Olfactory-driven beta band entrainment of limbic circuitry during neonatal development.

Cognitive processing relies on the functional refinement of the limbic circuitry during the first two weeks of life. During this developmental period, when the auditory, somatosensory and visual systems are still largely immature, the sense of olfaction acts as 'door to the world', providing an important source of environmental inputs. However, it is unknown whether early olfactory processing shapes the activity in the limbic circuitry during neonatal development. Here, we address this question by combining simultaneous in vivo recordings from the olfactory bulb (OB), lateral entorhinal cortex (LEC), hippocampus (HP) and prefrontal cortex (PFC) with olfactory stimulation as well as opto- and chemogenetic manipulations of mitral/tufted cells in the OB of non-anaesthetized neonatal mice of both sexes. We show that the neonatal OB synchronizes the limbic circuity in the beta frequency range. Moreover, it drives neuronal and network activity in LEC, as well as subsequently, HP and PFC via long-range projections from mitral cells to HP-projecting LEC neurons. Thus, OB activity shapes the communication within limbic circuits during neonatal development. KEY POINTS: During early postnatal development, oscillatory activity in the olfactory bulb synchronizes the limbic circuit. Olfactory stimulation boosts firing and beta synchronization along the olfactory bulb-lateral entorhinal cortex-hippocampal-prefrontal pathway. Mitral cells drive neuronal and network activity in the lateral entorhinal cortex (LEC), as well as subsequently, the hippocampus (HP) and the prefrontal cortex (PFC) via long-range projections from mitral cells to HP-projecting LEC neurons. Inhibition of vesicle release on LEC targeting mitral cell axons reveals direct involvement of LEC in the olfactory bulb-driven oscillatory entrainment of the limbic circuitry.

Coordinated electrical activity in the olfactory bulb gates the oscillatory entrainment of entorhinal networks in neonatal mice.

Although the developmental principles of sensory and cognitive processing have been extensively investigated, their synergy has been largely neglected. During early life, most sensory systems are still largely immature. As a notable exception, the olfactory system is functional at birth, controlling mother-offspring interactions and neonatal survival. Here, we elucidate the structural and functional principles underlying the communication between olfactory bulb (OB) and lateral entorhinal cortex (LEC)-the gatekeeper of limbic circuitry-during neonatal development. Combining optogenetics, pharmacology, and electrophysiology in vivo with axonal tracing, we show that mitral cell-dependent discontinuous theta bursts in OB drive network oscillations and time the firing in LEC of anesthetized mice via axonal projections confined to upper cortical layers. Acute pharmacological silencing of OB activity diminishes entorhinal oscillations, whereas odor exposure boosts OB-entorhinal coupling at fast frequencies. Chronic impairment of olfactory sensory neurons disrupts OB-entorhinal activity. Thus, OB activity shapes the maturation of entorhinal circuits.

Bursting mitral cells time the oscillatory coupling between olfactory bulb and entorhinal networks in neonatal mice.

KEY POINTS: During early postnatal development, mitral cells show either irregular bursting or non-bursting firing patterns Bursting mitral cells preferentially fire during theta bursts in the neonatal olfactory bulb, being locked to the theta phase Bursting mitral cells preferentially fire during theta bursts in the neonatal lateral entorhinal cortex and are temporally related to both respiration rhythm- and theta phase Bursting mitral cells act as a cellular substrate of the olfactory drive that promotes the oscillatory entrainment of entorhinal networks ABSTRACT: Shortly after birth, the olfactory system provides not only the main source of environmental inputs to blind, deaf, non-whisking and motorically-limited rodents, but also the drive boosting the functional entrainment of limbic circuits. However, the cellular substrate of this early communication remains largely unknown. Here, we combine in vivo and in vitro patch-clamp and extracellular recordings to reveal the contribution of mitral cell (MC) firing to early patterns of network activity in both the neonatal olfactory bulb (OB) and the lateral entorhinal cortex (LEC), the gatekeeper of limbic circuits. We show that MCs predominantly fire either in an irregular bursting or non-bursting pattern during discontinuous theta events in the OB. However, the temporal spike-theta phase coupling is stronger for bursting than non-bursting MCs. In line with the direct OB-to-LEC projections, both bursting and non-bursting discharge augments during co-ordinated patterns of entorhinal activity, albeit with higher magnitude for bursting MCs. For these neurons, temporal coupling to the discontinuous theta events in the LEC is stronger. Thus, bursting MCs might drive the entrainment of the OB-LEC network during neonatal development.

Protocol for adeno-associated virus-mediated optogenetic activation of olfactory output neurons in neonatal mice.

Optogenetic manipulation has proven a powerful tool for investigating the mechanisms underlying the function of neuronal networks, but implementing the technique on mammals during early development remains challenging. Here, we present a comprehensive workflow to specifically manipulate mitral/tufted cells (M/TCs), the output neurons in the olfactory circuit, mediated by adeno-associated virus (AAV) transduction and light stimulation in neonatal mice and monitor neuronal and network activity with in vivo electrophysiology. This method represents an efficient approach to elucidate functional brain development. For complete details on the use and execution of this protocol, please refer to Chen et al.1,2,3.

