Spontaneous Ca2+ signals in adult-born juxtaglomerular neurons during their integration into the glomerular layer of the mouse olfactory bulb

Adult mammalian brain is able to generate new neurons throughout life. Adult neural stem cells reside along the walls of the brain lateral ventricles – in a region called subventricular zone (SVZ). In the rodent brain thousands of neuroblasts that are born in the SVZ migrate into the olfactory bulb every day via a pathway that in called rostral migratory stream (RMS), to become functional interneurons – granule cells or juxtaglomerular neurons (JGNs). In the developing nervous system spontaneous calcium transients are believed to be involved in the regulation of neuronal migration, differentiation and survival. In contrast to embryonic development, adult-born neurons have to integrate into the mature environment. It was shown that spontaneous calcium signals are present in adult-born neuroblasts migrating in the rostral extension of the olfactory bulb in-vitro. However, it was not known whether spontaneous calcium signals are present in the immature adult-born JGNs during their integration into mature neuronal circuit in vivo. In the present work in-vivo two-photon calcium imaging was used to study spontaneous calcium signals in adult-born JGNs during their integration into the glomerular layer of the bulb. Adult-born JGNs were labelled via injection of lentivirus encoding the ratiometric fluorescent calcium sensor Twitch-2B into the rostral migratory stream. Twitch-2B is comprised of two fluorescent proteins: mCerulean3 and cpVenusCD that are linked by a calcium-binding region. Therefore, the dynamics of the intracellular calcium concentration is reflected in the corresponding dynamics of the cpVenusCD/mCerulean3 ratio. In order to characterize properties of spontaneous calcium transients, values of the maximal cpVenusCD/mCerulean3 ratio, fraction of time spent in active state and normalized area under the curve were calculated from corresponding traces of spontaneous calcium signals. The results showed that majority of adult-born JGNs studied in awake state between 8th and 11th days post injection (DPI) exhibit spontaneous calcium transients. Acute in-vivo experiments performed under isoflurane revealed that spontaneous calcium signals are blocked by an addition of 2 µM tetrodotoxin (TTX) into the standard extracellular solution. Substantial fraction of immature adult-born JGNs (DPI 9-11) that were spontaneously active in awake sate, showed odorant-evoked calcium transients under 3 component anaesthesia. However, there was no significant difference in the properties of spontaneous calcium transients between odorant-responsive and odorant non-responsive cells. At DPI 9, the majority of adult-born JGNs expressed immature neuronal markers, while expression of the mature markers was very limited. At DPI 20-24, the expression of mature neuronal markers was clearly upregulated. Despite the maturation of the neurochemical properties of adult-born JGNs, there was no significant difference in the percentage of spontaneously active adult-born JGNs in awake state at DPI 8-11 vs. DPI 20-22. However, some maturational dynamics was seen in the pattern of spontaneous calcium signals. Thus, the present work provided in-vivo evidence of the presence of TTX-sensitive spontaneous calcium signals in immature adult-born JGNs; revealed substantial temporal overlap between spontaneous and sensory-evoked activity of immature adult-born JGNs and showed maturational dynamics of spontaneous calcium transients as well as neurochemical properties of adult-born JGNs during their integration into the mature environment of the adult brain