Neuronal integration in the adult olfactory bulb is a non-selective addition process

Adult neurogenesis is considered a competition in which neurons during a critical period for integration and survival. Moreover, newborn neurons are thought to replace preexisting ones that die. Despite a wealth of evidence supporting this model, systematic in vivo observations of the process are still scarce. We used 2-photon in vivo imaging combined with low dose thymidine analog pulse chase experiment to study neuronal integration and survival in the olfactory bulb (OB). We show that cell-loss in the OB occurs only at low levels. Neuronal death resembling a critical period was induced by standard doses of BrdU or EdU, but disappeared when low doses of EdU were used, demonstrating toxicity. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur during integration and argues against the existence of a critical period for survival. Moreover, the OB is not a “turnover” system but shows lifelong neuronal addition.


Introduction
Neurogenesis continues after birth in the hippocampus and olfactory bulb of rodents. During OB neurogenesis predetermined stem cell population along the walls of forebrain ventricles generate neuronal precursors that migrate via the rostral migratory stream (RMS) into the center of the OB. After their radial migration into the principal target layers, the granule cell (GCL) and glomerular layers (GL), cells integrate into the preexisting circuitry and function as interneurons using GABA and dopamine as their principal neurotransmitters (Whitman and Greer, 2007).
The currently available information indicates that OB neurogenesis is based on two key principles: First, neuronal integration in the adult is a competitive process, during which large numbers of newly arriving neurons compete for integration into the circuitry and ultimately survival. This competition is thought to occur during a defined critical window of 2-8 weeks after arrival and leads to the apoptotic elimination of about half of the initial population (Bergami and Berninger, 2012;Lledo et al., 2006;Mandairon et al., 2006;Petreanu and Alvarez-Buylla, 2002;Winner et al., 2002;Yamaguchi and Mori, 2005). Second, the OB represents a turnover system, in which newly integrating cells replace preexisting ones, leading to a relatively stable total number of neurons in the target layers (Bergami and Berninger, 2012;Imayoshi et al., 2008;Lledo et al., 2006). These two concepts are to a large extend based on lineage tracing experiments using thymidine analogs like bromodeoxyuridine (BrdU) or 3H-thymidine to label the DNA of dividing cells, (Mandairon et al., 2006;Petreanu and Alvarez-Buylla, 2002;Winner et al., 2002;Yamaguchi and Mori, 2005). A common observation in such experiments is a loss of labeled cells during the first few weeks after their arrival in the olfactory bulb, which led to the postulation of a selection mechanism allowing the remodeling of specific OB circuits during a critical window for survival (Petreanu and Alvarez-Buylla, 2002).

Alternatively, genetic approaches using CRE-inducible markers layers have been performed
and demonstrated an accumulation of adult born neurons in the OB over time (Imayoshi et al., 2008). In agreement with the turnover model this has been interpreted as a replacement of older neurons that died (Imayoshi et al., 2008). Only recently more direct approaches based on 2-photon in vivo imaging allowed studying OB neurons directly in the living animal (Mizrahi et al., 2006;Sailor et al., 2016;Wallace et al., 2017). Interestingly, long-term observation of either juxtaglomerular neurons in general (Mizrahi et al., 2006), or more specifically of dopaminergic neurons, demonstrated an increase in these populations over time (Adam and Mizrahi, 2011). While at first sight this finding contradicts a pure replacement model, it was interpreted as a change in the interneuron subtype composition of the OB (Adam and Mizrahi, 2011).
Thus, while the available data is still mostly indirect and fragmentary, the elegant model based on selection and replacement appears justified. However, to doubtlessly validate this model and to really understand the adult neurogenic process, all populations of integrating neurons have to be observed in the living animal from their arrival in the OB throughout the critical window until their disappearance.
Here we combined genetic birthdating and lineage tracing with long term in vivo microscopy to follow timed cohorts of postnatal and adult born neurons from their arrival in the OB for up to six months. Quantitative analyses demonstrate that neuronal loss during the critical period and at later stages is rare in all observed populations. We demonstrate that classically used doses of the tracers BrdU and 5-ethynyl-2'-deoxyuridine (EdU) induce cell loss resembling a critical window, but that this toxicity disappears when low doses of EdU are used. Finally, we show that neuronal addition merely than replacement occurs in the adult OB, leading to permanent growth of the structure.

