Universidade de Lisboa

Faculdade de Medicina

THE CONTRIBUTION OF ODOR INDUCED ACTIVITY TO

ADULT NEUROGENESIS

Behavioural and Morphological consequences of the different learning contexts

Inês Sofia Silva Vieira

Mestrado em Neurociências

Lisboa, 2012

 

 

Universidade de Lisboa

Faculdade de Medicina

THE CONTRIBUTION OF ODOR INDUCED ACTIVITY TO

ADULT NEUROGENESIS

Behavioural and Morphological consequences of the different learning contexts

Inês Sofia Silva Vieira Mestrado em Neurociências, 2012

Dissertação orientada por:

Internal supervisor: Profª. Doutora Ana Sebastião1,2 Supervisor: Doutor Pierre-Marie Lledo, Institut Pasteur3,4

Co-supervisor: Doutor Gilles Gheusi, Institut Pasteur3,4  

1.Institute of Pharmacology and Neuroscience, Faculty of Medicine; 2. Unit of Neuroscience, Institute of Molecular Medicine, University of Lisbon. 3. Institut Pasteur, Laboratory of Perception and Memory , Paris, France and 4. Centre National de la Recherche Scientifique(CNRS) Unité de Recherche Associée(URA) Paris, France

Todas as afirmações efectuadas no presente documento são de exclusiva responsabilidade do seu

autor, não cabendo qualquer responsabilidade à Faculdade de Medicina da Universidade de Lisboa

pelos conteúdos nele apresentado.

 

A realização desta dissertação foi aprovada pela Comissão Coordenadora do Conselho Científico da

Faculdade de Medicina da Universidade de Lisboa.

  I  

 Acknowledgments I would like, in this modest way, to mention and thank all of those that somehow contributed

throughout this year to this master’s thesis for the degree of Master in Neuroscience:

First of all, to Dr. Pierre Marie Lledo, my supervisor, for giving me the chance to be in the

laboratory and for the support and advices throughout this year.

To my co-supervisor, Dr. Gilles Gheusi, for introducing me into the field of behavior where all

the evidences are questionable, for the good moments working, for his patience and for all the

opinions, comments and help.

To Dr. Gabriel Lepousez not just for teaching me the hardest techniques, but also for

generously finding rapid solutions to all the troubles faced.

To Julien Grimaud, for his great methodic collaboration in the cell counting, morphological

analysis and image acquisition.

To Carine Moigneu for the help with the mice surgeries.

To Dr. M. M. Gabellec for helping with the immunohistochemistry.

To S. Wagner for ‘playing’ with the olfactometers and improving the behavioral boxes we used.

Thanks to all the passionate researchers and students of the laboratory that, without

exceptions, contributed to the exciting scientific discussions in the lab meetings, to the

constructive critiques for this project, and finally to the great environment of work.

To Dr. Ana Sebastião, my internal co-supervisor, for the teaching, for coordinating the Master

and for the help whenever it was necessary along this two years.

Lastly, to my family and friends, the ones that supported me in all the decisions and all the

stages of my life, without them nothing would be possible.

  II  

Abstract The Olfactory Bulb (OB) is the first relay of the main olfactory system. In the OB, newborn

neurons coming from the subventricular zone differentiate into interneurons and integrate

mainly in the granule cell layer. Bulbar neurogenesis is an activity dependent process that is

modulated by odors. Notably, odor learning in a critical time window is able to increase

neurogenesis in the OB, but whether odor exposure or learning per se shape neurogenesis is

not clearly understood. To clarify this question, we tested the effect on adult neurogenesis of

olfactory associative learning versus auditory associative learning (i.e. a different sensory

modality) versus passive exposure to odorants versus control animals. Using these four

experimental contexts of sensory stimulation, we then investigated the consequences on the

number and the cell morphology of a cohort of newborn cells labelled with BrdU and GFP.

Using BrdU, no difference was observed between conditions whereas using GFP, the olfactory

learning condition had a significant increase in the number of cells when compared with auditory

learning. Modest increase in cell survival was also observed after odor exposure and auditory

learning. Morphological analysis revealed a reduced main dendrite length after odor learning.

This study suggests that olfactory exposure and the process of learning per se cooperate to

boost the survival and the integration of newborn neurons into the OB.

Key-words: Olfactory bulb – bulbar neurogenesis – newborn cells - learning - morphology

  III  

Resumo O Bulbo Olfactivo (BO) é o primeiro transmissor de informação do sistema olfactivo principal.

No BO, os novos neurónios provenientes da zona subventricular diferenciam-se em

interneurónios e são integrados principalmente na camada de células granulares. A

neurogénese bulbar é um processo dependente de actividade e é modulado pela presença de

odores. A aprendizagem de odores num período de tempo crítico é responsável por aumentar

a neurogénese no BO, contudo, ainda está por clarificar de que forma a exposição a odores ou

o processo de aprendizagem por si modula a neurogénese. Assim, foram testados os efeitos

da aprendizagem associativa olfactiva versus da aprendizagem associative auditiva (i.e., de

uma modalidade sensorial diferente) versus da exposição passiva a odores versus grupo

controlo na neurogénese do adulto. Partindo destes quatro contextos experimentais de

estimulação sensorial, foram investigadas as consequências no número e na morfologia

celular de uma coorte de novos neurónios marcados com BrdU e GFP. Com BrdU, não foram

observadas diferenças entre as condições enquanto que utilizando GFP, o grupo de

aprendizagem olfactiva mostrou um aumento significativo no número de novos neurónios

comparativamente ao grupo de aprendizagem auditiva. Um ligeiro aumento na sobrevivência

celular foi observado após exposição passiva a odores ou a aprendizagem auditiva. A análise

morfológica demonstrou que no grupo de aprendizagem olfactiva, o comprimento da dendrite

principal é menor. Este estudo sugere que a exposição a odores e o processo de

aprendizagem por si cooperam para aumentar a sobrevivência e a integração de novos

neurónios no BO.

Palavras-chave: Bulbo olfactivo – neurogénese bulbar – novos neurónios - aprendizagem –

morfologia

  IV  

General contents

Introduction…………………………….....……………………………………….………………..…. 1 Synaptic organization of the Olfactory Bulb …………………………………………..……………………………………2 Bulbar adult neurogenesis ……………………………………………………………………………………………...……5 Synaptic plasticity of adult born neurons in the OB…………………………………………….……………………..….. 6 Time and sensory experience dependent survival of Adult-Born Neurons in the OB………..…………………………8 Functional role of adult born neurons in the OB ……………………………………………………………………………9 Odor perception…………………………………………………………………………………………………………………………...…..9 Odor discrimination …………………………………………………………………………………………………………………………10 Olfactory memory ………………………………………………………………………………………………………………………...…10

Experimental design ……………………………………………………………………………...…11 Material and Methods .............................................................................................................. 13 Animals …………………………………………………………………………………………………………………….….13 Injections……………………………………………………………………………………………………………………….13 Behavioral apparatus, training, and odorants ……………………………………………………………………………..15 Brain fixation and slices preparation …………………………………………………………………………………….…20 Immunohistochemistry ………………………………………………………………..……………………………………..20 Image acquisition …………………………………………………………………………..………………………………...22 Image analysis ……………………………………………………………………………...………………………………..23 Statistical analysis ……………………………………………………………………………….…………………………..24

Time course of the behaviour experiments ……………….…………………………...………..26 Results.……………………………………………………………………………………………...….28 BrdU positive cells - Olfactory discrimination learning does not crucially regulate survival of adult born neurons in the OB ………...……………………………………………………………………………………………………………….30 Sparse colocalization was observed between GFP+ and BrdU+ cells………………………………………………………….………………………………………………..………………..31 GFP positive cells - The survival of adult born neurons is differentially regulated by different sensory experience……………………………………………………………………………………………………………………..32 Odor learning induces a decrease in the proximal dendritic length of the newborn granule cells …………………..33

Discussion ………………………………………………………………………………......………..36 1)Behavioural performance………………………………………………………………………………………………….36 2)Effect of training in the cell density of adult born neurons in the OB ………………………………….……………..37 2.1) Counting of GFP+ cells ………………………………………………………………………………..……………….37 2.2) Counting of BrdU+ cells ………………………………………………………………………………………………..37 2.3) Colocalization of BrdU+ and GFP+ cells ………………………….,………………………………………………….39 3) Influence of training in the cell morphology …………………………..………………………………………………..39 4) Future work…………………………………………………………………………………………………………………40

Conclusion …………………………………………………………………………………………….43 References …………………………………………………………………………………………….44 Supplemental data …………………………………………………………………………………….a    

 

  V  

Index of Images

Figure 1: General scheme of the connections of the main olfactory system and the accessory

olfactory system.

Figure 2: Synaptic organization of the olfactory bulb.

Figure 3: Different steps of adult neurogenesis in the OB.

Figure 4: Stereotaxic injections.

Figure 5. Immunohistochemistry using two different techniques.

Figure 6. Sholl analysis methodology.

Figure 7: Performance of the mice of the groups OL (Odor Learning, n=14) and AL (Auditory

Learning, n=16) in session 1 and 2.

Figure 8: Representative images of 40µm coronal sections of the OB with the GL, EPL, GCL,

RMS and AOB delineated for cell counting with QUIA.

Figure 9: Graphics expressing the results of the counting of cells BrdU+ resulting from

immunohistochemistry with DAB.

Figure 10: Graphics expressing the results of the counting of cells BrdU+ resulting from

immunofluorescence.

Figure 11: Graphics expressing the results of the counting of cells GFP+ resulting from

immunofluorescence

Figure 12: Graphics expressing the results of the counting of cells double labelled GFP+

BrdU+.

Figure 13: Analysis of the different morphological parameters of the newborn GCs according to

the condition. The length is expressed in µm. The illustration above each graphic represents in

red what was measured.

  VI  

Supplemental Figure 1: Performance of the mice used for cell survival and morphological

analysis of the groups OL (Odor Learning, n=6) and AL (Auditory Learning, n=8) in session 1

and 2.

Supplemental Figure 2: Analysis of the different morphological parameters of the newborn

GCs according to the condition.

Supplemental Figure 3: Terminal of the main dendrite of a granule cell of an AL mouse.

