PirB regulates a structural substrate for cortical plasticity

Experience-driven circuit changes underlie learning and memory. Monocular deprivation (MD) engages synaptic mechanisms of ocular dominance (OD) plasticity and generates robust increases in dendritic spine density on L5 pyramidal neurons. Here we show that the paired immunoglobulin-like receptor B (PirB) negatively regulates spine density, as well as the threshold for adult OD plasticity. In PirB^−/− mice, spine density and stability are significantly greater than WT, associated with higher-frequency miniature synaptic currents, larger long-term potentiation, and deficient long-term depression. Although MD generates the expected increase in spine density in WT, in PirB^−/− this increase is occluded. In adult PirB^−/−, OD plasticity is larger and more rapid than in WT, consistent with the maintenance of elevated spine density. Thus, PirB normally regulates spine and excitatory synapse density and consequently the threshold for new learning throughout life.Experience generates both functional and structural changes in neural circuits. The learning process is robust at younger ages during developmental critical periods and continues, albeit at a lower level, into adulthood and old age (1–3). For example, young barn owls exposed to horizontally shifting prismatic spectacles can adapt readily to altered visual input, but adult owls cannot. The experience in the young owls results in a rearranged audiovisual map in tectum that is accompanied by ectopic axonal projections (1). Experience-dependent structural changes have also been observed in the mammalian cerebral cortex. Enriched sensory experience or motor learning are both associated with an increase in dendritic spine density, and a morphological shift from immature thin spines to mushroom spines which harbor larger postsynaptic densities (PSDs) and stronger synapses (4–7). On the flip side, bilateral sensory deprivation induces spine loss (8, 9). Abnormal sensory experience also results in structural modification of inhibitory synapses and circuitry that is temporally and spatially coordinated with changes in excitatory synapses on dendritic spines (10–13).These experience-driven spine changes are thought to involve synaptic mechanisms of long-term potentiation (LTP) and long-term depression (LTD). In hippocampal slices, induction of LTP causes new spines to emerge, as well as spine head enlargement on existing spines (14–16); induction of LTD results in rapid spine regression (14, 17). Importantly, the emergence or regression of spines starts soon after the induction of LTP or LTD, suggesting that these structural changes underlie the persistent expression of long-term plasticity (14, 17).Little is known about molecular mechanisms that restrict experience-dependent plasticity at circuit and synaptic levels and connect it to spine stability. Paired Ig-like receptor B (PirB), a receptor expressed in cortical pyramidal neurons, is known to limit ocular dominance (OD) plasticity both during the critical period and in adulthood (18). PirB binds major histocompatibility class I (MHCI) ligands, whose expression is regulated by visual experience and neural activity (19–21) and thus could act as a key link connecting functional to structural plasticity. If so, mice lacking PirB might be expected to have altered synaptic plasticity rules on the one hand and changes in the density and stability of dendritic spines on the other.     

Activity-dependent dendritic spine neck changes are correlated with synaptic strength

Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d’etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons.Dendritic spines are found in neurons throughout the central nervous system (1), and in pyramidal neurons receive the majority of excitatory inputs, whereas dendritic shafts are normally devoid of glutamatergic synapses (2–7). These facts suggest that spines are likely to play an essential role in neural circuits (1), although it is still unclear exactly what this role is (8, 9). Because of their peculiar morphology, hypotheses regarding the specific function of spines have focused on their role in biochemical compartmentalization, whereby a small spine head, where the excitatory synapse is located, is separated from the parent dendrite by a thin neck, isolating the spine cytoplasm from the dendrite (10). Indeed, spines are diffusionally restricted from dendrites (11–13) and compartmentalize calcium after synaptic stimulation (14–16). This local biochemistry and the high calcium accumulations observed following temporal pairing of neuronal input and output (14, 17, 18) are thought to be responsible for input-specific synaptic plasticity (19–21). However, besides this biochemical role, spines have also been hypothesized to play an electrical role, altering excitatory postsynaptic potentials (EPSPs) (22–30). Consistent with this idea, activating spines with two-photon uncaging of glutamate generates potentials whose amplitudes are inversely proportional to the length of the spine neck (31), and these responses are much larger in spines than in adjacent dendritic shafts (32). Also, spine conductances can be activated independently of dendritic ones (33–36). These data suggest that spines could serve as electrical compartments but, at the same time, raise the issue of the functional significance of the thousands of long-necked spines that cover the dendrites of pyramidal neurons, which would therefore have negligible somatic voltage contributions.In this study we first undertook a series of experiments to discern the potential effect that the spine neck length has on the synaptic potentials generated by minimal synaptic stimulation at identified spines. We find that EPSP amplitudes are inversely correlated with spine neck lengths and that, as also seen in glutamate uncaging experiments (31), long-necked spines do not appear to generate any significant somatic depolarizations. In a separate set of experiments, we used a spike timing-dependent long-term potentiation (STD-LTP) induction protocol to trigger rapid shortening of the stimulated spine neck, which was accompanied by increases in the amplitude of the evoked potentials. In essence, we thus found a way to rapidly increase the voltage contribution of long-necked spines. To dissect the plausible mechanisms of the effect, we conducted biophysical simulations in the software NEURON. Our models show that the observed phenomenon could be accounted for by rapid regulation of synaptic conductance or, alternatively, stem from electrical attenuation effects due to the changes in spine neck resistance associated with changes in neck length. The spine neck resistance values necessary to entirely account for such attenuation are at odds with reported estimates (13, 32), so one would be inclined to assume that a rapid increase in synaptic conductance leads to the observed changes in somatic EPSP size. However, because spine neck resistance values have so far been inferred only indirectly, one cannot rule out the possibility that a combination of (synaptic) conductance and (neck) resistance changes could contribute to the observed activity-dependent changes in somatic EPSP size.     