Long term in vivo imaging of postnatal and adult born OB neurons
We used 2-photon imaging to directly study the integration and survival of perinatal and adult born OB neurons at high spatial and temporal resolution in the living animal. We first focused on the perinatal period, when most OB interneurons are generated (Batista-Brito et al., 2008). Postnatal in vivo brain electroporation of the dorsal ventricular zone targets stem cell populations that generate neurons for the superficial layers of the OB (Fig 1a, b (de Chevigny et al., 2012b), which can be reliably reached by two-photon microscopy (Adam and Mizrahi, 2011). We used this dorsal targeting approach to introduce a CRE-expression plasmid into R26-RFP reporter mice (Fig 1a). Three weeks later, OB labeled neurons comprised a mixed population of 6% tyrosine hydroxylase expressing dopaminergic neurons, 12% calretinin positive purely GABAergic neurons, 22% other PGN (Fig 1b) and 60% mostly superficially positioned granule cells (GC).
An adaptation of the reinforced thin skull method allowed for frequent and long term imaging of awake mice while perturbing the physiology of the OB only minimally (Drew et al., 2010).
In agreement with previous observations (Xu et al., 2007), there was no detectable astroglia reaction or accumulation of microglia after thinning and window implantation ( Fig S1).
Three weeks after electroporation, when skull growth was sufficiently advanced, thin-skull preparation was performed and the same population of neurons in the glomerular layer (GL; Fig 1d-g) and the granule cell layer (GCL, Fig S2) were imaged in awake animals at high resolution over the following weeks and months. All analyzed neurons were individually identified in Z-stacks (Supplementary Video 1) based on relative position and morphology.
Neurons were numbered and revisited weekly during the critical period (Fig 1d- Fig S3a,b). After identification of the first cohort, smaller numbers of additional neurons appeared permanently in the observation window as a consequence of ongoing neurogenesis in the stem cell compartment (arrowheads in Fig 1d, 5wpi). These were numbered and followed like the first cohort. Neurons in the observation field showed stable relative positions over time (Fig 1d), however, in some cases minor positional adjustment were observed that could be followed over subsequent imaging sessions (Fig  1d,f). Generally, resolution was sufficient to observe even minor changes in dendritic organization of neurons over time (see neuron no. 7 in Fig 1d,f).

Cell death during the critical period: perinatal born neurons
Based on this direct and systematic imaging approach, we first focused on perinatally born neurons during the proposed critical period, thus until 8 weeks after their generation at the ventricles (Mandairon et al., 2006). Neurons that were present during the first observation time point (3 weeks after electroporation of the respective stem cells) were followed over the next 5 weeks. Among 755 periglomerular neurons (PGN) in 11 mice only 5.1% were lost over the proposed critical period (Fig 2a, see circles for lost cells no. 14 and 17 in Fig 1d,g). The percentage of lost neurons was very similar between individual animals and was independent of the density of labeled cells in the observation window (between 18 and 100 neurons; Fig 2a).
Next, we investigated newborn granule cells (GCs) in the underlying GCL in 6 mice with particularly high-quality and stable window preparations ( Fig S2). Out of 178 RFP positive neurons observed between 3 and 8 weeks after their birth not a single cell disappeared over the subsequent imaging sessions (Fig 2b). We conclude that perinatally generated OB interneurons in both, the GL and the GCL are rarely eliminated during the proposed critical period.

Cell death during the critical period: adult born neurons
We then investigated the stability of adult born neurons during the critical period. First, we focused on PGN that can be reliably imaged after thin skull preparation. As in vivo electroporation is inefficient in adult mice we crossed the Rosa-RFP line with Nestin Cre-ER T2 mice (Lagace et al., 2007) and induced a heterogeneous cohort of labeled newborn neurons by tamoxifen injection at 2 months of age (Ninkovic et al., 2007) (Fig S4 a,b). One week after induction virtually all RFP positive cells in the RMS and about 30% in the OB layers expressed the immature neuron marker doublecortin (Fig S4c,d). Reinforced thin skull preparation was routinely performed at 1-week post induction (wpi). Weekly observations of individually identified PGN in the GL were performed as described above (Supplementary Video 2,Fig S3c,d). Analyses of 538 periglomerular neurons of the first cohort in 8 animals showed that only 1.5% disappeared over the 7-weeks period after their first identification ( Fig. 2c). Next we asked if neuron loss could be detected in non-physiological situations. It has been shown that olfactory sensory deprivation induces cell death in adult born OB neurons (Mandairon et al., 2006;Saghatelyan et al., 2005;Yamaguchi and Mori, 2005). To investigate if increased cell death could be observed in our imaging paradigm we performed naris closure in adult Nestin Cre-ER T2 /Rosa-RFP mice one week after tamoxifen induction (Fig 4a). Analysis of RFP positive PGN over the following 8 weeks revealed a significant increase in cell loss (Fig 4bc; p=0.03, 4 control and 3 occluded animals, 151 cells).
In conclusion, under physiological conditions newly born neurons in the perinatal and adult OB show very little cell loss during the proposed critical period. However, significant cell loss during this period was found after blocking sensory input, demonstrating that cell death could be detected with our approach.

