Adeno-associated viral-based strategy for morphological and functional dissection of olfactory dopaminergic neurons

Dopaminergic (DA) neurons of the olfactory bulb (OB) represent one of the largest forebrain DA populations, and increasing evidence highlights their role in shaping odor representations through presynaptic gain control, lateral inhibition, and circuit plasticity. These neurons are heterogeneous, comprising small, axonless cells generated throughout life and larger, axon-bearing cells whose generation is limited to embryonic and perinatal stages. Establishing functional distinctions between these subtypes has been hindered by limited tools for selective targeting. Here, we developed an adeno-associated viral (AAV) strategy using the tyrosine hydroxylase (TH) promoter to drive Cre recombinase expression (AAV-rTH-Cre), enabling temporally controlled labeling of OB DA neurons. Intracerebroventricular injections at embryonic day 14.5 preferentially enriched the targeted DA population in large cells, while adult injections predominantly targeted small DA neurons, consistent with their developmental origins. Neonatal injections yielded reduced specificity, likely reflecting developmental and technical constraints on TH promoter activity. To assess functional properties, we combined AAV-rTH-Cre with Cre-dependent GCaMP7s and performed two-photon calcium imaging in adult mice. Our data revealed significant heterogeneity in spontaneous activity, showing a negative correlation between soma size and both spiking probability and event frequency. Thus, smaller DA neurons exhibited higher spontaneous activity, whereas larger neurons were less active despite their higher intrinsic excitability reported previously. These findings validate AAV-rTH-Cre as a versatile tool for dissecting OB DA subtypes and provide direct in vivo evidence that their morphological diversity translates into distinct spontaneous activity patterns, supporting the hypothesis of complementary roles in olfactory processing.