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.

Blood pressure pulsations modulate central neuronal activity via mechanosensitive ion channels.

The transmission of the heartbeat through the cerebral vascular system causes intracranial pressure pulsations. We discovered that arterial pressure pulsations can directly modulate central neuronal activity. In a semi-intact rat brain preparation, vascular pressure pulsations elicited correlated local field oscillations in the olfactory bulb mitral cell layer. These oscillations did not require synaptic transmission but reflected baroreceptive transduction in mitral cells. This transduction was mediated by a fast excitatory mechanosensitive ion channel and modulated neuronal spiking activity. In awake animals, the heartbeat entrained the activity of a subset of olfactory bulb neurons within ~20 milliseconds. Thus, we propose that this fast, intrinsic interoceptive mechanism can modulate perception-for example, during arousal-within the olfactory bulb and possibly across various other brain areas.

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.

Gamma oscillations provide insights into cortical circuit development.

Rhythmic coordination in gamma oscillations provides temporal structure to neuronal activity. Gamma oscillations are commonly observed in the mammalian cerebral cortex, are altered early on in several neuropsychiatric disorders, and provide insights into the development of underlying cortical networks. However, a lack of knowledge on the developmental trajectory of gamma oscillations prevented the combination of findings from the immature and the adult brain. This review is intended to provide an overview on the development of cortical gamma oscillations, the maturation of the underlying network, and the implications for cortical function and dysfunction. The majority of information is drawn from work in rodents with particular emphasis on the prefrontal cortex, the developmental trajectory of gamma oscillations, and potential implications for neuropsychiatric disorders. Current evidence supports the idea that fast oscillations during development are indeed an immature form of adult gamma oscillations and can help us understand the pathology of neuropsychiatric disorders.

Layer-specific impairment in the developing lateral entorhinal cortex of immune-challenged Disc1+/- mice.

Cognitive deficits in mental disorders result from dysfunctional activity in large-scale brain networks centred around the hippocampus and the prefrontal cortex. Dysfunctional activity emerges early during development and precedes the cognitive disabilities. The prefrontal-hippocampal network is driven by a prominent input from the lateral entorhinal cortex. We have previously shown that during early development, the entorhinal drive of the prefrontal-hippocampal network is impaired in a mouse model of mental disorders, yet the cellular substrate of this impairment is still poorly understood. Here, we address this question by a detailed characterization of projection neurons across the layers of the lateral entorhinal cortex in immune-challenged Disc1+/- mice at the beginning of the second postnatal week. We found that the activity and morphology of neurons in layers 2b and 3, which project to the hippocampus, are impaired. Neurons in layer 2b show increased spike-frequency adaptation, whereas neurons in layer 3 have reduced dendritic complexity but increased spike density. These findings identify the developmental alterations of entorhinal-hippocampal communication that underlie network dysfunction in immune-challenged Disc1+/- mice. KEY POINTS: Neonatal immune-challenged Disc1+/- mice show layer-specific changes in the lateral entorhinal cortex. Entorhinal layer 2b pyramidal neurons have increased spike-frequency adaptation. Reduced dendritic complexity but increased spine density characterize layer 3 pyramidal neurons.

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.

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.

Rapid odor processing by layer 2 subcircuits in lateral entorhinal cortex.

Olfactory information is encoded in lateral entorhinal cortex (LEC) by two classes of layer 2 (L2) principal neurons: fan and pyramidal cells. However, the functional properties of L2 cells and how they contribute to odor coding are unclear. Here, we show in awake mice that L2 cells respond to odors early during single sniffs and that LEC is essential for rapid discrimination of both odor identity and intensity. Population analyses of L2 ensembles reveal that rate coding distinguishes odor identity, but firing rates are only weakly concentration dependent and changes in spike timing can represent odor intensity. L2 principal cells differ in afferent olfactory input and connectivity with inhibitory circuits and the relative timing of pyramidal and fan cell spikes provides a temporal code for odor intensity. Downstream, intensity is encoded purely by spike timing in hippocampal CA1. Together, these results reveal the unique processing of odor information by LEC subcircuits and highlight the importance of temporal coding in higher olfactory areas.

A transient developmental increase in prefrontal activity alters network maturation and causes cognitive dysfunction in adult mice.