Dose dependent toxicity of thymidine analogues
The above findings were at odds with the existence of a critical period for survival during which, under normal conditions, about half of the adult born neurons are removed from the OB by cell death (Mandairon et al., 2006;Mouret et al., 2008;Petreanu and Alvarez-Buylla, 2002;Winner et al., 2002;Yamaguchi and Mori, 2005). This concept is to a large extent based on tracing of timed cohorts of newborn neurons using the integration of thymidine analogs, most often BrdU, into the DNA of dividing cells. To investigate if these differences were due to our particular experimental conditions we first repeated such pulse chase studies using commonly used doses of BrdU and following established protocols (Mandairon et al., 2006;Mouret et al., 2008;Whitman and Greer, 2007). Indeed, using four i.p. injections of 50 mg/kg BrdU every two hours into adult mice, we found an approximately 40% loss of labeled neurons in the OB between 2 and 6 weeks in the GL as well as in the GCL (Fig 5ab).
As in our direct imaging approach we focused on the dorsal aspect of the OB, we investigated if in this region BrdU positive cells showed a different behavior than in the rest of the structure. BrdU+ cell number in the dorsal OB showed the same 40% loss that was found in the entire bulb (Fig.5b).
Altogether, these DNA-labeling based findings were in full agreement with previous studies, showing a strong reduction of newborn cells during the critical period (Mandairon et al., 2006;Mouret et al., 2008;Petreanu and Alvarez-Buylla, 2002;Whitman and Greer, 2007;Winner et al., 2002;Yamaguchi and Mori, 2005). However, they strongly contradicted our in vivo observations showing very little cell loss. As suggested before (Lehner et al., 2011), we considered the possibility that incorporation of modified nucleotides impacted on neuronal survival in the OB and developed an approach to test this hypothesis.
To allow immunohistological BrdU detection, tissue samples have to be subjected to strong denaturing conditions that break the complementary base-pairing of DNA, a prerequisite for efficient BrdU antibody binding. Such treatment invariably leads to sample degradation and negatively impacts on staining intensity (Salic and Mitchison, 2008). In concert with limitations due to antibody penetration this imposes the use of relatively high concentrations of BrdU, generally several injections of 50 to 200 mg/kg of body weight i.p., for reliable detection (Brown et al., 2003;Mandairon et al., 2006;Mouret et al., 2008;Petreanu and Alvarez-Buylla, 2002;Whitman and Greer, 2007;Winner et al., 2002). In contrast, EdU labeling of DNA is based on a click-reaction with fluorescent azides (Rostovtsev et al., 2002;Salic and Mitchison, 2008;Tornoe et al., 2002) that have much higher diffusion rates in tissue than antibodies. Moreover, the staining reaction can be can be repeated several times to increase signal strengths and DNA denaturation is dispensable, altogether allowing the use of considerably lower concentrations of EdU in comparison to BrdU (Salic and Mitchison, 2008). Based on this increased sensitivity, we asked if the concentration of altered nucleotides in the DNA impacts on cell survival of new neurons in the OB.
Four injections of 50 mg/kg EdU in two month old mice led to an about 40% loss of labeled cells in the GL and the GCL of the OB between 2 and 6 weeks, highly comparable to the results based on BrdU (Fig 5c,e). Four injection of 1 or 5 mg/kg EdU under the same conditions led to the detection of slightly lower amounts of newly generated cells in the OB layers after 2 weeks (Fig. 5de). Importantly, under these conditions the decrease in cell numbers between 2 and 6 weeks was not detectable anymore, both in the GL and the GCL indicating that thymidine induced toxicity did not pass via the apoptotic pathway (Fig. 5i).
In conclusion, the above results, showing that lineage tracing by high doses of thymidine analogues is associated with artificial cell loss in the OB, point to considerable toxicity of such DNA modifying agents. Moreover, the finding that at low EdU doses cell loss in the OB during the proposed critical period is non-detectable represents independent confirmation of our in vivo imaging based findings.