Supplemental Figure 4: Performance of the mice in the first part of a span capacity working

memory test.

Index of Tables

Table 1. Different groups of study according to the sensory modality and the type of exposition

evolved.

Supplemental Table 1. Number of mice trained within session and within condition according

to the aims of the study.

  VII  

List of abbreviations

AE Air Exposure

AL Auditory Learning

AOB Acessory Olfactory Bulb

AON Anterior Olfactory Nucleus

Ara–C Arabinofuranosyl Cytidine

BrdU 5-bromo-2'-deoxyuridine

CMV Cytomegalovirus

DAB 3,3′-Diaminobenzidine

DAPI 4',6-diamidino-2-phenylindole

DPI Days Post Injection

DG Dentate Gyrus

EC Enthorinal Cortex

EPL External Plexiform Layer

GC Granule cell

GCL Granule Cell Layer

GFP Green Fluorescent Protein

GL Glomerular Layer

ITI Intertrial Interval

LA Lateral Amygdala

LOT Lateral Olfactory Tract

MC Mitral Cell

MCL Mitral Cell Layer

MOE Main Olfactory Epithelium

NaCl Sodium chloride

NaOH Sodium hydroxide

OB Olfactory Bulb

OE Odor Exposure

OL Odor learning

ONL Olfactory Nerve Layer

OSN Olfactory Sensory Neurons

OT Olfactory Tubercle

PC Piriform Cortex

PBS Phosphate Buffered Saline

PFA Paraformaldehyde

PGC Periglomerular cell

RMS Rostral Migratory Stream

SAC Short Axon cell

SVZ Sub-ventricular zone

TF Tufted cell

SEM Standard Error of the Mean

VNO Vomeronasal Organ

WM Working Memory

WPRE woodchuck hepatitis post-

transcriptional regulatory element

 

  1  

Introduction

Humans and other animals share the capacity to learn. A decision to act is based on learning that a

particular stimulus predicts a reward if a particular action is pursued (Salzman et al., 2005). How

we react to a certain stimulus, to perform a task or how we learn a process by simple association

and which brain areas are involved is still unclear. Once perceiving a sensory stimulus, such as an

odorant, we are able to associate it automatically to events, persons or objects. This process

requires specific forms of learning and memory.

The olfactory system is the component of the nervous system responsible for processing the

millions of volatile molecules (odorants) present in the environment and mapping them onto a

mental representation in the central nervous system.

In mice and mammals overall, this system is composed of two pathways, the main olfactory system

and the accessory olfactory system. To each system belong respectively a sensory organ, the

Main Olfactory epithelium (MOE) and the Vomeronasal Organ (VNO) (Figure 1).

Figure 1: General scheme of the connections of the main olfactory system and the accessory olfactory system. Representation of sagittal sections of a mouse brain. A. The axons present in the MOE project to the main olfactory bulb (MOB), forming the olfactory nerve. The projection neurons from the OB send their axons (the lateral olfactory tract, LOT) to the different structures of the olfactory cortex, among them the anterior olfactory nucleus (AON), the olfactory tubercle (OT), the piriform cortex (PC), the lateral amygdala (LA) and the entorhinal cortex (EC). B. The axons arising from the vomeronasal organ (VNO) form the vomeronasal nerve and project to the accessory olfactory bulb (AOB). The projection neurons send their axons mainly to the Ventral Amygdala.

The main olfactory system detects and processes the vast majority of chemical cues that enter the

A B

 

  2  

nasal cavity while the accessory olfactory system is mainly responsible for integrating heavy-

molecular-weight, non volatile molecules responsible for reproductive and defensive behavior.

Over the past two decades the accessory olfactory system has drawn a great deal of attention

because of its essential role in pheromone detection and social communication. However, the

traditional opinion that the main olfactory system only detects volatile odorants and the accessory

olfactory system only detects non-volatile pheromones is no longer valid (Brennan PA. and Zufall

F. 2006; Baum MJ and Kelliher KR 2009).

The Olfactory Bulb (OB) is the principal component of the main olfactory system. With a very well

described organization, connection to other brain structures, and ease of acessibility in the mouse,

it provides an interesting model system for learning studies. In the adult brain, this organ is

continuously supplied with newborn cells coming from the sub ventricular zone (SVZ). This

process, bulbar neurogenesis, has a role in odor learning and processing of odor information.

Synaptic organization of the Olfactory Bulb  

In the main olfactory system, the information is processed from the periphery to the OB. The

olfactory sensory neurons (OSN) located in the olfactory epithelium, are the first neurons in contact

with the external environment. Those OSN will connect to the OB where each axon innervates

mainly principal cells (Firestein, 2001). The odorants, composed of a mixture of volatile molecules

in the air, bind to the olfactory receptors present on the cilia of the OSN. There are ~900 different

olfactory receptors in mice, each OSN only expresses one receptor and all the OSN expressing the

same receptor converge to the same spatial position on the surface of the OB, thus creating a

spatial map of odor receptors (Buck and Axel, 1991). This chemical signal is then transduced into

electric impulses, which propagate trough the axon of the OSN into the olfactory bulb.

Once in the bulb, this information goes to the output neurons (mitral and tufted cells), which receive

sensory inputs from the OSN and inhibitory inputs from local interneurons. The major part of the

 

  3  

interneurons are Granule Cells (GCs, GABAergic) and Periglomerular cells (PGCs, GABAergic and

dopaminergic). Those interneurons are the major neural population in the OB, GABAergic GCs

outnumber the output neurons by a factor of more than 10 (Shepherd et al. 2004).

In response to sensory inputs, mitral and tufted cells release glutamate onto GCs spines, that in

turn release GABA onto activated mitral and tufted cell dendrites. This dendrodendritic inhibition

mediates also lateral inhibition between neighbouring mitral cells and synchronization during odor

presentation (Rall and Shepherd 1968; Friedman and Strowbridge 2000; Schoppa 2006). Lastly,

bulbar interneurons also receive inhibitory inputs from other types of local OB interneurons in the

Granule Cell Layer (GCL) (Eyre et al. 2008) and excitatory inputs from axons collaterals of the

mitral and tufted cells and from terminals of centrifugal projections (Balu et al. 2007) (Figure 2C).

According to the different cell types present in the OB, it is possible to identify five different

concentric layers with specific cells and connexions with afferent and efferent regions of the

olfactory system (Shipley et Ennis, 1996; Shepherd, 2004).

The most external layer, the Olfactory Nerve Layer (ONL), is where the axons of the OSN coming

from the olfactory epithelium are located. Then, the Glomerular Layer (GL) is composed by

glomeruli and surrounded by juxtaglomerular neurons. A glomerulus is made up of a globular

tangle of axons from the olfactory sensory neurons and dendrites from the mitral and tufted cells,

as well as from cells that surround the glomerulus such as the external tufted cells, periglomerular

cells, short axon cells and astrocytes. The cell bodies of the neurons and the astrocytes together

make a physical barrier for the diffusion of the neurotransmitters to the outside of the glomeruli.

Deeper in the bulb, the External Plexiform Layer (EPL) contains the dendrites of the principal

neurons and the apical dendrites of granule cells (Shepherd, 2004). In a smaller proportion, we find

also the cell bodies of short axon cells and tufted cells. A thinner layer deep to this one, the mitral

cell layer (MCL), contains mainly the cell bodies of the mitral cells.

 

  4  

The internal plexiform layer is composed of axons of the mitral and tufted cells, the dendrites of the

interneurons and centrifugal fibers. Finally, the GCL is the most internal layer and is composed by

granule cell somas and axons of mitral cells, tufted cells and centrifugal fibers. In the deepest part

of the GCL is situated the rostral migratory stream (RMS). This area contains the immature

neuroblasts, which are migrating and will differentiate into GCs in the GCL or periglomerular cells

in the GL (Figure 2A, 2B).

Figure 2: Synaptic organization of the olfactory bulb (OB). A. Coronal section of the OB B. Representation of the different layers of the OB: Olfactory Nerve Layer (ONL), Glomerular Layer (GL), External Plexiform Layer (EPL), Mitral Cell Layer (MCL), Internal Plexiform Layer (IPL), Granule Cell Layer (GCL) and Rostral Migratory Stream (RMS). C. Schematic cellular organization in the OB: The olfactory epithelium (OE) located in the nasal cavity is composed of olfactory sensory neurons (OSN), which project to the OB through their axons located in the Olfactory Nerve Layer (ONL). OSNs responding to an odorant project their axons to the main olfactory bulb into one of the glomeruli that form the Glomerular Layer (GL). In the GL, sensory neuron terminals synapse onto the apical dendrites of output neurons - the mitral cells (MC) and the tufted cells (TC). In addition, periglomerular cells (PGC), superficial short-axon cells (sSAC), and external tufted cells (eTC.) act on glomerular synaptic transmission exerting diverse functional effects. In the external plexiform layer (EPL), the lateral dendrites of mitral and tufted cells interact with the dendrites of granule cells (GC). Granule cells can also be subdivided into distinct subpopulations: superficial granule cells (GCS) that target the superficial lamina of the external plexiform layer and synapse with tufted cells. Deep granule cells (GCD) targeting the deep lamina of the external plexiform layer are connected to mitral cells. The soma of mitral cells are aligned and delineate the Mitral Cell Layer (MCL), and the soma of tufted cells are scattered in the EPL. Granule cell somas and also some deep short-axon cells (dSAC) compose the granule cell layer (GCL). Centrifugal fibers from other brain

 

  5  

regions innervate specific layers of the olfactory bulb, with respect to their brain origin. Lastly, output neuron axons fasciculate to form the lateral olfactory tract (LOT). All the cell types colored in orange are glutamatergic, GABAergic cells are in blue. Bulbar adult neurogenesis The OB shares with the dentate gyrus (DG) of the hippocampus the ability to continually generate

new neurons in the adult brain – adult neurogenesis. This cellular renewal is not static or merely

restorative; adult neurogenesis represents an adaptive response to challenges imposed by the

animal’s environment or its internal state. This fact raises some important questions about the role

of neurogenesis in mature neuronal circuits.

