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Fragile X syndrome (FXS) is the most prevalent inherited cause of autism and is accompanied by behavioral and sensory deficits. Errors in the wiring of the brain during early development likely contribute to these deficits, but the underlying mechanisms are unclear. Spontaneous activity patterns, which are required for fine-tuning neuronal networks before the senses become active, are perturbed in rodent models of FXS. Here, we investigated spontaneous network activity patterns in the developing visual cortex of the Fmr1 knockout mouse using in vivo calcium imaging during the second postnatal week, before eye opening. We found that while the frequency, mean amplitude and duration of spontaneous network events were unchanged in the knockout mouse, pair-wise correlations between neurons were increased compared to wild type littermate controls. Further analysis revealed that interneuronal correlations were not generally increased, rather that low-synchronization events occurred relatively less frequently than high-synchronization events. Low-, but not high-, synchronization events have been associated with retinal inputs previously. Since we found that spontaneous retinal waves were normal in the knockout, our results suggest that peripherally driven activity is underrepresented in the Fmr1 KO visual cortex. Therefore, we propose that central gating of retinal inputs may be affected in FXS and that peripherally and centrally driven activity patterns are already unbalanced before eye opening in this disorder.

Developing neurons form synapses at a high rate. Synaptic transmission is very energy-demanding and likely requires ATP production by mitochondria nearby. Mitochondria might be targeted to active synapses in young dendrites, but whether such motility regulation mechanisms exist is unclear. We investigated the relationship between mitochondrial motility and neuronal activity in the primary visual cortex of young mice in vivo and in slice cultures. During the first 2 postnatal weeks, mitochondrial motility decreases while the frequency of neuronal activity increases. Global calcium transients do not affect mitochondrial motility. However, individual synaptic transmission events precede local mitochondrial arrest. Pharmacological stimulation of synaptic vesicle release, but not focal glutamate application alone, stops mitochondria, suggesting that an unidentified factor co-released with glutamate is required for mitochondrial arrest. A computational model of synaptic transmission-mediated mitochondrial arrest shows that the developmental increase in synapse number and transmission frequency can contribute substantially to the age-dependent decrease of mitochondrial motility.

Spontaneous activity drives the establishment of appropriate connectivity in different circuits during brain development. In the mouse primary visual cortex, two distinct patterns of spontaneous activity occur before vision onset: local low-synchronicity events originating in the retina and global high-synchronicity events originating in the cortex. We sought to determine the contribution of these activity patterns to jointly organize network connectivity through different activity-dependent plasticity rules. We postulated that local events shape cortical input selectivity and topography, while global events homeostatically regulate connection strength. However, to generate robust selectivity, we found that global events should adapt their amplitude to the history of preceding cortical activation. We confirmed this prediction by analyzing in vivo spontaneous cortical activity. The predicted adaptation leads to the sparsification of spontaneous activity on a slower timescale during development, demonstrating the remarkable capacity of the developing sensory cortex to acquire sensitivity to visual inputs after eye-opening.

Neurofibromatosis type 1 (NF1) is a common monogenic neurodevelopmental disorder associated with physical and cognitive problems. The cognitive issues are thought to arise from increased release of the neurotransmitter γ-amino butyric acid (GABA). Modulating the signaling pathways causing increased GABA release in a mouse model of NF1 reverts deficits in hippocampal learning. However, clinical trials based on these approaches have so far been unsuccessful. We therefore used a combination of slice electrophysiology, in vivo two-photon calcium imaging and optical imaging of intrinsic signal in a mouse model of NF1 to investigate whether cortical development is affected in NF1, possibly causing lifelong consequences that cannot be rescued by reducing inhibition later in life. We find that in NF1 mice of both sexes, inhibition increases strongly during the development of the visual cortex and remains high. While this increase in cortical inhibition does not affect spontaneous cortical activity patterns during early cortical development, the critical period for ocular dominance plasticity is shortened in NF1 mice due to its early closure but unaltered onset. Notably, after environmental enrichment, differences in inhibitory innervation and ocular dominance plasticity between NF1 mice and wild-type litter mates disappear. These results provide the first evidence for critical period dysregulation in NF1 and suggest that treatments aimed at normalizing levels of inhibition will need to start at early stages of development.Significance:Neurofibromatosis type 1 is associated with cognitive problems for which no treatment is currently available. This study shows that in a mouse model of Neurofibromatosis type 1, cortical inhibition is increased during development and critical period regulation is disturbed. Rearing the mice in an environment that stimulates cognitive function overcomes these deficits. These results uncover critical period dysregulation as a novel mechanism in the pathogenesis of Neurofibromatosis type 1. This suggests that targeting the affected signaling pathways in Neurofibromatosis type 1 for the treatment of cognitive disabilities may have to start at a much younger age than has so far been tested in clinical trials.