Neuronal addition in the OB
The OB is considered to be a turnover system in which new neurons replace older ones, leading to a relatively stable size of the structure (Imayoshi et al., 2008;Petreanu and Alvarez-Buylla, 2002). In such a scenario cell loss has to be expected. As we did not observe considerable cell death during the proposed critical period, we asked if neurons disappear at later stages. Continuous long-term observations of perinatally generated PGN and GC provided no evidence for sustained cell loss after the initial 8 weeks time window (Fig 6a,b).
The same stability of the labeled population was evident when adult generated PGN were observed for up to 24 weeks after their generation (Fig. 6c). Moreover, as CRE-induced recombination in Nestin-CRE-ERT2 mice occurs often at the stem cell level (Imayoshi et al., 2008), recombined stem cells continue neurogenesis. In agreement, additional adult born neurons permanently appeared in the observation window (Fig 6d), leading to a more than doubling of the neuron population of adult generated PGN over an observation period of six months (Fig. 6e).
How does the OB deal with this permanent addition of neurons in the absence of considerable cell loss? Two potential consequences can be imagined: Either the OB grows in 1 0 about these parameters in the adult rodent OB is based on measurements of serial sections and the available data are fragmentary and in part contradictory (Hinds and McNelly, 1977;Imayoshi et al., 2008;Mirich et al., 2002 ;Petreanu and Alvarez-Buylla, 2002;Pomeroy et al., 1990;Richard et al., 2010).
First, we asked if a volume increase in the OB could be detected directly in the living brain during in vivo imaging experiments. We found that over time slightly larger image frames were necessary to accommodate the same group of neurons in our Z-maximum projections of the GCL and GL (Fig 1d; Fig S2b). Using this systematic imaging, we developed an approach to quantify local changes in OB volume over time based on measuring distance between individually identified neurons. Indeed, volumetric analysis of inter-neuronal space between groups of four neurons in X, Y, Z (thus an irregular pyramid) demonstrated that distance between neurons increased steadily between 2 and 5 months ( Next, we investigated the evolution of cell density in the more homogeneous granule cell layer using transparent brain tissues. To count all cells in the GCL we stained nuclei with the fluorescent marker TOPRO3. Quantification revealed that the density of nuclei was highly stable at all observed time points (Fig 7 h,i) while the density of astrocytes decreased and microglia density was unchanged (Fig. S5b,c).
Thus, both in vivo brain imaging and light sheet microscopy of fixed tissue demonstrate that the mouse OB grows significantly during adult life in the absence of detectable changes in 1 cell density. This is in strong support of the permanent addition of new neurons to a stable preexisting circuitry in the absence of substantial cell death.