While in the embryo bulbar interneurons are generated in the ganglionic eminence migrating to the

developing OB (Wichterle et al. 2001), in the adult brain, those interneurons are derived from the

sub-ventricular zone (SVZ), near the medial wall of the lateral ventricles of the forebrain where they

give rise to neuroblasts and migrate in the rostral migratory stream (RMS) to the OB.

The adult SVZ neural stem cells are capable of producing the three major cell types of the central

nervous system: neurons (about 95% GABAergic and 3% dopaminergic), astrocytes and

oligodendrocytes. In the SVZ, four main cell types are present (Doetsch et al. 1997). A layer of

ependymal cells (E) lines the lateral ventricle. Close to these cells, slow-dividing astrocytic stem

cells (type B cells) divide asymmetrically to generate clusters of type C cells, transit amplifying

cells, which in turn originate type A cells (neuroblasts), by symmetrical division, that start to migrate

in chain to the rostral migratory stream (RMS), 5 days after birth (Figure 3). Sequentially, the

neuroblasts coming from the RMS will differentiate into interneurons.

A question remaining to ask is how neuroblasts differentiate and integrate into fully functional

circuits. Recently, some of the molecular and cellular events that govern the synapse formation,

development and integration of the adult born neurons into the OB have begun to be elucidated.

 

  6  

The GC maturation comprises five stages, from migrating neuroblasts to GCs with a complex

dendritic tree (Petreanu and Alvarez-Buylla, 2002).

Class-1 cells represent cells migrating in the RMS, once they reach the OB, newborn neurons

(class 2) begin a radial migration and extend their apical dendrite through the GCL toward the

mitral cell layer to the EPL. Ten days after birth, newborn neurons start receiving the GABAergic

and glutamatergic synaptic inputs in the GCL (class 3) and then excitatory inputs in the EPL (class

4 cells). Ultimately, adult born neurons reach their final stage of development when they exhibit

distal branches with full spine density (class 5).

Figure 3: Different steps of adult neurogenesis in the OB. A. Representation of a sagittal slice of the adult mouse brain displaying the areas where proliferation, migration and differentiation of the newborn cells occur. Germinal zone, SVZ (sub-ventricular zone), RMS (rostral migratory stream) containing the migrating neuroblasts, and the OB (Olfactory Bulb), final destination of newborn neurons. B. The SVZ contains the ependymal cells (E) lining the lateral ventricle. The first neural progenitors, type B cells will give rise to fast-dividing type C cells which will originate type A cells – neuroblasts C. The neuroblasts take a long time to differentiate into interneurons following a specific period of morphological maturation. The process is similar for Granule and Periglomerular cells.

Synaptic plasticity of adult born neurons in the OB

Even after the final stage of development, the neural network of adult born neurons in the OB is

A

B C

 

  7  

highly dynamic and sensitive to changes at the level of sensory inputs. As an example, sensory

deprivation decreases synaptic wiring of adult-born neurons and this activity-dependent change is

restricted to a time window when adult-born neurons first develop their synapses (Kelsch et al.

2009). In addition, the different regions of the adult born GC dendrite are differentially affected by a

reduction of sensory input, while the distal and basal parts of the GC dendrite show a reduction of

excitatory inputs, the density of the glutamatergic synapses on the proximal region of the apical

dendrite increases. This dual regulation could represent a compensatory mechanism to a variation

in the sensory environment to preserve a minimal level of excitation on adult-born GCs and

therefore for survival (Sagathelyan et al. 2005; Kelsch et al. 2009).

In contrast to the idea that adult born neurons plasticity is restricted to a specific period of time,

Whitman and Greer (2007) have shown that between 28 and 56 days after birth, newborn GCs

exhibit a transient overproduction of spines followed by a drastic elimination. Also supporting this

idea, using time-lapse two-photon microscopy, both adult born GCs and PGCs demonstrates

plasticity several months after their maturation and integration into the OB (Livneh and Mizrahi,

2011).

The potential for synaptic plasticity over time is a specific contribution of adult-born neurons. The

comparison of the potential for synaptic organization of adult-born neurons with neurons generated

during the neonatal period has shown that neonatal GCs are largely variable in the density of their

synaptic inputs in contrast to the adult born interneurons which display stable synaptic connectivity

over time (Kelsch et al. 2012). The adult born GCs exhibit specific patterns of neural activity,

between 2 and 6 weeks after cell birth, GCs exhibit long-term potentiation of its proximal excitatory

inputs (Nissant et al. 2009) playing a critical role in the formation and maintenance of synapses in

newborn GCs.

 

  8  

Time and sensory experience dependent survival of Adult-Born Neurons in the OB

Sensory experience has a clear role for neuronal survival (Petreanu and Alvarez-Buylla 2002;

Rochefort et al. 2002).

Odor experiences are responsible for increasing the rate of cell survival and integration of newborn

neurons in the OB (Rochefort et al. 2002; Miwa and Storm 2005; Bovetti et al. 2009; Moreno et al.

2009; Veyrac et al. 2009), while sensory deprivation decreases the survival of newly generated

neurons (Corotto et al. 1994; Petreanu and Alvarez-Buylla 2002; Yamaguchi and Mori 2005).

Perceptual learning, a form of implicit memory, has been shown to increase the number of

newborn GCs in the OB (Moreno et al., 2009). Olfactory associative learning also promotes the

survival of adult-born neurons in the OB (Alonso et al. 2006; Mouret et al. 2008; Kermen et al.

2010; Sultan et al. 2010, Sultan et al. 2011a, b).

Even though synaptic plasticity is not strictly dependent on time, olfactory learning in different time

periods is able to increase or decrease the BrdU cell density in the OB in the adult brain. Mouret et

al. (2008) labeled adult born cells with BrdU and evaluated cell survival at different days post

injection. Using the same training conditions (trained mice subjected to one week of olfactory

training with a single odor nine days before perfusion) it was shown that learning increases cell

survival in the bulb when learning occurs between 18 and 30 days post BrdU injection.

The elimination of adult born neurons is essential for odor exploration and discrimination, and

blocking the elimination process disrupts olfactory discrimination (Mouret, et al. 2009). This

elimination is modulated by memory; newborn neurons are removed from the network when the

memory trace is no longer active, breaking the odor-reward association (Sultan et al, 2011).

A combination of events and mechanisms may determine the survival and integration of adult born

neurons. Three interrelated pathways mediate the survival of adult-born neurons: dendro-dendritic

synaptic changes, top–down glutamatergic inputs originating from cortical regions and centrifugal

 

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modulation by neuroamines and neuropeptides locally released in the OB. The increased or

decreased survival of adult born neurons in the OB has distinct functional consequences.

Functional role of adult born neurons in the OB

Olfactory experience (odor enrichment and odor learning) can regulate the maturation and survival

of adult-born neurons. Newborn neurons have different properties comparing to pre-existing

interneurons, for example, enhanced synaptic plasticity during a critical time window (Nissant et al.,

2009). Different studies have tested the hypothesis that adult neurogenesis contributes to improve

the plasticity of neuronal networks.

By ablating neurogenesis, using techniques such as anti-mitotic drugs, irradiation or transgenic

mouse models, it was possible to assess the functional contribution of adult neurogenesis.

Odor perception

The contribution of adult OB neurogenesis to odor detection thresholds has been examined in two

recent studies. Using a sniffing attraction task that consisted of recording the time spent by a

subject freely investigating an odorant, Breton-Provencher et al. (2009) reported that mice treated

with Ara-C showed higher detection thresholds and thus a reduced sensitivity of their odorant

perception. In contrast, Lazarini et al. (2009) did not find any impairment of odorant perception in

SVZ-irradiated mice trained to detect odors during a nose poke based go/no-go odor-discrimination

task.

Perceptual learning is an implicit (non associative) form of learning in which discrimination between

sensory stimuli is improved by previous experience (Gilbert et al, 2001). Moreno and collaborators

(2009) have shown that neurogenesis is necessary for perceptual learning by comparing animals

treated with AraC to animals treated just with saline exposed to the same combination of odorants.

In the AraC group, mice had a significant decrease of cell density in the GCL and no enrichment-

induced improvement of discrimination that occurs in the saline group was observed.

 

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Odor discrimination

The causal effect of adult neurogenesis on olfactory discrimination is not clear. Some studies have

reported that reduced neurogenesis impairs odor discrimination (Gheusi et al. 2000; Enewere et al.

2004; Bath et al. 2008). In contrast, other studies showed that ablating neurogenesis in the OB did

not interfere with odor discrimination, using similar protocols (Imayoshi et al. 2008; Breton-

Provencher et al. 2009; Lazarini et al. 2009; Sultan et al. 2010).

Olfactory memory

There are some convergent experiments for the role of neurogenesis for memory. The short-term

strength of the odor-cue fear-conditioned olfactory memory is dependent on adult bulbar

neurogenesis (Valley et al. 2009). Long-term memory is reduced in SVZ-irradiated mice compared

to controls (Lazarini et al. 2009) and it is required for long-term retention of reward-associated

odors (Sultan et al. 2010).

Thus, these results provide evidence of a direct and immediate causal contribution of adult born

olfactory neurons on the maintenance of the olfactory circuits and its behavioral outcomes.

Interestingly, not just olfactory stimuli are able to evoke a response in the olfactory system.

Auditory-evoked responses have been recorded in the olfactory tubercule (Wesson and Wilson,

2010) but not in the OB. In addition, Cohen and collaborators (2011) have shown that response to

pups’ body odor reshapes neuronal responses to pure tones and natural auditory stimuli. This

olfactory-auditory interaction appeared naturally in lactating mothers shortly after parturition and

was long lasting. Although auditory discrimination tests have been used to test the mice ability to

discriminate between different tones, none has been used to study adult bulbar neurogenesis

(Tsukano et al., 2011).

