, and release soluble

CRH. In this way, activity-dependent release of CRH, and

subsequent reception by granule cells that express CRHR1

both synaptically and/or extrasynaptically, may function as

a key mechanism toward modulating synaptogenesis and

plasticity via neuropeptide signaling, linking activity to circuit

integration via neuropeptidergic interneurons. Ultimately, this

interaction has the potential to shape whole circuit activity and

Figure 6. Constitutive CRHR Signaling in Granule Cells Promotes Syna

(A) Representative trace of CRHR1-EGFP+ granule cell before and after CRH ap

(B) Average mEPSCs before and after CRH.

(C and D) Quantification of the mEPSC frequency and amplitude before (C) and

(E) Representative mEPSC traces of granule cells from CRHR-Cre+/� mice inject

(F) Average mEPSCs of EGFP and (CA)CRHR1::EGFP+ granule cells.

(G and H) Quantified granule cell mEPSC frequency and amplitude (*p < 0.05 St

(I) Representative mIPSC traces of mitral cells from CRHR-Cre+/� mice injected

(J) Average mitral cell mIPSCs.

(K and L) Average frequency and amplitude of mitral cell mIPSCs in CRHR-Cre+

CRHR1::EGFP (*p < 0.05 Student’s t test, n = 13 mitral cells per group from thre

Developmen

plasticity. Although this model supports a role for local interac-

tions, it remains to be determined if other forms of neuromodu-

latory signals are conveyed specifically to EPL interneurons via

other local or centrifugal inputs.

Dual Roles of InterneuronsNeuropeptides are traditionally acknowledged for roles in

shaping whole-body physiological responses and/or modulating

systemic homeostatic mechanisms (Vale et al., 1981). Recently,

neuropeptides have received increasing attention for their roles

in shaping synapses and facilitating neuronal plasticity (Bayatti

et al., 2003; Fenoglio et al., 2006; Lipschitz et al., 2005). Inter-

estingly, however, interneurons have been most extensively

studied with respect to their traditional, GABAergic inhibitory

function, and the neuropeptides they express have served pri-

marily in categorizing the many different interneuron subtypes

throughout the brain (Ma et al., 2006; Rudy et al., 2011; Xu

et al., 2013). Although granule cells make up the majority of

inhibitory interneurons in the OB, previous work from our lab

and others has identified other types of interneurons with

GABAergic connections onto mitral cells that likely serve impor-

tant olfactory functions (Huang et al., 2013; Kato et al., 2013;

Miyamichi et al., 2013; Lepousez et al., 2010a, 2010b; Kosaka

and Kosaka, 2008). Here, we have identified a population of

inhibitory interneurons in the OB with a neuropeptidergic role

in promoting synaptic protein expression and circuit integration.

Interestingly, this interneuron population has a dual role in

shaping the OB circuitry and likely, olfaction. This interaction is

inhibitory onto mitral cells and neuromodulatory onto granule

cells, suggesting increased connectivity and/or signaling not

only between excitatory principal cells and inhibitory interneu-

rons, but also between different subtypes of interneurons. This

apparent dual functionality may indeed exemplify a prominent

mechanism to support neuronal plasticity, and a way to shape

circuitries by inhibitory neurotransmitter signaling onto mitral

cells, and neuromodulatory signaling onto granule cells. EPL in-

terneurons form reciprocal GABAergic connections with mitral

cells, and are in turn depolarized following odor stimulation

(Huang et al., 2013; Kato et al., 2013; Miyamichi et al., 2013).

This activation further strengthens mitral cell inhibition, but

may also facilitate CRH release, linking olfactory-dependent

activity to neuromodulation, and ultimately synaptogenesis

and circuit integration.

Local Neuropeptide Signaling, Synaptogenesis, andCircuit IntegrationOur data suggest that local neuromodulatory signaling by CRH+

EPL interneurons in the OB aids in granule cell circuit integration

ptic and Circuit Plasticity in the OB

plication (500 nM CRH).

after (D) CRH bath application (*p < 0.05, n = 13 cells from three animals).

ed with either AAV-flexed EGFP or AAV-flexed-(CA)CRHR1::EGFP.

udent’s t test, n = 11 granule cells per group from three animals).

with either AAV-flexed EGFP or AAV-flexed-(CA)CRHR1::EGFP.

/� OBs in which CRHR-expressing granule cells express either EGFP or (CA)

e animals). All data points represent mean ± SEM.


Figure 7. Optogenetic Activation of CRH+ EPL Interneurons Induces Synaptogenesis in the OB

(A) ChR2 expression pattern of CRH-Cre+/�; ROSALSL-ChR2+/� mice (scale bars represent 200 and 50 mm).

(B and C) Quantification of CRH concentration with hypothalamic or OB optogenetic activation in ROSALSL-ChR2+/� (control) or CRH-Cre+/�; ROSALSL-ChR2+/�

mice (*p < 0.05 Student’s t test, n = 3 animals per group).

(D) Experimental scheme for in vivo photostimulation of CRH+ EPL interneurons.

(legend continued on next page)

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Developmental Cell


by increasing synaptic protein expression and/or stabilizing

existing synapses. Removing CRH signaling decreases synaptic

protein expression, whereas enhancing CRH signaling induces

synaptic protein expression in theOB. Perhapsmore intriguingly,

the result that CRHR loss-of-function does not completely

abolish adult-born neuron survival and integration poses some

additional questions. Given that numerous neuropeptides,

including aomatostatin, vasointestinal peptide, cholecystokinin,

oxytocin, and neuropeptide Y are present in the olfactory bulb

(Gracia-Llanes et al., 2003; Lepousez et al., 2010a, 2010b; Ma

et al., 2006; Tobin et al., 2010), and that granule cells in turn

appear to express the cognate receptors, it remains to be seen

if other neuromodulators have the same effect on the OB cir-

cuitry, and if these neuropeptides act in concert to promote

synaptogenesis and circuit integration in the adult brain.