Disturbed neuronal activity in neuropsychiatric pathologies emerges during development and might cause multifold neuronal dysfunction by interfering with apoptosis, dendritic growth, and synapse formation. However, how altered electrical activity early in life affects neuronal function and behavior in adults is unknown. Here, we address this question by transiently increasing the coordinated activity of layer 2/3 pyramidal neurons in the medial prefrontal cortex of neonatal mice and monitoring long-term functional and behavioral consequences. We show that increased activity during early development causes premature maturation of pyramidal neurons and affects interneuronal density. Consequently, altered inhibitory feedback by fast-spiking interneurons and excitation/inhibition imbalance in prefrontal circuits of young adults result in weaker evoked synchronization of gamma frequency. These structural and functional changes ultimately lead to poorer mnemonic and social abilities. Thus, prefrontal activity during early development actively controls the cognitive performance of adults and might be critical for cognitive symptoms in neuropsychiatric diseases.

Gamma activity accelerates during prefrontal development.

Gamma oscillations are a prominent activity pattern in the cerebral cortex. While gamma rhythms have been extensively studied in the adult prefrontal cortex in the context of cognitive (dys)functions, little is known about their development. We addressed this issue by using extracellular recordings and optogenetic stimulations in mice across postnatal development. We show that fast rhythmic activity in the prefrontal cortex becomes prominent during the second postnatal week. While initially at about 15 Hz, fast oscillatory activity progressively accelerates with age and stabilizes within gamma frequency range (30-80 Hz) during the fourth postnatal week. Activation of layer 2/3 pyramidal neurons drives fast oscillations throughout development, yet the acceleration of their frequency follows similar temporal dynamics as the maturation of fast-spiking interneurons. These findings uncover the development of prefrontal gamma activity and provide a framework to examine the origin of abnormal gamma activity in neurodevelopmental disorders.

Resolving and Rescuing Developmental Miswiring in a Mouse Model of Cognitive Impairment.

Cognitive deficits, core features of mental illness, largely result from dysfunction of prefrontal networks. This dysfunction emerges during early development, before a detectable behavioral readout, yet the cellular elements controlling the abnormal maturation are still unknown. Here, we address this open question by combining in vivo electrophysiology, optogenetics, neuroanatomy, and behavioral assays during development in mice mimicking the dual genetic-environmental etiology of psychiatric disorders. We report that pyramidal neurons in superficial layers of the prefrontal cortex are key elements causing disorganized oscillatory entrainment of local circuits in beta-gamma frequencies. Their abnormal firing rate and timing relate to sparser dendritic arborization and lower spine density. Administration of minocycline during the first postnatal week, potentially acting via microglial cells, rescues the neuronal deficits and restores pre-juvenile cognitive abilities. Elucidation of the cellular substrate of developmental miswiring causing later cognitive deficits opens new perspectives for identification of neurobiological targets amenable to therapies.

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.

Transient Knock-Down of Prefrontal DISC1 in Immune-Challenged Mice Causes Abnormal Long-Range Coupling and Cognitive Dysfunction throughout Development.

Compromised brain development has been hypothesized to account for mental illness. This concept was underpinned by the function of the molecule disrupted-in-schizophrenia 1 (DISC1), which represents an intracellular hub of developmental processes and has been related to cognitive dysfunction in psychiatric disorders. Mice with whole-brain DISC1 knock-down show impaired prefrontal-hippocampal function and cognitive abilities throughout development and at adulthood, especially when combined with early environmental stressors, such as maternal immune activation (MIA). However, the contribution of abnormal DISC1-driven maturation of either prefrontal cortex (PFC) or hippocampus (HP) to these deficits is still unknown. Here, we use in utero electroporation to restrict the DISC1 knock-down to prefrontal layer II/III pyramidal neurons during perinatal development and expose these mice to MIA as an environmental stressor (dual-hit GPFCE mice, both sexes). Combining in vivo electrophysiology and neuroanatomy with behavioral testing, we show that GPFCE mice at neonatal age have abnormal patterns of oscillatory activity and firing in PFC, but not HP. Abnormal firing rates in PFC of GPFCE mice relate to sparser dendritic arborization and lower spine density. Moreover, the long-range coupling within prefrontal-hippocampal networks is decreased at this age. The transient prefrontal DISC1 knock-down was sufficient to permanently perturb the prefrontal-hippocampal communication and caused poorer recognition memory performance at pre-juvenile age. Thus, developmental dysfunction of prefrontal circuitry causes long-lasting disturbances related to mental illness.SIGNIFICANCE STATEMENT Hypofrontality is considered a main cause of cognitive deficits in mental disorders, yet the underlying mechanisms are still largely unknown. During development, long before the emergence of disease symptoms, the functional coupling within the prefrontal-hippocampal network, which is the core brain circuit involved in cognitive processing, is reduced. To assess to which extent impaired prefrontal development contributes to the early dysfunction, immune-challenged mice with transient DISC1 knock-down confined to PFC were investigated in their prefrontal-hippocampal communication throughout development by in vivo electrophysiology and behavioral testing. We show that perturbing developmental processes of prefrontal layer II/III pyramidal neurons is sufficient to diminish prefrontal-hippocampal coupling and decrease the cognitive performance throughout development.