Dynamics of dendritic spines in the mouse auditory cortex during memory formation and memory recall

Long-lasting changes in synaptic connections induced by relevant experiences are believed to represent the physical correlate of memories. Here, we combined chronic in vivo two-photon imaging of dendritic spines with auditory-cued classical conditioning to test if the formation of a fear memory is associated with structural changes of synapses in the mouse auditory cortex. We find that paired conditioning and unpaired conditioning induce a transient increase in spine formation or spine elimination, respectively. A fraction of spines formed during paired conditioning persists and leaves a long-lasting trace in the network. Memory recall triggered by the reexposure of mice to the sound cue did not lead to changes in spine dynamics. Our findings provide a synaptic mechanism for plasticity in sound responses of auditory cortex neurons induced by auditory-cued fear conditioning; they also show that retrieval of an auditory fear memory does not lead to a recapitulation of structural plasticity in the auditory cortex as observed during initial memory consolidation.Mammalian brains are characterized by a tremendous level of plasticity. This plasticity is believed to underlie the ability to extract and store information about past experiences and is crucial for animals and humans to interact adaptively in a changing environment. Therefore, detection and localization of a physical representation of a memory has been an intriguing aspect for a long time (1). Plastic changes in synapses are believed to be substrates of memory (2). The development of imaging techniques that allow chronic monitoring of dendritic spines, the morphological correlates of excitatory synapses on pyramidal neurons, in the living animal has provided valuable insights in the dynamics of neuronal circuits (3–5). It has recently been shown that not only chronic perturbations of sensory inputs (6, 7), but also temporally restricted learning experiences, impact the turnover of synaptic structures in the motor cortex and frontal association cortex of the mouse (8–10) and the high vocal center in zebra finches (11).Auditory-cued fear conditioning (ACFC) is an associative learning paradigm that has been widely used to analyze mechanisms of learning in the auditory modality (12). During a conditioning session, subjects quickly learn to associate a previously neutral sound cue [the conditional stimulus (CS)] with an aversive stimulus like a mild foot shock [unconditional stimulus (US)]. It is well established that memory traces after initial formation undergo several processes at different time scales that lead to their consolidation and render them to a stable state that is, e.g., resistant to trauma introduced by an electroconvulsive shock (13). Interestingly, similar molecular cascades are triggered not only during memory formation, but also when a memory trace is retrieved (14, 15). Furthermore, memory traces that were recently retrieved become sensitive again to manipulations like electroconvulsive shock (16), blockade of NMDA receptors (17), or blockade of protein synthesis (18). These similarities have been suggested to reflect remodeling of memory traces following recall (14, 19). However, data on the dynamics of synaptic structures during memory recall is lacking up to date.A number of brain structures have been identified mediating the formation of a memory induced by ACFC (12). Whereas inputs via the auditory cortex (ACx) to the amygdala, an essential brain structure for this learning paradigm, appear to be always sufficient to support fear conditioning (20), their necessity can depend on the spectrotemporal properties of the auditory CS (21, 22). The ACx as the primary sensory cortical area for the auditory modality has been extensively analyzed in the past during classical conditioning to sound stimuli (23–25) or pairing of sounds with artificial stimulation of the cholinergic system, which can substitute for aspects of the US (26–28). These paradigms lead to changes in the receptive fields of ACx neurons that are specific to the conditioned sound. There is evidence based on local pharmacological or optogenetic manipulations that plasticity of the ACx itself is necessary for experience-induced alterations in sound responses and does not simply reflect plasticity elsewhere in the auditory pathway (29, 30). Indeed, there is evidence based on electrophysiological recordings that synaptic plasticity at intracortical synapses can be induced by pairing of a sound with stimulation of the cholinergic system in vivo (28). Structural plasticity following ACFC has been observed in the frontal association cortex (10). However, it remains elusive if plasticity in sound responses in the ACx induced by ACFC (23–25) also has a structural correlate at the synaptic level.In this study, we asked two major questions: Does ACFC in behaving mice induce structural plasticity in synaptic circuits of the ACx? To what extent do memory formation and memory recall share similarities at the level of synaptic structures? We addressed these questions by combining sound-cued fear conditioning and memory testing with chronic in vivo imaging of dendritic spines in the ACx.     

