Astrocyte-dependent circuit remodeling by synapse phagocytosis

In the central nervous system, synaptic pruning, the removal of unnecessary synaptic contacts, is an essential process for proper circuit maturation in neurodevelopment as well as for synaptic homeostasis in the adult stage. Dysregulation of synaptic pruning can contribute to the initiation and progression of various mental disorders, such as schizophrenia and depression, as well as neurodegenerative diseases including Alzheimer's disease. In the past 15 years, pioneering works have demonstrated that different types of glial cells regulate the number of synapses by selectively eliminating them through phagocytic molecular machinery. Although a majority of findings have been focused on microglia, it is increasingly evident that astrocytes function as a critical player in activity-dependent synapse elimination in developing, adult, and diseased brains. In this review, we will discuss recent findings showing the mechanisms and physiological importance of astrocyte-mediated synapse elimination in controlling synapses and circuit homeostasis. We propose that astrocytes play dominant and non-redundant roles in eliminating synapses during the activity-dependent circuit remodeling processes that do not involve neuro-inflammation.     

Neuron–glia crosstalk in health and disease: fractalkine and CX3CR1 take centre stage

An essential aspect of normal brain function is the bidirectional interaction and communication between neurons and neighbouring glial cells. To this end, the brain has evolved ligand–receptor partnerships that facilitate crosstalk between different cell types. The chemokine, fractalkine (FKN), is expressed on neuronal cells, and its receptor, CX₃CR1, is predominantly expressed on microglia. This review focuses on several important functional roles for FKN/CX₃CR1 in both health and disease of the central nervous system. It has been posited that FKN is involved in microglial infiltration of the brain during development. Microglia, in turn, are implicated in the developmental synaptic pruning that occurs during brain maturation. The abundance of FKN on mature hippocampal neurons suggests a homeostatic non-inflammatory role in mechanisms of learning and memory. There is substantial evidence describing a role for FKN in hippocampal synaptic plasticity. FKN, on the one hand, appears to prevent excess microglial activation in the absence of injury while promoting activation of microglia and astrocytes during inflammatory episodes. Thus, FKN appears to be neuroprotective in some settings, whereas it contributes to neuronal damage in others. Many progressive neuroinflammatory disorders that are associated with increased microglial activation, such as Alzheimer''s disease, show disruption of the FKN/CX₃CR1 communication system. Thus, targeting CX₃CR1 receptor hyperactivation with specific antagonists in such neuroinflammatory conditions may eventually lead to novel neurotherapeutics.     

Phagocytosis in the developing CNS: more than clearing the corpses

Cell corpses generated during CNS development are eliminated through phagocytosis performed by a variety of cells, including mesenchyme-derived macrophages and microglia, or glial cells originating in the neurogenic ectoderm. Mounting evidence indicates that in different species, phagocytes not only clear cell corpses but also engulf still-living neural cells or axons, and thereby promote cell death or axon pruning. Knowledge of the mechanisms of corpse recognition by engulfing cells provides molecular signals to this new role for phagocytes. These observations support a conserved and instructive role for phagocytosis in the execution of regressive events during neurogenesis.     

Microglia in development: linking brain wiring to brain environment

Microglia are enigmatic non-neuronal cells that infiltrate and take up residence in the brain during development and are thought to perform a surveillance function. An established literature has documented how microglia are activated by pathogenic stimuli and how they contribute to and resolve injuries to the brain. However, much less work has been aimed at understanding their function in the uninjured brain. A series of recent in vivo imaging studies shows that microglia in their resting state are highly motile and actively survey their neuronal surroundings. Furthermore, new data suggest that microglia in their resting state are able to phagocytose unwanted synapses and in this way contribute to synaptic pruning and maturation during development. Coupled with their exquisite sensitivity to pathogenic stimuli, these data suggest that microglia form a link that couples changes in brain environment to changes in brain wiring. Here we discuss this hypothesis and propose a model for the role of microglia during development in sculpting brain connectivity.     

The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.     

The classical complement cascade mediates CNS synapse elimination

During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.     

Cell biology in neuroscience: Architects in neural circuit design: Glia control neuron numbers and connectivity

Glia serve many important functions in the mature nervous system. In addition, these diverse cells have emerged as essential participants in nearly all aspects of neural development. Improved techniques to study neurons in the absence of glia, and to visualize and manipulate glia in vivo, have greatly expanded our knowledge of glial biology and neuron–glia interactions during development. Exciting studies in the last decade have begun to identify the cellular and molecular mechanisms by which glia exert control over neuronal circuit formation. Recent findings illustrate the importance of glial cells in shaping the nervous system by controlling the number and connectivity of neurons.

Introduction

The nervous system is comprised of two main cell types: neurons and glia. Glia represent a heterogeneous population of nervous system cells that associate with neurons and have diverse roles in nervous system development, maintenance, and function. Major glial cell types in vertebrates include astrocytes, microglia, oligodendrocytes, and NG2⁺ oligodendrocyte precursor cells (OPCs) in the central nervous system, and Schwann cells in the peripheral nervous system. Active roles for glia in all major steps of neural development have been described in a variety of model organisms from worms to mammals. Among the first recognized (and perhaps the most well-known) developmental functions of glia is in helping to guide axons to their correct targets. Glia serve as contact-dependent guideposts and sources of numerous attractive and repulsive cues in many circuits (Chotard and Salecker, 2004). It is also now understood that glia serve as stem cells in both embryonic and adult vertebrate nervous systems (Doetsch et al., 1999; Noctor et al., 2001; Seri et al., 2001), in addition to providing important substrates for neuronal migration (Rakic, 1971). In recent years, new functions for glia in synaptogenesis and plasticity have revealed a very active role for glia in neural circuit formation. In this review, we highlight recent insights into active roles for glia during nervous system development, focusing primarily on glial shaping of circuit formation via control of neuron and synapse numbers.