 

  11  

Experimental design

Learning is the formation of associations. During learning multiple brain areas support a large

range of psychobiological processes such as selective attention, sensory processing, execution of

motor responses, reward expectancy, decision making, action selection, evaluation of outcomes

resulting from choices, etc. At a neurobiological level of analysis, all these processes are the

source of a long list of molecular, cellular and wiring changes of the plastic nervous system that

define a ‘brain learning state’. Some of these neurobiological events may non-specifically, but

significantly, influence the production and/or survival of adult-born neurons. However, to date, no

study has clearly examined what could be the relative contribution of the brain state associated

with learning to the processes governing adult bulbar neurogenesis. In the present study we

specifically addressed this question by comparing the rate of adult born neurons in the OB between

animals trained in an odor discrimination learning task (OL group) and animals performing exactly

the same task, except that they had to discriminate between different tones (auditory discrimination

learning – AL group) instead of different odors. Four groups of mice were used in this study: a

group of mice trained to discriminate between odorants (OL), a group of mice trained to

discriminate between tones (AL), a group of mice passively exposed to the same odors (OE) as

those used in the OL group (OL) and a group mice exposed to clean air (AE) (Table 1). Any

changes observed in the OL vs AL training comparison will be likely to reflect the specific

contribution of odor learning to the rate of survival of adult born neurons in the OB. Any changes

observed in the number of surviving adult born neurons between the OL and OE groups will

illustrate the effects of reward-driven odor conditioning vs passive odor exposure. Finally, any

changes observed in the AL and AE comparison will reveal the non-specific contribution of learning

to the rescue of newborn neurons from death.

 

  12  

Table 1. Different groups of study according to the sensorial modality and the type of exposition evolved.

By designing those experimental groups, we aimed to study how the different conditions were

influencing the survival and morphology of the newborn neurons and, as a second goal, evaluate

the performance of the different groups in a span capacity working memory task (Sup. Table 1).

 

Group   Sensorial  Modality   Type  of  exposition  

Odor  Learning  (OL)   Olfaction   Active  

Odor  Enrichment  (OE)   Olfaction   Passive  

Auditory  Learning  (AL)   Audition   Active  

Air  Exposure  (  AE  )   -­‐   -­‐  

 

  13  

Material and Methods

1. Animals. We used adult male mice C57/BL6 (8 weeks old, n=60), housed under a 12h light/dark

cycle at 22ºC with dry food and access to water ad libitum. During the experimental period the

animals were water restricted (1ml H2O/day) or food restricted (2,5g food/day) according to the

body weight that should be about 85% of the original body-weight.

The experiments were performed in two sessions, using the same conditions of accommodation (4

animals/cage).

2. Injections

BrdU injections.

5’-bromo-2’deoxyuridine (BrdU) is a  halogenated thymidine analog that permanently integrated into

the DNA of dividing cells during DNA synthesis in S-phase. BrdU can be immunohistochemically

detected in vitro and in vivo, allowing the identification of cells that were dividing during the period

of BrdU exposure.   This marker was used to study neuronal survival. In particular, the type C

progenitors are the main target of BrdU once they have a very fast cell cycle and they give birth to

neuroblasts.

Mice were injected intraperitoneally with BrdU (75mg/kg, Sigma-Aldrich) in a solution with NaCl (0,

9%) and NaOH (0,4N). Mice received BrdU on the day before the LV injection, four injections, 2h

apart (due to the length of the cell cycle).

Lentivirus injections.

To label the neuroblasts, a lentivirus-based lentiviral vector, plenty-CMV-GFP-WPRE, was used.

This WPRE (woodchuck hepatitis post-transcriptional regulatory element) is an amplifying

sequence (distal enhancer). This virus is non replicable and is driven by CMV which is a strong

promoter. The GFP (Green Fluorescent Protein), driven by the CMV (cytomegalovirus promoter),

 

  14  

emits fluorescence for the entire cell. The virus was diluted 1/20 from an initial concentration of

138ng/µl of p24 (protein from the viral capsid) to a final concentration of 6,9ng/ µl of p24.

The LV injections were stereotaxic injections. They were done bilaterally in the RMS with a

nanoliter injector (glass micropipette – rate of 23nl/s). The viral aliquots were stored at -80ºC and

thawed before pipette loading. The microinjector (Nanoject II) was programmed for delivery of 50nl.

The microinjector had to be attached onto a sterotaxic frame. The micropipette was loaded with 2µl

of virus solution onto a sterile piece of Parafilm.

Each mouse was anesthethized with 100mg/kg Ketamine and 10mg/kg Xylazine, diluted in sterile

saline. Before the injection, the hair was removed from the scalp of the animal using hair clippers

and razors. All surgical instruments were autoclaved and then disinfected with 70% ethanol. The

animal was placed in the stereotaxic frame with ear bars and nose bar. The eyes were hydrated

with NaCl to prevent drying. With a scalpel, the scalp was cut from between the eyes to between

the ears. The skin was pulled aside to expose the skull and then anchored with a pair of clamps.

The surface of the skull was cleaned. The stereotaxic apparatus is zeroed with the tip of the glass

pipette at bregma point. Then, the tip is positioned at the injection site (Antero-Posterior, +3.3mm;

Medio-Lateral, ±0.82mm, for both right and left hemispheres, Dorso-Ventral, -2.9mm from brain

surface) (Figure 4). The position of the nose bar was adjusted according to the calculation of the

stereotaxic coordinates. In our experiment, the nose bar was adjusted until bregma and the

injection site was aligned to the same height. After identifying the injection site, two holes were

carefully drilled into the skull. The remnants of thin bone were removed to expose the dura mater.

At this step, the pipette can be lowered and the Dorso-Ventral height set to zero when the tip of the

pipette touched the surface of the brain. After lowering the pipette in the tissue to the target point,

the virus were injected 4 times (50 nl of virus for a total of 200 nl) with a delay of 30s between each

injection (pressure equilibrium). One minute after the last injection, the pipette was slowly

 

  15  

withdrawn and this procedure was repeated for the other hemisphere. At the last step, the animal

was removed from the stereotaxic apparatus, the incision was cleaned and the skin stitched using

surgical threads. For recovery, the animals were accommodated on a warming pad before

returning to the cage and received a non-steroidal anti-inflammatory analgesic (Carprofen 4mg/kg).

Figure 4. Stereotaxic intections. On the left, representation of the coordinates of the stereotaxic injection in a mice skull (yellow dots) and on the right, a sagittal section showing the point of injection, on the RMS (rostral migratory stream).

3. Behavioral apparatus, training, and odorants.

Six apparatuses were used to test four different conditions.

Behavioral apparatus. The OL mice were trained in computer-controlled eight-channel

olfactometers (detailed description Bisulco and Slotnick, 2003). Briefly, solenoid pinch valves

controlled air streams and odors were generated by passing a 50 cc/minute stream of air over the

surface of mineral oil diluted odorants in disposable 50 ml centrifuge tubes. The 50 cc /minute

odorant vapor from the saturator tube was mixed with 1950 cc/minute clean air before its

introduction into an odor sampling tube in the mouse operant chamber.

The AL mice were trained in comparable olfactometers that were equipped with auditory stimuli

generators. No odorants were released but it was installed a speaker (Farnell) for tone delivery in

the cage for discrimination.

 

 

 

 

  16  

The OE mice were transported to a small cage where they were exposed to the same odorants as

the OL group but they were not submitted to any learning task. The boxes were equipped with a

fan so the odors were renewed every time a mouse received a different odorant (in a random

order).

The AE mice were moved to a cage with the same dimensions of the one for the animals of the OE

condition and no odorants were introduced. The tubes were filled with mineral oil without any

odorants in solution.

Between mice, the olfactometers, the ‘audiometers’ and the boxes of enrichment were washed with

95% ethanol and air-dried. Each odorant was maintained in its own saturator tube and the liquid

odorant/mineral oil solution was refreshed daily.

a) Training, odorants and tones

The odorants used and their rated purities were Anisole (99%), Cineole (98%), Linalool (97%) and

β-ionone (96%), all from Sigma-Aldrich (St. Louis, MO, USA). The odorant sources were prepared

in the odor saturation tubes, all the odorants were diluted on a per volume basis with odorless

mineral oil to the desired concentration (10-2) for a final volume of 10 ml of solution. Odorant

concentrations are given as the liquid dilution of the odorant in the saturator tubes and the stimuli

used in training were designated by the name of the odorant and its liquid dilution. The odor

concentration delivered to the sampling port was 2.5% of the headspace above the liquid odorant.

We did not check the odorant concentration of the headspace above the liquid solution, but gas

chromatographic analyses have shown that the headspace concentrations of various hydrocarbons

from mineral oil dilutions are proportional to their liquid dilution (Cometto-Muniz et al., 2003).

For the auditory discrimination tasks, no odors were present in the saturators, those were replaced

by tones and the delivery of the different tones was controlled by a ToneGenerator. The tones

selected were 8kHz, 20kHz, 12kHz and 17kHz. The auditory discrimination tasks were run

 

  17  

identically to the olfactory discrimination tasks except that no odors were present in the saturators.

Response requirements were exactly the same as for the olfactory tasks. Instead of the olfactory

stimuli, each trial involved the presentation of a specific auditory stimulus.

The OE mice were exposed to the same odorants as the OL group.

The AE mice were exposed to air puffs of mineral oil at the same rate as the odorants for the OE

group.

b) Protocol description

Pre-discrimination test - shaping.

The training sessions were conducted during the light cycle between 9:00 and 20:00. The four

animals housed in a same cage were belonging to a different condition. This was a way to avoid

social deprivation. The mice were handled and four days later, before the shaping period, they

started being partially water restricted. The amount of water given to the mice was adjusted to have

at the end all the mice with 85% of their original body-weight. The animals were water restricted

receiving 1ml of water/day.

In the period of shaping, firstly, mice were trained to lick on the water delivery tube to obtain a 3 μl

drop of water, then they were trained for nose pokes into the odor samping port in order to get the

reward in the water delivery tube. Then, obtaining a water reward became a more difficult task,

mice had to keep their nose in the odor sampling port for increasing lengths of time (until 1.2s) has

a condition to get the water reward. If the nose was kept in the odor port for the required amount of

time, any subsequent lick by the mouse after this on the left side water tube was reinforced with 3

μl of water. An ITI of 5 seconds was imposed between trials. Once the mice reached 85% of

correct responses, they started the go out discrimination test.

For auditory learning group, the period of shaping was similar but a tone was introduced at the last

 

  18  

trials.

For the OE and AE groups the animals were just moved to the ‘box of enrichment’.

Training

They were trained using an operant conditioning Go Out paradigm, as described by Slotnick B.

