Neuronal ensembles: Building blocks of neural circuits.

Neuronal ensembles, defined as groups of neurons displaying recurring patterns of coordinated activity, represent an intermediate functional level between individual neurons and brain areas. Novel methods to measure and optically manipulate the activity of neuronal populations have provided evidence of ensembles in the neocortex and hippocampus. Ensembles can be activated intrinsically or in response to sensory stimuli and play a causal role in perception and behavior. Here we review ensemble phenomenology, developmental origin, biophysical and synaptic mechanisms, and potential functional roles across different brain areas and species, including humans. As modular units of neural circuits, ensembles could provide a mechanistic underpinning of fundamental brain processes, including neural coding, motor planning, decision-making, learning, and adaptability.

Protocol to image and analyze hippocampal network dynamics in non-anesthetized mouse pups.

Two-photon calcium imaging is a powerful technique that has revolutionized our understanding of how neural circuit dynamics supports different behaviors and cognitive processes. However, performing imaging during development remains challenging. Here, we provide a protocol to image CA1 neurons in mouse pups as well as a pipeline of analysis to analyze and share the data. We describe steps for intracerebroventricular injection, cranial window surgery, two-photon calcium imaging, and analysis of imaging data. For complete details on the use and execution of this protocol, please refer to Dard et al.1 and Denis et al.2.

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Coupling optimized bending-insensitive multi-core fibers for lensless endoscopy.

We report a bending-insensitive multi-core fiber (MCF) for lensless endoscopy imaging with modified fiber geometry that enables optimal light coupling in and out of the individual cores. In a previously reported bending insensitive MCF (twisted MCF), the cores are twisted along the length of the MCF allowing for the development of flexible thin imaging endoscopes with potential applications in dynamic and freely moving experiments. However, for such twisted MCFs the cores are seen to have an optimum coupling angle which is proportional to their radial distance from the center of the MCF. This brings coupling complexity and potentially degrades the endoscope imaging capabilities. In this study, we demonstrate that by introducing a small section (1 cm) at two ends of the MCF, where all the cores are straight and parallel to the optical axis one can rectify the above coupling and output light issues of the twisted MCF, enabling the development of bend-insensitive lensless endoscopes.

Extrinsic control of the early postnatal CA1 hippocampal circuits.

The adult CA1 region of the hippocampus produces coordinated neuronal dynamics with minimal reliance on its extrinsic inputs. By contrast, neonatal CA1 is tightly linked to externally generated sensorimotor activity, but the circuit mechanisms underlying early synchronous activity in CA1 remain unclear. Here, using a combination of in vivo and ex vivo circuit mapping, calcium imaging, and electrophysiological recordings in mouse pups, we show that early dynamics in the ventro-intermediate CA1 are under the mixed influence of entorhinal (EC) and thalamic (VMT) inputs. Both VMT and EC can drive internally generated synchronous events ex vivo. However, movement-related population bursts detected in vivo are exclusively driven by the EC. These differential effects on synchrony reflect the different intrahippocampal targets of these inputs. Hence, cortical and subcortical pathways act differently on the neonatal CA1, implying distinct contributions to the development of the hippocampal microcircuit and related cognitive maps.

Cortical neuronal assemblies coordinate with EEG microstate dynamics during resting wakefulness.

The disruption of cortical assembly activity has been associated with anesthesia-induced loss of consciousness. However, the relationship between cortical assembly activity and the variations in consciousness associated with natural vigilance states remains unclear. Here, we address this by performing vigilance state-specific clustering analysis on 2-photon calcium imaging data from the sensorimotor cortex in combination with global electroencephalogram (EEG) microstate analysis derived from multi-EEG signals obtained over widespread cortical locations. We report no difference in the structure of assembly activity during quiet wakefulness (QW), non-rapid eye movement sleep (NREMs), or REMs, despite the latter two vigilance states being associated with significantly reduced levels of consciousness relative to QW. However, we describe a significant coordination between global EEG microstate dynamics and general local cortical assembly activity during periods of QW, but not sleep. These results suggest that the coordination of cortical assembly activity with global brain dynamics could be a key factor of sustained conscious experience.

The rapid developmental rise of somatic inhibition disengages hippocampal dynamics from self-motion.