Glia regulate neuron production

A key step in neural development is generating the appropriate number of neurons and glia at the correct time and in the right place. In both invertebrate and vertebrate systems, glial cells have been identified as crucial constituents of neural stem cell niches that modulate neurogenesis by dynamically regulating stem cell proliferation and precursor differentiation in response to a variety of changing developmental time points and functional needs.In Drosophila, neural stem cells called neuroblasts undergo a stereotyped period of quiescence during early larval stages before resuming divisions to give rise to the adult nervous system (Truman and Bate, 1988), and proper transition between these states is crucial for normal nervous system formation (Ebens et al., 1993). As with many other stem cell types, neuroblast exit from quiescence is tied to overall animal development through nutrient signaling. Signals from the larval fat body, which monitors nutrient status in the periphery, are required to activate neuroblasts to reenter the cell cycle (Britton and Edgar, 1998). Two recent studies have shown that glia are required to act as an intermediate to transduce this information to neuroblasts. In response to nutrient-dependent fat body signaling, glia secrete insulin/IGF-like peptides (Drosophila insulin-like peptides [dILPs]). These dILPs activate insulin receptor–PI3K/Akt signaling in neuroblasts, triggering reentry into the cell cycle (Chell and Brand, 2010; Sousa-Nunes et al., 2011). Neuroblast proliferation is significantly delayed in animals lacking dILPs, or when glia are prevented from signaling by inhibiting vesicle trafficking with a mutant form of the fly dynamin gene, shibire. Moreover, glial expression of dILPs is sufficient to induce precocious activation of neuroblasts under normal conditions or bypass the requirement for dietary amino acids in starvation conditions (Chell and Brand, 2010; Sousa-Nunes et al., 2011). Glia, therefore, play a specific role in receiving systemic signals and transmitting them to neural stem cells to modulate their growth. Using glia as an intermediary may allow for more stable and precisely timed regulation of stem cell activation by allowing glia to serve as an integration site for multiple cues, which clearly include nutritional status. Auto-regulation of their own activity also appears to modulate glial control of stem cell activity: there is evidence glia may cell-autonomously modulate their own dILP production through expression of the RNA-binding protein FMRP, and that this regulation is critical for proper timing of neuroblast reactivation (Callan et al., 2012). Thus, glia may not simply relay information, but serve as important integration or filtering sites for multiple signals that impinge on neuroblast biology.Glial cells also play important roles in regulating progenitor cell division and specification in the proliferative zones of embryonic and adult mammalian brains. Microglia derived from myeloid precursors in the yolk sac migrate into the brain as early as E10.5 (Hirasawa et al., 2005; Ginhoux et al., 2010), where they can modulate neural precursor proliferation and neural cell fate specification during embryonic cortical neurogenesis. Evidence from in vitro studies comparing isolated progenitors to those cultured with young microglia suggest that the presence of microglia enhances proliferation and biases progeny toward an astroglial fate (Antony et al., 2011). A novel role for microglia in the negative regulation of neurogenesis has also recently been described. Data from immunostaining of fixed tissue and imaging of microglia in cultured brain slices suggest that young microglia phagocytose cortical precursor cells as cortical neurogenesis nears completion, which may serve as a key mechanism to curb neuron production or reduce neural populations at this time (Cunningham et al., 2013). One intriguing feature of this finding is that microglia appear to engulf cells that lack several standard cell death markers and may even engulf actively dividing progenitors (Cunningham et al., 2013). These observations suggest that microglia may not simply be acting as scavengers, but might be initiating cell death via engulfment. There is evidence that strongly suggests that engulfment by microglia is required to fully execute partially activated developmental cell death programs in post-mitotic Purkinje neurons (Marín-Teva et al., 2004; see next section), and roles for engulfing cells in promoting final execution of apoptosis in target cells have been described in Caenorhabditis elegans (Hoeppner et al., 2001; Reddien et al., 2001). However, the above study goes further, suggesting that microglia somehow decide to engulf and destroy seemingly healthy neural precursors. Defining the molecular mechanisms that govern this choice will be an important focus for future work.Radial glia are a key feature of the developing cortex, long understood to serve as important scaffolds for neuronal migration in the developing embryo. Studies in the last decade have now demonstrated that radial glia cells are in fact neuronal progenitors in the developing brain (Noctor et al., 2001; Tamamaki et al., 2001). This important finding came on the heels of the identification of astrocytes as adult neural stem cells in the mammalian brain (Doetsch et al., 1999). In addition to serving as stem cells, mammalian astrocytes in adult proliferative zones can modulate progenitor division and differentiation through the release of secreted molecules (including FGF2) and contact-dependent mechanisms (such as Eph/ephrin signaling; Morrens et al., 2012). Only astrocytes from proliferative zones are capable of promoting neurogenesis; thus, astrocytes constrain where new neurons are generated in the adult (Song et al., 2002). In addition, astrocytes can also couple neurogenesis to physiological status or injury. For example, IGF-I, normally expressed by neurons, is strongly expressed in astrocytes only after brain injury, and may contribute to injury-induced increases in adult neurogenesis (Yan et al., 2006).These exciting new studies of neural stem cells and neurogenesis in different species and at different developmental stages demonstrate conserved and wide-ranging roles for glia as regulators of neuron production. Through mechanisms that remain to be defined, glia appear to serve as sites of integration that allow dynamic modulation of neurogenesis in response to changing physiological needs during development and after injury or disease in the adult. Understanding the cellular and molecular mechanisms by which glia monitor animal physiology and modulate neurogenesis should have important implications for the understanding and treatment of many neurodevelopmental disorders and neurodegenerative diseases.

Glia in developmental programmed cell death

Overproduction, followed by programmed cell death (PCD) of excess neurons, is a well-described and conserved feature of nervous system development across phyla that is designed to ensure a sufficient number of neurons are initially formed to accomplish neural circuit construction (see Dekkers et al., in this issue). Competition between neurons for limited environmental and target-derived trophic factors results in cell death of extraneous or weakly connected neurons (Levi-Montalcini, 1987). PCD most often occurs via apoptosis followed by phagocytosis of the cellular debris by microglia, the resident immune cells and phagocytes of the central nervous system. A number of recent studies in the rodent have challenged the view that microglia act as passive scavengers of cellular debris. Rather, accumulating evidence now demonstrates an active role for microglia and innate immune mechanisms in promoting developmental PCD (Fig. 1).Open in a separate windowFigure 1.Microglia promote cell death and clearance of neuronal debris in the developing brain. (A) Model of the role of microglia in promoting neuronal programmed cell death in the developing cerebellum and hippocampus. Caspase-3+ neurons that have initiated apoptosis express as-yet-unknown “kill me/eat me” signals that recruit microglia to engulf them. Contact between dying neurons and microglia expressing the integrin CD11b and the immunoreceptor DAP12 triggers production of reactive oxygen species (including O₂^.−) in microglia, which is locally released to promote completion of apoptosis in the dying neuron. CD11b and DAP12 are required in microglia for efficient ROS production and for normal levels of PCD, but it remains unclear whether CD11b/DAP12 act as a receptor for one of the elusive neuronal signal(s), or if other receptors indirectly activate this pathway. (B) Microglia are also required for the clearance of cellular debris generated by PCD. The neuronal signals and specific microglial receptors that mediate corpse recognition and phagocytosis also remain unknown. Proper clearance of corpses is required to prevent inflammation.Close association or partial engulfment of intact but caspase-3–expressing Purkinje cells and microglia is seen in fixed tissue and in cultured cerebellar slices from postembryonic day 3 (P3) mice, a time corresponding to high levels of Purkinje cell PCD in vivo (Marín-Teva et al., 2004). At this stage, microglia were observed to be amoeboid in shape, which is characteristic of phagocytic function. Despite the fact that many of the Purkinje cells at this stage are already positive for activated caspases, depletion of microglia from the slices using clodronate containing liposomes (which target phagocytic cells) dramatically increases the number of surviving Purkinje cells after 3 days in vitro (Marín-Teva et al., 2004). These data fit a model proposed by PCD studies in C. elegans that shows engulfment by phagocytes is not simply required for debris clearance, but is itself required for the completion of cell death, even in cells already expressing activated caspases (Hoeppner et al., 2001; Reddien et al., 2001).In the innate immune response, engulfing cells can release reactive oxygen species (ROS) in a process called “respiratory burst” to aid in killing pathogens (Chanock et al., 1994). Using pharmacology, Marín-Teva et al. (2004) found evidence that microglia produce such respiratory bursts upon engulfment of Purkinje cells that contributed to their death. Subsequent studies demonstrated similar requirements for microglia in mediating PCD in the developing hippocampus and identified two immune molecules expressed in microglia that have been co-opted from the innate immune system for use in developmental PCD (Wakselman et al., 2008). In peripheral immune cells, the cell surface molecules CD11b and DAP12 work together to target pathogens for destruction using ROS (Mócsai et al., 2006). In the developing hippocampus, CD11b and DAP12 are expressed exclusively by microglia, and examination of DAP12 or CD11b mutant mice showed reduced microglial superoxide production along with significantly reduced PCD in the hippocampus (Fig. 1 A; Wakselman et al., 2008). In both the cerebellum and hippocampus, caspase activation is still assumed to be cell-autonomously induced, and although greatly reduced, some PCD is still observed in the absence of microglial signaling. These data suggest that cell-autonomous mechanisms are capable of both inducing PCD and carrying it to completion in some cases, but that microglia play an important role in a majority of cases. Thus, microglia appear to have two important roles in developmental PCD. First, they can act to promote cell death via production of reactive oxygen species (and potentially additional mechanisms) to ensure the appropriately high levels of cell death that are required for normal development (Fig. 1 A). In addition, microglia act as phagocytes to clear the debris PCD generates (Fig. 1 B). It is assumed that dying neurons produce “kill me” and/or “eat me” signals that help recruit microglia and initiate CD11b/DAP12-dependent ROS production and engulfment, but these putative signals and their microglial receptors remain unknown.Whether microglia play such an active role in cell death outside of development is unclear. In uninjured adult hippocampus, for example, microglia lack DAP12 (Wakselman et al., 2008) and have very low levels of CD11b (Sierra et al., 2010), yet there are high levels of cell death among newly born neurons in the subgranular zone of the hippocampus (a site of adult neurogenesis; Sierra et al., 2010). This suggests that microglia may not play the same active role in driving apoptosis of the newly born neurons, but this remains to be tested directly. Interestingly, engulfment of these cells in the mature hippocampus is performed by the processes of ramified (resting) microglia that efficiently phagocytose dying neurons without any morphological signs of activation (Sierra et al., 2010). Engulfment earlier in development is generally performed by amoeboid-like microglia, so this morphological difference may suggest the molecular mechanisms engaged may also be distinct at different stages. On the other hand, microglial activity during PCD at all stages is clearly distinguished from microglial response to injury or infection, which causes inflammation. Rapid engulfment of neurons undergoing PCD is considered immunologically silent—actually preventing inflammation that might be caused by unattended cell corpses.

