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The olfactory critical period is determined by activity-dependent Sema7A/PlxnC1 signaling within glomeruli

  1. Nobuko Inoue
  2. Hirofumi Nishizumi
  3. Rumi Ooyama
  4. Kazutaka Mogi
  5. Katsuhiko Nishimori
  6. Takefumi Kikusui
  7. Hitoshi Sakano  Is a corresponding author
  1. Department of Brain Function, School of Medical Sciences, University of Fukui, Japan
  2. Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, Japan
  3. Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Japan
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Cite this article as: eLife 2021;10:e65078 doi: 10.7554/eLife.65078

Abstract

In mice, early exposure to environmental odors affects social behaviors later in life. A signaling molecule, Semaphorin 7A (Sema7A), is induced in the odor-responding olfactory sensory neurons. Plexin C1 (PlxnC1), a receptor for Sema7A, is expressed in mitral/tufted cells, whose dendrite-localization is restricted to the first week after birth. Sema7A/PlxnC1 signaling promotes post-synaptic events and dendrite selection in mitral/tufted cells, resulting in glomerular enlargement that causes an increase in sensitivity to the experienced odor. Neonatal odor experience also induces positive responses to the imprinted odor. Knockout and rescue experiments indicate that oxytocin in neonates is responsible for imposing positive quality on imprinted memory. In the oxytocin knockout mice, the sensitivity to the imprinted odor increases, but positive responses cannot be promoted, indicating that Sema7A/PlxnC1 signaling and oxytocin separately function. These results give new insights into our understanding of olfactory imprinting during the neonatal critical period.

Introduction

Mammalian sensory systems are generated by a combination of activity-dependent and -independent processes. The basic architecture of sensory systems is built before birth based on a genetic program (Hong and Luo, 2014; Luo et al., 2008; Yogev and Shen, 2014). However, the sensory maps and neural circuits are further refined in an activity-dependent manner (Espinosa and Stryker, 2012; Hensch, 2005; Hooks and Chen, 2007; Kirkby et al., 2013; Okawa et al., 2014). In the mouse olfactory system, a coarse map is generated independently from neuronal activity (Mori and Sakano, 2011). Axon targeting along the dorsal-ventral (D-V) axis is regulated by positional information of olfactory sensory neurons (OSNs) (Takeuchi et al., 2010). In contrast, targeting along the anterior-posterior (A-P) axis is instructed by odorant receptor (OR) molecules using cAMP as a second messenger (Imai et al., 2006). Each OR species generates a specific level of cAMP by intrinsic, non-neuronal OR-activity (Nakashima et al., 2013) that determines expression levels of A-P targeting molecules including Semaphorin (Sema) 3A, Neuropilin (Nrp) 1, and Plexin (Plxn) A1 (Imai et al., 2009). The map is then refined by axon-sorting molecules, for example, Kirrel 2/3 and Ephrin A/eph A receptors (Serizawa et al., 2006) whose expression is regulated by neuronal activity using cAMP generated by olfactory G protein (Golf) and cyclic-nucleotide-gated (CNG) channels (Sakano, 2010). It has been reported that this activity for the map refinement is intrinsic and not odor-evoked (Nakashima et al., 2019). Intrinsic OSN activity is also needed for synapse formation within glomeruli. Inoue et al., 2018 found that odor-evoked OSN activity further promotes synapse formation and dendrite selection in mitral/tufted (M/T) cells using Sema7A/PlxnC1 signaling.

In neonates, there is a narrow time frame, referred to as the critical period, that allows proper development in response to environmental inputs. This activity-dependent process is initially plastic but soon becomes irreversible. If the circuit is left unstimulated, the brain function served by that circuit is impaired. Thus, sensory inputs during the critical period are important to make the system functional. Unlike other sensory systems, the olfactory system constantly regenerates OSNs throughout the animal’s life span (Costanzo, 1991; Graziadei and Monti Graziadei, 1985; Leung et al., 2007). Although OSNs are replaced and form new connections with second-order neurons, proper circuits cannot be regenerated once the existing OSNs are completely ablated after the early neonatal period. This time frame of olfactory circuit formation has been referred to as the olfactory critical period (Ma et al., 2014; Tsai and Barnea, 2014). However, the olfactory critical period has not been defined at the molecular level, and the key process that makes this time-window critical has yet to be clarified.

In mammals, neonatal exposure to environmental odorants can affect odor perception and behavior later in life (Logan et al., 2012; Mennella et al., 2001; Sullivan et al., 2000; Wilson and Sullivan, 1994). Salmon and trout return to their home river based on olfactory memory (Nevitt et al., 1994). In the visual system, ducklings follow the first moving object upon hatching recognizing it as the parent bird (Horn, 1998; Nakamori et al., 2013). Although such phenomena are widely known, little is known about how imprinting is established and how the imprinted memory modulates innate behavioral decisions.

In an effort to address these unanswered questions about the olfactory critical period, we studied olfactory imprinting in mice by analyzing a pair of signaling molecules, Sema7A and PlxnC1, which induces post-synaptic events within glomeruli. We found that Sema7A/PlxnC1 signaling enlarges glomeruli resulting in increased sensitivity to experienced odors. Neonatal odor experience also induces positive responses to the imprinted odor memory. Separately from Sema7A/PlxnC1 signaling, oxytocin in neonates is responsible for imposing the positive quality on imprinted memory.

Results

Naris occlusion/reopen experiments

In order to precisely determine the critical period that causes plastic changes in synapse formation and odor detection, we performed naris occlusion/reopen experiments (Figure 1A and Figure 1—figure supplement 1). Unilateral naris occlusion at postnatal day 0 (P0) was continued through various time points, and resultant synapse markers within glomeruli were examined at P21. We found that the levels of a pre-synapse marker vGlut2 (Honma et al., 2004) and a post-synapse marker GluR1 (Montague and Greer, 1999), were normal when the occluded naris was reopened at P6 or before. However, both markers decreased and remained low when the occluded naris was reopened after P8.

Figure 1 with 1 supplement see all
Unilateral naris occlusion in the mouse neonates.

(A) Effects of naris occlusion on the expression of synapse markers within glomeruli. Mice were unilaterally occluded at P0 and the occluded naris was reopened at various time points. OB samples were isolated at P21 and analyzed by immunostaining for pre- and post-synapse markers, vGlut2 and GluR1, respectively. Relative staining levels of these markers in the glomerular layer are compared. Error bars are SD (n = 10 glomeruli for each condition). See also Figure 1—figure supplement 1. (B) Odor detection in the naris-occluded mice. Six-week-old mice used in this assay were unilaterally naris-occluded at P0 and the occluded naris was reopened at P6 (green) or P10 (red). Mice without occlusion (–) were analyzed as controls (black). Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Next, a filter paper spotted with the 1st odor was presented three times (detection trials 4–6). Then, a filter paper spotted with the 2nd odor was presented three times (detection trials 7–9). Investigation times (sec) observed during each presentation were measured. Odorant pairs examined were as follows: left, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and right, 62 mM (+)-CAR and 64 mM EUG. Error bars are SD (n = 15, 12, 10 animals, 10 litters). ***p<0.005 (Student’s t-test). n.s., not significant. CAR, carvone; EUG, eugenol.

Figure 1—source data 1

Odor detection in the naris-occluded mice.

Six-week-old mice used in this assay were unilaterally naris-occluded at P0 and the occluded naris was reopened at P6 or P10. Mice without occlusion (–) were analyzed as controls. Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Next, a filter paper spotted with the 1 st odor was presented three times (detection trials 4–6). Then, a filter paper spotted with the 2nd odor was presented three times (detection trials 7–9). Investigation times (s) observed during each presentation were measured. Odorant pairs examined were as follows: top, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and bottom, 62 mM (+)-CAR and 64 mM EUG. CAR, carvone; EUG, eugenol. The average values of investigation times are shown.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig1-data1-v1.xlsx

We then studied the effect of naris occlusion on odor detection in the habituation/dishabituation test (Figure 1B). The ability to sense odors using the occluded naris was tested by plugging the non-occluded naris at the 6th week of life (6w). When the occluded naris was reopened at P6, mice demonstrated normal abilities to detect odor information. In contrast, when the occlusion was continued to P10, the mice demonstrated reduced responsiveness to odorants and poor ability to discriminate enantiomers, (+) and (–) carvones (CARs). Taken together, the critical period in the mouse olfactory system appears to be restricted to the early neonatal period, approximately during the first week after birth.

Odor perception is specifically affected by the odorants exposed as neonates

In order to study whether neonatal odor experience can affect odor perception, we exposed pups to a particular odorant and analyzed their responses as adults. In the habituation/dishabituation test at 6w, the mice conditioned to vanillin (VNL) at P2~4 demonstrated increased responsiveness/sensitivity to VNL (Figure 2). Furthermore, unlike the unconditioned control, the VNL-conditioned mice showed lasting interests for VNL and were not fully habituated when VNL was presented a second time. Such conditioning effects were neither found in mice conditioned to VNL at P9~11, nor observed toward other unconditioned odorants, for example, eugenol (EUG).

Effects of neonatal odor experience on odor perception in adults.

Odor detection in the vanillin (VNL)-conditioned mice. Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Then, a filter paper spotted with 0.5 μl of 20 mM VNL or 6.4 M eugenol (EUG) was presented three times (detection trials 4–6). Investigation times (sec) observed during each presentation were measured. Mice were conditioned to VNL at P2~4 (green) or P9~11 (red), and were analyzed as adults at 6w. Mice without VNL conditioning (–) were analyzed as controls (black). Odorants with 10−3 dilution were also analyzed and shown in the right of each figure set. Error bars are SD (n = 5, 6, 7 animals, 5 litters).

Figure 2—source data 1

Odor detection in the vanillin (VNL)-conditioned mice.

Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Then, a filter paper spotted with 0.5 μl of 20 mM VNL or 6.4 M eugenol (EUG) was presented three times (detection trials 4–6). Investigation times (s) observed during each presentation were measured. Mice were conditioned to VNL at P2~4 or P9~11, and were analyzed as adults at 6 weeks (6w). Mice without VNL conditioning (–) were analyzed as controls. Odorants with 10−3 dilution were also analyzed. The average values of investigation times are shown.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig2-data1-v1.xlsx

Odor exposure promotes dendrite selection resulting in glomerular enlargement

As neonatal odor experience affects odor perception in adults, we examined whether any changes could be found in the glomeruli in response to odor exposure. We focused on an OR, MOR29A (also known as Olfr1510), whose ligand is known as VNL (Tsuboi et al., 2011). This is because the onset of its expression is relatively early during olfactory development and its intrinsic level of Sema7A is low compared with other OR species. Exposure to VNL at P2~4 increased Sema7A expression (Figure 3A and Figure 3—figure supplement 1A), which promotes post-synaptic events and dendrite selection in mitral/tufted (M/T) cells (Inoue et al., 2018). VNL stimulation also increased the levels of synaptic markers, vGlut2 and GluR1 (Figure 3B left and Figure 3—figure supplement 1B). We then examined dendrite maturation by injecting Lucifer yellow into the glomeruli to visualize a single connecting M/T cell (Figure 3—figure supplement 2). The selection of primary dendrites was promoted within the MOR29A glomeruli (Figure 3B right). In this experiment, M/T cells with a selected primary-dendrite were classified as mature, whereas those with multiple unselected dendrites in the glomerular layer were classified as immature.

Figure 3 with 3 supplements see all
Glomerular changes by odor exposure in neonates.