EXPERIMENTAL PROCEDURES

Experimental Animals

All experimental animals were treated in compliance with the United States

Department of Health and Human Services and the Baylor College of Medicine

IUACUC guidelines. ROSARITVA/RITVA (Takatoh et al., 2013), ROSALSL-tdTom

(Arenkiel et al., 2011), CRHR1-EGFP mice (Justice et al., 2008), and

CRHR1flox/flox mice (Kuhne et al., 2012) were previously described. CRH-Cre

(Taniguchi et al., 2011), ROSALSL-ChR2 (Madisen et al., 2012), CRH�/� (Muglia

et al., 1995), and CRHR1�/� (Smith et al., 1998) were obtained from Jackson

Laboratories and maintained on a C57BL/6 background. Generation of the

CRHR1-Cre line is described in the Supplemental Experimental Procedures.

Transsynaptic Tracing

Briefly, adult (6–8weeks old)ROSARITVA/RITVAmicewere injectedwith high titer

lentivirus encoding tdTomato-IRES-Cre stereotaxically into the RMS. Twenty-

eight days later, mice were injected with low titer SADG-EGFP RV (Wicker-

sham et al., 2007b) into the core of the OB and killed after 7 days for mapping

studies. Next, CRH-Cre+/�; ROSALSL-tdTom+/� pups were electroporated with

an expression construct for rabies-G and TVA as previously described (Are-

nkiel et al., 2011). Twenty-eight days postelectroporation, mice were injected

with SADG-EGFP RV as described above. See also Supplemental Experi-

mental Procedures.

Immunohistochemistry and Imaging

OB tissues were processed for immunohistochemistry as previously

described (Huang et al., 2013). Briefly, free-floating sections were stained

with the following primary antibodies: guinea pig anti-Parvalbumin, and rabbit

anti-CRH (kindly provided by Nick Justice), followed by washing and incuba-

tion with species-specific Alexa-633 secondary antibodies. Confocal image

analysis was performed using a Leica TCS SPE confocal microscope. See

also Supplemental Experimental Procedures

CRH–/– and CRHR1–/– Adult-Born Neuron Proliferation, Survival,

and Apoptosis

Age-matched adult (6–8 weeks old) male mice were injected with BrdU. For

SVZ-based proliferation studies, animals were killed 24 hr later as and 50-

mm-thick sections were taken throughout the SVZ and stained with mouse

anti-BrdU and rabbit anti-Ki67 to assess proliferating cells. To assay granule

cell survival, mice were killed 30 days later and processed for immunohisto-

chemistry using mouse anti-BrdU antibody. Images were taken in the middle

and outer granule cell layer, avoiding the RMS and IPL/MCL regions, and total

(E–H) Western blot analysis of synaptic protein expression of ROSALSL-ChR2+/� (c

animals per group).

(I) Model: CRH signaling between local EPL interneurons and granule cells prom

mean ± SEM.

See also Figure S6.

Developmen

cell numbers were counted through serial sections. See also Supplemental

Experimental Procedures.

Constitutively Active CRHR1 Overexpression

A constitutively active CRHR1::EGFP fusion construct (Nielsen et al., 2000)

was electroporated into the SVZ of P2 pups for morphology analysis using

Neurolucida software. The construct was further subcloned into a conditional

flexed adeno-associated-viral vector (Atasoy et al., 2008) and packaged into

viral particles. Virus was stereotaxically injected into the RMS of CRHR1-Cre

mice, and OB tissue was harvested 14 days postinjection. See also Supple-

mental Experimental Procedures.

Electrophysiology

Animals (P21–P35) were deeply anesthetized using isoflurane, and perfused

intracardially with ice-cold ACSF. Coronal olfactory bulb slices (300 mm)

were placed in a room-temperature chamber mounted on an Olympus upright

microscope (BX50WI), and perfused with oxygenated ACSF. Cells were

depicted using fluorescence and differential interference contrast imaging.

Synaptic currents were recorded using cesium-methanesulfonate based inter-

nal solutions. See also Supplemental Experimental Procedures.

In Vivo Photostimulation

Fiberoptics were generated and implanted directly over the olfactory bulb as

previously described (Ung and Arenkiel, 2012). Animals were allowed to

recover from the surgery for 3 days prior to photostimulation. Photostimulation

was performed for 3 hr using a blue laser source (CrystaLaser) controlled by a

Master-8 (A.M.P.I.). See also Supplemental Experimental Procedures.

Statistical Methods

Unless otherwise indicated, statistical comparisons between experimental

groups were made using Student’s t test and all error bars represent SEM.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures

and six figures and can be found with this article online http://dx.doi.org/10.

1016/j.devcel.2014.07.001.

ACKNOWLEDGMENTS

This work was supported by the McNair Medical Institute, NINDS grant

F31NS081805 to I.G. and NINDS R01NS078294 to B.R.A. We thank Drs.

Dona Murphey, Ian Davison, and Steve Shea for critical review of this

manuscript.

Received: December 8, 2013

Revised: May 2, 2014

Accepted: June 30, 2014

Published: September 4, 2014

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