Glia control connectivity by mediating developmental axon pruning

During development neurons often make inappropriate, excessive, or transient connections that must be eliminated as circuits mature. Though this process sometimes results in developmental cell death, selective pruning of axonal and dendritic branches is an important alternative method of neural circuit refinement and remodeling. Glia play key roles in two of the most well-characterized examples of developmental axon pruning—mammalian neuromuscular junction (NMJ) refinement and fly mushroom body γ-neuron remodeling.In early development, mammalian NMJs are innervated by multiple motor neuron axons. Activity-dependent competition between the axonal branches innervating the same NMJ eventually results in exactly one “winning” axon that maintains connectivity, while all the other “losing” branches are eliminated (Colman et al., 1997; Walsh and Lichtman, 2003). Detailed analysis of branch elimination using confocal and electron microscopy has revealed that losing axons retract from the NMJ by shedding pieces of themselves (termed axosomes) in a distal-to-proximal manner (Bishop et al., 2004; Song et al., 2008). Individual axon branches are ensheathed by perisynaptic Schwann cells—a specialized subtype of glia cell in the peripheral nervous system—before and during retraction. Shed axosomes are engulfed into the ensheathing perisynaptic Schwann cell where they undergo lysosomal degradation in the Schwann cell cytoplasm (Song et al., 2008). Thus, Schwann cells appear to play an essential role in at least the clearance of axonal debris, a feature that appears to be conserved at the fly NMJ (Fuentes-Medel et al., 2009).An unresolved question is whether axosomes are shed cell-autonomously by the retracting axon, or whether the ensheathing Schwann cell may contribute to the formation or “pinching off” of these structures in the losing axon branch. Some imaging evidence reveals thin Schwann cell processes around budding axosomes, suggesting glia might play an active role in axosome formation and/or shedding as they are engulfed (Bishop et al., 2004). Demonstrating such an active role is technically challenging due to the requirement of Schwann cell ensheathment for axon survival (Koirala et al., 2003). Schwann cell ablation does result in loss of nerve terminals, suggesting that retraction can take place in the absence of glia, but whether retraction in this situation is similar to activity-induced pruning is unclear (Reddy et al., 2003)—perhaps in the absence of Schwann cells axon terminals simply degenerate due to lack of support. Experiments in which Schwann cell endocytosis or membrane dynamics can be acutely disrupted during dual-color live imaging will likely be required to definitively resolve this question. Moreover, such an active role in eliminating weak synapses should require that Schwann cells are capable of discriminating and evaluating the relative synaptic strength of multiple inputs so that they target the correct input for elimination. A recent study using simultaneous glial calcium imaging and neuronal electrophysiological recording demonstrates that perisynaptic Schwann cells are capable of “sensing” the relative activity levels of each synapse at a multiply innervated NMJ. An individual PSC contacting two NMJs displays stronger calcium responses to “stronger” than to “weaker” synapses, an effect mediated by purinergic receptors on the glial cells (Darabid et al., 2013). If and how this responsiveness might affect synaptic competition or branch retraction awaits future studies in which glial responses are perturbed and the effects on innervation determined.In Drosophila, axon pruning during metamorphosis is important for the remodeling required to form mature adult circuits. Mushroom body γ-neurons prune their larval axons during early metamorphosis, followed by formation of new adult-specific connections as metamorphosis continues (Fig. 2; Lee et al., 1999). In addition to many cell-autonomous requirements for proper axon pruning, there are multiple essential roles for neighboring glia in mediating axon removal in this circuit. Initiation of pruning is triggered by a pulse of the molting hormone ecdysone just before metamorphosis (Lee et al., 2000). γ-Neuron responsiveness to this critical signal is controlled by surrounding cortex and astrocyte-like glia, which express the TGF-β ligand Myoglianin (Myo) during late larval stages (Awasaki et al., 2011). Glial-derived Myo must activate TGF-β signaling within γ-neurons to up-regulate expression of the ecdysone receptor B1 isoform (EcR-B1) so that γ-neurons can receive the hormonal signal that will trigger pruning (Zheng et al., 2003; Awasaki et al., 2011). Myo therefore represents perhaps the first glial-derived factor required for neurons to become competent to prune their axons (Fig. 2 A).Open in a separate windowFigure 2.Developmental pruning of mushroom body γ-neuron axons requires glia. (A) Before metamorphosis, cortex and astrocyte-like glia secrete the TGF-β ligand Myoglianin, which acts through γ-neuron baboon receptors to up-regulate expression of the ecdysone receptor EcR-B1. This makes γ-neurons competent to respond to the ecdysone pulse that will initiate pruning. (B) As metamorphosis begins, glia infiltrate the mushroom body neuropil. This infiltration is dependent on the glial cell surface receptor Draper. Arrival of glia coincides with axon blebbing and fragmentation, but it remains uncertain whether glia actively promote this fragmentation. (C) Glia are required for clearance of axonal debris. Recognition and phagocytosis of the debris is also mediated by the glial cell surface receptor Draper.γ-Neuron axons are pruned by local degeneration in which specific axonal branches fragment in place and are subsequently cleared by glia cells in a process primarily mediated by the glial cell surface receptor Draper (Fig. 2, B and C; Watts et al., 2003, 2004; Awasaki and Ito, 2004; Awasaki et al., 2006). Although glial clearance and degradation of axonal debris is required for complete pruning, it remains unclear if engulfing glia play active roles in promoting degeneration and fragmentation of pruned axons, or whether they simply respond to and clear cell-autonomously fragmented axons. Preventing glial infiltration by blocking membrane dynamics strongly suppresses axon pruning, providing support for an active role for glia in driving pruning (Awasaki and Ito, 2004). However, these results are inconclusive because the experiments lacked sufficient resolution to distinguish whether axons had been broken down but not cleared, or remained completely intact. Additional evidence in support of an active role is that glia infiltrate the mushroom body lobes before any observable fragmentation and can even infiltrate if fragmentation is suppressed via genetic mutations (Watts et al., 2004). Subsequent work showed that glial infiltration is dependent on the engulfment receptor Draper (Awasaki et al., 2006). Draper is believed to recruit engulfing cells to dying cells by responding to an unidentified “eat me” signal (Zhou et al., 2001; MacDonald et al., 2006). The requirement of Draper for infiltration, therefore, suggests glia are responding to axon-derived signals that may be generated independently of, or before, fragmentation. Microtubule disorganization occurs before fragmentation and can occur even in the absence of glial infiltration (Awasaki et al., 2006), suggesting that axons are already committed to pruning before visible fragmentation. Together, these findings suggest that initiation of axon degeneration (as defined by microtubule breakdown) may be cell-autonomous to neurons, but does not rule out a role for glia in driving fragmentation in addition to engulfment of debris. Live imaging experiments taking advantage of new techniques to independently label and genetically manipulate engulfing glia and mushroom body γ-neurons with single-cell resolution (Lai and Lee, 2006; Potter et al., 2010) will likely be necessary to fully resolve the role of glia in this process. If glia are indeed promoting axonal degeneration, the next exciting question would be—how do they choose which axons to destroy?