(A) Sema7A expression stimulated by VNL exposure. The ECFP-tagged MOR29A glomeruli were analyzed by immunohistochemistry after the exposure to VNL at P2~4. OB sections were immunostained at P4 using antibodies against Sema7A. Relative fluorescent signals within the MOR29A glomeruli were analyzed in the mice with (+) or without (–) VNL exposure. **p<0.01 (Student’s t-test). Error bars are standard deviation, SD (n = 6 animals for each condition). See also Figure 3—figure supplement 1A. (B) MOR29A glomeruli in the VNL-exposed mice. Left: Changes in the levels of synapse markers. Pups were exposed to VNL at different time periods, and the OB sections at P11 were immunostained for pre- and post-synapse markers, vGlut2 and GluR1, respectively. Signal intensities within the glomerular layer (GL) were normalized by those detected in the olfactory nerve layer (ONL) or external plexiform layer (EPL), and compared with the VNL-unexposed controls (–). **p<0.01 (Student’s t-test). n.s., not significant. Error bars indicate SD (n = 6, 3, 3, 5, 5 glomeruli for each condition). See also Figure 3—figure supplement 1B. Right: Dendrite selection within the MOR29A glomeruli. The mice conditioned to VNL (P2~4) and unconditioned (–) were analyzed. M/T cells at P4 were visualized by Lucifer yellow (LY) injection into the glomeruli (Figure 3—figure supplement 2). Intracellular LY injection was performed as previously described (Inoue et al., 2018). The numbers of M/T cells with one dendrite (mature) and those with multiple dendrites (immature) were counted in the MOR29A glomeruli. The ratios (%) of mature (dark blue) and immature (cyan) M/T cells are shown: VNL-cond., 12/17 (70.6 %); VNL-uncond., 4/16 (25.0 %). n = 6, 5 glomeruli. See also Figure 3—figure supplement 2. (C) VNL-responding cells connecting to the MOR29A glomeruli. Left: Detection of tufted and periglomerular cells activated by VNL. The MOR29A glomeruli were identified by ECFP signals. The sections were then counter-stained with DAPI. Levels of an immediate-early gene product EGR1 (red) surrounding the MOR29A glomeruli were analyzed at 3w by immunostaining of OB sections (25 μm-thick). The mice were conditioned to VNL at P2~4 or P9~11. Middle: Quantification of EGR1 signals. EGR1 signals in the glomerular layer (GL) and external plexiform layer (EPL) were counted as activated periglomerular cells and tufted cells, respectively. Mice without VNL exposure in neonates were analyzed as controls (–). Error bars are SD (n = 4, 4, 5 animals). The one-way ANOVA was applied on values. ***p<0.005 (Student’s t-test). n.s., not significant. Scale bar is 30 μm. Right: MOR29A-positive OSNs. ECFP-tagged MOR29A OSNs in the OE were counted at P21 after the exposure to VNL at P2~4 or P9~11. To identify the MOR29A-positive OSNs, OE sections were immunostained with antibodies against GFP. Relative numbers of MOR29A+ OSNs are compared for the mice with (P2~4, 9~11) or without (–) VNL exposure. n.s., not significant. Error bars are standard deviation, SD (n = 6, 5, 4 animals). See also Figure 3—figure supplement 1C. (D) Glomerular sizes with or without VNL conditioning. Relative sizes (ratios of diametral areas) of the MOR29A glomeruli were measured at 3w after the VNL exposure at different time periods in neonates. Glomeruli without VNL exposure (–) were analyzed as negative controls. Error bars are SD (n = 6, 3, 3, 5, 5 animals). VNL, vanillin; GL, glomerular layer; ONL, olfactory nerve layer; EPL, external plexiform layer.

We found that sizes of the MOR29A glomeruli increased with a ligand odor as previously reported for another OR, M72 (also known as Olfr160) (Liu et al., 2016; Todrank et al., 2011). When the pups were exposed to VNL at P2~4, the numbers of VNL-responsive cells surrounding the MOR29A glomeruli increased at P21 (Figure 3C left and Figure 3—figure supplement 1C), resulting in the enlargement of MOR29A glomeruli (Figure 3D). Interestingly, the number of MOR29A-expressing OSNs remained the same (Figure 3C right). These changes were not found when VNL was exposed at P7~9 or P9~11.

It is notable that in the olfactory bulb (OB) of naris-occluded mice, the levels of synaptic markers in the MOR29A glomeruli were lowered (Figure 3—figure supplement 3 left) and the glomerular sizes for MOR29A was reduced (Figure 3—figure supplement 3 middle). Furthermore, VNL-responding tufted cells and periglomerular cells connecting to the MOR29A glomeruli were reduced (Figure 3—figure supplement 3 right). These changes were observed when occlusion was performed at P0~4, but not at P7~11 (Figure 3—figure supplement 3 right). The time frame that allows for activity-dependent changes appears to be restricted to the early neonatal period, roughly during the first week after birth. It should be noted that the critical period as determined by naris occlusion, represents the maximum time frame covering for all OR species. In contrast, in the odor exposure experiment, the critical period is determined for a specific OR species and can be different among various ORs depending upon the onset of their expression.

Sema7A/PlxnC1 signaling defines the olfactory critical period

We next proceeded to study the key process that causes the neonatal time frame to become critical. Our previous study (Inoue et al., 2018) indicated that post-synaptic events and dendrite selection in M/T cells are regulated by a pair of signaling molecules, Sema7A (Pasterkamp et al., 2003) and its receptor PlxnC1 (Tamagnone et al., 1999) expressed in OSN axons and M/T-cell dendrites, respectively. We analyzed the cell-type-specific conditional knockout (cKO) of PlxnC1 for synapse formation. The M/T-cell-specific cKO was generated using the Pcdh21 promoter for the Cre driver. We also analyzed the Sema7A total knockout (KO) in parallel. It was found that post-synaptic density (PSD) is rarely formed at P5, however, targeting of OSN axons is not affected in these KOs (Inoue et al., 2018).

Expression of the Sema7a gene appears to be regulated in an activity-dependent manner, as Sema7A is diminished in the KO of CNG channels (Brunet et al., 1996) that generate OR-specific OSN activity (Figure 4A left and Figure 4—figure supplement 1A left). Supporting this observation, the mutant OR defective in G-protein coupling (Imai et al., 2006), also diminished Sema7a expression (Figure 4A right and Figure 4—figure supplement 1A right). Individual glomeruli possess unique but different levels of Sema7A expression, determined by the intrinsic activity of ORs, thereby forming the glomerular rank of Sema7A. Sema7A expression increased with a ligand odor and the glomerular rank of MOR29A became higher for Sema7A by VNL exposure (Figure 4B). In contrast to Sema7A, PlxnCl expression or its localization to the M/T-cell dendrites was not affected in the KO of Sema7A or CNG-A2 (Figure 4C and Figure 4—figure supplement 1B). Interestingly, however, its localization to the tuft structure of M/T cells was restricted to the first week after birth (Inoue et al., 2018; Figure 4D). Thus, Sema7A expression in OSNs supports the activity dependency, and PlxnC1 localization in M/T-cell dendrites limits the time frame of plastic changes within glomeruli during the olfactory critical period.

Figure 4 with 1 supplement see all
Expression of Sema7A and PlxnC1 in the neonatal OB.

(A) Activity-dependent expression of Sema7A. Left: Analysis of the CNG-A2+/- mice. Duplicated glomeruli of rI7 were analyzed for Sema7A expression in the CNG-A2+/- female mice at P5. EYFP-tagged rI7 glomeruli were detected by immunostaining with anti-GFP antibodies. OB sections were immunostained with antibodies against CNG-A2 and Sema7A. Relative signal intensities of Sema7A (CNG+/CNG-) are compared between the CNG+ and CNG- glomeruli. **p<0.05 (Student’s t-test). Error bars indicate SD (n = 8 animals). Right: Effects of the DRY-motif mutation of OR on Sema7A expression. OE sections expressing the WT and DRY-motif mutant (RDY) of rI7 were analyzed by in situ hybridization for Sema7a transcripts at P5. The DRY-motif mutant suppresses Sema7a transcription because the mutant receptor does not produce cAMP that generates CNG-channel activity. OSNs expressing the EYFP-tagged rI7 were detected by immunostaining with anti-GFP antibodies. Relative signal intensities of Sema7a transcripts (WT/DRY) are compared between the WT and DRY-motif mutant of rI7. ***p<0.005 (Student’s t-test). Error bars indicate SD (n = 4 animals). See also Figure 4—figure supplement 1A. (B) Ranking of glomeruli for Sema7A expression. Individual glomeruli possess unique but different levels of Sema7A expression determined by intrinsic activity of ORs, forming the glomerular rank of Sema7A expression. OB sections were immunostained with anti-Sema7A antibodies. Intensities of Sema7A signals were determined for each glomerulus and plotted in order. Glomerular rank of fluorescent signals is shown for 437 different glomeruli in the OB at P8. Expression levels of Sema7A are indicated for the rI7 (yellow), MOR29A (green), VNL-stimulated (P5~7) MOR29A (red), and CNG- rI7 glomeruli (gray). See also Figure 3—figure supplement 1A and Figure 4—figure supplement 1A. (C) Expression of Plxnc1 in the OB. Both CNG+/+ and CNG-/- mice were analyzed. OB sections were analyzed at P4 by in situ hybridization using the Plxnc1 probe. Mitral cell layers (MCL) are circled by dotted lines. n = 6 animals. Scale bar, 20 μm. See also Figure 4—figure supplement 1B. (D) Localization of PlxnC1 in the M/T-cell dendrites. To detect PlxnC1, a receptor for Sema7A, OB sections were immunostained with PlxnC1 antibodies. Relative signal intensities (GL/EPL) are shown for different time points in the neonatal period. Note that PlxnC1 is found in the M/T-cell dendrites only during the first week after birth. n = 2 animals except for P4 (n = 6). GL, glomerular layer; EPL, external plexiform layer.

Figure 4—source data 1

Ranking of glomeruli for Sema7A expression.

Individual glomeruli possess unique but different levels of Sema7A expression determined by intrinsic activity of ORs, forming the glomerular rank of Sema7A expression. OB sections were immunostained with anti-Sema7A antibodies. Intensities of Sema7A signals were determined for each glomerulus and plotted in order. Glomerular rank of fluorescent signals is shown for 437 different glomeruli in the OB at P8. Expression levels of Sema7A are indicated for the rI7, MOR29A, VNL-stimulated (P5~7) MOR29A, and CNG- rI7 glomeruli.

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Sema7A is sufficient to induce post-synaptic events in M/T cells

We wanted to determine whether there are any additional components for synapse formation, whose expression is downstream of OR-specific OSN activity. To address this question, we performed rescue experiments in the CNG-A2 KO. In the KO mice, synapse formation is significantly affected (Figure 5—figure supplement 1). Since the Cnga2 gene is located on chromosome X, duplicated glomeruli are formed in the hemizygous KO (Zhao and Reed, 2001) due to stochastic X-chromosome inactivation. In the CNG-negative glomeruli of rI7 (also known as Lofr226), both pre- and post-synaptic markers decreased and primary-dendrite selection was delayed in contrast to the CNG-positive glomeruli (Figure 5A). We then introduced the transgenic (Tg) Sema7a gene with the activity-independent promotor of MOR23 (also known as Olfr16) into the Cnga2+/- female mice with the Sema7A KO background. When the rI7 glomeruli were analyzed, constitutive expression of the Tg Sema7a alone restored synapse formation and dendrite selection in the CNG-negative glomeruli (Figure 5B). Compared with the endogenous Sema7A in the WT mice, expression levels of Tg-Sema7A were approximately 10% within the rI7 glomeruli in the Cnga2+/- mice with the Sema7A KO background (Figure 5—figure supplement 2A). The mutant Sema7A (Y213S) (Liu et al., 2010), which is incapable of interacting with PlxnC1, did not rescue the KO phenotype of the CNG channels (Figure 5C and Figure 5—figure supplement 2B). We, therefore, conclude that Sema7A-PlxnC1 interaction is sufficient to induce the activity-dependent post-synaptic events in M/T cells.