(2007). Standard operant conditioning methods were used to train mice to insert their snouts into

the odor sampling port. The presence of the positive stimulus (S+) was associated with a water

reward obtained when the mouse licked the adjacent water delivery tube. In the presence of the

negative stimulus (S-), the mouse received no water reward and had to refrain from licking the

water tube. The first snout insertion after a 5 s intertrial interval (ITI) initiated a trial. At the

beginning of the trial, the stimulus control valves and a valve directing the air stream away from the

sampling tube were functional. This resulted in the odorant vapor being combined with the main air

stream and the diversion of the main air stream to an exhaust path. The diversion valve relaxed 1 s

later, and the odor stimulus was presented to the odor sampling port. The stimulus valves relaxed

2 s later, thus terminating the delivery of the odor. Reward delivery depended on the mouse licking

the water delivery tube in the 2 s odor presentation period. Trials in which the mouse did not keep

its snout in the odor sampling port for at least 1 s after odor onset, were aborted and counted as

short sample trials. A 3μl water reward was delivered if the mouse satisfied the response criterion.

Odor discrimination learning. In each trial, a single stimulus (S+ or S-) was presented. If the

response criterion was met in S+ trials, a 3μl droplet of water was given as a reward and the trial

was scored as a hit, whereas failing to meet the response criterion was scored as a miss. Meeting

the response criterion in S- trials was scored as a false alarm and failing to make a criterion

response was scored as a correct rejection. S+ and S- trials were presented in a modified random

order, such that each block of 20 trials contained equal numbers of each type of trial and no one

 

  19  

type of trial was presented more than three times consecutively. The trial procedures were identical

to those used in the initial pretraining sessions. The percentage of correct responses was

determined for each block of 20 trials [(hits + correct rejections)/20 x 100]. Scores above 85%

implied that mice had correctly learned to assign the reward value of the S+ and the non-reward

value of the S-. The trained mice of the experiment had to perform two followed odor discrimination

tasks with two different odorant combinations for 7 days. They were trained in discrimination tasks

in which mice had to learn to discriminate between 1% Anisole and 1% Cineol solutions and then

between 1% Linalool and 1% β-ionone. In those discriminations, for the two pairs of odorants,

within the same group of mice, half had one of the odorants as S+ and the other half the same

odorant as S-. With this procedure it was avoided any type of bias associated with the odorant that

was S+.

Auditory discrimination learning. The same procedure was reproduced in this group; animals in an

olfactometer were submitted in each trial to the presence of different auditory stimulus (S+ or S-)

associated to a water reward, the tone was delivered every time the mouse did a nose poke in a

random order and the percentage of correct responses was determined for each block of 20 trials.

The first discrimination was between 8kHz and 20kHz and the second between 12kHz and 17kHz,

again, different mice of the same group had different tones associated to S+ and S-, the auditory

and odor discrimination tasks occurred in the same period of time, 7 days each.

Odor enrichment. The mice of this group stand on a box of enrichment during the same

approximated period as the animals of the learning groups. In those boxes, the odor is delivered

during 2 seconds and then a fan is activated to remove the odorant. Five seconds after, a new

odorant is introduced. According to this protocol, all the mice of this group were submitted to the

 

  20  

same odorants in a random order, as in the odor learning group, but no procedure of instrumental

learning occurred. They received 200 exposures (100 for each odorant) during each daily session.

Air exposure. The protocol of odor enrichment was reproduced for the air exposure group. Water

restricted animals which had to stay during approximately 40 min in a box of enrichment were

submitted to the same conditions but without being exposed to any odorant, just mineral oil.

4. Brain fixation and slices preparation. Mice were deeply anesthetized with an intraperitoneal

injection of sodium pentobarbital (100 mg/kg, Sanofi). They should be totally immobilized before

starting the transcardiac perfusion. Brains were dissected out after transcardiac perfusion (on the

left ventricle) with 0.9% NaCl at 37ºC during 5 min followed by a solution of paraformaldehyde

(PFA, 4% in a 0,1M phosphate buffer, pH=7,4) to fix the tissue, this takes approximately 15min with

a flux of 10ml/min. After dissection, brains were stored at 4°C in 4% PFA overnight, and then

transferred to phosphate buffer saline (PBS) containing 0.2% sodium azide. Forty-micron thick

coronal sections were cut using a vibrating microtome (VT1000S, Leica).

5. Immunohistochemistry. Immunostaining was performed on 40 µm coronal free-floating sections,

and slices processed for BrdU-GFP, GFP and for BrdU-DAB (Figure 5).

BrdU/GFP immunohistochemistry in free-floating slices. BrdU and

GFP immunohistochemistry were performed sequentially. All the steps were done in a shaker to

allow soft movements to the slices, at room temperature and non-directly exposed to light. The

first step consists of DNA denaturation by 30min incubation with HCl 2N solution at 37°C. After

rinsing with PBS, we performed permeabilization of the slices and blocking the non-specific

antigenic sites. The slices were floating in a solution of PBS Triton X-100 at 0,2% with a 10% goat

serum (blocking solution). The slices were in this medium during 1h. Following this step, the

primary antibodies were added. For this purpose, the slices were immersed into a solution of PBS

 

  21  

Triton 0,2% with goat serum 2%, a 4‰ polyclonal antibody solution of rat antibodies anti-BrdU

(1:200; Oxford Biotech, Kidlington, UK) during two days at 4°C. After incubation, the slices were

washed three times with PBS during 10 minutes. The third step consists of adding the secondary

antibodies. The slices were immersed in a solution of PBS Triton (0,2%) with goat serum at 5%

containing polyclonal A568-conjugated Goat anti-Rat (1:1000, Mol Probes). After a 2h period of

incubation, they were washed with PBS 3 times during 10 min. After BrdU labeling, the slices were

processed with the same protocol for GFP labeling using a polyclonal Chicken anti-GFP (1:1000,

Invitrogen) and A488-conjugated goat anti-chicken (1:1000, Mol Probes). The slices were then

incubated for 5min in a DAPI solution (4',6-diamidino- 2-phenylindole; 1:5000) in PBS. At a final

step, the slices were washed in PBS one time during 10min.

Slices were mounted between slide and the coverslip within a mounting medium (Mowiol). Those

slices were left drying in a slide until they change their color to translucid. The slides were kept at

4ºC avoiding direct contact with light.

GFP immunohistochemistry. GFP immunohistochemistry was performed as mentioned above with

omission of the DNA denaturation treatment. The primary antibody was Chicken Anti-GFP (1:1000;

Invitrogen) and the secondary antibody was Goat Anti-rabbit Alexa488 (Molecular Probes)

(1/1000).

BrdU/DAB immunohistochemistry. For BrDU revelation with DAB, slices were processed as

mentioned above for BrdU immunohistochemistry until the secondary antibody step. Following 3

rinses in PBS (1x), slices were incubated with biotin-conjugated goat anti-rat (1:1000; Chemicon) in

PBS Triton 0.2% containing 5% Normal Goat Serum during 2 hours at room temperature. After 3

rinses in PBS (1x), the ABC complex (avidine-biotine-peroxydase, VECTASTAIN Elite ABC kit,

Vector) was added and the slices were in this solution during one hour. After three new rinses in

 

  22  

PBS (1x) the revelation was done with DAB (a solution which as a chromogene which is oxidated

by the peroxydase to produce a dark brown agregate) followed by three rinses in PBS (1x). The

slices were mounted in slides and then they were left without coverslip until they were dry. Under

the hood, the slices were dehydrated by followed immersion in different ethanol solution (70 %,

80 % and two times at 100 % during two minutes) and then they were immersed three times in a

Xylene medium (30 seconds each). The slides were finally mounted with a hydrophobe mounting

medium (DEPEX) and covered with a coverslip.

Figure 5. Immunohistochemistry using two different techniques. On the left, BrdU/DAB and on the right, immunohistochemistry with fluorescence, used for GFP and for BrdU.

6. Image acquisition.

For light microscopy (BrdU/DAB staining), it was used a 20x objective to reconstruct images of

each section (Compix Imaging; Hamamatsu Photonics).

For the acquisition of fluorescence images, for BrdU/GFP staining, a microscope was used (Zeiss,

Germany) equipped with an Apotome. An oil objective of 25x was used to reconstruct images of

each section, Z-sectioning was performed at 5μm intervals; those optical sections were fused to

generate the final image. The light time of exposure was 50ms for GFP and 200ms for BrdU. For

the reconstruction of the different Z-sectioned layers and for final light adjustments, we used

Axovision 4.6 software.

 

  23  

For the acquisition of the fluorescence images for GFP staining, we used a confocal microscope

(Zeiss, Germany) equipped with the Zen software (Zeiss). The objective used was of 20x for the

cell analysis and for spines analysis a 40x oil objective. The parameters were adapted for each

slice.

7. Image analysis.

To all the images analyzed was attributed a code which was just revealed at the time of the statistic

analysis.

Analysis of BrdU/DAB labeled cells. For counting of BrdU+ cells, it was used a dedicated computer

program using a B3 wavelet filtering approach (Quia, de Chaumont et al., 2008). The method

enhances spots (corresponding to BrdU+ cells) while filtering out the background. For each animal,

counts were made for one in every three coronal sections of the same bulb (120 µm apart, 10-14

slices per animals). Anatomical landmarks within the OB were used to align coronal sections

across animals. The rostral landmark, defining the origin of the rostrocaudal axis, contained the

first clear mitral cell and external plexiform layers. The accessory olfactory bulb (AOB) was used as

the caudal landmark and the last section counted contained the first, clear AOB. The internal and

external borders of the GL, EPL, GCL and the border of the rostral migratory stream of the OB

(RMSOB) were outlined on the same image. The program then numbered cells detected in the GL,

EPL and GCL. Values were given as BrdU+

cell density (number of positive cells per mm2). The

same counting criteria were applied for all the light microscopy images of BrdU/DAB labeled cells.

Analysis of BrdU/GFP labeled cells. BrdU and GFP positive cells were counted automatically using

the same software (Quia, de Chaumont et al., 2008). For each animal, counts were made for one

in every three coronal sections of the OB (120 µm apart) using the same program and procedure

 

  24  

as for BrdU/DAB labeled cells. The program counted the cells detected in the various layers, the

same counting criteria were used for all the fluorescence images.