SummaryDuring early development, even before the senses are active, bursts of activity travel across the nervous system. This spontaneously generated activity drives the refinement of synaptic connections, preparing young networks for patterned sensory input. Synaptic fine-tuning relies not only on the presence of spontaneous activity, but also on the specific characteristics of these activity patterns, such as their frequency, amplitude and synchronicity. Here, we provide evidence that these crucial characteristics are shaped by the relative balance of excitation and inhibition, where patterns with distinct characteristics have different excitatory/inhibitory ratios. Inhibition can control whether cells participate during a spontaneous event, as pharmacogenetic suppression of the somatostatin (SST) expressing subtype of inhibitory interneurons increased cell recruitment and lateral spread of events.

Summary Spontaneous network activity shapes emerging neuronal circuits during early brain development prior to sensory perception. However, how neuromodulation influences this activity is not fully understood. Here, we report that the neuromodulator oxytocin differentially shapes spontaneous activity patterns across sensory cortices. In vivo, oxytocin strongly decreased the frequency and pairwise correlations of spontaneous activity events in the primary visual cortex (V1), but it did not affect the frequency of spontaneous network events in the somatosensory cortex (S1). Patch-clamp recordings in slices and RNAscope showed that oxytocin affects S1 excitatory and inhibitory neurons similarly, whereas in V1, oxytocin targets only inhibitory neurons. Somatostatin-positive (SST+) interneurons expressed the oxytocin receptor and were activated by oxytocin in V1. Accordingly, pharmacogenetic silencing of V1 SST+ interneurons fully blocked oxytocin’s effect on inhibition in vitro as well its effect on spontaneous activity patterns in vivo. Thus, oxytocin decreases the excitatory/inhibitory (E/I) ratio by recruiting SST+ interneurons and modulates specific features of V1 spontaneous activity patterns that are crucial for the wiring and refining of developing sensory circuits., Graphical Abstract, Highlights • Oxytocin modulates spontaneous activity event patterns in the developing V1 • Oxytocin increases inhibition by activating somatostatin+ interneurons • SST+ neuron activity reduces event frequency and correlations, but not amplitude • In S1, oxytocin increases excitation and inhibition and does not modulate frequency, Maldonado et al. uncover oxytocin’s role in developing sensory cortices. In vivo, they show that oxytocin decreases the frequency and correlations of spontaneous activity patterns in V1 by specifically activating somatostatin+ interneurons. In S1, oxytocin increases both excitation and inhibition and does not affect spontaneous activity frequency.

© 2021 The Author(s)During early development, before the eyes open, synaptic refinement of sensory networks depends on activity generated by developing neurons themselves. In the mouse visual system, retinal cells spontaneously depolarize and recruit downstream neurons to bursts of activity, where the number of recruited cells determines the resolution of synaptic retinotopic refinement. Here we show that during the second post-natal week in mouse visual cortex, somatostatin (SST)-expressing interneurons control the recruitment of cells to retinally driven spontaneous activity. Suppressing SST interneurons increases cell participation and allows events to spread farther along the cortex. During the same developmental period, a second type of high-participation, retina-independent event occurs. During these events, cells receive such large excitatory charge that inhibition is overwhelmed and large parts of the cortex participate in each burst. These results reveal a role of SST interneurons in restricting retinally driven activity in the visual cortex, which may contribute to the refinement of retinotopy.