Glia promote synapse formation

A key step in the formation of a functional nervous system is the establishment of appropriate synaptic connections between neurons. Unraveling how neurons make appropriate synaptic connections has been a major focus of neurodevelopment research for decades, and we now understand that synaptogenesis is a multistep process including recognition and selection of appropriate targets, assembly and localization of synaptic components, and stabilization of appropriate connections. Research in the last decade has identified important roles for glia in many of these steps.An important breakthrough was the development of methods to isolate and maintain purified neurons in culture in the absence of glia. Pfrieger and Barres (1997) developed a technique to purify retinal ganglion cell (RGC) neurons and keep them healthy in culture without glia. Intriguingly, they found that purified RGCs cultured alone formed sevenfold fewer functional excitatory synapses than RGCs cultured with astrocyte feeder layers as assayed by electrophysiological recordings, immunostaining for synaptic protein localization, and EM analysis (Pfrieger and Barres, 1997; Nägler et al., 2001; Ullian et al., 2001). Subsequent in vitro studies with other neuronal subtypes demonstrated that these findings were not just specific to rat RGCs (Peng et al., 2003; Ullian et al., 2004; Cao and Ko, 2007). Hints that astrocytes might play similar synaptogenic roles in vivo first came from observations that timing of synaptogenesis between RGCs and their in vivo targets correlated with glial infiltration of the area, even though RGC axons had reached their target region several days prior (Ullian et al., 2001).In culture, the synapse-promoting effects of glia did not require physical contact between the glia and neurons, implicating secreted factors as the primary mediators of glia-induced synaptogenesis (Fig. 3). Fractionation of astrocyte-conditioned media (ACM) was used to identify thrombospondins (TSPs)—extracellular matrix proteins with known roles in cell adhesion—as key glial-derived synaptogenic factors (Fig. 3 A; Christopherson et al., 2005). Purified TSP-1 or TSP-2 mimicked ACM to promote structural synapse formation, whereas depleting TSP-2 from ACM abrogated its synaptogenic effects on cultured neurons. With a specific molecule in hand, researchers could now ask whether a glial-derived molecule was required for synaptogenesis in vivo. Christopherson et al. (2005) showed that TSPs are expressed in astrocytes in vivo when synapses are forming and colocalize with synapse-associated astrocyte processes. Importantly, mice mutant for both TSP-1 and TSP-2 had significantly fewer synapses than wild-type animals (∼40% reduction at P8), demonstrating a requirement for an astrocyte-derived factor for normal synapse development in vivo (Christopherson et al., 2005).Open in a separate windowFigure 3.Model of astrocyte–neuron interactions during synapse formation and maturation. (A) Astrocytes secrete several factors to promote structural synapse formation. Astrocytes secrete thrombospondins (TSPs), which act through α2–δ1 calcium channel subunit/gabapentin receptors to drive formation of structurally intact glutamatergic synapses. The exact mechanism by which TSP binding to α2–δ1 promotes adhesion and structural synapse formation is unknown. Astrocytes also secrete hevin and SPARC. Hevin also promotes structural synapse formation, whereas SPARC antagonizes this function. Competition between hevin and SPARC for a common unknown binding partner on neurons may explain the antagonism. The synapses formed in response to TSPs or hevin are structurally intact with docked vesicles and PSD-95 correctly localized; however, they lack surface expression of AMPARs at the postsynapse and are therefore not fully functional. (B) Other astrocyte-secreted factors influence surface expression and clustering of glutamatergic AMPARs. Glypicans secreted from astrocytes act through an unidentified receptor to promote surface expression and clustering of AMPARs during development. Activity can also induce astrocytes to secrete SPARC, which can perturb integrin interactions with surface AMPARs, leading to decreased surface expression of AMPARs in response to excess activity.In an elegant follow-up study, Eroglu et al. (2009) identified the α2–δ1 calcium channel subunit/gabapentin receptor as the neuronal receptor for glial TSPs, which bind the receptor through their EGF-like domains. α2–δ1 colocalizes with both pre- and postsynaptic puncta, and in vitro overexpression experiments suggest it functions postsynaptically. The intracellular mechanisms by which TSP/α2–δ1 signaling promotes synapse assembly remains an open question, though α2–δ1 modulation of calcium channel function is not likely to be involved because perturbation of calcium channel expression or function is unable to interfere with TSP- or astrocyte-induced synaptogenesis (Eroglu et al., 2009).The extracellular matrix protein hevin is a second astrocyte-derived factor capable of inducing structural synapse formation in vitro (Fig. 3 A). Although purified hevin works just as well as TSPs at inducing glutamatergic synapses in culture, depletion of TSPs from ACM completely abolishes the synaptogenic activity of ACM despite the presence of hevin (Christopherson et al., 2005; Kucukdereli et al., 2011), implying that there might be ACM components that can antagonize hevin function. This inhibitory factor was identified as SPARC (secreted protein acidic and rich in cysteine), a hevin homologue that is also highly expressed in ACM. SPARC selectively interferes with hevin’s synapse-promoting ability, likely by competing with hevin for an as-yet-unidentified common binding partner on neurons. Hevin and SPARC are coexpressed in vivo, with peak expression in the superior colliculus at a time when RGCs are actively forming synapses. hevin mutant mice have reduced, and SPARC mutant mice have increased, RGC-collicular synapses, supporting antagonistic roles for these proteins in vivo. The overall positive effect of synaptogenesis in this circuit during development is likely due to a high hevin/SPARC ratio. (This ratio is low in ACM.) Expression of hevin and SPARC is maintained into adulthood, unlike TSPs (Christopherson et al., 2005), indicating that hevin and SPARC might also play changing roles in synapse maintenance and plasticity throughout life, potentially through dynamic regulation of their relative levels.Although TSPs and hevin play critical roles in promoting synapse formation, these molecules are only capable of promoting structural assembly but not functional maturation of synapses (Christopherson et al., 