Early electrophysiological brain oscillations recorded in preterm babies and newborn rodents are initially mostly driven by bottom-up sensorimotor activity and only later can detach from external inputs. This is a hallmark of most developing brain areas, including the hippocampus, which, in the adult brain, functions in integrating external inputs onto internal dynamics. Such developmental disengagement from external inputs is likely a fundamental step for the proper development of cognitive internal models. Despite its importance, the developmental timeline and circuit basis for this disengagement remain unknown. To address this issue, we have investigated the daily evolution of CA1 dynamics and underlying circuits during the first two postnatal weeks of mouse development using two-photon calcium imaging in non-anesthetized pups. We show that the first postnatal week ends with an abrupt shift in the representation of self-motion in CA1. Indeed, most CA1 pyramidal cells switch from activated to inhibited by self-generated movements at the end of the first postnatal week, whereas the majority of GABAergic neurons remain positively modulated throughout this period. This rapid switch occurs within 2 days and follows the rapid anatomical and functional surge of local somatic GABAergic innervation. The observed change in dynamics is consistent with a two-population model undergoing a strengthening of inhibition. We propose that this abrupt developmental transition inaugurates the emergence of internal hippocampal dynamics.

Step by step: cells with multiple functions in cortical circuit assembly.

It is often thought that the construction of cortical circuits occurs as the result of an elegantly designed process that unfolds sequentially as an animal develops until adult functional networks emerge. In reality, cortical circuits are shaped by evolutionary mechanisms, changes in developmental programmes driven by neuronal activity or epigenetic mechanisms and the need to adapt to the external world, and must pass through several important phases and timely checkpoints as they form. Some cortical cell types serve multiple functions during this developmental journey and are then reused (or 'recycled') to perform different functions in the adult cortex. Understanding the different stages of the cortical construction process and taking into account the ways in which cellular functions change across time and space is therefore essential if we are to build a comprehensive framework of cortical wiring in both health and disease.

How development sculpts hippocampal circuits and function.

In mammals, the selective transformation of transient experience into stored memory occurs in the hippocampus, which develops representations of specific events in the context in which they occur. In this review, we focus on the development of hippocampal circuits and the self-organized dynamics embedded within them since the latter critically support the role of the hippocampus in learning and memory. We first discuss evidence that adult hippocampal cells and circuits are sculpted by development as early as during embryonic neurogenesis. We argue that these primary developmental programs provide a scaffold onto which later experience of the external world can be grafted. Next, we review the different sequences in the development of hippocampal cells and circuits at anatomical and functional levels. We cover a period extending from neurogenesis and migration to the appearance of phenotypic diversity within hippocampal cells and their wiring into functional networks. We describe the progressive emergence of network dynamics in the hippocampus, from sensorimotor-driven early sharp waves to sequences of place cells tracking relational information. We outline the critical turn points and discontinuities in that developmental journey, and close by formulating open questions. We propose that rewinding the process of hippocampal development helps understand the main organization principles of memory circuits.

CA1 pyramidal cell diversity is rooted in the time of neurogenesis.

Cellular diversity supports the computational capacity and flexibility of cortical circuits. Accordingly, principal neurons at the CA1 output node of the murine hippocampus are increasingly recognized as a heterogeneous population. Their genes, molecular content, intrinsic morpho-physiology, connectivity, and function seem to segregate along the main anatomical axes of the hippocampus. Since these axes reflect the temporal order of principal cell neurogenesis, we directly examined the relationship between birthdate and CA1 pyramidal neuron diversity, focusing on the ventral hippocampus. We used a genetic fate-mapping approach that allowed tagging three groups of age-matched principal neurons: pioneer, early-, and late-born. Using a combination of neuroanatomy, slice physiology, connectivity tracing, and cFos staining in mice, we show that birthdate is a strong predictor of CA1 principal cell diversity. We unravel a subpopulation of pioneer neurons recruited in familiar environments with remarkable positioning, morpho-physiological features, and connectivity. Therefore, despite the expected plasticity of hippocampal circuits, given their role in learning and memory, the diversity of their main components is also partly determined at the earliest steps of development.

The organization and development of cortical interneuron presynaptic circuits are area specific.