SummarySpontaneous activity fine-tunes neuronal connections in the developing brain. To explore the underlying synaptic plasticity mechanisms, we monitored naturally occurring changes in spontaneous activity at individual synapses with whole-cell patch-clamp recordings and simultaneous calcium imaging in the mouse visual cortex in vivo. Analyzing activity changes across large populations of synapses revealed a simple and efficient local plasticity rule: synapses that exhibit low synchronicity with nearby neighbors (

During development, activity-dependent synaptic plasticity refines neuronal networks with high precision. For example, spontaneous activity helps sorting synaptic inputs with similar activity patterns into clusters to enhance neuronal computations in the mature brain. Here, we show that TrkB activation and postsynaptic brain-derived neurotrophic factor (BDNF) are required for synaptic clustering in developing hippocampal neurons. Moreover, BDNF and TrkB modulate transmission at synapses depending on their clustering state, indicating that endogenous BDNF/TrkB signaling stabilizes locally synchronized synapses. Together with our previous data on proBDNF/p75NTR signaling, these findings suggest a push-pull plasticity mechanism for synaptic clustering: BDNF stabilizes clustered synapses while proBDNF downregulates out-of-sync synapses. This idea is supported by our observation that synaptic clustering requires matrix-metalloproteinase-9 activity, a proBDNF-to-BDNF converting enzyme. Finally, NMDA receptor activation mediates out-of-sync depression upstream of proBDNF signaling. Together, these data delineate an efficient plasticity mechanism where proBDNF and mature BDNF establish synaptic clustering through antagonistic modulation of synaptic transmission. Niculescu et al. found that synaptic clustering requires BDNF/TrkB signaling, activity of the proBDNF-to-BDNF conversion enzyme MMP9, and NMDA receptor activation. Their study delineates a push-pull plasticity mechanism where BDNF stabilizes clustered synapses while its precursor, proBDNF, depresses unclustered synapses, together ensuring robust clustering of synaptic inputs in developing neurons.

The presence of direct, cytoplasmatic, communication between neurons in the brain of vertebrates has been demonstrated a long time ago. These gap junctions have been characterized in many brain areas in terms of subunit composition, biophysical properties, neuronal connectivity patterns, and developmental regulation. Although interesting findings emerged, showing that different subunits are specifically regulated during development, or that excitatory and inhibitory neuronal networks exhibit various electrical connectivity patterns, gap junctions did not receive much further interest. Originally, it was believed that gap junctions represent simple passageways for electrical and biochemical coordination early in development. Today, we know that gap junction connectivity is tightly regulated, following independent developmental patterns for excitatory and inhibitory networks. Electrical connections are important for many specific functions of neurons, and are, for example, required for the development of neuronal stimulus tuning in the visual system. Here, we integrate the available data on neuronal connectivity and gap junction properties, as well as the most recent findings concerning the functional implications of electrical connections in the developing thalamus and neocortex.

The brain is programmed to drive behaviour by precisely wiring the appropriate neuronal circuits. Wiring and rewiring of neuronal circuits largely depends on the orchestrated changes in the strengths of synaptic contacts. Here, we review how the rules of synaptic plasticity change during development of the brain, from birth to independence. We focus on the changes that occur at the postsynaptic side of excitatory glutamatergic synapses in the rodent hippocampus and neocortex. First we summarize the current data on the structure of synapses and the developmental expression patterns of the key molecular players of synaptic plasticity, N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, as well as pivotal kinases (Ca(2+)/calmodulin-dependent protein kinase II, protein kinase A, protein kinase C) and phosphatases (PP1, PP2A, PP2B). In the second part we relate these findings to important characteristics of the emerging network. We argue that the concerted and gradual shifts in the usage of plasticity molecules comply with the changing need for (re)wiring neuronal circuits.

Calcium imaging has become a widely used technique to probe neuronal activity on the cellular and subcellular levels. In contrast to standard electrophysiological methods, calcium imaging resolves sub- and suprathreshold activation patterns in structures as small as fine dendritic branches and spines. This review highlights recent findings gained on the subcellular level using calcium imaging, with special emphasis on synaptic transmission and plasticity in individual spines. Since imaging allows monitoring activity across populations of synapses, it has recently been adopted to investigate how dendrites integrate information from many synapses. Future experiments, ideally carried out in vivo, will reveal how the dendritic tree integrates and computes afferent signals. For example, it is now possible to directly test the concept that dendritic inputs are clustered and that single dendrites or dendritic stretches act as independent computational units.

