The awakening of dormant neuronal precursors in the adult and aged brain

Abstract

Beyond the canonical neurogenic niches, there are dormant neuronal precursors in several regions of the adult mammalian brain. Dormant precursors maintain persisting post-mitotic immaturity from birth to adulthood, followed by staggered awakening, in a process that is still largely unresolved. Strikingly, due to the slow rate of awakening, some precursors remain immature until old age, which led us to question whether their awakening and maturation are affected by aging. To this end, we studied the maturation of dormant precursors in transgenic mice (DCX-CreER^T2 /flox-EGFP) in which immature precursors were labelled permanently in vivo at different ages. We found that dormant precursors are capable of awakening at young age, becoming adult-matured neurons (AM), as well as of awakening at old age, becoming late AM. Thus, protracted immaturity does not prevent late awakening and maturation. However, late AM diverged morphologically and functionally from AM. Moreover, AM were functionally most similar to neonatal-matured neurons (NM). Conversely, late AM were endowed with high intrinsic excitability and high input resistance, and received a smaller amount of spontaneous synaptic input, implying their relative immaturity. Thus, late AM awakening still occurs at advanced age, but the maturation process is slow.

Keywords: action potential; aged brain; axon initial segment; dormant precursor; doublecortin; neurogenesis; neuronal precursor; synapse.

FIGURE 1

Designation and definition of experimental groups. (a) Graph highlights the designation of three experimental groups used to define age‐related effects on morphological traits of adult‐matured neurons (AM). Tamoxifen was administered at 2–3 months of age. Ex vivo analysis was carried out at 9 months (T1), 15 months (T2) and > 24 months (T3). (b) Graph highlights the designation of two experimental groups used to determine effects of age of maturation onset in relation to AM morphology and function. Tamoxifen was administered at 2–3 months of age (AM) or at 9 months of age (late AM). Analysis was carried out ex vivo at 15 months in both groups.

FIGURE 2

Morphological characteristics of adult‐matured neurons (AM) according to age. (a) The density, morphology, and neurite branching of AM was comparable between adult (T1) and aged brain (T3). (b) Subtle morphological differences were revealed by analysis of individual cells at different time points, for example, the axon initial segment (AIS; outlined by the detection of Ank‐G, highlighted by yellow arrows). (c) Density of labelled cells did not change significantly across ages. (d) Soma size of AM decreased significantly with age. (e) AIS length, as outlined by the scaffolding protein Ank‐G, decreased significantly with age in neurons expressing EGFP. (f) Dendrite length of labelled neurons decreased significantly between T1 and T2. (g) Juxtaposition of synaptophysin puncta (red) and EGFP+ synaptic spines on AM at different age allowed to estimate the degree of pre‐postsynaptic contacts. Top and bottom inset highlight a spine with and a spine without postsynaptic puncta respectively. (h) The density of dendritic spines did not change significantly with age. (i) The percentage of synaptophysin+ puncta juxtaposed to dendritic synapses increased with age, with significant difference detected between T1 and T2. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Morphological differences according to age of maturation onset. (a) Typical morphology of AM labelled at 3 months and analyzed at 15 months. Note the higher abundance of labelled cells in comparison to late AM in (b). (b) Typical morphology of AM with late onset of maturation, following labelling of dormant precursors at 9 months and analysis at 15 months. (c) Cell density of AM was significantly higher (by about 20‐fold) than that of late AM. (d) Soma size of AM was significantly smaller than that of late AM. (e) AIS length of AM and late AM were not significantly different. (f) Dendrite length of AM and late AM were not significantly different. (g) Density of dendritic spines in AM was significantly smaller than that of late AM. (h) The percentage of synaptophysin+ puncta juxtaposed to dendritic synapses in AM and late AM were not significantly different. *p < 0.05; **; ***p < 0.001.

FIGURE 4

Action potential (AP) firing and intrinsic excitability of NM, AM, and late AM. (a) Typical patterns of action potential (AP) firing in NM and AM, evoked by chronic injection of current during single‐cell current clamp experiments. (b) Relation between the amplitude of input current injected and frequency of action potential firing showed the slightly higher intrinsic excitability of AM and late AM in comparison to NM; the differences were not significant. (c) The minimum input current necessary to elicit action potentials in NM was slightly larger compared with AM (not significant) and significantly larger compared with late AM. (d) The input resistance (R _in) of NM was slightly smaller (not significant) compared with AM and significantly smaller than that of late AM. (e) The ohmic relation between R _in and rheobase is highlighted by individual data distribution showing coincidence of higher rheobase and lower R _in. (f) The capacitance of NM was slightly larger (not significant) than that of AM and late AM. (g) The resting membrane potential (RMP) of NM, AM, and late AM were not significantly different. (h) Analysis of the first derivative of voltage over time (V/s) highlights the different kinetics (AP slope) in the AP of NM and AM. AP kinetics are displayed in relation to membrane voltage (E _M). (i) The maximum slope of AP in NM was significantly higher than that of AM and significantly higher than that of late AM. (j) The AP thresholds of NM, AM, and late AM were not significantly different.

FIGURE 5

Spontaneous excitatory currents (PSC) received by NM, AM, and late AM measured near RMP (−70 mV) in single‐cell voltage‐clamp experiments. (a) Representative traces display spontaneous synaptic input in NM (black) and AM (light green) and late AM (dark green). (b) Histogram shows the distribution of PSC amplitude and frequency in NM, AM, and late AM. (c) The PSC frequencies of NM and AM are not significantly different. The PSC frequency of late AM is significantly lower than that of AM (d). The PSC amplitudes of NM and AM are not significantly different. However, the PSC amplitude of late AM is significantly larger than that of NM and AM.