Parvalbumin and somatostatin inhibitory interneurons gate information flow in discrete cortical areas that compute sensory and cognitive functions. Despite the considerable differences between areas, individual interneuron subtypes are genetically invariant and are thought to form canonical circuits regardless of which area they are embedded in. Here, we investigate whether this is achieved through selective and systematic variations in their afferent connectivity during development. To this end, we examined the development of their inputs within distinct cortical areas. We find that interneuron afferents show little evidence of being globally stereotyped. Rather, each subtype displays characteristic regional connectivity and distinct developmental dynamics by which this connectivity is achieved. Moreover, afferents dynamically regulated during development are disrupted by early sensory deprivation and in a model of fragile X syndrome. These data provide a comprehensive map of interneuron afferents across cortical areas and reveal the logic by which these circuits are established during development.

Hippocampal hub neurons maintain distinct connectivity throughout their lifetime.

The temporal embryonic origins of cortical GABA neurons are critical for their specialization. In the neonatal hippocampus, GABA cells born the earliest (ebGABAs) operate as 'hubs' by orchestrating population synchrony. However, their adult fate remains largely unknown. To fill this gap, we have examined CA1 ebGABAs using a combination of electrophysiology, neurochemical analysis, optogenetic connectivity mapping as well as ex vivo and in vivo calcium imaging. We show that CA1 ebGABAs not only operate as hubs during development, but also maintain distinct morpho-physiological and connectivity profiles, including a bias for long-range targets and local excitatory inputs. In vivo, ebGABAs are activated during locomotion, correlate with CA1 cell assemblies and display high functional connectivity. Hence, ebGABAs are specified from birth to ensure unique functions throughout their lifetime. In the adult brain, this may take the form of a long-range hub role through the coordination of cell assemblies across distant regions.

DeepCINAC: A Deep-Learning-Based Python Toolbox for Inferring Calcium Imaging Neuronal Activity Based on Movie Visualization.

Two-photon calcium imaging is now widely used to infer neuronal dynamics from changes in fluorescence of an indicator. However, state-of-the-art computational tools are not optimized for the reliable detection of fluorescence transients from highly synchronous neurons located in densely packed regions such as the CA1 pyramidal layer of the hippocampus during early postnatal stages of development. Indeed, the latest analytical tools often lack proper benchmark measurements. To meet this challenge, we first developed a graphical user interface (GUI) allowing for a precise manual detection of all calcium transients from imaged neurons based on the visualization of the calcium imaging movie. Then, we analyzed movies from mouse pups using a convolutional neural network (CNN) with an attention process and a bidirectional long-short term memory (LSTM) network. This method is able to reach human performance and offers a better F1 score (harmonic mean of sensitivity and precision) than CaImAn to infer neural activity in the developing CA1 without any user intervention. It also enables automatically identifying activity originating from GABAergic neurons. Overall, DeepCINAC offers a simple, fast and flexible open-source toolbox for processing a wide variety of calcium imaging datasets while providing the tools to evaluate its performance.

Assemblies of Perisomatic GABAergic Neurons in the Developing Barrel Cortex.

The developmental journey of cortical interneurons encounters several activity-dependent milestones. During the early postnatal period in developing mice, GABAergic neurons are transient preferential recipients of thalamic inputs and undergo activity-dependent migration arrest, wiring, and programmed cell-death. Despite their importance for the emergence of sensory experience and the role of activity in their integration into cortical networks, the collective dynamics of GABAergic neurons during that neonatal period remain unknown. Here, we study coordinated activity in GABAergic cells of the mouse barrel cortex using in vivo calcium imaging. We uncover a transient structure in GABAergic population dynamics that disappears in a sensory-dependent process. Its building blocks are anatomically clustered GABAergic assemblies mostly composed by prospective parvalbumin-expressing cells. These progressively widen their territories until forming a uniform perisomatic GABAergic network. Such transient patterning of GABAergic activity is a functional scaffold that links the cortex to the external world prior to active exploration. VIDEO ABSTRACT.

GABAergic Restriction of Network Dynamics Regulates Interneuron Survival in the Developing Cortex.

During neonatal development, sensory cortices generate spontaneous activity patterns shaped by both sensory experience and intrinsic influences. How these patterns contribute to the assembly of neuronal circuits is not clearly understood. Using longitudinal in vivo calcium imaging in un-anesthetized mouse pups, we show that spatially segregated functional assemblies composed of interneurons and pyramidal cells are prominent in the somatosensory cortex by postnatal day (P) 7. Both reduction of GABA release and synaptic inputs onto pyramidal cells erode the emergence of functional topography, leading to increased network synchrony. This aberrant pattern effectively blocks interneuron apoptosis, causing increased survival of parvalbumin and somatostatin interneurons. Furthermore, the effect of GABA on apoptosis is mediated by inputs from medial ganglionic eminence (MGE)-derived but not caudal ganglionic eminence (CGE)-derived interneurons. These findings indicate that immature MGE interneurons are fundamental for shaping GABA-driven activity patterns that balance the number of interneurons integrating into maturing cortical networks.