From the Cover: Longer or shorter spines: Reciprocal trait evolution in stickleback via triallelic regulatory changes in Stanniocalcin2a

Vertebrates have repeatedly modified skeletal structures to adapt to their environments. The threespine stickleback is an excellent system for studying skeletal modifications, as different wild populations have either increased or decreased the lengths of their prominent dorsal and pelvic spines in different freshwater environments. Here we identify a regulatory locus that has a major morphological effect on the length of stickleback dorsal and pelvic spines, which we term Maser (major spine enhancer). Maser maps in a closely linked supergene complex that controls multiple armor, feeding, and behavioral traits on chromosome IV. Natural alleles in Maser are differentiated between marine and freshwater sticklebacks; however, alleles found among freshwater populations are also differentiated, with distinct alleles found in short- and long-spined freshwater populations. The distinct freshwater alleles either increase or decrease expression of the bone growth inhibitor gene Stanniocalcin2a in developing spines, providing a simple genetic mechanism for either increasing or decreasing spine lengths in natural populations. Genomic surveys suggest many recurrently differentiated loci in sticklebacks are similarly specialized into three or more distinct alleles, providing multiple ancient standing variants in particular genes that may contribute to a range of phenotypes in different environments.

Similar ecological conditions often result in parallel evolution of the same phenotypic traits in independent populations (1–3). However, ecological conditions typically vary in detail between locations, leading to the evolution of interesting phenotypic differences among evolving populations (4, 5). The contrast between convergence and divergence during adaptive radiations has contributed to decades of work seeking to better understand the principles underlying evolution (6, 7).The threespine stickleback (Gasterosteus aculeatus) provides an opportunity to study the mechanisms that contribute to both parallel and divergent evolution. Migratory marine stickleback have been colonizing and adapting to new freshwater environments for millions of years, with the most recent wide-scale radiation occurring in the countless new freshwater environments generated by glacial recession since the last Ice Age, ∼12 Kya (8). Newly derived freshwater populations typically evolve similar phenotypic changes, including reduced bony armor plates and less robust spines. However, characteristic differences also evolve repeatedly among populations in diverse freshwater environments. Decades of work have analyzed the diverging ecological pressures between lakes and streams (9), large and small lakes (10), benthic and limnetic trophic niches within a lake (11), habitats with different water chemistry and light environments (12), and presence or absence of different types of predators (13). Consequently, freshwater stickleback exhibit exceptional phenotypic diversity, including changes in body size, body shape, color, feeding structures, armor plates, and bony dorsal and pelvic spines (8). Despite recent progress on the genetics of some stickleback traits, the molecular mechanisms underlying many phenotypic specializations remain poorly understood.A key unanswered question is whether diverse evolutionary outcomes occur by modifying different genes in different environments or by modifying the same genes in different ways. Ancient alleles have been identified at particular loci that allow rapid evolution of common marine-freshwater differences by repeated selection of standing variants that already preexist at low frequencies in marine ancestors (14–18). Repeated fixation of preexisting variants favors the reuse of not only the same gene, but also the same freshwater haplotypes in derived populations that share traits. However, it is still not clear whether the distinct phenotypes seen among many freshwater populations are controlled by additional alleles of the same loci that control common marine–freshwater differences, by changes in additional loci, or both.Phenotypic variability among different stickleback populations is particularly pronounced in the dorsal and pelvic spines for which the species is named. Ossified spines are a key evolutionary innovation that spurred a massive radiation of acanthomorph fish, the remarkably diverse fish group that contains about one-third of all living vertebrate species (19, 20). Threespine stickleback typically have three eponymous dorsal spines, but their length can differ greatly among populations and some populations have more than three while others have fewer than three (8, 21). Paired pelvic fins or hindlimbs are found in both fish and tetrapods. In stickleback, the pelvic fin consists of one fin ray and a large, serrated, locking pelvic spine that articulates with an underlying pelvis and can be raised and lowered as a defense against predators (22). The length of the pelvic spine varies dramatically among stickleback populations, and is sometimes lost entirely (8, 21). Although the Pitx1 locus has been identified as a major locus controlling major reduction and even complete loss of the pelvic apparatus in stickleback (23, 24), the genes controlling quantitative variation in pelvic spine length in pelvic-complete individuals are still largely unknown. Based on the significance and diversity of dorsal and pelvic spine phenotypes in Gasterosteus and other fish, we decided to further investigate the genetic mechanisms underlying spine evolution and development.     

Rehabilitation drives enhancement of neuronal structure in functionally relevant neuronal subsets