Studying changing synaptic activity patterns during development provides a wealth of information on how activity-dependent processes shape synaptic connectivity. In this chapter we introduce a method that combines whole-cell electrophysiology with calcium imaging to map functional synaptic sites on the dendritic tree and follow their activity over time. The key strength of this method lies in its ability to distinguish between synaptic and non-synaptic calcium signaling by their coincidence with synaptic currents measured at the soma. Next to the required materials and protocols that are necessary to perform these experiments, we thoroughly discuss how the acquired data can be analyzed. Since this method can be employed in many neuronal systems we believe that it can be a valuable tool to study developmental changes in synaptic connectivity.

In order to accurately process incoming sensory stimuli, neurons must be organized into functional networks, with both genetic and environmental factors influencing the precise arrangement of connections between cells. Teasing apart the relative contributions of molecular guidance cues, spontaneous activity and visual experience during this maturation is on-going. During development of the sensory system, the first, rough organization of connections is created by molecular factors. These connections are then modulated by the intrinsically generated activity of neurons, even before the senses have become operational. Spontaneous waves of depolarisations sweep across the nervous system, placing them in a prime position to strengthen correct connections and weaken others, shaping synapses into a useful network. A large body of work now supports the idea that, rather than being a mere side-effect of the system, spontaneous activity actually contains information which readies the nervous system so that, as soon as the senses become active, sensory information can be utilized by the animal. An example is the neonatal mouse. As soon as the eyelids first open, neurons in the cortex respond to visual information without the animal having previously encountered structured sensory input (Cang et al., 2005a; Ko et al., 2013; Rochefort et al., 2011; Zhang et al., 2012). In vivo imaging techniques have advanced considerably, allowing observation of the natural activity in the brain of living animals down to the level of the individual synapse. New (opto)genetic methods make it possible to subtly modulate the spatio-temporal properties of activity, aiding our understanding of how these characteristics relate to the function of spontaneous activity. Such experiments have had a huge impact on our knowledge by permitting direct testing of ideas about the plasticity mechanisms at play in the intact system, opening up a provocative range of fresh questions. Here, we intend to outline the most recent descriptions of spontaneous activity patterns in rodent developing sensory areas, as well as the inferences we can make about the information content of those activity patterns and ideas about the plasticity rules that allow this activity to shape the young brain.

During brain maturation, neurons form specific connections with each other to establish functional neuronal circuits. The processes underlying the development of connectivity, such as the selection of synaptic partners and the fine-tuning of neuronal networks, act with single-synapse precision. Calcium is an intracellular secondary messenger that operates with remarkable spatio-temporal specificity and regulates functional and structural adaptations at the level of individual synapses. Although the structure, molecular composition and function of an emerging synapse changes dramatically during its development, the single-synapse specificity of calcium signaling is maintained at every step of synapse formation: when the first contacts between axons and dendrites form, during the onset of synaptic function and later, when spine synapses emerge. Here, we describe the mechanisms that help developing neurons to confine calcium signaling to individual synapses, and discuss how these local calcium dynamics facilitate the development of accurate neuronal connections at each step of synapse maturation.

