The olfactory bulb (OB), together with the hippocampus, is one of the few regions of the brain that retains adult neurogenesis by receiving a constant influx of neuroblasts during the lifetime of an individual (Bayer, 1985; Brann and Firestein, 2014; Whitman and Greer, 2009). After birth, neuroblasts targeted to the OB are generated in the subventricular zone (SVZ), a neurogenic layer located in the walls of the lateral ventricles, and migrate to the OB via the rostral migratory stream (RMS) to differentiate into several classes of OB interneurons (INs) (Lledo et al., 2008; Lois and Alvarez-Buylla, 1994). The production of OB INs begins embryonically in the dorsal part of a fetal brain structure called the lateral ganglionic eminence (LGE) (Guo et al., 2019; Stenman et al., 2003; Wichterle et al., 2001), the majority of OB INs are generated at perinatal ages and production declines to only about 2% during adulthood (Kim et al., 2020). The continuous addition of INs into the circuitry is the basis for suggesting that the OB is one of the most plastic environments of the brain, a characteristic that is crucial for essential functions such as feeding, social interactions, reproduction, memory formation, and many other behaviors (Lazarini and Lledo, 2011; Naffaa, 2025; Sakamoto et al., 2011). This process may reflect an evolutionary response to unexpected and diverse stimuli throughout life (Alvarez-Buylla et al., 2001), and disruptions in early IN development is seen in various neurodevelopmental disorders such as autism and Tourette's syndrome, and has implications for mental health (Dejou et al., 2024; Lyons-Warren et al., 2021; Meller et al., 2023).

The continuous addition of INs into the OB results in a very high ratio of INs to projection neurons (100:1) relative other brain regions, providing the OB with an extraordinary coding power to process olfactory information (Shepherd et al., 2021). The modulation of olfactory information is segregated across the OB layers, where distinct subpopulations of OB INs, which are often anaxonic, establish feedforward and feedback inhibitory circuits with projection neurons (Shepherd et al., 2021; Whitman and Greer, 2007b). As a result, depending on the location their soma, the OB INs can be classified in four groups (Nagayama et al., 2014): first are the periglomerular cells (PGCs), located in the vicinity of the glomerular layer (GL). PGCs are considered a subset of juxtaglomerular neurons (Kosaka and Kosaka, 2007; Kosaka and Kosaka, 2016; Nagayama et al., 2014). Subgroups of PGCs receive synaptic input from olfactory sensory neuron axons and make reciprocal dendrodendritic synapses with the OB projection neurons, mitral and tufted cells (M/Tc) (Shepherd et al., 2021; Wachowiak and Shipley, 2006). Second, there are INs in the external plexiform layer (EPL) that make reciprocal connections with the mitral cells, and include the superficial short-axon neurons (sSA) and multipolar neurons, among others (Nagayama et al., 2014). The third group are superficial granule cells (GCs), which are located in the mitral cell layer (MCL) and account for the majority of cells in this layer (Ennis and Hayar, 2008). GCs here are generally believed to connect predominately with tufted cells via reciprocal dendrodendritic synapses (Greer, 1987; Woolf et al., 1991). Fourth, are GCs in the granule cell layer (GCL), which contains the vast majority of OB INs, accounting for ∼94% of the total (Lledo and Valley, 2016). GCs in the GCL project their dendrites into the EPL where they interact with either mitral or tufted cells via reciprocal dendrodendritic synapses (Greer et al., 2008; Nagayama et al., 2014; Whitman and Greer, 2007b). It is generally agreed that these distinct subpopulations of INs contribute differentially to the OB activity by tuning the strength of the olfactory signal that is processed across the OB layers (Mori and Sakano, 2021).

An important aspect of the OB INs is that their function and integration into circuits is impacted by the timing of generation, particularly between embryonic and postnatal stages, making the neuronal birthdate a fundamental factor to study OB function (Batista-Brito et al., 2008; Fritz et al., 1996; Galliano et al., 2018; Kim et al., 2020; Lemasson et al., 2005; Takahashi et al., 2018). For instance, INs generated early or late in the perinatal development have different roles in regulating fear behaviors in mice exposed to aversive olfactory cues (Muthusamy et al., 2017). In addition, INs generated early in postnatal development migrate into outer regions of the OB compared with those generated at later times, which target deeper regions of the OB (Batista-Brito et al., 2008; Lemasson et al., 2005). Most recently, this has been explored by our lab where timing of neurogenesis in perinatal mice influences the placement of GCs in the OB (Liberia et al., in press). Similar patterns have been reported in projection neurons in the OB (M/Tc), which are distributed differentially within the OB axes based on the timeframe of neurogenesis, as well as showing stereotyped projections to olfactory cortex depending on their birthdates (Chon et al., 2020; Imamura et al., 2011; Imamura and Greer, 2015).

While adult neurogenesis in the OB has generated a great deal of interest and study, we sought here to focus on the development and differentiation of OB INs generated embryonically that survive into adulthood. We compared these cells with those generated in postnatal animals to assess how timing of generation influences IN placement in the OB across embryonic development and into adulthood. We tracked neurogenesis and INs positioning in the OB using thymidine analogs and immunohistochemistry, complementing our prior studies in perinatal OB (Liberia et al., in press). We also assessed embryonic OB IN migration and differentiation by labeling progenitor cells in the rostral LGE (rLGE) using in utero injection and electroporation (IUE) of the piggyBac transposon (Martin-Lopez et al., 2019a; Martin-Lopez et al., 2019b). After the analysis of the final position of embryonic INs in the adult OB following migration, we established a preference of INs to occupy the lateral OB regions compared to the medial. Furthermore, our data supported that OB INs in the GCL generated at the earliest embryonic ages occupied the outer regions of the GCL prior to the deeper regions, similarly to what occurs postnatally (Liberia et al., in press). We also analyzed their phenotypes at their final destinations in the adult OB, finding that at these ages they did not exhibit the most stereotyped phenotypes of subpopulations of INs, identified by common antibodies (Batista-Brito et al., 2008). We compared these phenotypes with INs generated in postnatal mice giving rise to similar results. Finally, the embryonic study of neuroblast morphologies and their migration from the rLGE showed a rapid population of the embryonic GCL at E13 and the formation of a prospective GL by E14. We established E15 as the age at which OB neuroblasts transitioned from migrating into differentiating INs. In addition, the formation of a primitive RMS was observed at E16. Collectively, this analysis shows the importance of timing of embryonic neurogenesis in relation to anatomical position and cell characterization in the adult OB.