2005; Eroglu et al., 2009; Kucukdereli et al., 2011). Synapses formed in response to TSPs and hevin are “silent” synapses: synapses that look ultrastructurally normal, but lack postsynaptic AMPA glutamate receptors (AMPARs), rendering them incapable of neurotransmission. Astrocytes or ACM are sufficient to induce the formation of functional synapses, as well as strengthen newly formed synapses by increasing the amplitude and frequency of miniature excitatory postsynaptic potentials (mEPSPs; Ullian et al., 2001). Thus, there must be additional glia-derived factors that work in conjunction with TSPs and hevin to specifically instruct the addition of AMPARs at the postsynapse. This was an outstanding question in the field until the glypicans Gpc-4 and Gpc-6, which are part of the heparan sulfate proteoglycan family, were identified as ACM factors that fulfill this role (Fig. 3 B; Allen et al., 2012). When supplied to isolated neurons in conjunction with TSPs, Gpc-4 and Gpc-6 were each sufficient to increase synaptic activity in cultured RGCs, primarily by increasing surface expression and clustering of postsynaptic AMPAR GluA1 subunits. Synapse function is impaired in gpc-4 knockout mice, as evidenced by reductions in the amplitude of mEPSPs in hippocampal slices, supporting an in vivo role for glypicans in promoting functional synapse formation (Allen et al., 2012). The mechanism by which glypicans induce surface expression and clustering of GluA1 AMPARs is still unknown. In other contexts, glypicans have roles in concentrating morphogens to enhance signaling pathways, but examination of several glypican interactors (including FGFs, Sonic hedgehog, and Wnts) failed to show roles for these molecules in AMPAR regulation. This suggests that Gpc-4 and Gpc-6 might act directly on a postsynaptic receptor to mediate their effects. Identification of the synaptic glypican receptor or binding partner, and of additional molecules that regulate the surface expression and clustering of AMPAR subunits, will be important for determining how astrocytes contribute to synaptic maturation and the plasticity of neural circuits in vivo.It is unlikely the above-described molecules represent the sum of pro-synaptogenic factors supplied by glia. For example, recent work on inhibitory GABAergic synapse formation indicates the effect of TSPs is specific to glutamatergic synapses, having virtually no effect on inhibitory synapse development. Nonetheless, ACM is required for normal inhibitory synapse development (Hughes et al., 2010); thus, ACM must contain additional synaptogenic factor(s) that can affect inhibitory synapses that have yet to be identified. In addition, astrocytes are not the only glial cell type to support synapse formation—peripheral glia also promote NMJ synapse formation and growth in vertebrates and Drosophila through secretion of TGF-β ligands. Schwann cell–derived TGF-β1 can drive synapse formation in Xenopus motor neuron/muscle co-cultures by acting on presynaptic neurons to increase expression of a protein called agrin, which is required to cluster acetylcholine receptors at neuron/muscle contact sites (Feng and Ko, 2008). In Drosophila, the role of glial TGF-β signaling on synapse growth is distinct. In this system, glia express the TGF-β ligand Maverick (Mav), which acts on postsynaptic muscle to modulate muscle release of a distinct TGF-β ligand (Glass bottom boat) that acts directly on neurons to drive synaptic growth (Fuentes-Medel et al., 2012). Thus, although a synaptogenic role for glia cells is highly conserved, diverse mechanisms exist to drive this function that may be specific to species, glial cell type, or synapse type.Several important questions emerge from these studies. First, are the signals discussed above nonspecific permissive signals that allow synapses to form with specificity being driven by entirely distinct factors, or are they capable of selectively driving the formation of specific, appropriate synapses in vivo? Implication of mainly secreted factors as mediators of synapse promotion from in vitro studies suggests the former. There are some studies, however, that have demonstrated close association and dynamic interactions of glial processes and developing synaptic spines that suggest that glia can and do act in synapse-specific ways as well. Whether similar or different molecular mechanisms are involved in these interactions remains to be determined.A second question is how glial synapse promotion is regulated and modulated during and after development. The effects of TSPs and hevin are robust in vitro, and aside from controlling the general timing of expression or the coexpression SPARC and hevin (mechanisms that seem relatively imprecise and slow), it remains unclear how synapse formation is regulated, particularly in vivo both during development and throughout life. Most glial cells are capable of sensing and responding to neural activity, but how activity affects the production or secretion of synaptogenic factors (particularly before synapse formation) has not been fully resolved. It has been recently reported that SPARC is up-regulated in astrocytes in response to neural activity. In this context though, SPARC seems to have a distinct primary function from inhibiting hevin—negative regulation of AMPAR levels at synapses via inhibition of neuronal β-integrin function (Fig. 3 B; Jones et al., 2011). Thus, SPARC appears to play at least two distinct roles in synapse development and plasticity at different times. At early stages, SPARC limits the number of synapses by abrogating hevin function before synapse assembly. After synapses have formed, SPARC can refine synaptic strength by interfering with integrin localization and function that normally serves to stabilize AMPARs at the membrane. Thus, production of SPARC in response to activity can indirectly limit AMPAR clustering at highly active synapses (Jones et al., 2011). Glial regulation of synapse formation, function, and plasticity throughout life is likely to be a rich and fruitful area of future study.Finally, we need to understand how these astrocyte-derived pro-synaptogenic factors function in vivo to coordinate synapse formation and plasticity in different brain regions. This is clearly a complex issue—for example, TSP knockout mice have an ∼40% reduction in cortical synapses and hevin knockout mice show an ∼35% reduction in collicular synapses. These observations indicate that there are likely additional partially redundant glial-derived factors yet to be discovered. Determining how these glial signals interact or synergize with the molecular cues required between pre- and postsynaptic neurons in vivo will be an important future goal.