We determined whether rehabilitation after cortical injury also drives dynamic dendritic and spine changes in functionally distinct subsets of neurons, resulting in functional recovery. Moreover, given known requirements for cholinergic systems in mediating complex forms of cortical plasticity, including skilled motor learning, we hypothesized that cholinergic systems are essential mediators of neuronal structural and functional plasticity associated with motor rehabilitation. Adult rats learned a skilled forelimb grasping task and then, underwent destructive lesions of the caudal forelimb region of the motor cortex, resulting in nearly complete loss of grasping ability. Subsequent intensive rehabilitation significantly enhanced both dendritic architecture and spine number in the adjoining rostral forelimb area compared with that in the lesioned animals that were not rehabilitated. Cholinergic ablation markedly attenuated rehabilitation-induced recovery in both neuronal structure and motor function. Thus, rehabilitation focused on an affected limb robustly drives structural compensation in perilesion cortex, enabling functional recovery.Studies over the past decade have indicated that the adult brain is structurally dynamic (1–3). Indeed, dendritic spines dynamically turn over in the adult brain (3, 4), and learning of novel tasks is associated with further increases in spine turnover (4). Moreover, total and stable increases in spine number together with enhanced dendritic complexity can be detected when analyses are focused specifically on neuronal subpopulations that are functionally related to a newly learned motor skill (5). For example, we recently reported that cortical layer V pyramidal neurons, which project to spinal segment C8 and are specifically engaged when learning a skilled forelimb grasping task, elaborate a 22% increase in apical dendritic spines and exhibit significant increases in dendritic branching and total dendritic length (5); an adjoining control population of cortical layer V pyramidal neurons that project to C4, which are not specifically shaped by the skilled motor task, exhibits no change in spines or dendritic complexity when the same task is learned (5). The detection of stable structural increases in neurons engaged by skilled motor learning in contrast to a lack of change in adjacent neurons that are not engaged by learning advances our understanding of mechanisms underlying experience-dependent cortical plasticity.Damage to the adult CNS also generates adaptive brain plasticity. For example, focal cortical lesions evoke cortical map plasticity (6, 7), extension of new axonal connections (7, 8), and neurogenesis (9). A very important and unresolved question in the neural plasticity and injury fields is whether rehabilitation—that is, specific retraining of injured neural circuits—can drive, alter, or enhance neural plasticity subsequent to brain lesions. Whereas extensive literature has shown that rehabilitation can increase the numbers of dendritic spines and dendritic complexity in the cortical hemisphere opposite a brain lesion (10–13) and is associated with improved skill in the limb unaffected by the lesion, effects of rehabilitation on neuronal structure in perilesioned cortex have not been described. Indeed, some studies suggest either stability or early loss of dendritic structure in perilesion cortex (14–16). However, knowing whether rehabilitation can drive adaptive brain plasticity could be essential in improving outcomes of numerous CNS disorders acquired in adulthood, including stroke, traumatic brain injury, and spinal cord injury.Prior studies that have sought to interrogate neuronal structure after injury have been limited by their use of nonspecific cellular sampling methods, such as Golgi–Cox staining or EM; these approaches lack the ability to specifically sample structural changes in neurons associated with specific tasks that are practiced in rehabilitation. Sampling from subpopulations of neurons mediating specific behaviors, such as skilled grasping in the motor cortex, may yield far more sensitive measures of changes in dendritic structure and spine number as a function of rehabilitation, fundamentally advancing our understanding of the role of experience and rehabilitation on structural neuronal plasticity.Another consideration in understanding cortical mechanisms underlying plasticity after CNS injury is the contribution of subcortical systems that modulate cortical activity, including cholinergic inputs. Studies have identified an essential role for cholinergic activation in modulating cortical plasticity associated with learning (17–19) and motor map plasticity that is evoked after lesions of the caudal forelimb region of the motor cortex (6, 20). These observations raise the possibility that cholinergic inputs to the motor cortex are also essential for generating neuronal structural adaptations in response to rehabilitation training after injury.In this study, we hypothesized that rehabilitation after injury to the adult brain drives adaptive plasticity, rebuilding spines and enhancing dendritic architecture in neurons surrounding the lesion site. We further hypothesize that these changes are cholinergic-dependent. We examined specific subpopulations of layer V cortical neurons directly related to the learning, loss, and subsequent relearning of skilled forelimb grasping, allowing detailed and specific sampling of structural parameters among subpopulations of neurons specifically engaged in the skilled grasping task.     

Proximity proteomics of synaptopodin provides insight into the molecular composition of the spine apparatus of dendritic spines

The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus–specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.