Figure 5 with 2 supplements see all
Sema7A is a key to synapse formation within glomeruli.

(A) Synapse markers in the hemizygous female KO of CNG-A2. Duplicated glomeruli (CNG+ and CNG-) of rI7 were analyzed at P5 (n = 8 pairs). Levels of both pre- and post-synapse markers (vGlut2 and GluR1) were reduced in the CNG- glomerulus compared with the CNG+ (left). Dendrite selection was delayed in the CNG- glomerulus (right). Dendrites were visualized by LY injection as shown in Figure 3—figure supplement 2. The ratios of M/T cells with one primary dendrite (mature) and those with multiple branched dendrites (immature) are compared in the rI7 glomeruli: CNG+, 14/17 (82.3 %); CNG-, 3/14 (21.4 %). See also Figure 5—figure supplement 1. (B) Rescue of synapse formation in the CNG-A2+/- mice. The Tg Sema7a gene was introduced into the hemizygous female mice of CNG-A2 with the Sema7A KO background. Activity-independent, constitutive expression of the Tg Sema7a was sufficient to rescue the defective phenotype of synapse formation (left) and dendrite selection (right) in the CNG-A2 KO glomeruli (n = 6 pairs). The ratios of mature and immature M/T cells are: Tg-Sema7A, CNG+, 15/18 (83.3 %); Tg-Sema7A, CNG-, 13/16 (81.3 %). (C) Rescue experiments with the interaction mutant of Sema7A in the CNG- A2+/- mice. The mutant Sema7A (Y213S) incapable of binding to PlxnC1 was used for the rescue experiment (n = 5 glomeruli). *p<0.05, **p<0.01 (Student’s t-test). n.s., not significant. The ratios of mature and immature M/T cells are: Tg-Sema7A (Y213S), CNG+, 3/13 (23.0 %); Tg-Sema7A (Y213S), CNG-, 2/9 (22.2 %). n = 8, 7 glomeruli. For the synapse markers, relative signal intensities were calculated for the glomerular layer (GL) in comparison to those in the olfactory nerve layer (ONL) or external plexiform layer (EPL). See also Figure 5—figure supplement 2.

Figure 5—source data 1

Dendrite selection within the rI7 glomeruli.

M/T cells at P5 were visualized by Lucifer yellow (LY) injection into the glomeruli (Figure 3—figure supplement 2). Intracellular LY injection was performed as previously described (Inoue et al., 2018). The numbers of M/T cells with one dendrite (mature) and those with multiple dendrites (immature) were counted in the MOR29A glomeruli. The ratios of M/T cells with one primary dendrite (mature) and those with multiple branched dendrites (immature) are compared in the rI7 glomeruli: CNG+, 14/17 (82.3 %); CNG-, 3/14 (21.4 %) in (A), Tg-Sema7A, CNG+, 15/18 (83.3 %); Tg-Sema7A, CNG-, 13/16 (81.3 %) in (B), and Tg-Sema7A (Y213S), CNG+, 3/13 (23.0 %); Tg-Sema7A (Y213S), CNG-, 2/9 (22.2 %) in (C).

https://cdn.elifesciences.org/articles/65078/elife-65078-fig5-data1-v1.xlsx

Blockage of Sema7A/PlxnC1 signaling affects social responses later in life

We then studied how mice are affected when Sema7A/PlxnC1 signaling is blocked. Since the Sema7A KO is a total KO and would not be appropriate for behavioral analyses (Carcea et al., 2014), we chose the M/T-cell-specific cKO of PlxnC1. Absence of Plxnc1 expression was confirmed by in situ hybridization and immunostaining in the PlxnC1 cKO (Inoue et al., 2018). Similar to naris-occluded mice (Figure 1B), odor detection was lowered (Figure 6A), but not entirely abolished because the cKO mice remained attracted to food smells (Figure 6B). It appears that the basic olfactory circuitry is established by intrinsic OSN activity separately from odor-evoked activity (Gire et al., 2012).

Blockage of Sema7A/PlxnC1 signaling affects social behaviors later in life.

(A) Odor detection of the PlxnC1 cKO in the habituation/dishabituation test. Investigation times for odors were measured in the PlxnC1 cKO and WT male mice at 6w. Please see the legend to Figure 1B for the experimental procedure. Odorant pairs examined are as follows: left, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and right, 62 mM (+)-CAR and 64 mM EUG. Error bars are SD (n = 15, 6 animals, 6 litters). CAR, carvone; EUG, eugenol. (B) Social responses in the PlxnC1 cKO. Beds with the odor of unfamiliar mice were presented to the 6w-old WT male or to the PlxnC1 cKO where Sema7A signaling is blocked. The samples were presented in a plastic cup to avoid direct contacts with the mouse nose. Times (sec) spent in the room with or without a test odorant were measured during the 5-min test period. Differences of staying times in the two rooms (A - B) are shown. Mouse food (SLC Japan, Inc) and fresh beds without the mouse scents were used as positive and negative controls, respectively. Error bars are SD (n = 18, 7 animals, 7 litters). (C) Three-chamber test. The male WT and PlxnC1 cKO were analyzed at 6w. The mouse being tested was placed in the center chamber (2). An empty cage was placed to the right (3) and an unfamiliar male mouse in a plastic cage was to the left (1). Time duration (sec) spent in each room was measured during a 15-min test period. Error bars are SD (n = 5, 4 animals, 4 litters). (D) Stress-induced hyperthermia test in the VNL-conditioned PlxnC1 cKO. Pups of the WT (red and green) and PlxnC1 cKO (light blue) were exposed to VNL at P2~4 or P9~11 and analyzed at 6w. Immediately after the transfer to a new cage, a filter paper spotted with VNL was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without VNL exposure (–) were analyzed as negative controls (black). PPA was used as an attractive-odor control (gray). Temperature differences before (T) and after (Tx) the transfer are compared. Error bars are SD (n = 3, 4, 4, 3, 3 animals). *p<0.05; ***p<0.005 (Student’s t-test). VNL, vanillin; PPA, propionic acid.

Figure 6—source data 1

Odor detection of the PlxnC1 cKO in the habituation/dishabituation test.

Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Next, a filter paper spotted with the 1st odor was presented three times (detection trials 4–6). Then, a filter paper spotted with the 2nd odor was presented three times (detection trials 7–9). Investigation times for odors were measured in the PlxnC1 cKO and WT male mice at 6w. Odorant pairs examined are as follows: left, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and right, 62 mM (+)-CAR and 64 mM EUG. CAR, carvone; EUG, eugenol. The average values of investigation times are shown.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig6-data1-v1.xlsx
Figure 6—source data 2

Stress-induced hyperthermia test in the VNL-conditioned PlxnC1 cKO.

Pups of the WT and PlxnC1 cKO were exposed to VNL at P2~4 or P9~11 and analyzed at 6 w. Immediately after the transfer to a new cage, a filter paper spotted with VNL was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without VNL exposure (–) were analyzed as negative controls. PPA was used as an attractive-odor control (gray). Temperature differences before (T) and after (Tx) the transfer are compared. Error bars are SD (n = 3, 4, 4, 3, 3 animals). *p<0.05; ***p<0.005 (Student’s t-test). VNL, vanillin; PPA, propionic acid.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig6-data2-v1.xlsx

In the PlxnC1 cKO, social responses in adults were perturbed. While male mice normally demonstrate strong curiosity for unfamiliar mouse scents of both genders, the cKO showed avoidance behavior toward them (Figure 6B). A similar impairment in social interactions was observed in the three-chamber test, a standard method to detect autism spectrum disorders (ASD) in rodents (Moy et al., 2004). In the three-chambered box, the mouse being tested was placed in a center chamber, an unfamiliar mouse in a plastic cage was to the left, and an empty cage was to the right (Figure 6C). Time duration spent in each room was measured during a 15-min test period. Although the WT mouse spent most of its time in the left chamber inspecting the stranger, the PlxnC1 cKO spent longer times in the right chamber, thereby avoiding social interactions with the unfamiliar mouse. These results indicate that Sema7A/PlxnC1 signaling is involved in establishing positive imprinted memory in neonates, whose defect causes the impairment of social responses in adulthood. While the cKO mice fail to demonstrate positive interest toward the odor of unfamiliar mice, they are still able to detect food smells.

Mice demonstrate stress responses in an unfamiliar environment. When they are transferred to a new cage, the rectal temperature rises and remains elevated for about 40 min (Spooren et al., 2002). However, in the mice conditioned to VNL at P2~4, stress was eased by VNL at 6w (Figure 6D). We tested propionic acid (PPA) as a positive control, whose quality is innately attractive (Figure 6D). PPA lowers the stress, but its effect is more moderate compared with the imprinted VNL whose quality is innately neutral. It is notable that such a conditioning effect was not observed in the mice exposed to VNL after the critical period at P9~11. In the PlxnC1 cKO, stress was not eased by the conditioned odor VNL in the hyperthermia test (Figure 6D).

Oxytocin in neonates is needed for smooth social interactions in adults

Increased sensitivity is a form of imprinted memory that can be established at the level of the glomeruli, and its blockage in the PlxnC1 cKO can be explained by the failure of odor-evoked synapse formation. However, lasting interest (Figure 2) or easing of stress (Figure 6D) is another example of conditioned memory that needs to be further formed in the central brain; a neuronal level not affected by the PlxnC1 cKO. How is it then that the perceptive change takes place for the exposed odors? What is responsible for establishing smooth social interactions by imposing the positive quality on imprinted odor memory? We therefore, turned our attention to various neuronal hormones, including norepinephrine, dopamine, and oxytocin; all of which are known to induce positive mental status and mediate attractive social behaviors (Andersson, 2000; de Wied et al., 1993). Among them, oxytocin appeared to be promising to us because it is highly expressed in the neonatal brain (Sannino et al., 2017) and promotes attractive social interactions (Bosch and Young, 2018; Insel, 2010). Furthermore, it has been reported that the KO of oxytocin (Nishimori et al., 1996) or its receptor (Takayanagi et al., 2005) affects social interactions and causes ASD-like responses. We, therefore, studied whether oxytocin is needed in neonates by the rescue experiment with the oxytocin KO (Smith et al., 2019).

To examine a possible effect of neonatal oxytocin on smooth social interactions later in life, we analyzed the oxytocin-administrated KO mice for social memory. In the oxytocin KO, increase in the sensitivity to the imprinted odor can still be seen (Figure 7A) and Sema7A expression is not affected by the KO (Figure 7—figure supplement 1). However, the positive quality is not imposed on the imprinted odor, as observed by loss of lasting interest (Figure 7A) and stress-reducing effect in the hyperthermia test (Figure 7B). In the social memory test, male mice demonstrate a characteristic decline in the time spent investigating an ovariectomized female during repeated pairings (Ferguson et al., 2000). In the male KO of oxytocin, time duration of sniffing did not decrease in the subsequent presentations of the same female mouse.