Discussion
Our work combining long term in vivo observations, pulse chase experiments and 3D morphometric analyses, leads to three main conclusions: First, the level of neuronal cell death among perinatal and adult born OB interneurons is very low. Second, the adult OB is not a homeostatic, but a developing and growing brain structure. Third, classical lineage tracing approaches based on thymidine analogs are associated with unwanted side effects and have to be interpreted with care.
Using a non-invasive long-term imaging approach combined with lineage tracing approaches using low concentrations of the thymidine analogue EdU, we were unable to detect However, both BrdU and 3H-dT are toxic (Breunig et al., 2007;Ehmann et al., 1975;Kolb et al., 1999;Kuwagata et al., 2007;Nowakowski and Hayes, 2000;Sekerkova et al., 2004;Taupin, 2007) and studies in both rodents (Lehner et al., 2011;Webster et al., 1973) and primates (Duque and Rakic, 2011) pointed to unwanted, and hard to interpret, long term effects associated to their use. Accordingly, warnings concerning the interpretation of such data have been issued (Costandi, 2011;Lehner et al., 2011).
In agreement with the existing literature we observed massive loss of newborn neurons in the OB during the critical period when standard doses (4x50 mg/kg) of BrdU or EdU were used for tracing. Interestingly, in the presence of considerably lower concentrations of EdU (4x1 mg/kg) neurogenesis was still obvious but cell loss during the critical period was not detectable anymore. This finding is in perfect agreement with our in vivo observations, in which we find almost no neuronal death during the critical period. Thus, death of newborn neurons during the critical period appears to be tightly linked to the concentration of thymidine analogues used for their detection.
These results lead to the conclusion that a selection step in which an overproduced precursor population is matched to the needs of the target structure, does not occur during synaptic integration in the OB. While this observation is unexpected it is not completely isolated. For example, in Bax-KO mice, in which apoptotic cell death is blocked, the general structure and size of the OB neuron layers are indistinguishable (Kim et al., 2007). However, Bax-mutants show a strong disorganization and accumulation of neuronal precursors in the RMS, pointing to the possibility that neuronal selection occurs more at the level of recruitment from the RMS than at the level of integration in the target layers. Such a scenario of "early selection" is also supported by the observation that the density of apoptotic cells is much higher in the RMS than in the OB proper (Biebl et al., 2000). However, other scenarios, like an impact of altered migration on survival in the OB, cannot be excluded.
Alternatively, integration or death of OB interneurons might be intrinsically encoded. It has been shown that in developing cortical interneurons neuronal survival is largely independent of signals from the local environment but that about 40% of the total population is predestined to undergo Bax-dependent apoptosis (Southwell et al., 2012). In such a scenario cell death would be expected to occur already in the SVZ/RMS.
Our results demonstrate that neuronal death is a rare event not only during the postulated critical selection period, but at all observed time points. However, the permanent arrival of new neurons in the absence of considerable cell removal is not compatible with the current idea that the OB represents a turnover system of constant size (Bergami and Berninger, 2012;Imayoshi et al., 2008). Growth has to be expected, and our in vivo imaging and light sheet microscopy studies clearly demonstrate a 40% volume increase during the first year of adulthood in the absence of detectable changes in cell density. Indeed, growth of the adult OB in mice has been observed in other studies, although considerable variation and dependence on genetic background have been reported (Mirich et al., 2002;Richard et al., 2010). Other studies did not find considerable differences in total OB size or specific sublayers (Imayoshi et al., 2008;Petreanu and Alvarez-Buylla, 2002;Pomeroy et al., 1990).
What could be the reason underlying these contradictory findings? Past approaches were based on the 2D analysis of a subset of tissue sections and the extrapolation of the total volume based thereon. However, the OB is not a simple radial symmetric globule, but a complex multi-layered structure that shows huge variations along the rostro-caudal and dorso-ventral axes (see Supplementary Video 3). Measuring a limited amount of tissue sections may not sufficiently consider longitudinal growths and intra-bulbar variations.
Light sheet microscopy is suited to overcome many of these limitations as the OB is imaged and measured in its entirety. Extrapolations can be avoided and the selection of comparable levels for layer analyses is simple and reliable. Increases in OB lengths are directly obvious and do not have to be deduced from varying numbers of sections that can be generated from a given bulb. As a consequence inter-animal variations are minor and growth of the structure becomes evident. Independently from the light-sheet approach we show that during in vivo long-term observations constantly larger frames are needed to accommodate the same group of cells and that the distance of individually identified neurons measurably increases.
The latter finding is in full agreement with the finding that the distance between specific glomeruli increases with age of the animal (Richard et al., 2010). Altogether, these data clearly demonstrate growth of the mouse OB during the entire first year of the animal's life.
In conclusion, we show here that neuronal cell death is rare in the OB and that neuronal         Nestin-CreER T2 X rosa-RFP mice were used between 2 and 3 month old at the time of surgery.

In vivo labeling of neurons
In vivo electroporation was performed as previously described (Boutin et al., 2008). Briefly, 1day-old pups were anaesthetized by hypothermia and 1μl of a pCAG-CRE) plasmid (Platel et al., 2010) at 4μg/μl) was injected in the lateral ventricle. Electrical pulses were applied to target the dorsal V-SVZ.