The neuronal endoplasmic reticulum (ER) is an intricate continuous network of membrane tubules and cisterns that runs throughout neuronal processes with region-specific specializations. One such specialization of the smooth ER is the spine apparatus (SA) that is located in a subset of dendritic spines. The SA consists of stacks of flat cisterns that are connected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft (1–3) (Fig. 1A and SI Appendix, Fig. S1A). Morphological changes in the SA have been reported after long-term potentiation (4) and also, in a variety of human disorders, including several neurodegenerative conditions (5–9). While the first observation of the SA by electron microscopy (EM) was reported in 1959 by Gray (1), our understanding of this organelle remains fairly limited. Its molecular characterization has proven to be challenging due to the difficulty of its biochemical isolation and its absence in organisms suitable for genetic screens.Open in a separate windowFig. 1.The localization of synaptopodin (indicated as Synpo in all figures) in cultured hippocampal neurons overlaps with the localization of the SA in dendritic spines of cortical slices. (A) SA as visualized by transmission EM. (B and C) SA and ER reconstructed by a semiautomated algorithm from 3D volumes acquired by FIB-SEM. An SA is shown in B, while C shows a portion of a dendritic shaft with spines containing (magenta arrows) and not containing (white arrows) an SA. The plasma membrane (PM) is shown in blue, the ER is in red, and the post-synaptic density (PSD) is in yellow. (D) Cisternal organelle (CO), as observed in an FIB-SEM optical section, at an axonal initial segment. Note that the stacks of ER cisterns are similar to those characteristics of the SA. (E) mRFP-synaptopodin coexpressed with cytosolic EGFP as a marker of the entire dendritic volume. Note in the zoomed-in views of the region enclosed by a rectangle (the lower three fields) that synaptopodin is concentrated near the spine neck, where the SA is localized. (F) Localization by immunofluorescence of endogenous synaptopodin showing strong overlap with a pool of F-actin labeled by phalloidin-Alexa488. Also, in this sample, the magnified views (the lower three fields) show enrichment of synaptopodin, relative to actin, at the spine neck. (G) mRFP-synaptopodin coexpressed with an ER marker, EGFP-VAPB, showing colocalization of the two proteins. In the zoomed-in views (the lower three fields), red arrows show a spine positive for both the ER marker and synaptopodin, and the blue arrows show a spine with the ER marker but lacking synaptopodin. (H) Percentage of ER-positive spines that also contain synaptopodin and percentage of synaptopodin-positive spines that contain ER quantified in cultured hippocampal neurons expressing mRFP-synaptopodin and the ER markers EGFP-VAPB or EGFP-Sec61β. Each data point represents at least 99 spines from a single neuron. (I) Spine of a synaptopodin KO mouse that lacks the SA but contains the ER. (J) Quantification of the number of spines where ER was visible in the plane of the section with or without an SA in the brain of wild type (WT) vs. synaptopodin mutant mice (n≥800 spines per genotype).The only known protein enriched at the SA and required for its formation is synaptopodin, a protein without transmembrane regions localized in the cytosolic space (10). Neuronal synaptopodin specifically localizes to dendritic spines and to the axonal initial segment, where another specialization of the ER similar to the SA (stack of flattened cisterns) called the cisternal organelle is present (11–14). Lack of synaptopodin in synaptopodin knock-out (KO) mice correlates with the lack of SA and of the cisternal organelle, as well as with a reduction in Hebbian plasticity and spatial memory (11, 15–18). A longer isoform of synaptopodin is expressed in the foot processes of podocytes, where it functions as a regulator of the actin cytoskeleton (19, 20). Synaptopodin binds to and bundles actin (21) and interacts with several actin binding proteins, such as α-actinin (13, 21, 22). While more is known about the interactors of synaptopodin in podocytes, its binding partners at the SA remain unknown.The goal of this work was to gain insight into the molecular composition of the SA. To this aim, we used synaptopodin as a starting point for our analysis. We identified some of its binding partners by an in vivo proximity biotinylation approach and characterized the specific localization of a subset of these proteins in neurons and their interaction with synaptopodin in an exogenous system.     

Dendrite tapering actuates a self-organizing signaling circuit for stochastic filopodia initiation in neurons

How signaling units spontaneously arise from a noisy cellular background is not well understood. Here, we show that stochastic membrane deformations can nucleate exploratory dendritic filopodia, dynamic actin-rich structures used by neurons to sample its surroundings for compatible transcellular contacts. A theoretical analysis demonstrates that corecruitment of positive and negative curvature-sensitive proteins to deformed membranes minimizes the free energy of the system, allowing the formation of long-lived curved membrane sections from stochastic membrane fluctuations. Quantitative experiments show that once recruited, curvature-sensitive proteins form a signaling circuit composed of interlinked positive and negative actin-regulatory feedback loops. As the positive but not the negative feedback loop can sense the dendrite diameter, this self-organizing circuit determines filopodia initiation frequency along tapering dendrites. Together, our findings identify a receptor-independent signaling circuit that employs random membrane deformations to simultaneously elicit and limit formation of exploratory filopodia to distal dendritic sites of developing neurons.

A crucial first step toward a functional neuronal network is to establish contact with the correct synaptic partners. To accomplish this daunting task, individual neurons undergo a phase of intense transcellular sampling (1, 2). To augment the sampling volume, and thus the total number of possible encounters, neurons rely on actin-rich exploratory filopodia that form along dendritic arbors (1, 3–5). From this overabundance of transcellular contacts, only connections with compatible surface identities mature into synapses (6), while mismatched interfaces are rapidly aborted (1, 2).Seminal work over the past decades has elucidated the machinery driving filopodial dynamics in neuronal and nonneuronal cells. These studies established an intricate signaling network that regulates actin polymerization during filopodial growth and retraction. In addition, several extra- and intracellular signaling cues have been identified that trigger filopodia formation (7–9). Yet, given its exploratory purpose and that sampling behavior is not only observed in brain slices (2) but also in sparsely cultured neurons that lack neighbors (1), suggests that filopodial initiation also occurs in the absence of causative signaling cues. Indeed, stochastic membrane deformations induced by actin polymerization were described to initiate finger-like protrusions reminiscent of filopodia in vitro (10–12). Similarly, filopodia emerge at lamellipodia from cone-shaped precursors in cells (13), establishing membrane curvature as central element at the onset of filopodial formation. If and how cells utilize membrane geometry to control filopodia initiation in neurons, however, is not fully understood.Combining quantitative light and electron microscopy of primary hippocampal neurons with chemical approaches and computational modeling, we find that binding of curvature-sensitive proteins alters the lifetime of stochastically deformed membrane sections. Intriguingly, these proteins not only enrich at nascent filopodia but also alter local polymerization dynamics. This dual ability to sense and induce membrane deformations is relevant, as it allows the formation of a self-organizing signaling circuit. As this circuit is curvature sensitive, dendrite tapering augments exploratory filopodia initiation at thin, distal ends. Our results present a receptor-independent mechanism used by neurons to nucleate signaling hubs and facilitate transcellular sampling in a curvature-dependent fashion.     