Glia prune exuberant synaptic connections

Activity-dependent mechanisms ensure that appropriate connections are strengthened while inappropriate or weak connections are eliminated, but the cellular and molecular mechanisms that underlie experience-dependent synapse elimination are largely unknown. A flurry of research over the last several years has identified microglia as key mediators of developmental synaptic pruning by phagocytosing excessive presynaptic inputs.Microglia were once assumed to be relatively quiescent in healthy brains, but advances in live imaging techniques have revealed “resting” microglia to be highly active cells that constantly extend and retract their processes to make contact with neighboring neurons, glia, and synapses (Davalos et al., 2005; Nimmerjahn et al., 2005; Wake et al., 2009). Moreover, microglia activity changes in response to perturbations in neurotransmission, indicating that microglia might be actively monitoring local neural activity, perhaps playing a role in activity-dependent plasticity. High-resolution imaging studies combining live imaging with electron microscopy soon revealed that microglia made frequent contact with dendritic spines during periods of plasticity and remodeling. This contact has consequences on spine morphology and stability with microglia contact frequently predicting the loss of individual spines (Tremblay et al., 2010).Evidence of synaptic elements contained within microglia cytoplasm combined with the known role of microglia as phagocytes, and a report implicating the classical complement cascade with synaptic pruning provided highly suggestive evidence that microglia were involved in activity-dependent synapse elimination via phagocytosis (Stevens et al., 2007; Tremblay et al., 2010; Paolicelli et al., 2011; Schafer et al., 2012). In an elegant study, Schafer et al. (2012) used the mammalian retinogeniculate system to clearly demonstrate the in vivo role for microglia in mediating activity-dependent synaptic pruning that relies on signaling from the classical complement system. This circuit undergoes a well-characterized period of robust activity-dependent synaptic elimination during postnatal development with a clear readout. RGC axons from both eyes form synapses in the lateral geniculate nucleus (LGN) of the thalamus. Due to exuberant connectivity, distinct ipsilateral and contralateral eye domains are poorly defined until approximately postnatal day 5, at which point activity-dependent synaptic pruning results in clearly segregated eye-specific domains (Fig. 4 A). Injecting unique fluorescent anterograde tracers in each eye labels RGC axons and synapses independently, allowing a clear readout of the success of activity-dependent synaptic pruning (Huberman et al., 2008). GFP-expressing microglia in the LGN were closely associated with synapses, and were often found with tracer-labeled presynaptic membrane within their processes and soma during the period (∼P5) of robust synaptic pruning, similar to previous findings in the developing cortex and hippocampus (Tremblay et al., 2010; Paolicelli et al., 2011).Open in a separate windowFigure 4.Microglia refine circuits by complement-mediated phagocytosis of weak synapses. Schematic of retinogeniculate connectivity in wild-type (A) and complement receptor CR3 knockout (B) animals. Inputs from each eye are completely segregated in the LGNs of wild-type animals (A, separate blue and green regions). Synaptic debris from inappropriate connections is observed within local microglia (brown cells) during the pruning process. In CR3 knockout animals (B), segregation of inputs in the LGN is incomplete, resulting in regions with overlapping inputs from both eyes (blue/green regions). Significantly less synaptic debris is observed in microglia in these animals. (C) Proposed model for complement-dependent synaptic pruning by microglia. Weak synapses are tagged for elimination by recruitment of the classical complement molecule C1q. C1q can recruit and activate C3, which then serves as a ligand for the CR3 receptor (expressed exclusively by microglia) to recruit microglia to clear the synapse. How C1q and C3 are localized to and activated at weak synapses is still unknown.To test whether microglia were indeed ingesting synapses destined for elimination, the authors injected one eye with tetrodotoxin (TTX) to create a strong imbalance of activity between the RGCs from each eye. As expected, the authors found significantly more engulfed synapses that originated from the TTX-injected eye as compared with the control-injected eye, indicating that microglia were pruning synapses in an activity-dependent manner. A wealth of anatomical data supported the idea that microglia engulf weak synapses for elimination, but what were the molecular mechanisms that controlled this engulfment? A good candidate came from an earlier study that identified the classical complement components C1q and C3 as synaptically localized molecules required for activity-dependent pruning of retinogeniculate synapses (Stevens et al., 2007). As microglia are the only cell type in the brain that express the complement receptor CR3, it was hypothesized that binding between synaptic C3 and microglial CR3 might mediate synapse–microglia interactions required for engulfment (Fig. 4 C). To test this hypothesis, Schafer et al. (2012) examined CR3 knockout mice and found that CR3-deficient microglia showed an ∼50% reduction in phagocytosed presynaptic material. This in turn resulted in incomplete pruning, as evidenced by incomplete segregation of eye-specific domains in the LGN, and demonstrated a clear role for complement signaling in microglia pruning of synapses (Fig. 4 B).Among the key questions this work raises is how weak synapses are tagged for removal by the complement system. How the diffusible molecules C1q and activated C3 are localized to or activated at specific synapses is unclear, so the identification of complement system binding partners, inhibitors, and activators at synapses and determining how they are regulated by neuronal activity will be an important focus of future studies (Stevens et al., 2007). In addition, although complement signaling plays a major role in synaptic pruning, loss of complement signaling does not completely prevent pruning, causing only an ∼50% reduction. Thus, it is likely that other molecules and signaling pathways contribute to microglial synaptic pruning. One candidate pathway suggested by studies in fly is that of Draper (Megf10 in mouse), which has been shown to mediate developmental axon pruning (Awasaki et al., 2006) and can function in peripheral glia and muscle to mediate engulfment of shed synapses at the NMJ (Fuentes-Medel et al., 2009). This study from Drosophila also elegantly demonstrates the importance of synaptic clearance by glia. Failure to clear synaptic debris leads to severe defects in synapse growth at the NMJ, indicating that the elimination of synaptic debris by glia is an important event required for synaptic plasticity (Fuentes-Medel et al., 2009).In addition to synaptic pruning by microglia, other glial subtypes play important roles in synaptic plasticity. For example, as discussed above, astrocyte-secreted factors including SPARC can influence synaptic plasticity. SPARC expression in the postnatal hippocampus is regulated by activity. SPARC affects clustering and stabilization of glutamate receptor subunits at hippocampal synapses during postnatal development, and is required for normal synaptic plasticity during this time (Jones et al., 2011). Visual cortex plasticity is also strongly affected by glia, for example implantation of immature astrocytes into the brains of older animals can restore ocular dominance plasticity (Müller and Best, 1989). Maturation of oligodendrocytes might also affect the duration of ocular dominance plasticity via releasing factors that bind to neuronal Nogo receptors, as knockout of these receptors can lead to an extension of the plastic period in mice (McGee et al., 2005). These are just a few of the contributions of different glial subtypes to activity-dependent plasticity throughout development (and into adulthood) that have been described thus far. Continued work in this field should reveal many more unexpected roles for glia in modulating plasticity and activity in neural circuits.