Figure 7 with 1 supplement see all
Odor imprinting in the oxytocin (Oxt) KO.

(A) Odor responsiveness in the Oxt KO. Sniffing times (sec) were measured in the habituation/dishabituation test. The WT and Oxt KO were conditioned to 4MT at P2~4. Mice without conditioning (–) were analyzed as negative controls (n = 4 animals). Odorants used were as follows: 100 mM and 10 μM for 4MT (left); 6.4 M and 6.4 mM for EUG (right). Error bars are SD (n = 4, 5 animals). See also Figure 1B for the procedure. (B) Stress-induced hyperthermia test in the 4MT-conditioned Oxt KO. Pups of the WT (green) and Oxt KO (red) were exposed to 4MT at P2~4 and analyzed at 6w. Immediately after the transfer to a new cage, a filter paper spotted with 4MT was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without 4MT exposure (–) were analyzed as negative controls (black). Temperature differences before (T) and after (Tx) the transfer are compared. Error bars are SD (n = 4, 3, 4 animals). ***p<0.005 (Student’s t-test). 4MT, 4-methyl-thiazole; EUG, eugenol.

Figure 7—source data 1

Odor responsiveness in the Oxt KO.

Sniffing times (s) were measured in the habituation/dishabituation test. The WT and Oxt KO were conditioned to 4MT at P2~4. Mice without conditioning (–) were analyzed as negative controls (n = 4 animals). Odorants used were as follows: 100 mM and 10 μM for 4MT (top); 6.4 M and 6.4 mM for EUG (bottom). The average values of total sniffing times are shown.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig7-data1-v1.xlsx
Figure 7—source data 2

Stress-induced hyperthermia test in the 4MT-conditioned Oxt KO.

Pups of the WT and Oxt KO were exposed to 4MT at P2~4 and analyzed at 6w. Immediately after the transfer to a new cage, a filter paper spotted with 4MT was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without 4MT exposure (–) were analyzed as negative controls. Temperature differences before (T) and after (Tx) the transfer are compared. 4MT, 4-methyl-thiazole; EUG, eugenol.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig7-data2-v1.xlsx

We then injected oxytocin into the KO pups by intraperitoneal administration (Mizuno et al., 2015) and tested their social memory after 10w. In the KO male treated with oxytocin at P0~6, sniffing times progressively decreased for the subsequent presentations (trials 1–5) of the same female mouse, but increased for a newly introduced female (trial 6) (Figure 8A). This rescue effect was not observed when the oxytocin was administrated after the critical period, at P8~14 (Figure 8B). Taken together, oxytocin in early neonates appears to be needed for smooth social interactions as adults by imposing positive quality on imprinted odor memory. Thus, imprinting (increase in the sensitivity) and positive memory formation are separately regulated by Sema7A/PlxnC1 signaling and oxytocin, respectively, during the neonatal critical period.

Social memory in the Oxt KO.

The KO mice were administrated with oxytocin (Oxt) or saline (SAL) by intraperitoneal injection at P0~6 (A) or P8~14 (B). In the Oxt KO treated with SAL, time duration of sniffing did not change for the subsequent presentations of the same unfamiliar female mouse (trials 1–5). In contrast, in the KO treated with Oxt, sniffing time progressively decreased for the subsequent presentations of the same female (trials 1–5), but increased for the newly introduced female (trial 6), as found in the WT control. Data are shown as mean ± standard error. The male mice used were (P0~6, 8~14): WT-SAL (n = 6, 7), WT-Oxt (n = 8, 8), KO-SAL (n = 6, 9), and KO-Oxt group (n = 6, 9).

Figure 8—source data 1

Social memory in the Oxt KO.

The KO mice were administrated with oxytocin (Oxt) or saline (SAL) by intraperitoneal injection at P0~6 (A) or P8~14 (B). Time duration of sniffing are shown after the presentations of the same unfamiliar female mouse (trials 1–6). Data also show mean ± standard error.

https://cdn.elifesciences.org/articles/65078/elife-65078-fig8-data1-v1.xlsx

Discussion

During development, an olfactory map is generated by a combination of A-P and D-V targeting using distinct sets of axon guidance molecules (Nishizumi and Sakano, 2020). These processes are independent from the neuronal activity of OSNs. The map is then refined by glomerular segregation molecules (Serizawa et al., 2006) whose expression is regulated by the intrinsic neuronal activity reported by Reisert, 2010 (Nakashima et al., 2019). It appears that the intrinsic activity also mediates synapse formation within glomeruli. The olfactory map is further modified by the odor-evoked activity in neonates by enlarging the responsive glomeruli. This process is plastic and mediated by Sema7A/PlxnC1 signaling during the critical period. Activity-dependent Sema7A expression in OSNs triggers post-synaptic events in M/T cells via PlxnC1 (Inoue et al., 2018). Our present study demonstrates that Sema7A/PlxnC1 signaling is key to imprinting the neonatal odor experience during the critical period in mice. Rescue experiments with the Tg Sema7a gene in the CNG KO indicate that Sema7A is sufficient to promote activity-dependent synapse formation and dendrite selection within glomeruli. In M/T cells, PlxnC1 is localized to the dendrites only during the first week after birth. Thus, Sema7A supports the activity-dependency of plastic changes within glomeruli and PlxnC1 limits the olfactory critical period to the early neonatal stage. It will be interesting if we can see shifts in the time window for the olfactory critical period by changing the time frame of PlxnC1 localization in the M/T-cell dendrites.

Sema7A expression increases in the responding glomeruli when newborns are exposed to a particular odorant. As a result, post-synaptic events are enhanced in the connecting M/T cells and dendrite selection is promoted. Unstimulated dendrites that connect other glomeruli are likely to be removed by synaptic competition. Thus, the number of primary dendrites increases in the stimulated glomerulus causing it to become larger. A specific set of enlarged glomeruli may be recognized as an imprinted odor code in adults. Sema7A/PlxnC1 signaling appears to have a prime role in adapting pups to environmental odorants and generating imprinted memory with lasting effects later in life. How does this imprinting occur in the context of circuit formation for adaptive odor responses? It has been reported that postnatal odor exposure heightens odor-evoked mitral-cell responses (Liu and Urban, 2017). It is likely that the enlarged glomeruli generate stronger signals of exposed odorants, which is then transmitted to the olfactory cortex, increasing the possibility that imprinted memory is established in neonates.

KO studies indicate that oxytocin is involved in imposing the positive quality on imprinted odor memory and is necessary for smooth social interactions. In order to determine when oxytocin is needed to achieve normal social skills, we attempted the rescue experiment for the oxytocin KO by intraperitoneal injection of oxytocin in neonates. Defective phenotypes of the KO mice were partly restored by this treatment when oxytocin was administrated during the critical period. The establishment of smooth social interactions by imprinted memory may be associated with a pleasant mental state in the nursed neonates. It is notable that in the oxytocin KO, Sema7A expression is not affected and the sensitivity to the imprinted odor still increases. Thus, imprinting and positive memory formation are independently regulated by Sema7A/PlxnC1 signaling and oxytocin, respectively, during the olfactory critical period. There are three major molecules involved in olfactory imprinting during the critical period. They are Sema7A which supports activity-dependency of the imprinting process, PlxnC1 which restricts the time frame of the critical period to the first week after birth, and oxytocin which is responsible for imposing the positive quality on imprinted memory.

It has been reported that early exposure to environmental odors affects social behavior later in life (Logan et al., 2012; Mennella et al., 2001; Sullivan et al., 2000; Wilson and Sullivan, 1994). Our present study suggests that odor-induced Sema7A/PlxnC1 signaling in neonates is essential for establishing odor imprinting, whose impairment cannot be recovered in adults. Male mice normally demonstrate strong curiosity toward unfamiliar mice of both genders. If Sema7A/PlxnC1 signaling is blocked during the critical period, the mice do not respond in their usual manner but demonstrate avoidance response to the stranger’s scent. Correlating to this observation, there is a report showing that micro-deletion in the human chromosome 15q24 covering the Sema7a locus is associated with ASD (McInnes et al., 2010). It is well known that social stress induces the release of adrenocorticotropic hormone (Backström and Winberg, 2013). We assume that mice innately avoid stressful interactions with unfamiliar mice. However, imprinting mediated by Sema7A/PlxnC1 signaling may be necessary for the pups to adapt to their community. It will be interesting to study whether similar neonatal imprinting as that described in the mouse olfactory system can be found in humans, not only in the olfactory system, but also in other sensory systems. It is also important to determine when the critical period starts and how long it lasts in humans.

Our present study demonstrates that the imprinted odor induces positive responses even when its quality is innately aversive. In such a situation, where the hard-wired decision is modified by imprinting to elicit the final response, how does the amygdala arbitrate the conflicting decisions, one which is innate and the other which is modified by imprinted memory? In order to reconcile the two decisions, there may be a cross talk between the positive and negative valence cells in the amygdala (Mori and Sakano, 2021). These studies will give new insights into our understanding of decision making in humans and will shed light on the neurodevelopmental disorders, such as ASD and attachment disorders, that may be caused by improper sensory inputs during the critical period.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(M. musculus)
Cnga2 KOJackson LaboratoryStock #: 002905
RRID:MGI:3717661
PMID:15071119
Genetic reagent
(M. musculus)
Sema7a KOJackson LaboratoryStock #: 005128
RRID:MGI:2683896
PMID:12879062
Genetic reagent
(M. musculus)
Oxytocin KOPMID:8876199RRID:MGI:3603795Dr. Katsuhiko Nishimori (Tohoku University)
Genetic reagent
(M. musculus)
BAC Olfr1510/1511 TgPMID:21105914Dr. Hitoshi Sakano
(University of Fukui)
Genetic reagent
(M. musculus)
pOlfr16- Lofr226PMID:16990513RIKEN BRC (RBRC02931)
Genetic reagent
(M. musculus)
pOlfr16- Lofr226 (RDY)PMID:16990513RIKEN BRC (RBRC02933)
Genetic reagent
(M. musculus)
Plxnc1flox/floxPMID:29743476RIKEN BDR (Acc. #: CDB0908K)
Genetic reagent
(M. musculus)
pOlfr16- Lofr226-Sema7aThis paperDr. Hitoshi Sakano
(University of Fukui)
Genetic reagent
(M. musculus)
pOlfr16- Lofr226-Sema7a (Y213S)This paperDr. Hitoshi Sakano
(University of Fukui)
Genetic reagent
(M. musculus)
Tg(Pcdh21-cre)BYokoPMID:16106355RRID:MGI:4940883RIKEN BRC (RBRC02189)
antibodyanti-Sema7AR and D SystemsCat. #: AF-1835IF(1:3000)
antibodyanti-PlxnC1AbcamdiscontinuedIF(1:3000)
antibodyanti-CNG-A2Alomone LabsCat. #:APC-045IF(1:200)
antibodyanti-vGlut2MilliporeCat. #: AB2251-lIF(1:1000)
antibodyanti-GluR1AbcamCat. #: ab51092IF(1:1000)
antibodyanti-GFPThermo Fisher ScientificCat. #: A-10260IF(1:1000)
antibodyanti-Lucifer yellowThermo Fisher ScientificCat. #: A-5750IF(1:2000)
antibodyanti-EGR1AbcamCat. #: ab6054IF(1:1000)

Contact for reagent and resource sharing

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Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Hitoshi Sakano (sakano.hts@gmail.com).