Analysis of GFP labeled cells. The pictures that were taken of the granule cells (n = 5-10 per

animal) at the confocal microscope were used for morphological analysis of the granule cells. For

this purpose, we used ImageJ software with two plugins, NeuronJ and Advanced Sholl Analysis.

NeuronJ gave us the possibility to analyze different parameters such as the main dendrite length

(from the soma to the first branch point), total dendritic length and number of branch points. After

tracing the dendritic tree, from the soma to the most distal branch of the main dendrite, we did a

Sholl analysis to analyze the complexity of the dendritic tree. Using the scale of 10µm, the software

draws, for each circle, concentric circles starting in the soma and calculates the number of

crossings between branches. The result gives us the number of branches in function of the

distance from the soma (Figure 6). Those tools allow us to visualize the configuration of the

dendritic tree and the level of complexity.

Figure 6. Sholl analysis methodology. A. Confocal picture of the granule cell for analysis; B. Traces of the GC. C. The software draws concentric circles to count the number of intersections of the tracing for each of the concentric circle starting from the soma. D. The data then is transferred for a graphic.

8. Statistical analysis. The level of significance of the tests were done for p=0.05. To test the

 

  25  

independence of distributions between conditions (OL, AL, OE, AE), a Kruskal-Wallis test (non-

parametric ANOVA) was performed. We thus tested for each layer (GCL, EPL, GL), the following

parameters: surface area, density of BrdU/DAB + cells, density of GFP+ cells, density of BrdU+

cells, percentage of colocalization/number of BrdU+ cells, percentage of colocalization/ number of

GFP+ cells and percentage of colocalization given the total number of immunostained cells. When

the test indicated a significant difference, the value of the decision variable were reported in the

corresponding graph of the form: H ([number of samples] -1, N = [size of all samples]) = [value of

the decision variable], p = probability associated. We then tested two by two population

distributions within each condition with a post-hoc test - Wilcoxon-Mann-Whitney.

The same approach was applied to the results of morphology: proximal dendrite length, total length

of the dendritic tree and the number of branch points. For the Scholl analysis, it was done a two-

factor ANOVA (condition as independent variable and distance as a dependent variable) to analyze

the effect of the two factors, conditions and radius of the circle for the different means.

The statistical analysis was performed in all cases for the four conditions of training (OL, AL, OE

and AE) not taking into consideration mice from behavioral session 1 and 2 separately. Data were

expressed as mean values ± SEM.

 

  26  

Time course of the behaviour experiments

The behavioural experiments were performed in two sessions due to the high number of mice

belonging to each condition; the following steps were executed in the exact same way and the time

course of the experiments was reproducible between sessions.

At the beginning of the experiments, the mice were aged 8 weeks. Two days after the arrival of the

mice in the animal facility, all mice were injected with BrdU to label the dividing cells in the SVZ.

On the following day, half of the mice (the mice used for cell survival and morphological studies)

were injected with the lentivirus GFP in the RMS to label the neuroblasts in migration to the OB.

One day is approximately the time necessary for the cells coming from the SVZ arrive into the RMS

(Petreanu & Alvarez-Buylla, 2002, Carleton et al., 2003). Therefore, this delay between BrdU and

LV injections was important to make sure we labelled the same neuronal population.

After three days of recovery from the surgery, the mice were moved to the behaviour animal facility

where they were marked with a different tail colour according to the condition. Within a cage there

was one animal of each condition, OL, AL, OE and AE. Those animals were handled during two

days. Thirteen days after BrdU injection, mice started the behavioural interventions. The first five

days corresponded to the shaping period (essential to habituate the mice to the new apparatus and

the rules of the tasks they had to execute). Afterwards, they started the first discrimination (Anisol

vs Cineol, OL group and 8kHz vs 20kHz, AL group) and then the second discrimination (Linalool vs

β-ionone, OL group and 12khz vs 17khz, AL group), each discrimination task occurred during

seven days. Simultaneously, OE mice were exposed on the first 7 days to Anisol and Cineol and

1st  discrimination   2nd  discrimination  

 

  27  

then the last 7 days to Linalool and β-ionone while the AE mice were exposed to clean air during

the 14 days. Two days after the behavioural conditioning, 34 days post injection (dpi) of BrdU, half

of the mice were isolated in different cages with water ad libitum to recover to the original body

weight for a working memory test and the other half were perfused. After perfusing, slicing and

sorting, the immunohistochemistry was done, the slices were kept on the fridge and once we had

the slices of both sessions, we did the acquisition of the pictures and the counting for the slices

immunolabelled with DAB (BrdU+) and the ones with immunofluorescence for BrdU+ and GFP+.

For morphological analysis we used slices GFP+ (due to the BrdU+ treatment, the slices BrdU+

GFP+ were not used for morphology).

   

 

  28  

Results The mice of the four conditions, OL, AL, OE and AE were submitted to different training conditions

during the same period. On the first discrimination task, the mice of the Odor Learning group had to

discriminate between Anisol and Cineol and after two days they reached a performance of 95% of

correct responses. For the AL group, the mice had to discriminate between 8kHz and 20kHz, but in

contrast to the OL group, they couldn’t discriminate after two days, the best performance was only

achieved at the last day of training - 75% of correct responses (Figure 7), as reported in others

studies (e.g. Bathelier et al., 2012).

For the second discrimination task, we expected to see a better performance for OL and AL once

they were already habituated to the test (Slotnick BM and Katz H, 1974; Slotnick BM et al., 2000).

However, even though the OL group easily distinguished between those two odorants after two

days of training, reaching 95% of performance, the AL mice were slower and in fact there was

almost no progress in the performance achieved. After 7 days, the percentage of correct responses

was about 60%.

     

 

  29  

Figure 7:   Performance of the mice of the groups OL (Odor Learning, n=14) and AL (Auditory Learning, n=16) in session 1 and 2. Each dot corresponds to the mean +/- sem of all the mice in that block (20 trials), to remind 10 blocks were performed within a day. The dashed line represents chance level (50%). A. First odor discrimination task of the OL group, Anisol (10 -2 ) vs Cineol(10 -2 ) . B. Second discrimination task of the OL group, Linalool (10-2) vs β-ionone (10 -2 ). C. First auditory discrimination task of the AL group, 8kHz vs 20kHz. D. Second discrimination task of the AL group, 12kHz vs 17kHz.

 From the mice of the AL and OL group, we repeated the analysis just for the OL and AL cohort of

mice used to study cell survival and morphology. The performances obtained for this cohort of mice

in the first and the second discrimination for OL and AL conditions were similar (Sup. Fig. 1).

The main purpose of this project was to study how learning affects neurogenesis. To evaluate it, it

was necessary to count the number of cells within different conditions. As we labelled with different

cell markers, we did the counting for BrdU and for GFP. For BrdU, we counted in two different

ways, according to the immunohistochemistry we did on the slices (Figure 8).

 

D C

Anisol (10-2) vs Cineol (10-2)

Blocks (x20 trials)

Perc

ent o

f cor

rect

resp

onse

s (m

ean

+/- s

em)

40

50

60

70

80

90

100

Linalool (10-2) vs b-damascenone (10-2)

Blocks (x20 trials)

Per

cent

of c

orre

ct re

spon

ses

(mea

n +/

- sem

)

40

50

60

70

80

90

100

8 Khz vs 20 Khz

Blocks (x20 trials)

Perc

ent o

f cor

rect

resp

onse

s (m

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Anisol (10-2) vs Cineol (10-2) Linalool (10-2) vs β-ionone (10-2)

A B

C D

 

  30  

Figure 8: Representative images of 40μm coronal sections of the OB with the GL, EPL, GCL, RMS and AOB delineated for cell counting with QUIA. A. Slice treated with immunofluorescence for BrdU+(red) and GFP+(green) cell counting. B. Slice treated for BrdU/DAB for BrdU counting. BrdU positive cells - Olfactory discrimination learning does not crucially regulate survival of adult born neurons in the OB.

Eighteen days before the period of training the mice were injected with BrdU. By quantifying BrdU+

cells in the OB, we investigated whether adult neurogenesis might be affected by this training using

the same software for counting and analyzing the three layers, the GL, EPL and GCL (not the

RMS). Even though previous studies have shown that in this period the cell survival is increased

(Mouret et al. 2008), the difference in the cell density within different layers is not significant

between conditions. However, as already reported (Alonso et al. 2006), the cell density within the

granule cell layer is significantly higher when compared to other layers.

The cell density of BrdU +cells between conditions was around 180 cells/mm2 (average of the four

conditions in the GCL) and the different sensory experiences did not affect neuronal survival.

Thus, there was no significant difference in the BrdU+ cell density between groups (Figure 9), for

the GCL (H (3,28) =1,80; p=0,614), for the EPL (H (3,28)=0,42; p=0,936) and for the GL (H

GL  EPL  GCL  RMS+AOB  

 

  31  

(3,28)=0,90;p=0,824).

Figure 9: Graphics expressing the results of the counting of cells BrdU+ resulting from immunohistochemistry with DAB. In each graphic, the four conditions were separated and for each, it is indicated the mean (+- SEM) density (nº of cells/mm2) of BrdU+ cells. A. Density of BrdU+ cells by condition in the Granule Cell Layer (GCL). B. Density of BrdU+ cells by condition in the External Plexiform Layer (EPL). C. Density of BrdU+ cells by condition in the Glomerular cell layer(GL). Those results were in contrast with previous publications (Mouret et al., 2008; Alonso et al., 2006;

Moreno et al., 2012). The similar results between conditions in the three layers were not just

observed with DAB, it was also confirmed when the slices were counted using a different treatment

- BrdU+ fluorescence, indeed, the density of BrdU+ cells were similar between techniques (Figure

10).    

Figure 10: Graphics expressing the results of the counting of cells BrdU+ resulting from immunofluorescence. In each graphic, the four conditions were separated and for each, it is indicated the mean (+- SEM) density (nº of cells/mm2) of BrdU+ cells. A. Density of BrdU+ cells by condition in the Granule Cell Layer (GCL). B. Density of BrdU+ cells by condition in the External Plexiform Layer (EPL). C. Density of BrdU+ cells by condition in the Glomerular Cell Layer (GL).