Internal representation of hippocampal neuronal population spans a time-distance continuum.

The hippocampus plays a critical role in episodic memory: the sequential representation of visited places and experienced events. This function is mirrored by hippocampal activity that self organizes into sequences of neuronal activation that integrate spatiotemporal information. What are the underlying mechanisms of such integration is still unknown. Single cell activity was recently shown to combine time and distance information; however, it remains unknown whether a degree of tuning between space and time can be defined at the network level. Here, combining daily calcium imaging of CA1 sequence dynamics in running head-fixed mice and network modeling, we show that CA1 network activity tends to represent a specific combination of space and time at any given moment, and that the degree of tuning can shift within a continuum from 1 day to the next. Our computational model shows that this shift in tuning can happen under the control of the external drive power. We propose that extrinsic global inputs shape the nature of spatiotemporal integration in the hippocampus at the population level depending on the task at hand, a hypothesis which may guide future experimental studies.

Temporal Embryonic Origin Critically Determines Cellular Physiology in the Dentate Gyrus.

The dentate gyrus, the entry gate to the hippocampus, comprises 3 types of glutamatergic cells, the granule, the mossy and the semilunar granule cells. Whereas accumulating evidence indicates that specification of subclasses of neocortical neurons starts at the time of their final mitotic divisions, when cellular diversity is specified in the Dentate Gyrus remains largely unknown. Here we show that semilunar cells, like mossy cells, originate from the earliest stages of developmental neurogenesis and that early born neurons form age-matched circuits with each other. Besides morphology, adult semilunar cells display characteristic electrophysiological features that differ from most neurons but are shared among early born granule cells. Therefore, an early birthdate specifies adult granule cell physiology and connectivity whereas additional factors may combine to produce morphological identity.

Modeling driver cells in developing neuronal networks.

Spontaneous emergence of synchronized population activity is a characteristic feature of developing brain circuits. Recent experiments in the developing neo-cortex showed the existence of driver cells able to impact the synchronization dynamics when single-handedly stimulated. We have developed a spiking network model capable to reproduce the experimental results, thus identifying two classes of driver cells: functional hubs and low functionally connected (LC) neurons. The functional hubs arranged in a clique orchestrated the synchronization build-up, while the LC drivers were lately or not at all recruited in the synchronization process. Notwithstanding, they were able to alter the network state when stimulated by modifying the temporal activation of the functional clique or even its composition. LC drivers can lead either to higher population synchrony or even to the arrest of population dynamics, upon stimulation. Noticeably, some LC driver can display both effects depending on the received stimulus. We show that in the model the presence of inhibitory neurons together with the assumption that younger cells are more excitable and less connected is crucial for the emergence of LC drivers. These results provide a further understanding of the structural-functional mechanisms underlying synchronized firings in developing circuits possibly related to the coordinated activity of cell assemblies in the adult brain.

Mouse subthalamic nucleus neurons with local axon collaterals.

The neuronal population of the subthalamic nucleus (STN) has the ability to prolong incoming cortical excitation. This could result from intra-STN feedback excitation. The combination of inducible genetic fate mapping techniques with in vitro targeted patch-clamp recordings, allowed identifying a new type of STN neurons that possess a highly collateralized intrinsic axon. The time window of birth dates was found to be narrow (E10.5-E14.5) with very few STN neurons born at E10.5 or E14.5. The fate mapped E11.5-12.5 STN neuronal population included 20% of neurons with profuse axonal branching inside the nucleus and a dendritic arbor that differed from that of STN neurons without local axon collaterals. They had intrinsic electrophysiological properties and in particular, the ability to generate plateau potentials, similar to that of STN neurons without local axon collaterals and more generally to that of classically described STN neurons. This suggests that a subpopulation of STN neurons forms a local glutamatergic network, which together with plateau potentials, allow amplification of hyperdirect cortical inputs and synchronization of the STN neuronal population.