Tapered Fibers Combined With a Multi-Electrode Array for Optogenetics in Mouse Medial Prefrontal Cortex.

Optogenetics offers many advantages in terms of cell-type specificity, allowing to investigate functional connectivity between different brain areas at high spatial and neural population selectivity. In order to obtain simultaneous optical control and electrical readout of neural activity, devices called "optrodes" are employed. They are typically composed of a linear array of microelectrodes integrated on a slender probe shafts combined with flat-cleaved optical fibers (FF) placed above the recording sites. However, due to tissue absorption and scattering, light delivered by the FF unevenly illuminates the region of interest. This issue is of particular relevance when cellular populations are disposed along the dorso-ventral axis, such as in medial prefrontal cortex (mPFC) where cortical layers are aligned vertically. The study presented here aims at using tapered optical fibers (TFs) in combination with a 16-electrode neural probe to better access neural populations distributed along the dorso-ventral axis in the mPFC of newborn mice, restricting light delivery over a specific portion of the cortical layer of interest. Half of the TF surface is coated with a reflecting metal blocking the light to enable light delivery from one side of the probe's shaft only, with the probe base being designed to host the fiber without interfering with the wire-bonds that connect the recording sites to a printed circuit board. Monte-Carlo simulations have been implemented to define the relative TF-probe position and to identify the light intensity distribution above the recording sites. In vivo recordings indicate that simultaneous optical stimulation and electrical readout of neural activity in the mPFC benefit from the use of the engineered TF-based optrode in terms of a more uniform light distribution along the dorso-ventral axis and the possibility of restricting light delivery to a subset of electrical recording sites of interest.

Glutamatergic drive along the septo-temporal axis of hippocampus boosts prelimbic oscillations in the neonatal mouse.

The long-range coupling within prefrontal-hippocampal networks that account for cognitive performance emerges early in life. The discontinuous hippocampal theta bursts have been proposed to drive the generation of neonatal prefrontal oscillations, yet the cellular substrate of these early interactions is still unresolved. Here, we selectively target optogenetic manipulation of glutamatergic projection neurons in the CA1 area of either dorsal or intermediate/ventral hippocampus at neonatal age to elucidate their contribution to the emergence of prefrontal oscillatory entrainment. We show that despite stronger theta and ripples power in dorsal hippocampus, the prefrontal cortex is mainly coupled with intermediate/ventral hippocampus by phase-locking of neuronal firing via dense direct axonal projections. Theta band-confined activation by light of pyramidal neurons in intermediate/ventral but not dorsal CA1 that were transfected by in utero electroporation with high-efficiency channelrhodopsin boosts prefrontal oscillations. Our data causally elucidate the cellular origin of the long-range coupling in the developing brain.

Methodological Approach for Optogenetic Manipulation of Neonatal Neuronal Networks.

Coordinated patterns of electrical activity are critical for the functional maturation of neuronal networks, yet their interrogation has proven difficult in the developing brain. Optogenetic manipulations strongly contributed to the mechanistic understanding of network activation in the adult brain, but difficulties to specifically and reliably express opsins at neonatal age hampered similar interrogation of developing circuits. Here, we introduce a protocol that enables to control the activity of specific neuronal populations by light, starting from early postnatal development. We show that brain area-, layer- and cell type-specific expression of opsins by in utero electroporation (IUE), as exemplified for the medial prefrontal cortex (PFC) and hippocampus (HP), permits the manipulation of neuronal activity in vitro and in vivo. Both individual and population responses to different patterns of light stimulation are monitored by extracellular multi-site recordings in the medial PFC of neonatal mice. The expression of opsins via IUE provides a flexible approach to disentangle the cellular mechanism underlying early rhythmic network activity, and to elucidate the role of early neuronal activity for brain maturation, as well as its contribution to neurodevelopmental disorders.