Preconfigured architecture of the developing mouse brain.

In the adult brain, structural and functional parameters, such as synaptic sizes and neuronal firing rates, follow right-skewed and heavy-tailed distributions. While this organization is thought to have significant implications, its development is still largely unknown. Here, we address this knowledge gap by investigating a large-scale dataset recorded from the prefrontal cortex and the olfactory bulb of mice aged 4-60 postnatal days. We show that firing rates and spike train interactions have a largely stable distribution shape throughout the first 60 postnatal days and that the prefrontal cortex displays a functional small-world architecture. Moreover, early brain activity exhibits an oligarchical organization, where high-firing neurons have hub-like properties. In a neural network model, we show that analogously right-skewed and heavy-tailed synaptic parameters are instrumental to consistently recapitulate the experimental data. Thus, functional and structural parameters in the developing brain are already extremely distributed, suggesting that this organization is preconfigured and not experience dependent.

Olfactory bulb activity shapes the development of entorhinal-hippocampal coupling and associated cognitive abilities.

The interplay between olfaction and higher cognitive processing has been documented in the adult brain; however, its development is poorly understood. In mice, shortly after birth, endogenous and stimulus-evoked activity in the olfactory bulb (OB) boosts the oscillatory entrainment of downstream lateral entorhinal cortex (LEC) and hippocampus (HP). However, it is unclear whether early OB activity has a long-lasting impact on entorhinal-hippocampal function and cognitive processing. Here, we chemogenetically silenced the synaptic outputs of mitral/tufted cells, the main projection neurons in the OB, during postnatal days 8-10. The transient manipulation leads to a long-lasting reduction of oscillatory coupling and weaker responsiveness to stimuli within developing entorhinal-hippocampal circuits accompanied by dendritic sparsification of LEC pyramidal neurons. Moreover, the transient silencing reduces the performance in behavioral tests involving entorhinal-hippocampal circuits later in life. Thus, neonatal OB activity is critical for the functional LEC-HP development and maturation of cognitive abilities.

Postnatal Development of Centrifugal Inputs to the Olfactory Bulb.

Processing in primary sensory areas is influenced by centrifugal inputs from higher brain areas, providing information about behavioral state, attention, or context. Activity in the olfactory bulb (OB), the first central processing stage of olfactory information, is dynamically modulated by direct projections from a variety of areas in adult mice. Despite the early onset of olfactory sensation compared to other senses, the development of centrifugal inputs to the OB remains largely unknown. Using retrograde tracing across development, we show that centrifugal projections to the OB are established during the postnatal period in an area-specific manner. While feedback projections from the piriform cortex (PIR) are already present shortly after birth, they strongly increase in number during postnatal development with an anterior-posterior gradient. Contralateral projections from the anterior olfactory nucleus (AON) are present at birth but only appeared postnatally for the nucleus of the lateral olfactory tract (nLOT). Numbers of OB projecting neurons from the lateral entorhinal cortex (LEC), ventral hippocampus, and cortical amygdala (CoA) show a sudden increase at the beginning of the second postnatal week and a delayed development compared to more anterior areas. These anatomical data suggest that limited top-down influence on odor processing in the OB may be present at birth, but strongly increases during postnatal development and is only fully established later in life.

How the sense of smell influences cognition throughout life.

Although mostly unaware, we constantly navigate a complex landscape of airborne molecules. The perception of these molecules helps us navigate, shapes our social life, and can trigger emotionally charged memories transporting us back to the past within a split second. While the processing of olfactory information in early sensory areas is well understood, how the sense of smell affects cognition only recently gained attention in the field of neuroscience. Here, we review links between olfaction and cognition and explore the idea that the activity in olfactory areas may be critical for coordinating cognitive networks. Further, we discuss how olfactory activity may shape the development of cognitive networks and associations between the decline of olfactory and cognitive abilities in aging. Olfaction provides a great tool to study large-scale networks underlying cognitive abilities and bears the potential for a better understanding of cognitive symptoms associated with many mental disorders.

Neural Correlates of Anesthesia in Newborn Mice and Humans.

Monitoring the hypnotic component of anesthesia during surgeries is critical to prevent intraoperative awareness and reduce adverse side effects. For this purpose, electroencephalographic (EEG) methods complementing measures of autonomic functions and behavioral responses are in use in clinical practice. However, in human neonates and infants existing methods may be unreliable and the correlation between brain activity and anesthetic depth is still poorly understood. Here, we characterized the effects of different anesthetics on brain activity in neonatal mice and developed machine learning approaches to identify electrophysiological features predicting inspired or end-tidal anesthetic concentration as a proxy for anesthetic depth. We show that similar features from EEG recordings can be applied to predict anesthetic concentration in neonatal mice and humans. These results might support a novel strategy to monitor anesthetic depth in human newborns.