Mutant mice

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The Cnga2 KO (No. 002905) (Lin et al., 2000) and Sema7a KO (No. 005128) (Pasterkamp et al., 2003) were purchased from the Jackson Laboratory. The KO mice for oxytocin (Nishimori et al., 1996) was obtained from Tohoku University. BAC Tg mice containing the MOR29A (also known as Olfr1510) and MOR29B (also known as Olfr1511) genes (Tsuboi et al., 2011), Tg mice expressing the rI7 (also known as Lofr226) gene with the MOR23 (also known as Olfr16) promoter (Imai et al., 2006), and rI7 (RDY)-ires-gap-YFP mice (Imai et al., 2006) were generated in our group. For the cell-type-specific cKOs of Plxnc1, we cloned the exon 5 of Plxnc1 into the double-floxed, inverted open-reading frame plasmid, DT-A/Conditional FW (http://www2.clst.riken.jp/arg/cassettes.html). The cKOs mice were generated according to the published protocol (http://www2.clst.riken.jp/arg/gene_targeting.html). To generate the Tg mice carrying the rI7-ires-Sema7a-ires-YFP or the rI7-ires-Sema7a (Y213S)-ires-YFP, an EagI cassette containing the transgene was inserted into the EagI site in the pMOR23-rI7-ires-gap-YFP construct. All five transgenic lines obtained for each, successfully expressed Tg-Sema7A in the rI7-positive OSNs, although expression levels of Tg-Sema7A were ~10% compared with the endogenous Sema7A. The Pcdh21-Cre mouse (Nagai et al., 2005) was obtained from the RIKEN Bioresource Research Center (RBRC02189) and crossed with the Plxnc1flox/flox mice.

All animals were housed under a standard 12 hr light/dark cycle (light on from 8 am to 8 pm), constant temperature (22 ± 2°C), and humidity (50 ± 10%). Food and water were provided ad libitum. All animal experiments were approved by the Animal Care Committees at University of Fukui and Azabu University.

Naris occlusion

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Unilateral naris occlusion was performed at P0 on the right nostril with an electrical cautery, SURE SX-10C (Ishizaki, Japan). The occluded pups were examined daily to ensure blockade of the occluded nostril by checking the scar formation. The right-side naris was reopened at various time points during the postnatal period using a 31G needle. In some experiments, occlusion was initiated after P0 for different time lengths during the neonatal period.

Antibodies

Antibodies used in this study are as follows: Sema7A (goat, 1:3000, #AF-1835, R and D Systems); PlxnC1 (goat, 1:3000, discontinued, Abcam); CNG-A2 (rabbit, 1:200, #APC-045, Alomone Labs); vGlut2 (guinea pig, 1:1000, #AB2251-l, Millipore); GluR1 (rabbit, 1:1000, #ab51092, Abcam); GFP (rabbit, 1:1000, #A-10260, Thermo Fisher Scientific); Lucifer yellow (rabbit, 1:2000, #A-5750, Thermo Fisher Scientific); and EGR1 (rabbit, 1:1000, #ab6054, Abcam).

Immunostaining

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For immunohistochemistry, OB sections were fixed with 4% paraformaldehyde in PBS and treated with the primary and secondary antibodies. The sections were then photographed with a fluorescence microscope, Model IX70 (Olympus), coupled to a cooled CCD camera, C4742-95-12ERG (Hamamatsu Photonics).

Intensity measurement

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For fluorescent signals of immunostaining, digital images were taken with a digital CCD camera, C4742-95-12ERG (Hamamatsu Photonics), or with two-photon microscopy (Olympus FV1000). Tone was reversed and a monochrome image was used for the measurement. For staining of the OB sections, digital images were taken with a digital CCD camera, Model DP70 (Olympus). To quantify the staining level of each glomerulus, the mean pixel intensity within the region surrounded by the periglomerular-cell nuclei was measured using Scion Image (Scion Corp.).

Neonatal odor conditioning

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Before conditioning, mother mice were pre-exposed to the odor for 10 min, three times a day for 3 consecutive days. A filter paper (2×2 cm) spotted with a test odorant was placed near the mice. For conditioning, pups were exposed to the odor for 10 min with the foster mother, three times a day for 3 consecutive days. Odor concentration was 0.5 μl of 20 mM for vanillin (VNL) or 100 mM for 4-methyl-thiazole (4MT) (Wako).

Habituation/dishabituation test

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For the test, adult littermates (6w-old male) were kept individually in a new cage (26×40×18 cm) containing a plain filter paper for 5 min prior to the experiment. This treatment was to habituate the mice to a filter paper and to evaluate the net curiosity to a novel odor in the following filter presentation. During the treatment, about 8% of total mice did not demonstrate any interest in the plain filter paper. We precluded such mice for further experiments. In the test, a filter paper spotted with 0.5 μl of distilled water was intermittently presented for 3 min. This operation was repeated three times with 1 min intervals. Next, a filter paper spotted with the 1st test odorant was presented three times as in the presentation of water-spotted filter paper (habituation). Then, the 2nd test odorant was presented three times in the same way (dishabituation). Investigation times (s) observed during each odorant presentation were measured. In this test, the mice were used only once not to confound the data due to the previous learning. The test was blinded to avoid unconscious bias and to insure the objectivity. The mouse behavior was recorded with a digital video camera, DCR-SR100 (SONY). In this assay, investigation was defined as a nasal contact with filter papers within 1 mm of distance. Odorants used were (+)-carvone (CAR) (Sigma-Aldrich), (–)-CAR (Sigma-Aldrich), eugenol (EUG) (Tokyo-Kasei), and vanillin (VNL) (Wako).

Stress-induced hyperthermia test

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For the stress-induced hyperthermia test (Spooren et al., 2002), mice (6w-old) were individually habituated to the cage and their body temperature was checked to be normal. The mice were then transferred to a new cage and exposed to a testing odorant. The rectal temperature was measured with a digital thermistor, BAT-12 (Physitemp Instruments Inc) by inserting the probe to a length of 10–20 mm until stable reading was obtained. Differences of rectal temperatures before (T) and after (Tx) the transfer were individually measured every 20 min. Odor concentrations were 0.5 μl of 20 mM for vanillin (VNL), 100 mM for propionic acid (PPA) (Tokyo-Kasei), and 100 mM for 4-methyl-thiazole (4MT) (Wako).

In situ hybridization

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To prepare cRNA probes, DNA fragments of 500–3,000 bp were amplified by PCR from the OB cDNA of C57BL/6 mice. Only unique sequences were amplified for each gene. PCR products were subcloned into the pGEM-T vector (Promega) and used as templates to make cRNA probes. Hybridization was performed according to the standard method (Inoue et al., 2018).

Electron microscopy

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Mice were transcardially perfused with 2% paraformaldehyde (PFA) and 2.5% glutaraldehyde (GA) in 0.1 M PB. The mice were then immersed in the same buffer at 4°C for 1–2 hr and rinsed in 0.1 M PB prior to being sectioned transversely into 50 μm slices with a vibratome, LinearSlicer PRO7 (D.S.K.). The sections for electron microscopy were incubated in 2% osmium tetroxide for 60 min, dehydrated through an ascending ethanol series, and embedded in EPON. Thin sections (70–100 nm) were then sliced with EM UC7 (Leica), mounted on Cu grids, and examined in a transmission electron microscope VE-9800 (KEYENCE). Images were photographed at primary magnification.

Odor preference test

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For the social response test, 6w-old adult littermates (male mice) were individually habituated in a new cage (26×40×18 cm). Then, food pellet (Japan SLC, Inc) or beds with unfamiliar mouse odor were carefully placed not to disturb the mice in a corner of the test cage. To avoid direct contacts of the mouse nose, the food pellet or bed samples were presented in a small plastic cup. The cup positions were alternated to avoid side preference. This apparatus evaluates a simple social interest in the volatile smell of unfamiliar mice, excluding visual, auditory, and vomeronasal inputs. Times (s) spent in the room with or without a test odorant were measured during the 5-min test period. The mice were used only once to avoid possible bias attribute to learning. The test was blinded to avoid unconscious bias to insure the objectivity. The mouse behavior was recorded with a digital video camera, DCR-SR100 (SONY).

Three-chamber social interaction test

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Littermate mice (6w-old male) were used in the experiment. The three-chamber test was conducted as described by Moy et al., 2004. Before the test, a mouse was allowed to explore all three chambers divided with plastic walls with a loophole in the apparatus for 10 min. Only those mice moving actively in the cage were used for the test. Then, the test mouse was placed in the center chamber, an empty plastic cage was to the right, and an unfamiliar mouse (male C57BL/6J) in a cage was to the left. The mouse being tested was allowed to move freely and explore all three chambers for 15 min. The mouse behavior was recorded with a digital video camera, DCR-SR100 (SONY). Time duration spent in each room was measured during the 15-min test period. The positions of the empty cage and unfamiliar mouse were alternated to avoid left/right side preference.

Social memory test

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All pups in each litter received either an intra-peritoneal injection of oxytocin (2 μg/20 μl for P0~6 and 5 μg/20 μl for P8~14) or saline (SAL, 20 μl) on postnatal days 0, 2, 4, and 6, or 8, 10, 12, and 14. The same cohort of male mice that were examined for alloparental responsiveness, was tested by the habituation/dishabituation paradigm (Ferguson et al., 2000 and Ferguson et al., 2001). In this paradigm, mice exposed to the same stimulus animal should decrease their investigation times with each exposure (habituation) and increase their investigation time when a novel animal is introduced (dishabituation). One male subject was transferred from its group to an individual cage of the same size as the breeding cage for 7–10 days before testing for the establishment of a home-cage territory. Testing began with a stimulus mouse, an ovariectomized female unfamiliar to the subject, was introduced into the home cage of each subject for a 1 min confrontation. At the end of the 1 min trial, the stimulus was removed and returned to its home cage. This trial was repeated for five times with 10 min intertrial intervals, and each stimulus was introduced to the same male subject in all five trials. In the 6th dishabituation trial, an ovariectomized female mouse different from the previous one was introduced into the home cage of the subject mouse. Each trial was recorded with a CCD camera connected to a video recorder. The duration of sniffing the stimulus’ anogenital area was measured by a well-trained observer who was blinded to both the mice genotypes and treatments.

Statistical analyses

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All statistical analyses were performed using Excel 2016 (Microsoft) with the Statcel2 add-on (OMS).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

References

Decision letter

  1. Stephen Liberles
    Reviewing Editor; Harvard Medical School, United States
  2. Gary L Westbrook
    Senior Editor; Oregon Health and Science University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This manuscript investigates a role for Sema7A/PlxnC1 in an olfactory critical period. The authors observed that mice who encounter odors as neonates (before ~P7) display altered behavioral and neuronal responses to those odors as adults. Neonatal-experienced odors are investigated more vigorously, and mildly reduce stress, perhaps akin to familiar nest odors. Disruption of Plxn1 alters neuronal circuitry and eliminates the behavioral effects of neonatal odor exposure.