A stable proportion of Purkinje cell inputs from parallel fibers are silent during cerebellar maturation

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.

Typical central excitatory synapses are formed onto dendritic spines, the distinctive morphology of which enables their unambiguous identification (1–3). It has generally been assumed that the presence of a dendritic spine equates to the existence of a functional excitatory transmission (4). Based on this assumption, the observation of spine motility in several brain areas (motor cortex and somatosensory cortex) has been considered to reflect synaptic plasticity (5). Indeed, a number of studies have established a correlation between learning and spine formation (6–8) or pruning (9, 10). However, in some conditions, morphological and synaptic plasticity have been shown to be dissociated (11), in line with the view that morphology does not provide all the information necessary to infer synaptic function.The cerebellum contains the majority of brain neurons (12, 13) and the predominant excitatory synapses found in this structure connect granule cells (GC) to Purkinje cells (PC). These synapses are formed on typical spines borne by PC dendrites (14), the majestic shape of which is likely related to the huge amount of independent inputs they receive. The GC-to-PC synapse is generally acknowledged to be an essential site for plasticity (15–19). However, in sharp contrast to other parts of the brain such as motor and somatosensory cortices, PC spines appear to be constitutive (20, 21), i.e., they appear to be an inherent property of PCs, independent of external factors. Indeed, pruning of these synapses has not been reported. Novel spine formation has been reported, but likely as a result of dendritic tree expansion (22, 23). The high density of spines along PC dendritic branchlets (5 to 17 per linear micrometer in rat; refs. 14 and 24–27) and their regular ordering in a helical pattern (28) support the idea that they optimize space occupancy with little room for spine addition, in accordance with their constitutive nature.The apparent morphological homogeneity of PC spines is in sharp contrast with the spectacular heterogeneity observed in the strength of GC-to-PC synapses. An in vivo study has reported that the receptive field of a PC was much smaller than that of the GCs putatively connecting to it (29), suggesting that most GC-to-PC synapses are electrically silent. This has been confirmed by an in vitro report (30) showing that synaptic transmission between paired-recorded GC and PC was detected nearly 10 times less frequently than expected from the occurrence of morphologically defined synaptic connections predicted by anatomical data (14, 31–33). Taken together, these two studies conducted in adult rats suggest that most (85% according to ref. 30) morphologically and molecularly defined GC-to-PC synapses are silent, i.e., they do not transmit any detectable electrical signal.If silent synapses do not transmit information, what is their role? Are they a reserve for additional information storage? Or do they result from information storage optimization (34)? According to this latter proposal (17, 34), since the requirement for optimized information storage is more and more critical as the amount of learned information increases, one might expect that the proportion of silent synapses increases with the amount learning. As previously suggested (34), this hypothesis could be tested by comparing the proportion of silent synapses in young versus adult animals. Indeed, the mouse cerebellar circuitry is not fully in place until the third postnatal week. Then, for at least 3 wk, the mouse acquires basic skills (eating, walking, and social interactions), adapts to changes in muscle strengths and sensitivity to stimuli, and improves its agility (35). Although the amount of cerebellar learning occurring over this maturation period is unclear, it can be reasonably assumed that cerebellar operation continuously optimizes. Here, we investigate how the proportion of silent synapses changes over this period of maturation.We determine the proportion of silent GC-to-PC synapses by a method based on the determination of the average postsynaptic response per activated synapse (average synaptic weight, w¯) in superfused acute slices. Thanks to the geometrical and repetitive architecture of the cerebellar cortex, calcium imaging is used to quantitatively map GC inputs. This mapping, combined with postsynaptic recording of transmission, and the determination of two cerebellar anatomical features (the average synapse density and spacing between PC planar dendritic trees) enables the determination of w¯. By comparison with the properties of synapses that produce an electrical postsynaptic response (investigated by paired recording and quantal analysis), we show that the proportion of silent synapses is higher than 70% and stable between the postnatal stages of interest. This suggests that cerebellar maturation has insignificant impact on the proportion of silent synapses.     

Neuronal profilins in health and disease: Relevance for spine plasticity and Fragile X syndrome

