Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics.

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

In vivo live imaging of postnatal neural stem cells.

Neural stem cells (NSCs) are maintained in specific regions of the postnatal brain and contribute to its structural and functional plasticity. However, the long-term renewal potential of NSCs and their mode of division remain elusive. The use of advanced in vivo live imaging approaches may expand our knowledge of NSC physiology and provide new information for cell replacement therapies. In this Review, we discuss the in vivo imaging methods used to study NSC dynamics and recent live-imaging results with respect to specific intracellular pathways that allow NSCs to integrate and decode different micro-environmental signals. Lastly, we discuss future directions that may provide answers to unresolved questions regarding NSC physiology.

The Role of Adult-Born Neurons in the Constantly Changing Olfactory Bulb Network.

The adult mammalian brain is remarkably plastic and constantly undergoes structurofunctional modifications in response to environmental stimuli. In many regions plasticity is manifested by modifications in the efficacy of existing synaptic connections or synapse formation and elimination. In a few regions, however, plasticity is brought by the addition of new neurons that integrate into established neuronal networks. This type of neuronal plasticity is particularly prominent in the olfactory bulb (OB) where thousands of neuronal progenitors are produced on a daily basis in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) towards the OB. In the OB, these neuronal precursors differentiate into local interneurons, mature, and functionally integrate into the bulbar network by establishing output synapses with principal neurons. Despite continuous progress, it is still not well understood how normal functioning of the OB is preserved in the constantly remodelling bulbar network and what role adult-born neurons play in odor behaviour. In this review we will discuss different levels of morphofunctional plasticity effected by adult-born neurons and their functional role in the adult OB and also highlight the possibility that different subpopulations of adult-born cells may fulfill distinct functions in the OB neuronal network and odor behaviour.

Distinct forms of structural plasticity of adult-born interneuron spines in the mouse olfactory bulb induced by different odor learning paradigms.

The morpho-functional properties of neural networks constantly adapt in response to environmental stimuli. The olfactory bulb is particularly prone to constant reshaping of neural networks because of ongoing neurogenesis. It remains unclear whether the complexity of distinct odor-induced learning paradigms and sensory stimulation induces different forms of structural plasticity. In the present study, we automatically reconstructed spines in 3D from confocal images and performed unsupervised clustering based on morphometric features. We show that while sensory deprivation decreased the spine density of adult-born neurons without affecting the morphometric properties of these spines, simple and complex odor learning paradigms triggered distinct forms of structural plasticity. A simple odor learning task affected the morphometric properties of the spines, whereas a complex odor learning task induced changes in spine density. Our work reveals distinct forms of structural plasticity in the olfactory bulb tailored to the complexity of odor-learning paradigms and sensory inputs.

Live imaging of adult neural stem cells in freely behaving mice using mini-endoscopes.

During adulthood, the activation of adult neural stem cells (NSCs) has been mostly studied ex vivo in post-mortem tissues or in vivo in anesthetized animals. This protocol presents an approach that allows for the long-term and minimally invasive investigation of adult NSC activation and physiology in freely behaving animals. By combining specific NSC labeling and mini-endoscopic microscopy, live imaging of NSC division and Ca2+ activity can be performed continuously for 2-3 days and even up to several months. For complete details on the use and execution of this protocol, please refer to Gengatharan et al. (2021).

Deciphering Brain Function by Miniaturized Fluorescence Microscopy in Freely Behaving Animals.

Animal behavior is regulated by environmental stimuli and is shaped by the activity of neural networks, underscoring the importance of assessing the morpho-functional properties of different populations of cells in freely behaving animals. In recent years, a number of optical tools have been developed to monitor and modulate neuronal and glial activity at the protein, cellular, or network level and have opened up new avenues for studying brain function in freely behaving animals. Tools such as genetically encoded sensors and actuators are now commonly used for studying brain activity and function through their expression in different neuronal ensembles. In parallel, microscopy has also made major progress over the last decades. The advent of miniature microscopes (mini-microscopes also called mini-endoscopes) has become a method of choice for studying brain activity at the cellular and network levels in different brain regions of freely behaving mice. This technique also allows for longitudinal investigations while animals carrying the microscope on their head are performing behavioral tasks. In this review, we will discuss mini-endoscopic imaging and the advantages that these devices offer to research. We will also discuss current limitations of and potential future improvements in mini-endoscopic imaging.