Decision letter after peer review:

Thank you for submitting your article "The Olfactory Critical Period is Defined by Activity-Dependent Synapse Formation Induced by Sema7A/PlxnC1 Signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gary Westbrook as the Senior Editor. The reviewers have opted to remain anonymous. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Sensory systems develop with “critical periods” where sensory experience causes long-lasting structural changes within the brain with lasting impact on adult behavior. Here, the authors identify key development factors (Sema7A/PlxnC1) that are required for an olfactory critical period in mice, and loss of these factors, or the hormone oxytocin, impairs olfactory learning. The reviewers all agreed that the manuscript presented important findings about the roles of Sema7a and PlxnC1 in odor imprinting that will be of broad interest. However, there were also substantial concerns from all reviewers that results were not all coherent and thematically connected. Additional experiments are required, as detailed below in Essential revisions, to justify conclusions drawn from the data.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Essential revisions:

1) All reviewers agree that a link between oxytocin and Sema7a/PlxnC1 is lacking. Do the authors observe alterations in Sema7a signaling in oxytocin knockout mice? If so, the data would strengthen the manuscript considerably; if not, it is advised that the paper could be strengthened by removing the disconnected oxytocin data.

2) The reviewers request testing Sema7a mutants for behavioral effects in odor paradigms presented (including those in Figure 2 and Figure 6).

3) The authors should improve description and validation of several mouse models.

Reviewer #1:

This manuscript investigates a role for Sema7A/PlxnC1 in an olfactory critical period. The authors observe that mice who encounter odors as neonates (before ~P7) display altered behavioral and neuronal responses to those odors as adults. Neonatal-experienced odors are investigated more vigorously, and mildly induce stress, perhaps akin to familiar nest odors. The authors report that neonatal odor exposure increases Sema7a expression, enlarges target glomeruli through increased numbers of periglomerular cells, and increases the percentage of mature mitral cells. The authors then show that knockout of Plxn1 or oxytocin impairs social odor recognition. There are many interesting findings here, and the manuscript is well written. Yet, some of the key findings are not thematically connected and additional experiments are needed to strengthen links between Sema7A signaling, oxytocin, and behavioral responses.

1) The authors show that neonatal-experienced odors are more attractive to odors, and that neonatal odors upregulate Sema7A in responsive neurons. Yet, a role for Sema7A/PlxnC1 in enhanced neonatal odor investigation is not reported. Do Sema7a knockout mice lose effects of neonatal-experienced odors described in Figure 2? Are they rescued by re-expression of Sema7A in olfactory sensory neurons (using Tg-Sema7a)?

2) The connection between oxytocin and Sema7A signaling is unclear. Does oxytocin alter expression/localization of PlxnC1 and/or Sema7A? Can the authors propose and test models for how oxytocin impacts olfactory circuits?

3) As the authors have demonstrated beautifully in prior studies, activity through Gs is needed for map establishment while Golf/CNGa2 activity is needed for odor-evoked activity and maintenance. The paper would benefit by further discussion of the roles of “activity” and that whether Sema7a contributes only to maintenance and not map establishment.

4) Provide additional explanation in the main text and validation of the Tg-Sema7A line. What % of olfactory sensory neurons re-express Sema7a? Likewise, additional validation of the mitral cell-specific PlxnC1 knockout is needed.

5) Representative images should be shown for all data presented as a signal intensity, including Figures 1A, 3A, B, 4, and 5. Also, images showing the differences between mature/immature dendrite selection are needed (3B).

6) A home cage nest odor would be a nice positive control for Figure 2B, for context to understand the magnitude of the observed behavioral response.

7) The authors claim (Results) that both tufted and periglomerular cells increase in number, but Figure 3C only provides supporting data for periglomerular cells.

Reviewer #2:

1) It is not clear exactly what the relationship is between the oxytocin experiments and the rest of this study. First, do oxytocin mutants show alterations Sema7a expression, or PlexC1 temporal distribution in the tuft structure? Why is a different behavioral assay used for the oxytocin experiments as compared to the social interaction assay used in the Plexc1 conditional mutants.

Overall, there remain questions regarding the behavioral experiments such that if the oxytocin experiment was not part of this study, it would not in my view reduce interest or impact of the work.

2) Does loss of Sema7a in ORs lead to the same behavioral phenotypes observed in the cKO M/T PlexC1 mutants? And is there any way to show that the loss-of-function (LOF) experiment involving PlexC1 is dependent upon LOF during the critical period? For this current study, addressing the Sema7a requirement genetically in this behavioral assay seems to be an issue that should be tackled in the context of the social interaction assay, if it is to be presented here.

3) I do not have many comments on the first part of the study, however, and find it to be quite interesting. I do wonder:

a) If in the conditional CNG mutants, whether or not there is any change in the temporal localization of PlexC1 with respect to the presented postnatal localization in the tuft structure-is this, too, activity dependent and/or regulated by Sema7a during the critical period.

b) Is there any evidence that Sema7a OR expression normally follows a time course that directly links it to the critical period. This seems like a point that could be easily addressed.

Reviewer #3:

1) The conclusion of the paper appears to be based on the following logical connection: activity driven changes in synaptic function in the olfactory bulb is restricted to the first week after birth because activities drive the expression of Sem7A expression, which interacts with PlxnC1, expressed only during the critical period. This is not explicitly stated, nor extensively discussed. Although the model is very attractive, the experimental evidence is correlational. The conclusion that Sema7A/PlxnC1 support the olfactory critical period is largely based on the restricted dendritic expression PlxnC1 during the first postnatal week.

2) Naris occlusion and odor exposure experiments are complementary to each other to provide support for activity dependent regulation of Sema7A expression, but there is a discrepancy in the results. Naris occlusion experiment show that the level of synaptic proteins is normal if occlusion is reopened before P7. However, odor exposure using vanillin between P2 and P4 altered odor habituation and stress response. The authors have not explained the discrepancy in the experimental design and results with regard of the difference in timing.

3) The CNGA2 KO experiments lend support for activity-dependent expression of Sema7A, and the Sema7A rescue experiment provides further support that Sema7A could induced synaptic protein expression. However, different glomeruli exhibit different levels of Sema7A expression. Is this difference intrinsic to different types of OSNs, or is it caused by differences in neural activity?

4) The conditional PlxnC1 KO mice need more rigorous testing. If activity-dependent expression of Sema7A and Sema7A/PlxnC1 interaction are important for synapse formation in the olfactory glomeruli, odor detection and discrimination are expected to be affected. No experiments have been conducted to address this prediction. It is also not known whether the KO abolishes the phenotypes observed in Figure 2, which are associated with vanillin exposure during the critical period. Moreover, it is not clearly stated how the three-chamber assay is related olfactory imprinting. A likely explanation of the observed impairment of odor preference and social behavior is that the overall olfactory sense has been compromised.

5) The oxytocin KO experiment is interesting. However, the behavioral phenotype is difficult to interpret. WT mice exhibit a decline in investigation of an ovariectomized female whereas the oxy-/- mice show persistent investigation. The authors interpret the decline as an indication of social memory, but the lack of decline/persistent investigation in the oxy-/- mice could be interpreted as prosocial interactions. This interpretation would be consistent with the decrease of investigation in WT mice after P0-P6 oxytocin injection. Importantly, the authors have not addressed how this experiment is related to activity-dependent modulation of glomerular connectivity.

6) The authors present data with quantification, but primary data (images) are not presented. This makes it difficult to assess the accuracy of the quantification. Specifically, primary images demonstrating the selective induction of Sema7A expression in the MOR29A glomeruli after vanillin exposure should be compared with no-odor control side-by-side.

https://doi.org/10.7554/eLife.65078.sa1

Author response

Essential revisions:

1) All reviewers agree that a link between oxytocin and Sema7a/PlxnC1 is lacking. Do the authors observe alterations in Sema7a signaling in oxytocin knockout mice? If so, the data would strengthen the manuscript considerably; if not, it is advised that the paper could be strengthened by removing the disconnected oxytocin data.

The editor points out that a link between oxytocin and Sema7A/PlxnC1 is lacking, which is a theme raised by all the reviewers. Our description was insufficient and we should have provided a better explanation in our manuscript.

We continued our investigation into oxytocin, after the completion of PlxnC1 cKO experiments. This is because two of the three observations we found were behaviors that could not solely be explained at the neuronal level influenced by Sema7A/PlxnC1 signaling. In the PlxnC1 cKOs, the normal behavioral response to odors, which the mice were previously exposed to during the critical period, (1) increased sensitivity, (2) lasting interest, and (3) easing of stress were all abolished. Increased sensitivity is a form of imprinted memory, and its blockage can be explained by the failure of synapse formation within the glomeruli in the PlxnC1 cKO. However, lasting interest and easing of stress are examples of positive memories that need to be further formed in the central brain – a neuronal level not affected by the PlxnC1 cKO.

We therefore, turned our attention to various neuronal hormones that may influence the formation of conditioned memory, and homed-in on oxytocin. In the oxytocin KO studies, we observed that the two conditioned memories, lasting interest and easing of stress, were both perturbed, but increased sensitivity was not. This indicated that Sema7A/PlxnC1 signaling is necessary for odor imprinting, reflected by the formation of all three learning responses. However, oxytocin is then necessary for the further establishment of positive memory (lasting interest and easing of stress).

To the question of whether the authors observed alterations in Sema7A signaling in oxytocin knockout mice: Sema7A expression is not affected in the oxytocin KO (Figure 7—figure supplement 1). In the KO, imprinting still occurs as evidenced by an increase in odor sensitivity (Figure 7A), but conditioned learning does not. In other words, positive quality is not imposed on the imprinted odor, as observed by a negative response to the hyperthermia test. New data for the stress-reducing experiment is now shown in Figure 7B. We conclude that imprinting and conditioned memory formation are independently regulated by Sema7A/PlxnC1 and oxytocin, respectively, during the critical period. These observations also demonstrate that Sema7A/PlxnC1 signaling is not downstream of oxytocin signaling, although oxytocin is needed for establishing positive memory of imprinted odors.

These are now described in the revised text with new figures (Figure 7 and Figure 7—figure supplement 1). As both signaling systems play essential roles in establishing imprinted memory during the critical period, we would like to keep the description of oxytocin in the manuscript.

2) The reviewers request testing Sema7a mutants for behavioral effects in odor paradigms presented (including those in Figure 2 and Figure 6).

The editor makes mention of behavioral effects in the Sema7A KO. At present, we only have a total KO line for Sema7A, but not its cKO specific to OSNs. The total KO of Sema7A would not be appropriate for behavioral tests, because Sema7A is expressed in various brain regions (Carcea et al., 2014). Fortunately, however, we have the cKO of PlxnC1 specific to M/T cells, which blocks Sema7A signaling in the neonatal glomeruli. We have previously reported that the effects of Sema7A total KO and M/T-cell-specific PlxnC1 cKO are essentially the same in synapse formation and dendrite selection within glomeruli (Inoue et al., 2018).

We analyzed the PlxnC1 cKO mice for their responses to the conditioned odor in the habituation/dishabituation test and hyperthermia test. In the PlxnC1 cKO, neither an increase in the sensitivity, nor interest toward the conditioned odor was observed (Figure 6A). Stress was not eased by the conditioned odor in the hyperthermia test (Figure 6D). For their social responses, results in the three-chamber test are already shown in Figure 6C, using the PlxnC1 cKO where the sema7A signaling is blocked in M/T cells. In the cKO, abnormal social behavior is observed, avoiding social interactions with unfamiliar mice.

These are now described in the text with new figures (Figure 6A and D).

3) The authors should improve description and validation of several mouse models.

As advised by the editor, we described additional information for the mouse lines for Tg-Sema7A (Figure 5—figure supplement 2A) and PlxnC1 cKO in the text (Results and Materials and methods).