Layer-specific optogenetic activation of pyramidal neurons causes beta-gamma entrainment of neonatal networks.

Coordinated activity patterns in the developing brain may contribute to the wiring of neuronal circuits underlying future behavioural requirements. However, causal evidence for this hypothesis has been difficult to obtain owing to the absence of tools for selective manipulation of oscillations during early development. We established a protocol that combines optogenetics with electrophysiological recordings from neonatal mice in vivo to elucidate the substrate of early network oscillations in the prefrontal cortex. We show that light-induced activation of layer II/III pyramidal neurons that are transfected by in utero electroporation with a high-efficiency channelrhodopsin drives frequency-specific spiking and boosts network oscillations within beta-gamma frequency range. By contrast, activation of layer V/VI pyramidal neurons causes nonspecific network activation. Thus, entrainment of neonatal prefrontal networks in fast rhythms relies on the activation of layer II/III pyramidal neurons. This approach used here may be useful for further interrogation of developing circuits, and their behavioural readout.

Spindle Activity Orchestrates Plasticity during Development and Sleep.

Spindle oscillations have been described during early brain development and in the adult brain. Besides similarities in temporal patterns and involved brain areas, neonatal spindle bursts (NSBs) and adult sleep spindles (ASSs) show differences in their occurrence, spatial distribution, and underlying mechanisms. While NSBs have been proposed to coordinate the refinement of the maturating neuronal network, ASSs are associated with the implementation of acquired information within existing networks. Along with these functional differences, separate synaptic plasticity mechanisms seem to be recruited. Here, we review the generation of spindle oscillations in the developing and adult brain and discuss possible implications of their differences for synaptic plasticity. The first part of the review is dedicated to the generation and function of ASSs with a particular focus on their role in healthy and impaired neuronal networks. The second part overviews the present knowledge of spindle activity during development and the ability of NSBs to organize immature circuits. Studies linking abnormal maturation of brain wiring with neurological and neuropsychiatric disorders highlight the importance to better elucidate neonatal plasticity rules in future research.

Oscillatory activity in developing prefrontal networks results from theta-gamma-modulated synaptic inputs.

The hippocampus-driven entrainment of neonatal prefrontal circuits in theta-gamma oscillations contributes to the maturation of cognitive abilities, yet the underlying synaptic mechanisms are still unknown. Here we combine patch-clamp recordings from morphologically and neurochemically characterized layer V pyramidal neurons and interneurons in vivo, with extracellular recordings from the prelimbic cortex (PL) of awake and lightly anesthetized neonatal rats, to elucidate the synaptic framework of early network oscillations. We demonstrate that all neurons spontaneously fire bursts of action potentials. They receive barrages of fast and slow glutamatergic as well as GABAergic synaptic inputs. Oscillatory theta activity results from long-range coupling of pyramidal neurons, presumably within prelimbic-hippocampal circuits, and from local interactions between interneurons. In contrast, beta-low gamma activity requires external glutamatergic drive on prelimbic interneurons. High-frequency oscillations in layer V are independent of interactions at chemical synapses. Thus, specific theta-gamma-modulated synaptic interactions represent the substrate of network oscillations in the developing PL.

Oscillatory coupling within neonatal prefrontal-hippocampal networks is independent of selective removal of GABAergic neurons in the hippocampus.

GABAergic neurons have been proposed to control oscillatory entrainment and cognitive processing in prefrontal-hippocampal networks. Co-activation of these networks emerges already during neonatal development, with hippocampal theta bursts driving prefrontal oscillations via axonal projections. The cellular substrate of neonatal prefrontal-hippocampal communication and in particular, the role of GABAergic neurons, is still unknown. Here, we used saporin-conjugated anti-vesicular GABA transporter antibodies to cause selective immunotoxic lesion of GABAergic neurons in the CA1 area of the hippocampus during the first postnatal week. Without affecting the somatic development of rat pups, the lesion impaired the generation of hippocampal sharp waves, but not of theta bursts during neonatal development. Moreover, the oscillatory entrainment and firing of neonatal prefrontal cortex as well as the early prefrontal-hippocampal synchrony were largely independent of GABAergic neurotransmission in the hippocampus. Thus, hippocampal interneurons are critical elements for the ontogeny of hippocampal sharp waves, but seem to not control the directed oscillatory coupling between the neonatal prefrontal cortex and hippocampus.