Conclusions and future directions

There has been remarkable progress in our understanding of how glia function in the developing nervous system to shape neuronal connectivity. In particular, a number of cellular and molecular mechanisms by which glia regulate neural stem cell biology, and neural fate, survival, and connectivity have begun to be elucidated, but there is undoubtedly much more for us to learn about these remarkable cells. Exciting and fundamental questions about neuron–glia interactions await exploration and molecular or functional insight. For example, determining whether synaptogenic signals from astrocytes are perceived as permissive or instructive will be important, and if they are instructive for specific connections, how is specificity embedded in the message? In the context of neural circuit refinement we need to understand cell–cell communication events and how they are integrated with circuit function. How are “loser” synapses that need to be eliminated molecularly tagged? How does neuronal activity regulate glial pruning function or synaptic tagging during synapse elimination? Are microglia the only glia pruning synapses in mammals? What are the functional consequences of failed pruning in different brain regions? More broadly, we must explore how neural activity shapes glial developmental events, or neuron–glia interactions. It is well known that many types of glia express neurotransmitter receptors that could allow them to “listen in” on neuronal activity if they are in close contact with synapses. What are these used for? Is this a primary way that neurons signal to glia? If so, how does neurotransmitter signaling to glia shape their biology?In contrast to the growing list of astrocyte and microglial functions during embryonic and postnatal development, relatively little is known about specific roles for oligodendrocytes or their precursors (NG2⁺ cells) in the developing nervous system. Oligodendrocytes mediate myelination during the latest stages of development, though the cellular interactions between axons and glia that mediate myelination are still largely unknown. In vivo ablation studies indicate that loss of oligodendrocytes from the developing cerebellum near the onset of myelination disrupts overall cellular architecture and connectivity and alters the gene expression patterns of neurons, suggesting there may be a number of as-yet-identified functions for these cells during development (Mathis et al., 2003; Doretto et al., 2011). Intriguingly, NG2⁺ OPCs receive synapses from neurons during development (Bergles et al., 2000). Why these cells receive direct synaptic input is unclear, as is the exact function of NG2⁺ OPCs, but they are seemingly poised to respond rapidly to neural activity via their direct synaptic connections and generate new oligodendrocytes (De Biase et al., 2010; Mangin et al., 2012). Determining what these cells do in the developing and mature brain is a major question for the field in the future.Finally, although most communication between neurons and glia is likely bi-directional, many of our current models have elucidated only one half of the conversation. In several examples discussed above we have some knowledge of a glial-derived molecule, but many receptors and downstream signaling pathways remain to be identified. It is also notable that the expression of many glial-derived signals often changes over developmental time (e.g., TSPs and Gpcs). Is this the result of intrinsic changes in glial pro-synaptic potential, or is it the result of feedback signaling from neurons? Misregulation of several of the processes and/or molecules discussed above that modulate neural circuit construction are associated with disease. Understanding these functions more deeply and how they are altered in disease may be crucial for generating new therapeutic strategies to program appropriate levels of neural circuit plasticity.     

The information theory of developmental pruning: Optimizing global network architectures using local synaptic rules

During development, biological neural networks produce more synapses and neurons than needed. Many of these synapses and neurons are later removed in a process known as neural pruning. Why networks should initially be over-populated, and the processes that determine which synapses and neurons are ultimately pruned, remains unclear. We study the mechanisms and significance of neural pruning in model neural networks. In a deep Boltzmann machine model of sensory encoding, we find that (1) synaptic pruning is necessary to learn efficient network architectures that retain computationally-relevant connections, (2) pruning by synaptic weight alone does not optimize network size and (3) pruning based on a locally-available measure of importance based on Fisher information allows the network to identify structurally important vs. unimportant connections and neurons. This locally-available measure of importance has a biological interpretation in terms of the correlations between presynaptic and postsynaptic neurons, and implies an efficient activity-driven pruning rule. Overall, we show how local activity-dependent synaptic pruning can solve the global problem of optimizing a network architecture. We relate these findings to biology as follows: (I) Synaptic over-production is necessary for activity-dependent connectivity optimization. (II) In networks that have more neurons than needed, cells compete for activity, and only the most important and selective neurons are retained. (III) Cells may also be pruned due to a loss of synapses on their axons. This occurs when the information they convey is not relevant to the target population.     

Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila

Although synapses are assembled in a highly regulated fashion, synapses once formed are not static structures but continue to expand and retract throughout the life of an organism. One second messenger that has been demonstrated to play a critical role in synaptic growth and function is cAMP. Here, we have tested the idea that signaling through the heterotrimeric G protein, Gs, plays a coincident role with increases in intracellular Ca(+2) in the regulation of adenylyl cyclases (ACs) during synaptic growth and in the function of synapses. In larvae containing a hypomorphic mutation in the dgs gene encoding the Drosophila Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization, as well as defects in the facilitation of synaptic transmission. Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha either pre- or postsynaptically rescued the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. In addition, this Gs alpha mutation interacted with fasII, dnc, and hyperexcitability mutants in a manner that revealed a coincident role for Gs alpha in the regulation of cAMP and FASII levels required during growth of these synapses. Our results demonstrate that Gs alpha-dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.     

Microglia-mediated synaptic pruning as a key deficit in neurodevelopmental disorders: Hype or hope?

There is a consensus in the field that microglia play a prominent role in neurodevelopmental processes like synaptic pruning and neuronal network maturation. Thus, a current momentum of associating microglia deficits with neurodevelopmental disorders (NDDs) emerged. This concept is challenged by rodent studies and clinical data. Intriguingly, reduced numbers of microglia or altered microglial functions do not necessarily lead to overt NDD phenotypes, and neuropsychiatric symptoms seem to develop primarily in adulthood. Hence, it remains open for discussion whether microglia are truly indispensable for healthy neurodevelopment. Here, we critically discuss the role of microglia in synaptic pruning and highlight area- and age dependency. We propose an updated model of microglia-mediated synaptic pruning in the context of NDDs and discuss the potential of targeting microglia for treatment of these disorders.     

The Extent of Synaptic Stripping of Motoneurons after Axotomy Is Not Correlated to Activation of Surrounding Glia or Downregulation of Postsynaptic Adhesion Molecules

Synapse elimination in the adult central nervous system can be modelled by axotomy of spinal motoneurons which triggers removal of synapses from the cell surface of lesioned motoneurons by processes that remain elusive. Proposed candidate mechanisms are removal of synapses by reactive microglia and astrocytes, based on the remarkable activation of these cell types in the vicinity of motoneurons following axon lesion, and/or decreased expression of synaptic adhesion molecules in lesioned motoneurons. In the present study, we investigated glia activation and adhesion molecule expression in motoneurons in two mouse strains with deviant patterns of synapse elimination following axotomy. Mice deficient in complement protein C3 display a markedly reduced loss of synapses from axotomized motoneurons, whereas mice with impaired function of major histocompatibility complex (MHC) class Ia display an augmented degree of stripping after axotomy. Activation of microglia and astrocytes was assessed by semiquantative immunohistochemistry for Iba 1 (microglia) and GFAP (astrocytes), while expression of synaptic adhesion molecules was determined by in situ hybridization. In spite of the fact that the two mouse strains display very different degrees of synapse elimination, no differences in terms of glial activation or in the downregulation of the studied adhesion molecules (SynCAM1, neuroligin-2,-3 and netrin G-2 ligand) could be detected. We conclude that neither glia activation nor downregulation of synaptic adhesion molecules are correlated to the different extent of the synaptic stripping in the two studied strains. Instead the magnitude of the stripping event is most likely a consequence of a precise molecular signaling, which at least in part is mediated by immune molecules.     