Learning and memory, to a large extent, depend on functional changes at synapses. Actin dynamics orchestrate the formation of synapses, as well as their stabilization, and the ability to undergo plastic changes. Hence, profilins are of key interest as they bind to G-actin and enhance actin polymerization. However, profilins also compete with actin nucleators, thereby restricting filament formation. Here, we provide evidence that the two brain isoforms, profilin1 (PFN1) and PFN2a, regulate spine actin dynamics in an opposing fashion, and that whereas both profilins are needed during synaptogenesis, only PFN2a is crucial for adult spine plasticity. This finding suggests that PFN1 is the juvenile isoform important during development, whereas PFN2a is mandatory for spine stability and plasticity in mature neurons. In line with this finding, only PFN1 levels are altered in the mouse model of the developmental neurological disorder Fragile X syndrome. This finding is of high relevance because Fragile X syndrome is the most common monogenetic cause for autism spectrum disorder. Indeed, the expression of recombinant profilins rescued the impairment in spinogenesis, a hallmark in Fragile X syndrome, thereby linking the regulation of actin dynamics to synapse development and possible dysfunction.The immense computational power of the central nervous system depends on the formation of functional neuronal networks, which are further refined and adapted to environmental changes by processes of neuronal plasticity throughout the entire life span of an individual. The majority of synapses in highly plastic regions, such as the neocortex and hippocampus, are located at dendritic spines, tiny protoplasmatic membrane protrusions that build the postsynaptic compartment. Changes in spine shape are directly associated with the dynamic actin cytoskeleton, which is highly enriched in dendritic spines (1–6). In fact, up to 80% of actin filaments turn over in less than 2 min in the spine head (7). Hence, an understanding of the detailed molecular machinery and identification of key molecules that control actin polymerization in space and time will help to reveal details of spine function and plasticity, and might eventually also provide a better understanding of neurological disorders characterized by defects in spinogenesis and spine maintenance (8, 9).The small actin-binding protein profilin—present in the mammalian CNS in two different isoforms, profilin1 (PFN1) and profilin2a (PFN2a) (10)—has been described as such a promising candidate because its activity-dependent translocation into dendritic spines could be shown both in vitro and in vivo (11–13). However, recent studies exploiting knockout animals for either PFN1 or PFN2a demonstrated a surprising lack of a spine phenotype for both isoforms (14, 15). One explanation might reside in the crucial importance of tightly restricted actin dynamics for virtually all aspects of neuronal function that might be preserved in knockout animals by means of compensational effects acting on the expression or regulation of other actin-binding molecules. This theory is supported by work from our group showing that an acute knockdown of PFN2a actually revealed an important function in dendritic spines (16).In this study, we took advantage of an acute interference RNA (RNAi)-mediated loss-of-function approach, which allowed us to provide evidence that despite the fact that profilins are biochemically very similar, the two brain isoforms perform astonishingly diverse functions. Our results indicate that the ubiquitous isoform PFN1 is of great importance for spine formation. Furthermore, we can show that the expression of PFN1 is developmentally down-regulated in the hippocampus. In contrast to this, we found the evolutionary most-recent and brain-specific isoform PFN2a to be involved in synapse function, spine stabilization, and activity-dependent structural plasticity. Most notably, both isoforms were differentially engaged in regulating actin dynamics in dendritic spines. In line with a role of PFN1 for spine formation during development, we provide evidence that, of the brain profilin isoforms, only the mRNA of PFN1, comparable to the Drosophila homolog chickadee (17), is bound by the Fragile X mental retardation protein (FMRP). Similarly, PFN1 but not PFN2a levels were altered in the mouse model of the neurodevelopmental disorder Fragile X syndrome (FXS), a hallmark of which is an apparent defect in spine formation and maturation (18–20).Our results therefore point toward intriguingly different functions of profilin isoforms in the brain with a juvenile expression profile, indicating a major role of PFN1 during spinogenesis and a mature expression profile favoring PFN2a as the predominant isoform crucial for spine stabilization, synaptic function, and spine plasticity.     

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17β-estradiol or an estrogen receptor (ER)-α (but not ER-β) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently “pruned” by experience.Estradiol rapidly and dramatically increases hippocampal dendritic spine and synapse density within minutes of application (1–4). There is a strong correlative association between estrogen-induced spinogenesis and improvements in cognition (5); however, the relationship of these structural changes to estrogen-induced alterations in hippocampal function is unclear. Our laboratory recently reported that the density of hippocampal CA1 pyramidal dendritic spines increases very rapidly after systemic treatment with 17β-estradiol or estrogen receptor (ER) -selective agonists in ovariectomized female mice, changes that are paralleled by learning enhancements (2, 3). Estrogen-induced rapid structural changes are substantial, increasing spine density by 30–50% within 15–40 min of hormone application (1–3, 6). As a result, adult rodents can experience the addition of thousands of CA1 synapses within a span of minutes after exposure to estradiol. These effects of estrogens reproduce the changes occurring during the 4-d estrous cycle of female rodents, which include the induction of CA1 spines (7).How these processes contribute to the behavioral changes observed after estradiol treatment is not understood. Estradiol enhances excitatory neurotransmission throughout the hippocampus (8–10), and activates BDNF signaling in the mossy fiber system (11). Dendritic spines turn over more rapidly in the hippocampus than in the neocortex (12), particularly in the case of estradiol-induced spines (13). Such rapid, transient, and apparently indiscriminate increases in excitatory synapse formation would seem, at first sight, to be more likely to interfere with preexisting brain circuits and impair normal information processing than to enhance cognitive function.How then, does enhancement of spine formation lead to improved cognitive function? To address this question, we focused specifically on the effects of estradiol in the dorsal CA1 hippocampus, as a site mediating the rapid improving effects of estrogens on learning. Estradiol application, via a mechanism involving ERα, rapidly increases dendritic spine density in the CA1 pyramidal stratum oriens and stratum radiatum subregions. However, contrary to our initial assumptions based on previous work in this field, increased spinogenesis did not result in increased CA1 excitatory input. Rather, estrogen-induced formation of hippocampal spines was associated with a decrease in the ability of CA1 neurons to respond to AMPA receptor (AMPAR) activation, resulting in decreased excitatory input to CA1 neurons. This appears to be the result of AMPAR internalization from the synaptic membrane (6). Taken together, our data suggest that estrogens induce formation of “silent” or immature synapses that act as a substrate for the storage of new memories (14, 15). This finding explains estrogens’ ability to rapidly improve learning without precipitating uncontrolled activation of the hippocampal circuitry. These effects of estradiol on CA1 pyramidal neurons phenotypically resemble the structural and functional properties of neurons during development, when neurons with higher levels of spine density and higher numbers of silent/immature synapses are present before the activity dependent refinement of neural circuitry.     