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of the formation of functional neuronal networks. BDNF signaling regulates neuronal development not only globally, at the level of entire neurons or networks, but also at a subcellular level and with high temporal specificity; however, the spatiotemporal characteristics of intrinsic BDNF signaling are essentially unknown. Here, we used calcium imaging to directly observe intrinsic BDNF signaling in developing hippocampal neurons. We found that blocking intrinsic BDNF signaling with function-blocking BDNF antibodies (αBDNF) or K252-a reduced the frequency of spontaneously occurring fast and localized calcium rises in dendrites. Conversely, focal application of BDNF evoked fast and local dendritic calcium transients, which required activation of TrkB (tropomyosin-related kinase B) receptors as well as activation of voltage-gated sodium and calcium channels. Virus-mediated expression of PSD-95:CFP (postsynaptic density-95 tagged with cyan fluorescent protein) revealed that spontaneous local calcium transients occurred frequently at postsynaptic sites along the dendrite. The frequency of synaptically localized calcium transients was specifically reduced by blocking intrinsic BDNF signaling, whereas nonsynaptic calcium rises were not affected. Furthermore, focal BDNF delivery evoked localized and fast calcium elevations specifically at postsynaptic sites. Together, our results demonstrate that BDNF-dependent calcium signaling in developing hippocampal neurons is fast and occurs at synapses. These temporal and spatial characteristics of intrinsic BDNF signaling as well as its relative abundance renders BDNF an ideal signaling molecule in the establishment of specific synaptic connectivity and functional neuronal networks.

During development, dendrites, and in particular dendritic filopodia, undergo extensive structural remodeling, presumably to help establish synaptic contacts. Here, we investigated the role of calcium signaling in dendritic plasticity by simultaneously recording calcium dynamics and filopodial growth in rat hippocampal slice cultures. Local calcium transients occurred in dendritic filopodia and shafts, often at putative synaptic sites. These events were highly correlated with filopodial motility: comparatively rare when individual filopodia emerged from the dendrite, they became more frequent after filopodia started growing, finally causing them to halt. Accordingly, an experimental reduction of the frequency of local calcium transients elicited filopodial growth and, conversely, calcium uncaging reduced filopodial motility. Our observations suggest that low levels of local calcium transients facilitate filopodial outgrowth, whereas high levels inhibit the formation of filopodia and stabilize newly formed ones. This process may facilitate synapse formation and may serve as a homeostatic mechanism distributing synapses evenly along developing dendrites.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 150–162, 2001

In vitro models have frequently been employed to investigate the specificity of the formation of axonal projections during both development and regeneration. Such studies demonstrated pathway, target, and laminar specificity, yet they did not tackle the problem of topography. Here, we addressed the issue of regeneration of spatial specificity at the topographic level by lesioning a precisely organized projection from the auditory system of neonatal rats in organotypic slice culture and by analyzing regeneration capacity. Lesioning had no effect on the survival of axotomized neurons or the structure of the auditory nuclei. Anterograde and retrograde biocytin tracing demonstrated that the projection regenerated topographically at the supracellular level. Whole-cell patch-clamp recordings revealed that the regenerated projection was functional. Topographic regeneration was not impaired by blocking spike activity with tetrodotoxin or glycinergic transmission with strychnine. However, if lesioning was performed after the slices had been incubated for 1 week, regeneration capacity was lost despite good survival of neurons. The loss of the regeneration capacity in vitro occurs at a developmental stage that corresponds to the age when the capacity for axonal reorganization is lost in vivo. We conclude that the developmental processes occurring in vivo and in vitro are comparable in this system, which is why we think that essential aspects of the loss of regeneration capacity may be addressed with our culture model in the future.

Despite its complexity, the neural circuitry in the auditory brainstem of vertebrates displays a fascinating amount of order. How is this order established in such a precise manner during ontogeny? In this review, we will summarize evidence for both activity-independent and activity-dependent processes involved in the generation of the auditory brainstem circuitry of birds and mammals. An example of activity-independent processes is the emergence of crude topography, which, most probably, is determined by molecular markers whose expression is genetically controlled. On the other hand, neuronal activity supports cell survival, affects dendritic and axonal growth, and influences fine tuning of maps. It appears that various types of neuronal activity, namely spontaneous versus acoustically evoked, bilateral versus unilateral, uncoordinated versus patterned, play a role during different aspects of development and cooperate with the activity-independent processes to ensure the proper formation of neuronal circuitry.