Reviewer #1:

This manuscript investigates a role for Sema7A/PlxnC1 in an olfactory critical period. The authors observe that mice who encounter odors as neonates (before ~P7) display altered behavioral and neuronal responses to those odors as adults. Neonatal-experienced odors are investigated more vigorously, and mildly induce stress, perhaps akin to familiar nest odors. The authors report that neonatal odor exposure increases Sema7a expression, enlarges target glomeruli through increased numbers of periglomerular cells, and increases the percentage of mature mitral cells. The authors then show that knockout of Plxn1 or oxytocin impairs social odor recognition. There are many interesting findings here, and the manuscript is well written. Yet, some of the key findings are not thematically connected and additional experiments are needed to strengthen links between Sema7A signaling, oxytocin, and behavioral responses.

1) The authors show that neonatal-experienced odors are more attractive to odors, and that neonatal odors upregulate Sema7A in responsive neurons. Yet, a role for Sema7A/PlxnC1 in enhanced neonatal odor investigation is not reported. Do Sema7a knockout mice lose effects of neonatal-experienced odors described in Figure 2? Are they rescued by re-expression of Sema7A in olfactory sensory neurons (using Tg-Sema7a)?

The mice demonstrate positive responses to the conditioned odor as adults. Stress is lowered by the imprinted odor in the hyperthermia test. This stress-easing effect cannot be seen when Sema7A signaling is blocked in the cKO of PlxnC1 (Figure 6D). A role of Sema7A/PlxnC1 signaling in odor imprinting is now discussed more in the text with new behavioral data of the PlxnC1 cKO (Figure 6A and D).

The reviewer also suggests a rescue experiment in the Sema7A KO using the Tg-Sema7A. This would be an excellent experiment to strengthen the Sema7A data. However, the Tg-Sema7A described in Figure 5 can be analyzed only in the rI7-expressing OSNs, because of its MOR23 promoter for co-expression. Furthermore, our Sema7A KO is a total KO and unfortunately would not be appropriate for behavioral analyses because Sema7A is expressed in various brain areas (Carcea et al., 2014). For these reasons, the rescue experiment advised by the reviewer cannot be performed without generating a new cKO line.

2) The connection between oxytocin and Sema7A signaling is unclear. Does oxytocin alter expression/localization of PlxnC1 and/or Sema7A? Can the authors propose and test models for how oxytocin impacts olfactory circuits?

As also pointed out by the editor and other reviewers, we agree that a link between the two signaling systems, Sema7A/PlxnC1 and oxytocin is not clearly described in the manuscript. With the addition of new data, we hope that this point is made clearer in our revised manuscript.

As mentioned in the responses to other reviewers/editor, these two signaling systems are not directly linked to one another in imprinting. Separately from Sema7A/PlxnC1 signaling, oxytocin in neonates is needed for imposing the positive quality on neonatal odor memory and positive responses to the imprinted odor is not observed in the oxytocin KO (Figure 7B). Interestingly, however, increase in the sensitivity to the experienced odor can still be seen in the oxytocin KO (Figure 7A). Furthermore, Sema7A expression is not affected in the oxytocin KO (Figure 7—figure supplement 1). Our observations demonstrate that changes in the odor sensitivity and odor quality are separately regulated by Sema7A/PlxnC1 and oxytocin, respectively. These are now described in the text with new data (Figure 7) and discussed more in the text.

3) As the authors have demonstrated beautifully in prior studies, activity through Gs is needed for map establishment while Golf/CNGa2 activity is needed for odor-evoked activity and maintenance. The paper would benefit by further discussion of the roles of “activity” and that whether Sema7a contributes only to maintenance and not map establishment.

We agree that this is an important point. The basic architecture of the olfactory circuit is generated by the genetic program, then further refined and modified by neuronal activity. During development, a coarse olfactory map is generated by the combination of anterior/posterior and dorsal/ventral targeting using separate sets of axon guidance molecules (Sakano, 2010). These processes are independent from the neuronal activity of OSNs. The map is then refined by axon-sorting molecules whose expression is regulated by neuronal activity of OSNs. It has been reported that this activity is not odor-evoked, but is caused by the spontaneous firing of OSNs (Reisert, 2010; Nakashima et al., 2019). The intrinsic activity of OSNs also mediates basic synapse formation within glomeruli in the absence of environmental odorants. The olfactory map is further modified by the odor-evoked neuronal activity of OSNs by enlarging the responding glomeruli. This process is plastic and mediated by Sema7A/PlxnC1 signaling during the neonatal critical period. Sema7A expression is induced by OR-specific activity and promotes post-synaptic events within glomeruli (Inoue et al., 2018). Although the blockage of Sema7A/PlxnC1 signaling diminishes odor-evoked synapse formation, targeting of OSN axons to the OB is not affected. These are now described in the Introduction and Discussion.

4) Provide additional explanation in the main text and validation of the Tg-Sema7A line. What % of olfactory sensory neurons re-express Sema7a? Likewise, additional validation of the mitral cell-specific PlxnC1 knockout is needed.

For the Tg-Sema7A mice, we obtained five transgenic lines, all of which successfully expressed Tg-Sema7A in the rI7-positive glomeruli. Compared with the endogenous Sema7A in the WT, expression levels of Tg-Sema7A were ~10% within the rI7 glomeruli in the CNG/Sema7A KO (Results) (Figure 5—figure supplement 2A). Since the staining signals of Sema7A were not strong enough within individual cells for counting, exact % of OSNs re-expressing Sema7A were difficult to determine (Materials and methods).

As for the M/T-specific cKO of PlxnC1, it was generated using the Pcdh21-Cre mouse as a KO driver (Inoue et al., 2018). Absence of PlxnC1 expression was confirmed by in situ hybridization and immunostaining. These are now mentioned in the text (Results and Materials and methods).

5) Representative images should be shown for all data presented as a signal intensity, including Figures 1A, 3A, B, 4, and 5. Also, images showing the differences between mature/immature dendrite selection are needed (3B).

As advised, representative images are now shown in the new supplementary figures as follows:

Figure 1—figure supplement 1 is for the data in Figure 1A.

Figure 3—figure supplement 1A is for Figure 3A.

Figure 3—figure supplement 1B and Figure 3—figure supplement 2 are for Figure 3B.

Figure 3—figure supplement 1A and Figure 4—figure supplement 1 are for Figure 4.

Figure 5—figure supplement 2 is for Figure 5.

6) A home cage nest odor would be a nice positive control for Figure 2B, for context to understand the magnitude of the observed behavioral response.

As the reviewer advised, home-cage nest would be a nice positive control. Although we did not use it in our hyperthermia experiment, we tested propionic acid (PPA) as a positive control, whose odor quality is innately attractive (Figure 6D). PPA lowers stress, but its stress-reducing effect is more moderate compared with the imprinted VNL odor whose quality is innately neutral. This is now described in the text.

7) The authors claim (Results) that both tufted and periglomerular cells increase in number, but Figure 3C only provides supporting data for periglomerular cells.

We apologize to the reviewer for the poor resolution of the PDF photos for periglomerular cells. Pictured areas were also too restricted. We, therefore, replaced the figures with high resolution photos that cover larger areas of the OB (Figure 3C and Figure 3—figure supplement 1C).

Reviewer #2:

1) It is not clear exactly what the relationship is between the oxytocin experiments and the rest of this study. First, do oxytocin mutants show alterations Sema7a expression, or PlexC1 temporal distribution in the tuft structure? Why is a different behavioral assay used for the oxytocin experiments as compared to the social interaction assay used in the Plexc1 conditional mutants.

Overall, there remain questions regarding the behavioral experiments such that if the oxytocin experiment was not part of this study, it would not in my view reduce interest or impact of the work.

We apologize that the relationship between the Sema7A/PlxnC1 signaling and oxytocin experiments were not clearly described in the manuscript. This was also pointed out by the other reviewers. We observed that if the pups are exposed to a particular odorant during the critical period, they demonstrate increased sensitivity and positive responses to the conditioned odor as adults. We found that Sema7A and its receptor PlxnC1 are involved in this imprinting. Odor imprinting appears to be established by enlarging the responded glomeruli during the neonatal critical period, which is accomplished by promoting post-synaptic events within the glomeruli. In the gain-of-function and loss-of-function experiments, it became clear that Sema7A/PlxnC1 signaling is essential for imprinting (increase in the sensitivity to the conditioned odorant). However, it was not clear to us whether the Sema7A/PlxnC1 signaling is also involved in imposing positive quality on the imprinted odor.

We, therefore, searched for candidate hormones expressed in the neonatal brain that may be responsible for conditioned learning and found that oxytocin KO mice fail to demonstrate positive responses to imprinted odorants. As examples, lasting interest in the imprinted odor is not seen in the habituation/dishabituation test (Figure 7A) and stress in the KO is not eased by imprinted odor memory as observed in the hyperthermia test (Figure 7B). Interestingly, however, increase in the sensitivity can still be found for the conditioned odor.

As advised by the reviewer, we confirmed that Sema7A expression is not affected in the oxytocin KO mice (Figure 7—figure supplement 1). Our results indicate that Sema7A signaling is not downstream of oxytocin. Taken together, Sema7A is responsible for imprinting, while oxytocin is separately involved in imposing quality onto imprinted memory without an effect on Sema7A function. These are now described in the text and additional data are shown in Figure 7 and Figure 7—figure supplement 1.

The reviewer also points out why the behavioral assay used for oxytocin (old Figure 6) is different from that for PlxnC1 cKO. Separately from other social experiments, the social memory test for oxytocin KO was performed in a different laboratory in collaboration with Dr. Kikusui’s group at Azabu University. In attempts to link these two sets of experiments, we added new data of the stress-reducing experiments for both the oxytocin KO and PlxnC1 cKO (Figures 6D and 7B).

2) Does loss of Sema7a in ORs lead to the same behavioral phenotypes observed in the cKO M/T PlexC1 mutants? And is there any way to show that the loss-of-function (LOF) experiment involving PlexC1 is dependent upon LOF during the critical period? For this current study, addressing the Sema7a requirement genetically in this behavioral assay seems to be an issue that should be tackled in the context of the social interaction assay, if it is to be presented here.

The reviewer asks whether we could obtain the same behavioral phenotypes in the Sema7A KO as observed in the PlxnC1 cKO. We agree that this is an important point. However, the Sema7A KO used in our study is a total KO and cannot be used for behavioral experiments as Sema7A is also expressed in various brain areas other than the olfactory system. Thus, we would need to generate the cKO of Sema7A to address the reviewer’s comment. At least for synapse formation and dendrite selection of M/T cells, effects of PlxnC1 cKO are essentially the same as that of Sema7A KO (Inoue et al., 2018). This is because both molecules are membrane-bound proteins and function together by interacting with one another.

The reviewer also suggests interesting experiments of loss-of-function and gain-of-function for PlxnC1 by changing its localization time-frame. This is an elegant experiment to link the functions of PlxnC1 at the molecular and behavioral levels. It would be nice if we could see the shift of time window for the olfactory critical period. One difficulty here is that it would not be easy to regulate not only the expression of PlxnC1, but also its localization to M/T-cell dendrites. This experiment would require new knock-in mice containing the plasmid that directs on and off of PlxnC1 localization at various time points. We would like to incorporate this experiment in our future project. In the revised manuscript, the reviewer’s point is mentioned in the text.

3) I do not have many comments on the first part of the study, however, and find it to be quite interesting. I do wonder:

a) If in the conditional CNG mutants, whether or not there is any change in the temporal localization of PlexC1 with respect to the presented postnatal localization in the tuft structure-is this, too, activity dependent and/or regulated by Sema7a during the critical period.

b) Is there any evidence that Sema7a OR expression normally follows a time course that directly links it to the critical period. This seems like a point that could be easily addressed.