GFP positive cells

- The survival of adult born neurons is differentially regulated by different sensory

experience.  

A C B

B

   A B C

 

  32  

The same slices labelled in red for BrdU were also labelled for GFP. Surprisingly, even though we

observed no difference between groups when labelling with BrdU, when the cells were labelled

with GFP, we observed a significant difference in the density of newborn cells between OL and AE

groups, as previous results with BrdU/DAB have shown. Even though the only significant difference

is between OL and AE (for the GL, H (3,28) =10.02 p=0,018; EPL, H (3,28) =9.04 p=0,028 and for the

GCL, H (3,28) =13,80 p=0,003), the AL and OE conditions have shown a slight increase in the cell

density when compared to the control group, AE (Figure 11).

Figure 11: Graphics expressing the results of the counting of cells GFP+ resulting from immunofluorescence. In each graphic, the four conditions were separated and for each, it is indicated the mean (+- SEM) density (nº of cells/mm2) of GFP+ cells; *p=0.05. A. Density of GFP+ cells by condition in the Granule Cell Layer (GCL). B. Density of GFP+ cells by condition in the External Plexiform Layer (EPL). C. Density of GFP+ cells by condition in the Glomerular Cell Layer (GL).

 Sparse colocalization was observed between GFP+ and BrdU+ cells. It was expected to see colocalization between those two ways of labelling neurons. However, the

number of cells labelled with both markers was extremely low, about 2% of the total number of

cells, and similar between conditions. Even though just a small number of cells were labelled with

GFP+ and BrdU+, there was a significant difference between OL and AE in the GCL when the

percentage of colocalization was given according to the total number of BrdU+ cells (H (3,28) =10,05

p=0,018). However, when this percentage of colocalization was given in function of the total

number of cells or total number of GFP+ cells, no significant differences between conditions were

A B C

 

  33  

observed. To sum up, the number of cells labelled with both markers is low and similar between

conditions; therefore the number of cells incorporating both markers is not affected by training

(Figure 12).

Figure 12: Graphics expressing the results of the counting of cells double labelled GFP+ BrdU+. In function of the BrdU+ cells in the GCL (A), EPL (B) and GL (C), in function of the GFP+ cells in the GCL (D), EPL (E) and GL (F) and finally in function of the total number of cells for the three layers, GCL (G), EPL (H) and GL (I), respectively. *p=0.05

Odor learning induces a decrease in the proximal dendritic length of the newborn granule

cells

The study of the morphology of the newborn granule cells was done to answer different questions.

Is the length and complexity of those GCs variable/shaped according to different patterns of

learning? For this purpose, we measured the length of the proximal dendrite, from the soma to the

first branch point, the number of branch points and the length of the entire dendritic tree of GFP+

B

A

C

D G

E HB

I F

 

  34  

newborn cells that populate the OB.

As it is represented on Figure 13B and C, the number of branches and the total dendritic length of

the tree did not vary significantly according to the different conditions. However, the length of the

proximal dendrite of the newborn GCs tend to be shorter in the OL group than in the other groups,

(H(3,28)= 6.88, p = 0.07) (Figure 13A).

Using a Sholl analysis, we then aimed to determine whether the different sensory experiences

were also regulating the complexity of the dendritic tree of newly generated GCs in the different

groups.

We did two types of analysis, one starting in the soma (Figure 13D) and the other starting in the

first branch point (Figure 13E), using this analysis, it was possible to evaluate the complexity of the

dendritic tree and to reduce the variability in the analysis caused by the length of the proximal

dendrite. In both cases, we found no significant differences between the four groups, either starting

the analysis from the soma or from the first branch point, the complexity of the dendritic tree is not

affected by different patterns of learning.

In order to reduce bias associated to the type of GCs, we did the analysis taking into account just

the GCs with the morphological criterion of more than one branch point (Sup. Fig. 2) and the

results were similar, no significant differences between conditions except for the proximal dendritic

length, shorter for the OL group.

 

  35  

Figure 13: Analysis of the different morphological parameters of the newborn GCs according to the condition. The length is expressed in µm. The illustration above each graphic represents in red what was measured. A. Proximal dendritic length (first segment from the soma to the first branch point) of the different conditions expressed by the mean length +- sem of the group, in µm. B. Total dendritic tree length of the different conditions expressed by the mean length +- sem of the group, in µm. C. Number of branch points of the four conditions expressed as the mean number of the group (+- sem). D. Quantification by Sholl analysis of the number of intersections between dendritic segments and virtual concentric lines centered on the soma and spaced by 10µm. Each curve represents the mean of the mice of each condition. E. Quantification by Sholl analysis of the number of intersections between dendritic segments and virtual concentric lines centered on the first branch point and spaced by 10µm. Each curve represents the mean of the mice of each condition.

To sum up, the number of branches, the length of the total dendritic tree and the complexity of the

newborn GCs are the same between groups. The only parameter that significantly differed

between the four conditions was the length of the main dendrite. This length is shorter for OL than

for the other conditions and the dendritic tree is similar between conditions. Thus, odor learning

affects the length of the primary dendrite, suggesting that when becoming mature these newborn

cells tend to develop earlier their dendritic tree.

Proximal dendritic lenght

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  36  

Discussion Adult neurogenesis in the OB is an activity-dependent process. Here, we have investigated how

cell survival and morphology could be modulated by different sensory experiences. These

environmental and behavioural interventions were performed between 14 and 32 days after BrdU

administration, in order to be consistent with the critical time window (18 to 30 days of age) when

the survival of adult born neurons is increased in a sensory and learning experience-dependent

manner (Mouret et al., 2008).

1) Behavioural performance

While in the OL group mice easily reached 95% of correct responses, in the AL group, for auditory

discrimination, the maximum of correct responses obtained was 80% at the end of the first auditory

discrimination task. In contrast to humans, mice can hear high frequencies, they communicate

mainly with ultrasonic vocalizations, above 20kHz. The tones selected were in the range of the

auditory spectrum for C57BL6 mice (Tsukano et al., 2011) consequently they were able to perceive

the different tones used. Intriguingly, even extending the period of learning to seven days (the

maximum conceivable according to the critical period for survival), during the second auditory

discrimination, the AL mice didn’t increase their performance. We expected that, as it happened in

the OL group, the mice would increase their performance in a shorter period of time. However, it

was shown that in a similar auditory discrimination task in rats (Guo et al, 2012), the maximal

percentage of correct responses was achieved just after 15 days of training.

Taking into account that the highest performance reached by the AL mice in the auditory

discrimination task of the second pair of tones was about 60%, being constant during the seven

days of training, we considered that learning occurs once the mice perform the task and it is not

 

  37  

defined by the final percentage of correct responses, as this performance is achieved in a different

number of sessions according to the sensory modality.

2) Effect of training in the cell density of adult born neurons in the OB.

2.1) Counting of GFP+ cells

The specificity of the GFP+ cells labelled is driven by the position of the injection and not by the

vector or the promoter used. For this reason, the injection was done in the RMS. Almost all the

cells in the RMS are neuroblasts migrating en route the OB.

The counting of GFP+ cells has shown an increase in the survival of newborn neurons in the GL

and in the GCL in the mice exposed to odor learning condition when compared to the control group

(AE).

Although to a lesser extent, a relative increase of newborn cell survival has also been observed in

the AL and OE groups compared to control mice (AE). Apparently, what leads to a significant

increase in newborn cell survival in the OL group is an additive contribution of the effect of learning

(as shown with the increase in the AL group) with the effect of exposure to an enriched odor

environment (OE).

Based on this data, we can conclude that the mice exposure to the conditions tested is responsible

for increasing the cell survival rate of newborn neurons by establishing and facilitating contact with

output or input neurons.

2.2) Counting of BrdU+ cells

The BrdU is a cell marker integrated not just in the dividing cells but also in the cells being

repaired. Therefore, this marker labels all the dividing cells in the mouse.

As previously described, we used two techniques to count BrdU positive cells,

immunofluorescence and immunohistochemistry with DAB. The results obtained were similar and

 

  38  

they don’t reveal any significant difference between conditions in any of the three layers. These

findings show some controversy, why aren’t they in accordance to the ones obtained for GFP+

cells?

Regarding this results, we tried to identify a possible problem for the quantification with BrdU. We

first thought it might be a problem related to some bias in the counting, however, the slices BrdU+

and BrdU/DAB were counted by different persons and the results were similar. Then, we thought it

might have been a result of an error in the preparation of the solution to inject; this hypothesis was

discarded, as the cell density in the AE group is comparable with previous studies. Also, we

discarded the hypothesis that the immunohistochemistry protocol was not optimized because the

cell bodies were visible.

Those results are also distinct from unpublished and published results of the laboratory (Mouret et

al., 2008) showing that odor learning increases BrdU/DAB cell survival in the GL and in the GCL. It

is important to notice that in those studies, the protocol for olfactory learning was different. For

example, while in our study the mice just had to discriminate between two pairs of odorants during

approximately two weeks, other previous experiments in the laboratory used a higher number of

odor pairs and in Mouret et al., 2008, the number of odorants used for discrimination was two and

the training period reduced to one week. The way mice are accommodated in the cage is also a

variable to take into account. In our experiments, to reduce social deprivation, mice were not

isolated but grouped four by cage. Finally, another possible reason for this difference is that in the

paper previously mentioned, the control group was constituted by a group of sedentary mice that

were handled and stand always on his “home cage” and in our study, the mice of the AE group

were a group of mice exposed to clean air identically to the mice of the OE group except that no

odors were present in the saturators. To test this idea, it would be interesting to add another control

group of water-deprived and sedentary mice.

 

  39  

2.3) Colocalization of BrdU+ and GFP+ cells

The proportion of newborn cells co-labelled with BrdU and GFP is low. This low percentage of

colocalization (3% as a maximum under the total number of cells labelled) raises the question if we

are labelling the same cellular population. To clarify this, other experiments needs to be done.

The low level of colocalization can be due to the different timings used to inject both markers, thus,

the delay of 24h between GFP injection and the BrdU intraperitoneal injection should be

reconsidered. A possible solution would be to inject both markers at the same time point or with

different delays to determine the timing necessary for integration of the cells coming from the RMS

into the OB. Also, we could inject the GFP into the SVZ, where the cells are generated, however,

the SVZ is lining the third ventricle and is thus a large region which make the injection highly

variable from one mouse to the other.