WE investigated whether glycinergic transmission develops organotypically in auditory brain stem cultures. Slices of the medial nucleus of the trapezoid body and the lateral superior olive were incubated in medium with a raised extracellular K + concentration. As in vivo, glycine receptor α1 subunit immunoreactivity increased and became clustered on somata and proximal dendrites. Together with organotypic expression of glycine transporter GLYT2, this indicates that molecular components of glycinergic synapses form properly. In contrast, glycinergic synaptic currents did not develop as in vivo: after 7 days in vitro they were still similar to those at the time of culture preparation. We suggest that for organotypic development of glycine receptors and transporters, Ca 2+ influx due to elevated K + is sufficient. The development of functional synaptic transmission, however, may require patterned electrical activity.

During nervous system development, the formation of synapses between pre- and postsynaptic neurons is a remarkably specific process. Both structural and functional plasticity are critical for the selection of synaptic partners and for the establishment and maturation of synapses. To unravel the respective contributions of structural and functional mechanisms as well as their interactions during synaptogenesis, it is important to directly observe structural changes and functional signaling simultaneously. Here, we present an imaging approach to simultaneously follow changes in structure and function. Differential labeling of individual cells and the neuronal network with distinct dyes allows the study of structural plasticity and changes in calcium signaling associated with neural activity at the same time and with high resolution. This is achieved by bulk loading of neuronal populations with a calcium-sensitive indicator in combination with electroporation of individual cells with a calcium indicator and an additional noncalcium-sensitive dye with a different excitation spectrum. Recordings of the two differently labeled structures can be acquired simultaneously using confocal microscopy. Thus, structural plasticity and calcium dynamics of the individually labeled neuron and the surrounding network can be related to each other. This combined imaging approach can be applied to virtually all systems of neuronal networks to study structure and function. We provide a comprehensive description of the labeling procedure, the imaging parameters, and the important aspects of analysis for simultaneous recordings of structure and function in individual neurons.

Parvalbumin (PV), calretinin (CR), and calbindin (CB) are calcium-binding proteins which are presumably involved in the regulation of the intracellular calcium concentration. Within the rat auditory system, CB is transiently expressed in several nuclei during the period of synapse refinement, indicating a specific function of CB during development, yet little is known in this regard about PV and CR. In order to gather more information about calcium-binding proteins during development, we analyzed the spatiotemporal distribution of PV and CR in the rat auditory brainstem using immunocytochemistry. In the adult, PV was heavily present in somata and neuropil of all nuclei and in fibers of all tracts. CR was found in somata of the cochlear nucleus and peripheral aspects of the inferior colliculus as well as in fibers extending into the superior olivary complex and the nuclei of the lateral lemniscus. The developmental expression of PV was characterized by a relatively late appearance in somata (at postnatal day 8), followed by a rapid increase to adult levels. In contrast, CR immunoreactivity was already strong two days before birth, yet the number and intensity of labeled neurons subsequently decreased and CR disappeared almost completely in the superior olivary complex, nuclei of the lateral lemniscus, and central aspects of the inferior colliculus. These data, together with those on CB, show that CR, CB, and PV are sequentially expressed during auditory brainstem development. They also suggest that the presence of the three proteins can be correlated with definite developmental stages.

In order to contribute to a functional network a neuron must make specific connections and integrate the synaptic inputs that it receives in a meaningful way. Previous modeling and experimental studies have predicted that this specificity could entail a subcellular organization whereby synapses that carry similar information are clustered together on local stretches of dendrite. Recent imaging studies have observed exactly this kind of synaptic clustering during development and learning in different neuronal circuits. Interestingly, this organization is found to be dependent on synaptic activity through a mechanism that most likely involves local plasticity mechanisms. Here we discuss these new insights and give an overview of the candidate plasticity mechanism that could be involved.

SummaryDuring brain development, before sensory systems become functional, neuronal networks spontaneously generate repetitive bursts of neuronal activity, which are typically synchronized across many neurons. Such activity patterns have been described on the level of networks and cells, but the fine-structure of inputs received by an individual neuron during spontaneous network activity has not been studied. Here, we used calcium imaging to record activity at many synapses of hippocampal pyramidal neurons simultaneously to establish the activity patterns in the majority of synapses of an entire cell. Analysis of the spatiotemporal patterns of synaptic activity revealed a fine-scale connectivity rule: neighboring synapses (