Microglial interactions with synapses are modulated by visual experience

Microglia are the immune cells of the brain. In the absence of pathological insult, their highly motile processes continually survey the brain parenchyma and transiently contact synaptic elements. Aside from monitoring, their physiological roles at synapses are not known. To gain insight into possible roles of microglia in the modification of synaptic structures, we used immunocytochemical electron microscopy, serial section electron microscopy with three-dimensional reconstructions, and two-photon in vivo imaging to characterize microglial interactions with synapses during normal and altered sensory experience, in the visual cortex of juvenile mice. During normal visual experience, most microglial processes displayed direct apposition with multiple synapse-associated elements, including synaptic clefts. Microglial processes were also distinctively surrounded by pockets of extracellular space. In terms of dynamics, microglial processes localized to the vicinity of small and transiently growing dendritic spines, which were typically lost over 2 d. When experience was manipulated through light deprivation and reexposure, microglial processes changed their morphology, showed altered distributions of extracellular space, displayed phagocytic structures, apposed synaptic clefts more frequently, and enveloped synapse-associated elements more extensively. While light deprivation induced microglia to become less motile and changed their preference of localization to the vicinity of a subset of larger dendritic spines that persistently shrank, light reexposure reversed these behaviors. Taken together, these findings reveal different modalities of microglial interactions with synapses that are subtly altered by sensory experience. These findings suggest that microglia may actively contribute to the experience-dependent modification or elimination of a specific subset of synapses in the healthy brain.     

Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. We show that draper (drpr) and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. We propose that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism.     

The role of glial cells in synapse elimination

Excessive synapses generated during early development are eliminated extensively to form functionally mature neural circuits. Synapses in juvenile and mature brains are highly dynamic, and undergo remodeling processes through constant formation and elimination of dendritic spines. Although neural activity has been implicated in initiating the synapse elimination process cell-autonomously, the cellular and molecular mechanisms that transduce changes in correlated neural activity into structural changes in synapses are largely unknown. Recently, however, new findings provide evidence that in different species, glial cells, non-neuronal cell types in the nervous system are crucial in eliminating neural debris and unwanted synapses through phagocytosis. Glial cells not only clear fragmented axons and synaptic debris produced during synapse elimination, but also engulf unwanted synapses thereby actively promoting synapse elimination non-cell autonomously. These new findings support the important role of glial cells in the formation and maintenance of functional neural circuits in development as well as in adult stages and neurodegenerative diseases.     

CYY-1/cyclin Y and CDK-5 differentially regulate synapse elimination and formation for rewiring neural circuits

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.     

Complement-mediated microglial clearance of developing retinal ganglion cell axons

In many parts of the developing vertebrate nervous system, axons are pruned to establish mature patterns of connectivity. In this issue of Neuron, Schafer et al. (2012) show that microglia may play a role in developmental axon pruning in the thalamus by engulfing presynaptic retinal ganglion cell terminals via a C3- and CR3-dependent mechanism.     

The development of intrinsic excitability in mouse retinal ganglion cells

Activity-dependent refinement of synaptic connections occurs throughout the developing nervous system, including the visual system. Retinal ganglion cells (RGCs) overproduce synapses then refine them in an activity-dependent manner that segregates RGC connections into multicellular patterns, such as eye-specific regions and retinotopic maps. Ferrets additionally segregate ON and OFF retinogeniculate pathways in an activity-dependent manner. It was unknown whether differences in ON versus OFF intrinsic and spontaneous activity occur in postnatal mouse. The work reported here measured the intrinsic properties and spontaneous activity of morphologically identified postnatal mouse RGCs, and tested the hypothesis that mouse ON and OFF RGCs develop differences in spontaneous activity. We found developmental changes in resting potential, action potential threshold, depolarization to threshold, action potential width, action potential patterns, and maximal firing rates. These results are consistent with the maturation of the intrinsic properties of RGCs extending through the first three postnatal weeks. However, there were no differences among mouse ON, OFF, and multistratified RGCs in intrinsic excitability, spontaneous synaptic drive or spontaneous action potential patterns. The absence of differences between ON and OFF activity patterns is unlike the differences that arise in ferrets. In contrast to the ferret, the ON and OFF target neurons in the mouse are organized in a random pattern, not layers. This supports the hypothesis that the absence of systematic differences in activity results in the nonlayered distribution of retinogeniculate connections.     

Neuronal Synapse Formation Induced by Microglia and Interleukin 10

Recently, it was found that microglia regulated synaptic remodeling of the developing brain, but their mechanisms have not been well understood. In this study, the action of microglia on neuronal synapse formation was investigated, and the primary target of microglial processes was discovered. When the developing microglia were applied to cultured hippocampal neurons without direct contact, the numbers of dendritic spines and excitatory and inhibitory synapses significantly increased. In order to find out the main factor for synaptic formation, the effects of cytokines released from microglia were examined. When recombinant proteins of cytokines were applied to neuronal culture media, interleukin 10 increased the numbers of dendritic spines in addition to excitatory and inhibitory synapses. Interestingly, without external stimuli, the amount of interleukin 10 released from the intact microglia appeared to be sufficient for the induction of synaptic formation. The neutralizing antibodies of interleukin 10 receptors attenuated the induction of the synaptic formation by microglia. The expression of interleukin 10 receptor was newly found in the hippocampal neurons of early developmental stage. When interleukin 10 receptors on the hippocampal neurons were knocked down with specific shRNA, the induction of synaptic formation by microglia and interleukin 10 disappeared. Pretreatment with lipopolysaccharide inhibited microglia from inducing synaptic formation, and interleukin 1β antagonized the induction of synaptic formation by interleukin 10. In conclusion, the developing microglia regulated synaptic functions and neuronal development through the interactions of the interleukin 10 released from the microglia with interleukin 10 receptors expressed on the hippocampal neurons.     

Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.     

Role of calcineurin in activity-dependent pattern formation in the dorsal lateral geniculate nucleus of the ferret

In the retinogeniculate pathway of the ferret, in addition to the separation of the inputs from the two eyes to form eye-specific layers, there is also an anatomical segregation of the terminal arbors of on-center retinal ganglion cells from the terminal arbors of off-center retinal ganglion cell axons to form on/off sublaminae. Sublamination normally occurs during postnatal weeks 3-4 and requires the activity of retinal afferents, N-methyl-D-aspartate receptors, nitric oxide synthase, and a target of nitric oxide, cyclic guanosine monophosphate. Calcineurin is a calcium/calmodulin dependent serine, threonine protein phosphatase suggested to mediate NMDA-receptor dependent synaptic plasticity in the hippocampus. We have examined whether calcineurin plays a role during on/off sublamination in the dorsal lateral geniculate nucleus (dLGN) of the ferret. Immunohistochemistry showed that calcineurin expression is transiently up-regulated in dLGN cells and neuropil during the period of on/off sublamination. A functional role for calcineurin during sublamination was investigated by blocking the enzyme locally via intracranial infusion of FK506. Treatment with FK506 during postnatal weeks 3-4 disrupted the appearance of sublaminae. These results suggest that calcineurin may play a role during this process of activity-dependent pattern formation in the visual pathway.