3) Influence of training in the cell morphology

Using a virus encoding cytosolic GFP we captured the whole morphology of adult-born neurons.

We analysed the main dendrite length, the total dendritic length, the number of branch points and

the dendritic tree ‘complexity’. We didn’t observe differences between conditions for all the

parameters, except for the main dendrite length, the dendrite length was shorter in mice of the odor

learning group.

An explanation for this can be that, in contrast to the survival, the morphology of the newborn

granule cells is more resistant to the different forms of sensory experience we used in this study. It

is possible to wonder that a combination of both learning and olfaction stimuli will lead to an

inhibitory signal capable of inducing the crossing on the proximal dendrite, causing an earlier

ramification of the main dendrite.

 

  40  

In contrast to our results, an unpublished work done in the laboratory has found that olfactory

learning increases the number of dendritic nodes and ends and the dendritic length of adult-born

neurons in the OB.

4) Future work

Our analysis was done for granule cells with different morphologies and spatial locations in the

bulb. To reduce the bias, it will be interesting to redo an analysis taking into account just one type

of morphologies of the newborn granule cells. It will be also pertinent to specify the analysis to a

particular spatial location in the OB. Here, for this analysis, we considered all the newborn cells

without branch points in the proximal domain and that we could isolate, nonetheless, the most

superficial and the deeper cells may have a different time of birth or be affected by different inputs.

As previously mentioned in the introduction, neonatal GCs have a differential synaptic development

from the adult born GCs, and those cells are highly sensitive to changes in sensory inputs. Kelsch

and collaborators, in 2008, have shown that while neonatal GCs generate synapses

simultaneously in the proximal and distal domains during maturation, the adult born GCs first

develop synapses in the proximal domain and then on the distal and basal domains. When mice

are sensory deprived (Kelsch et al., 2009), the synaptic development is changed. Reduced sensory

input during synaptic development changed synaptic densities in all the dendritic domains. It is

observed an increase in the density of the glutamatergic input synapses in the proximal domain,

when the sensory deprivation occurs during the specific period for synapses formation. Even at

later stages, Mizrahi and Livneh in 2012, have shown that the adult olfactory bulb is continuously

supplied with newborn neurons that undergo experience-dependent plasticity long after maturation

and integration, as evidenced by the stabilization of synaptic turnover rates. Those studies

suggests that the highly dynamic adult born GCs are likely to be sensitive to different types of

 

  41  

learning.

According to this hypothesis, we did the acquisition of the fluorescence images of the GCs to see

whether the spine density was variable between the different groups. Are those cells branching

faster also more mature than the others? How do they communicate with the other neurons? We

couldn’t finish all the work by the end of my master’s internship and the quantification of the spines

of the newborn granule cells of the four conditions (Sup. Fig. 3) needs to be done. This analysis

may also reveal some regional increase in spines specific to each modality, like OL and OE more

on the apical (see Livneh et al., 2011) and auditory learning more on proximal and basal.

Also highly regulated by learning is adult hippocampal neurogenesis. It occurs in the SGZ, an area

enriched with different nerve terminals and subjected to dynamic circuit activity-dependent

regulation through different neurotransmitters.

Briefly, proliferating radial and nonradial precursors give rise to intermediate progenitors, which in

turn generate neuroblasts. Immature neurons migrate into the inner granule cell layer and

differentiate into dentate granule cells in the hippocampus. Within days, newborn neurons extend

dendrites toward the molecular layer and project axons through the hilus toward the CA3 (Zhao et

al., 2006). New neurons follow a sequential process for synaptic integration into the existing

circuitry (Ge et al., 2008). They are initially tonically activated by ambient GABA released from local

interneurons, followed by GABAergic synaptic inputs and finally glutamatergic synaptic inputs and

mossy fiber synaptic outputs to hilar and CA3 neurons (Ming and Song, 2011).

We wanted to study if those behavioural paradigms (processes of learning / passive exposure)

could affect adult hippocampal neurogenesis, for this, we took pictures to calculate in the future the

hippocampal cell density of Brdu+ cells.

Concerning the other purpose of this study – to evaluate the performance of the different training

groups in a working memory task – we initiated a span capacity working memory test in order to

 

  42  

see if the process of learning could have some consequences for memory.

The concept of Working Memory (WM) refers to a limited capacity system for the temporary

storage and manipulation of information. It is a system which holds information to do verbal and

non verbal tasks as those that involve monitoring and manipulation of information in the face of

interfering processes and distractions, this process requires integration followed by processing,

disposal and then retrieval of information (Baddeley et al., 2012). Surprisingly, it remains unknown

the contribution of OB neurogenesis for odor working memory (the ability to temporarily retain an

odor information).

The span capacity working memory test performed consists of different steps. Firstly, the food

restricted mice had to learn the non matching to sample rule (NMTS) (Sup. Fig. 3A). In each

session, the mouse was moved to a platform where he had firstly to dig into a cup with a specific

scent and find the cereal (reward) and then, he returns to the same platform where he has to

discriminate between the new and the old scent and dig into the new one to find the reward. This

procedure is repeated for 12 pairs of odorants within a session. Between sessions, to avoid spatial

learning, the cups were moved to different places of the platform and the pairs of odorants

changed. The number of sessions was conditioned by the mice performance, within a condition,

they had to reach a steady state accuracy of 85% of correct responses.

Once reaching the criteria, mice had to use the NMTS rule previously acquired with different delays

(odor delayed NMTS) (Rushforth et al. 2011). They had to remember for different periods of time

the rule acquired and use it to dig into the correct cup and find the reward. The mice performance

in this test would reveal for how long can a mouse remember a specific odorant and if the different

training conditions can have a consequence on memory.

The preliminary results of half of the animals in the acquisition of the NMTS rule are represented in

the supplemental data (Sup. Fig. 3B).

 

  43  

Conclusion

This study provided some insights into the role of learning for adult neurogenesis.

Our results have confirmed the good performance of the mice in an odor discrimination task and for

the first time in an auditory discrimination task. As a consequence for cell survival, the number of

newborn cells GFP positive was increased in the three conditions tested (odor learning, auditory

learning and odor enrichment) when compared to the AE group. However, the increase was just

significant in the OL group. Would we obtain the same significant increase in cell survival of the OL

group if the AL mice reached the same performance in the auditory discrimination task as the OL

mice in the odor discrimination task? Or on the other hand, is the biggest increase in cell survival in

the OL group due to an addictive effect of learning and olfactory exposure?

The significant rise in cell survival in the OL group is apparently a consequence of a combination of

the learning and odor exposure slight increase on cell survival.

The impact of auditory learning on OB neurogenesis suggests that bulbar neurogenesis can be

boosted without olfactory inputs and in a same level as a simple exposure to odorants. The fate of

the adult born cells in the OB is sensitive to the changes induced by learning even if this learning

process relies on a different sensory modality than olfaction.

In the same vein, it will be interesting to do a similar study but analysing the potential

consequences of the integration of different sensory stimuli in the bulbar neurogenesis.

Whether or not the cell morphology is shaped by the different behavior conditions is another

intriguing question that was raised in this study. Surprisingly, the different behaviour paradigms

used didn’t cause a strong impact on cell morphology as on cell survival with odor learning being

the only condition able to induce a change in cell morphology by decreasing the length of the adult

born GC main dendrite.

 

 

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  a  

Supplemental data

Supplemental Table 1. Number of mice trained within session and within condition according to the aims of the study.

 

  b  

Supplemental Figure 1: Performance of the mice used for cell survival and morphological analysis of the groups OL (Odor Learning, n=6) and AL (Auditory Learning, n=8) in session 1 and 2. Each dot corresponds to the mean +/- sem of all the mice in that block (20 trials), to remind 10 blocks were performed within a day. The dashed line represents chance level (50%). A. First odor discrimination task of the OL group, Anisol (10 -2 ) vs Cineol(10 -2 ). B. Second discrimination task of the OL group, Linalool (10-2) vs β-ionone (10 -2 ). C. First auditory discrimination task of the AL group, 8kHz vs 20kHz. D. Second discrimination task of the AL group, 12kHz vs 17kHz.

A B Anisol (10-2) vs Cineol (10-2)

Blocks (x20 trials)

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Linalool (10-2) vs b-damascenone (10-2)

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chance level chance level

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Anisol (10-2) vs Cineol (10-2) Linalool (10-2) vs β-ionone (10-2)

C D

 

  c  

Supplemental Figure 2: Analysis of the different morphological parameters of the newborn GCs according to the condition. We took into consideration for this analysis only the newborn GCs with more than one branch point. The length is expressed in μm. The illustration above each graphic represents in red what was measured. A. Proximal dendritic length (first segment from the soma to the first branch point) of the different conditions expressed by the mean length +- sem of the group, in μm. B. Total dendritic tree length of the different conditions expressed by the mean length +- sem of the group, in μm. C. Number of branch points of the four conditions expressed as the mean number of the group (+- sem). D. Quantification by Sholl analysis of the number of intersections between dendritic segments and virtual concentric lines centered on the soma and spaced by 10μm. Each curve represents the mean of the mice of each condition. E. Quantification by Sholl analysis of the number of intersections between dendritic segments and virtual concentric lines centered on the first branch point and spaced by 10μm. Each curve represents the mean of the mice of each condition.

Proximal dendritic lenght

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Number of branch points

olfactorylearning

auditorylearning

odorexposure

airexposure

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auditorylearning

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D E

A B C

 

  d  

Supplemental Figure 3: Terminal of the main dendrite of a granule cell of an AL mouse. In yellow, is visible a detail of a dendrite. In a red circle, it is visible a spine.

 

  e  

Supplemental Figure 4: Performance of the mice in the first part of a span capacity working memory test. A. Schematic representation of the odor non matching to sample task, each session ends after 12 trials with different odorants, in each trial the mouse has to recognize the new odorant and dig in the sand to find the food reward (cereal). B. Performance of the mice of the different conditions, correct responses according to the number of sessions.

B

Odor non matching to sample task

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1 2 3 4 5

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OL Group AL Group OE Group AE Group

A