During the development of the brain, synaptic connections between nerve cells are being established with remarkable specificity. This is achieved by a series of steps: first, axons grow to their terminal areas. Second, axons and dendrites contact each other and select among potential synaptic partners. Third, after synapses have become functional, the fine-tuning of synaptic connections optimizes emerging networks to perform their specific functions. Here, I summarize the evidence for a central role of intracellular calcium signaling in all three stages of the development of specific synaptic connections. In particular, calcium signaling has the capacity to integrate information from a wide array of extracellular factors that are known to regulate neuronal development, such as molecular cues or neuronal activity. Calcium signaling, in turn, directs structural as well as functional adaptations in individual neurons that underlie the establishment of synaptic specificity. Importantly, evidence is accumulating that errors in calcium-dependent network maturation are associated with neurodevelopmental disorders. Therefore, understanding the role of calcium in setting up brain networks may not only advance our insights into mechanisms of normal brain development, but also help identifying the causes of diseases such as autism or mental retardation.

Summary Synapse elimination is an important process underlying the establishment of functional neuronal networks during development. Here, we tested the idea that neurons select among potential synaptic partners already during initial contact formation between dendritic filopodia and axons—well before mature synapses are established. We show that filopodia frequently make contact with axons, and while some contacts are selectively stabilized, many are short-lived. More specifically, we demonstrate that contacts with a certain population of GABAergic axons never get stabilized, indicating that filopodia already early on select between different types of axons. Local dendritic calcium transients that are independent of glutamate occur within seconds after contact formation, and their frequency is high where contacts become stabilized and low at short-lived contacts. Thus, filopodia are capable of choosing between potential synaptic partners well before a mature synapse is established.

Inhibitory and excitatory connections of remarkably precise topographic order are characteristic features of the mammalian auditory system, particularly within the superior olivary complex (SOC). Little is known about the requirements for the correct development of these specific connections. Previous in vivo experiments have demonstrated a high expression of calcium-binding proteins in this system during development, pointing to the need for precise calcium regulation. Here, we have employed an organotypic slice culture from the above neuronal network and analyzed the requirements for the maintenance and development of this system in vitro. When slices from neonatal rats were incubated in standard culture medium for up to 7 days, we found no organotypic features. Only if 25 mM KCl was added to the culture medium, the cytoarchitecture of the nuclei, the neuronal morphology, and the specificity and topography of internuclear connections were indistinguishable from that in vivo. The addition of calcium channel blockers (MgCl2 and nifedipine) to the high-KCl medium reduced organotypicity drastically, indicating that a depolarization-induced increase of intracellular calcium is indispensable. Furthermore, the temporal course of the expression of the calcium-binding protein parvalbumin in culture under high KCl mimics that in vivo, demonstrating developmental processes during incubation. The need for calcium influx into neurons of this auditory network in vitro (which is not seen in other slice culture systems) strengthens the hypothesis that an optimal calcium concentration is exceptionally important in auditory neurons. The effect of KCl in the slice cultures may substitute for input activity regulating intracellular calcium in auditory neurons in vivo.

Calcium imaging has been used extensively to explore the role of action potential (AP) firing in the development of neuronal structure and synaptic function because increases in intracellular calcium ([Ca2+]i) reliably and, within a certain range, linearly reflect neuronal spiking activity. Patterns of APs in individual cells can be deduced from calcium recordings, which have typically been performed at the level of cell bodies. However, neurons are particularly susceptible to phototoxicity when they are illuminated at the soma. Furthermore, for some imaging experiments (e.g., those that address the interactions between dendrites and axons during synapse formation), the cell body of a given neuron may simply not be in the field of view. In these situations, it would be helpful to determine the spiking patterns of a neuron from the calcium activity in its subcellular compartments such as stretches of dendrites or axons. Here, we describe an approach for determining the relationship between AP firing and dendritic calcium transients by simultaneously imaging calcium transients in small dendritic stretches of hippocampal pyramidal neurons in slice cultures from neonatal rats and recording spiking activity with whole-cell patch-clamp recordings in these neurons. These experiments allow us to correlate the electrophysiological spiking pattern with the accompanying changes in the calcium concentration in individual dendritic segments.