The reviewer asks whether the PlxnC1 localization is affected by OR activity or by Sema7A expression in OSNs. We are finding that PlxnC1 localization is not affected in the Sema7A KO or in the CNG-A2 KO where Sema7A expression is severely lowered. Although Sema7A and PlxnC1 function together by interacting with one another, PlxnC1 expression is not affected by its partner molecule. These are now stated in the text and shown in Figure 4—figure supplement 1B.

The reviewer then asks whether there is any evidence that Sema7A and OR expression follow a time course linking them to the critical period. Each OR species has own onset time for its expression during development, which is restricted to the time frame of the critical period. Also, Sema7A or oxytocin expression is not restricted to the first week after birth. Among the three key molecules (Sema7A, PlxnC1, and oxytocin) involved in imprinting, only PlxnC1’s localization to M/T-cell dendrites is directly correlated to the time frame of the olfactory critical period. This is now mentioned in the text.

Reviewer #3:

1) The conclusion of the paper appears to be based on the following logical connection: activity driven changes in synaptic function in the olfactory bulb is restricted to the first week after birth because activities drive the expression of Sem7A expression, which interacts with PlxnC1, expressed only during the critical period. This is not explicitly stated, nor extensively discussed. Although the model is very attractive, the experimental evidence is correlational. The conclusion that Sema7A/PlxnC1 support the olfactory critical period is largely based on the restricted dendritic expression PlxnC1 during the first postnatal week.

There are three major molecules involved in olfactory imprinting in neonates: They are (1) Sema7A that supports odor-induced activity dependency of the imprinting process, (2) PlxnC1 that is a receptor of Sema7A, which restricts the time frame of the critical period to the first week after birth, and (3) Oxytocin that is needed in imposing the positive quality on imprinted odor memory. As also pointed out by the other reviewers, we agree that functional connections of these molecules are not explicitly stated nor extensively discussed. In response to these comments, we added supporting information in the text.

2) Naris occlusion and odor exposure experiments are complementary to each other to provide support for activity dependent regulation of Sema7A expression, but there is a discrepancy in the results. Naris occlusion experiment show that the level of synaptic proteins is normal if occlusion is reopened before P7. However, odor exposure using vanillin between P2 and P4 altered odor habituation and stress response. The authors have not explained the discrepancy in the experimental design and results with regard of the difference in timing.

We apologize that this point was not carefully explained in the text. In neonates, they need to be exposed to environmental odors to make the olfactory system functional. If such stimuli are blocked by naris occlusion throughout the critical period, odor perception is affected as adults. By changing the re-open timing of occluded naris started at P0, we determined the critical period to be from P0 to P7.

We also performed a gain-of-function experiment by exposing pups to a particular odorant. Unlike the naris occlusion experiment, this experiment does not examine all glomeruli as a whole, but only a particular OR. Since the onset of OR genes varies among different OR species, timing for gain-of-function (glomerular enlargement/imprinting) is different from glomerulus to glomerulus. Furthermore, synapse formation occurs using not only odor-evoked but also intrinsic OR activity. Since the early-onset OR with low intrinsic activity is convenient for our gain-of-function experiment, we chose MOR29A whose ligand is known as VNL. We found that exposure to VNL only for a few days during the critical period enhanced synapse formation in the responding glomeruli and increased the sensitivity to VNL. In the case of VNL-responsive MOR29A, P2~4 was most efficient for imprinting, but P5~7 was much less effective (Figure 3B and D).

The reviewer points out the discrepancy between the loss-of-function and gain-of-function experiments for the timing of critical period. It should be noted that in the loss-of-function experiment (naris occlusion), the critical period covers the maximum time frame for all OR species, while in the gain-of-function experiment, (odor exposure) critical periods are narrower and different among OR species depending upon their onset. These are now explained in the text.

3) The CNGA2 KO experiments lend support for activity-dependent expression of Sema7A, and the Sema7A rescue experiment provides further support that Sema7A could induced synaptic protein expression. However, different glomeruli exhibit different levels of Sema7A expression. Is this difference intrinsic to different types of OSNs, or is it caused by differences in neural activity?

Our experiments of KO, naris occlusion, and odor exposure all support the involvement of OR-derived neuronal activity in synapse formation mediated by Sema7A. As pointed out, it was not made clear in our previous manuscript whether the OR-derived activity is intrinsic or odor-evoked. We assume that both types of activities are involved in synapse formation within glomeruli. Since unique levels of Sema7A are detected in each glomerular species in unstimulated mice (please see glomerular ranking for Sema7A expression in Figure 4B), intrinsic OR activity appears to be responsible for forming basic circuitry.

However, synapses are further strengthened and glomerular structures enlarged by odor-evoked OR activity in neonates. This plastic process in neonates induced by environmental odors is responsible for the establishment of imprinted odor memory during the critical period. Thus, different levels of Sema7A, as shown in the glomerular rank in the unstimulated mice, are due to the differences in intrinsic OR activity, while differences in Sema7A expression in the odor-stimulated glomeruli are due to odor-evoked neural activity. These are now described is the Results and discussed more in the Discussion.

4) The conditional PlxnC1 KO mice need more rigorous testing. If activity-dependent expression of Sema7A and Sema7A/PlxnC1 interaction are important for synapse formation in the olfactory glomeruli, odor detection and discrimination are expected to be affected. No experiments have been conducted to address this prediction. It is also not known whether the KO abolishes the phenotypes observed in Figure 2, which are associated with vanillin exposure during the critical period. Moreover, it is not clearly stated how the three-chamber assay is related olfactory imprinting. A likely explanation of the observed impairment of odor preference and social behavior is that the overall olfactory sense has been compromised.

PlxnC1 cKO failed to demonstrate stress-reducing effect to VNL as adults in the hyperthermia test (Figure 6D). In the three-chamber test, WT mice demonstrate curiosity responses to foreign mouse scents, but the PlxnC1 cKO avoid them (Figure 6C). It is possible that the mice innately avoid foreign mouse scents that may be stressful to them, but odor-induced imprinting helps them feel comfortable to the surrounding mice for smooth social interactions. We assume that this is due to the lack of odor imprinting mediated by odor-evoked OR-activity.

As pointed out, in the PlxnC1 cKO where Sema7A signaling is blocked, odor detection and discrimination are lowered (Figure 6A). However, it is notable that these mice still demonstrate attractive responses to food smells and aversive behavior toward foreign mouse scents (Figure 6B). It appears that even in the absence of Sema7A signaling in the cKO, basic olfactory circuits, particularly to induce innate responses, can be established based on a genetic program, e. g., formation of gap junctions and connections to interneurons (Gire et al., 2012). In the CNG-A2 KO, synapse formation and dendrite selection within glomeruli are initially impaired, but later recover presumably using intrinsic OSN activity.

We, therefore, do not think that the observed impairment in odor preference and social behavior is due to the overall compromise of olfactory sensing as the reviewer points out. As we agree that this is an important point, we added some discussion in the new text.

5) The oxytocin KO experiment is interesting. However, the behavioral phenotype is difficult to interpret. WT mice exhibit a decline in investigation of an ovariectomized female whereas the oxy-/- mice show persistent investigation. The authors interpret the decline as an indication of social memory, but the lack of decline/persistent investigation in the oxy-/- mice could be interpreted as prosocial interactions. This interpretation would be consistent with the decrease of investigation in WT mice after P0-P6 oxytocin injection. Importantly, the authors have not addressed how this experiment is related to activity-dependent modulation of glomerular connectivity.

In the social-memory test shown in Figure 8, WT mice exhibit a decline in investigation of female mice while the oxytocin KO demonstrates persistent investigation. We assume that this is due to the lack of positive imprinting to become friendly to fellow mice, which is mediated by oxytocin in neonates. Oxytocin KO is not accustomed to the unfamiliar mouse scents in adults. This is supported by our observation that in the oxytocin KO, the sensitivity to the imprinted odor is increased but the positive quality is not imposed on the conditioned odor (Figure 7). Thus, Sema7A/PlxnC1 signaling is needed for imprinting (increase in the sensitivity to the imprinted odor), but oxytocin is separately needed to add the positive quality on the imprinted memory of mouse scents to establish social memory. These are now described in the text.

6) The authors present data with quantification, but primary data (images) are not presented. This makes it difficult to assess the accuracy of the quantification. Specifically, primary images demonstrating the selective induction of Sema7A expression in the MOR29A glomeruli after vanillin exposure should be compared with no-odor control side-by-side.

The reviewer points out that primary data are needed to assess the accuracy of the quantification. We now added primary images (Figure 1—figure supplement 1, Figure 3—figure supplement 1, Figure 4—figure supplement 1, and Figure 5—figure supplement 2), specifically for the selective induction of Sema7A in the VNL-exposed glomeruli (Figure 3—figure supplement 1A).

https://doi.org/10.7554/eLife.65078.sa2

Article and author information

Author details

  1. Nobuko Inoue

    Department of Brain Function, School of Medical Sciences, University of Fukui, Matsuoka, Japan
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  2. Hirofumi Nishizumi

    Department of Brain Function, School of Medical Sciences, University of Fukui, Matsuoka, Japan
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6083-9233
  3. Rumi Ooyama

    Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared
  4. Kazutaka Mogi

    Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan
    Contribution
    Data curation, Formal analysis, Validation, Investigation
    Competing interests
    No competing interests declared
  5. Katsuhiko Nishimori

    Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan
    Present address
    Department of Obesity and Internal Inflammation, Fukushima Medical University, Fukushima, Japan
    Contribution
    Resources
    Competing interests
    No competing interests declared
  6. Takefumi Kikusui

    Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, Sagamihara, Japan
    Contribution
    Data curation, Supervision, Validation, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3134-5859
  7. Hitoshi Sakano

    Department of Brain Function, School of Medical Sciences, University of Fukui, Matsuoka, Japan
    Contribution
    Conceptualization, Supervision, Funding acquisition, Validation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    sakano.hts@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5333-1722

Funding

JSPS (KAKENHI 24000014&17H06160)

  • Hitoshi Sakano

JSPS (KAKENHI 17K19386 & 20K06909)

  • Hirofumi Nishizumi

Japan Foundation for Applied Enzymology (J180000322)

  • Nobuko Inoue

The Naito Foundation

  • Hirofumi Nishizumi

MEXT (KAKENHI 17H05943 & 19H04745)

  • Hirofumi Nishizumi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to H Naritsuka for her advice in single-cell labeling and H Sagara for his help in EM analysis. We thank members of our laboratory for valuable suggestions and discussion. This research was supported by the JSPS KAKENHI (Grant Numbers 24000014 and 17H06160 to HS; 17K19386 and 20K06909 to HN) and the MEXT KAKENHI (Grant Numbers 17H05943 and 19H04745 to HN). This work was also supported by the Japan Foundation for Applied Enzymology (J180000322 to NI) and the Naito Foundation (HN).

Ethics

Animal experimentation: the Animal Care Committees at University of Fukui and Azabu University.

Senior Editor

  1. Gary L Westbrook, Oregon Health and Science University, United States

Reviewing Editor

  1. Stephen Liberles, Harvard Medical School, United States

Publication history

  1. Received: November 21, 2020
  2. Accepted: March 18, 2021
  3. Version of Record published: March 29, 2021 (version 1)

Copyright

© 2021, Inoue et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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