Surgical preparation
Implantation of an observation window was performed as previously described (Drew et al., 2010) but with minor modifications. Briefly, mice were anaesthetized by intraperitoneal (ip.) injection of ketamine/ xylazine (125/12,5 mg/kg). Dexaméthasone (0.2 mg/kg) and buprenorphine (0.3 mg/mL) were injected subcutaneously and lidocaine was applied locally onto the skull. The pinch withdrawal reflex was monitored throughout the surgery, and additional anesthesia was applied if needed. Carprofen (5 mg/kg) was injected ip. after the surgery. A steel bar was added during this step to allow fixation of the animal to the microscope. The skull overlying the OB was carefully thinned with a sterile scalpel blade until a thickness of 10-20 μ m was reached. A thin layer of cyanoacrylate (superglu3, Loctite) was applied and a 3mm round coverslip was apposed and sealed with dental cement (superbond, GACD). A first microscopic observation was performed on these anesthetized mice.
Efficiency of occlusion was checked the following day and before each imaging session. At the end of the experiment immunostaining against tyrosine hydroxylase was performed to confirm the efficiency of occlusion.

In vivo two-photon imaging
We used a Zeiss LSM 7MP two-photon microscope modified to allow animal positioning under a 20X water immersion objective (1.0 NA, 1.7mm wd) and coupled to a femtosecond pulsed infrared tunable laser (Mai-Tai, SpectraPhysics). After two-photon excitation, epifluorescence signals were collected and separated by dichroic mirrors and filters on 4 independent non-descanned detectors (NDD). Images were acquired using an excitation wavelength of 950 nm. RFP was first collected between 605-678. In addition, we collected an additional RFP signal between 560-590 that was voluntarily saturated to allow a better identification of subcellular structures like dendrites.
In general, image acquisition lasted about 10 min. Mice could potentially move on a treadmill during imaging, but rarely did so. On consecutive observation, the same field of view was Analysis: Z stacks taken with a 1 or 2 µm z-step were used for tracking cells over weeks.
Maximum intensity projections were created and annotated manually in ImageJ and crossreferenced with z-stacks to confirm that the dendritic structure and location of a cell allowed unambiguous identification. Each line in 3C represents cells tracked for different lengths of time, and multiple lines may correspond to a single field of view. For example, this imaged field of view corresponds to the two lines representing 3 cells tracked over time, with one line ending at 5 weeks and one ending at 7 weeks (due to the final z stack not extending deep enough to include the first 3 cells). The newcomers arrived at different times, so they have different lines. For example, some of the cells with asterisks arrived at 3 weeks and others arrived at 4 weeks. Incoming cells not marked with asterisks had cell bodies that either were not fully included within the z stack or we were not able to track them for more than one imaging session and so were not quantified.

Lightsheet microscopy
To render brains transparent we followed the Cubic protocol (Susaki et al., 2014). Briefly, brains where incubated in Cubic1 solution for 10 days at 37°C using gentle agitation. After clearing, brains were incubated for 1 day in the red nuclear dye TOPRO3 (1/1000) in PBS, 0.01% Tween 20, 0.01% sodium Azide at 37°C. The brains were then re-incubated for 3 hours in Cubic1 solution and subsequently placed in Cubic2 solution for 2 days at 37°C.
Timing of all steps was carefully monitored.
We used a lightsheet Z1 microscope (Zeiss) with a 5x/0.16NA objective to image the transparized OB and UltraMicroscope II (LaVision BioTec) with LWDO 2x/0.14NA for whole brain imaging. OB layers were easily distinguishable using the nuclear staining of the TOPRO3. The OB was imaged every 5.9 μ m in Z with a xy pixel dimension of 2.5 μ m and the whole brain with 30 µm steps in Z and a xy resolution of 3.03 µm, respectively. We used Imaris software (Bitplane, Germany) for reconstruction of the total volume based on the nuclear TOPRO3 staining. To determine total forebrain size we measured the entire volume from the caudal end on the OB to the caudal end of the neocortex. All measurements were normalized to the mean obtained on 2 month old brains.

Measurement of cell density
We measured cell density in the granule cell layer by imaging the same transparized brain with the 2-photon microscope used for in vivo imaging to obtain a better resolution. We acquired Z stacks of 200 μ m with 2μm resolution in Z and 0.3 μ m in xy in the central part of the OB. These images were first de-noised in Fiji using a 3D mean filter. Then the volumetric density of nuclei was quantified using Imaris software: We use the cell detection module to detect nuclei in the granule cell layer. We used 4 μm as a seed point value to split the connected objects.

Immunohistochemistry
Stainings were done on 50 μm floating vibratome sections as described before (