The role of calretinin-expressing granule cells in olfactory bulb functions and odor behavior.

The adult mouse olfactory bulb is continuously supplied with new neurons that mostly differentiate into granule cells (GCs). Different subtypes of adult-born GCs have been identified, but their maturational profiles and their roles in bulbar network functioning and odor behavior remain elusive. It is also not known whether the same subpopulations of GCs born during early postnatal life (early-born) or during adulthood (adult-born) differ in their morpho-functional properties. Here, we show that adult-born calretinin-expressing (CR+) and non-expressing (CR-) GCs, as well as early-born CR+ GCs, display distinct inhibitory inputs but indistinguishable excitatory inputs and similar morphological characteristics. The frequencies of inhibitory post-synaptic currents were lower in early-born and adult-born CR+ GCs than in adult-born CR- neurons. These findings were corroborated by the reduced density of gephyrin+ puncta on CR+ GCs. CR+ GCs displayed a higher level of activation following olfactory tasks based on odor discrimination, as determined by an immediate early gene expression analysis. Pharmacogenetic inhibition of CR+ GCs diminished the ability of the mice to discriminate complex odor mixtures. Altogether, our results indicate that distinct inhibitory inputs are received by adult-born CR+ and CR- GCs, that early- and adult-born CR+ neurons have similar morpho-functional properties, and that CR+ GCs are involved in complex odor discrimination tasks.

CaMKIIα Expression Defines Two Functionally Distinct Populations of Granule Cells Involved in Different Types of Odor Behavior.

Granule cells (GCs) in the olfactory bulb (OB) play an important role in odor information processing. Although they have been classified into various neurochemical subtypes, the functional roles of these subtypes remain unknown. We used in vivo two-photon Ca2+ imaging combined with cell-type-specific identification of GCs in the mouse OB to examine whether functionally distinct GC subtypes exist in the bulbar network. We showed that half of GCs express Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα+) and that these neurons are preferentially activated by olfactory stimulation. The higher activity of CaMKIIα+ neurons is due to the weaker inhibitory input that they receive compared to their CaMKIIα-immunonegative (CaMKIIα-) counterparts. In line with these functional data, immunohistochemical analyses showed that 75%-90% of GCs expressing the immediate early gene cFos are CaMKIIα+ in naive animals and in mice that have been exposed to a novel odor and go/no-go operant conditioning, or that have been subjected to long-term associative memory and spontaneous habituation/dishabituation odor discrimination tasks. On the other hand, a perceptual learning task resulted in increased activation of CaMKIIα- cells. Pharmacogenetic inhibition of CaMKIIα+ GCs revealed that this subtype is involved in habituation/dishabituation and go/no-go odor discrimination, but not in perceptual learning. In contrast, pharmacogenetic inhibition of GCs in a subtype-independent manner affected perceptual learning. Our results indicate that functionally distinct populations of GCs exist in the OB and that they play distinct roles during different odor tasks.

Plasticity in the olfactory bulb of the maternal mouse is prevented by gestational stress.

Maternal stress is associated with an altered mother-infant relationship that endangers offspring development, leading to emotional/behavioral problems. However, little research has investigated the stress-induced alterations of the maternal brain that could underlie such a disruption of mother-infant bonding. Olfactory cues play an extensive role in the coordination of mother-infant interactions, suggesting that motherhood may be associated to enhanced olfactory performances, and that this effect may be abolished by maternal stress. To test this hypothesis, we analyzed the impact of motherhood under normal conditions or after gestational stress on olfactory functions in C57BL/6 J mice. We report that gestational stress alters maternal behavior and prevents both mothers' ability to discriminate pup odors and motherhood-induced enhancement in odor memory. We investigated adult bulbar neurogenesis as a potential mechanism of the enhanced olfactory function in mothers and found that motherhood was associated with an increased complexity of the dendritic tree of newborn neurons. This motherhood-evoked remodeling was totally prevented by gestational stress. Altogether, our results may thus provide insight into the neural changes that could contribute to altered maternal behavior in stressed mothers.