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2’s activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

Although the molecular pathogenesis of schizophrenia (SCZ) is poorly understood, altered structure and function of neural networks in the brain have been implicated (1–5). In developing brains, neural circuitry is formed through neurogenesis, differentiation, axon guidance, dendritogenesis, synaptogenesis, and activity-dependent and microglial refinement of immature synapses (6). Because synapses are the sites of neurotransmitter signal transduction, dendritic spine dysfunction may play an important etiological role in SCZ. Indeed, genetic linkage studies have suggested that aberrations in genes responsible for synapse formation and maturation may be SCZ risk factors (4, 6–8). The lack of reliable, pathogenetically related, clinically accessible biomarkers hinders not only understanding underlying etiological mechanisms, but also the diagnosis of SCZ. Enhancing the speed, sensitivity, and specificity of diagnosing early-stage SCZ would facilitate that systematic examination of genes across larger more homogenous patient populations, improve clinical management, and accelerate the development of new therapeutic options.Collapsin response mediator protein 2 (CRMP2), also known as Dihydropyrimidinase-like 2 (DPYSL2), is a master regulator of axon guidance, dendritic branching, and spine formation: hence, a neural network modulator. It was first identified as an intracellular molecule that mediates the signaling of Semaphorin3A, a repulsive axon guidance molecule (9), but has since been recognized as playing a much larger role in neural development and maintenance of homeostasis in the adult nervous system. The CRMP family of proteins are now known to consist of five homologous cytosolic proteins, CRMP1 through CRMP5 (10, 11). CRMP2 actively binds cytoskeletal elements in its nonphosphorylated active state; phosphorylation of CRMP2—which is a two-step process—inactivates it and induces it to release cytoskeletal elements. Cdk5 first phosphorylates CRMP2 at Ser522, priming it for glycogen synthase kinase 3β (GSK3β) to phosphorylate it at Thr514 and Ser518 (12, 13). We now know that “toggling” between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is an ongoing physiologic adaptive mechanism for preventing abnormal neuronal sprouting. Overall, a balance exists between active and inactive CRMP2. We previously described how this balance is pivotal for proper dendritic branching and spine organization in vivo (14). Dendritic spines are the point of contact for interneuronal synaptic communication. Nonphosphorylated CRMP2 is expressed throughout the neuron, including the dendritic spines; phosphorylated CRMP2 is not expressed in the spines, suggesting that when CRMP2 becomes inactivated, it leaves or is excluded from the spines. There appears to be a continuous dynamic in which CRMP2 enters and fills the spines when it is activated/dephosphorylated and is absent from or leaves the spines when it becomes inactivated/phosphorylated. Agents known to decrease inactivated CRMP2 (e.g., lithium) also increase dendritic spine volume and density, an action abrogated by the elimination of CRMP2. Constitutive Crmp2 knockout mice are characterized by defects in dendritic morphology, including diminished spine density and dendritic length, in regions where CRMP2 is expressed (e.g., the cerebral cortex, hippocampus, cerebellum, striatum).The processes in which we’ve learned CRMP proteins play critical roles are also now recognized as pivotal to the network abnormalities central to neuropsychiatric disorders (10, 11, 13–19). Phenotypic analysis of crmp1 and crmp2 gene-deficient mice (crmp^−/− and crmp2^−/−, respectively) revealed that both of these gene-deficient animals show behavioral abnormalities that model neuropsychiatric symptoms (15–17). We recently implicated aberrant CRMP2 posttranslational regulation as central to the pathogenesis of lithium-responsive (LiR) bipolar disorder (BPD) (14, 20). Specifically, the “set-point” for the ratio of phosphorylated (inactive)-to-nonphosphorylated (active) CRMP2 was abnormally high in LiR BPD human brains and patient-specific human induced pluripotent stem cell (hiPSC)-derived neurons; lithium, a CRMP2 pathway modulator, normalized that set-point by reducing the levels of phosphorylated CRMP2 (pCRMP2) and, concomitantly, dendritic structural pathology and neuronal hyperexcitability. Hence, we viewed BPD as a disorder not of a gene per se, but rather of the posttranslational regulation of a developmentally critical molecule. The specificity of the elevated pCRMP2:CRMP2 ratio for LiR BPD—an elevation not seen in unaffected controls, in other psychiatric and neurological disorders, or even in lithium-nonresponsive (LiNR) BPD patients—suggested that it might serve as a biomarker for that disorder.A series of recent genome-wide association studies have, intriguingly, shown BPD to cluster with cognitive disorders, such as SCZ, more so than with mood disorders (21–23). Given CRMP2’s role as a master regulator of cytoskeletal and hence neural network modulation, we investigated whether its activity state might serve as a potential biomarker for SCZ as well, a condition also suspected of being characterized by abnormal synaptogenesis, neuritogenesis, and neural network activity. We, indeed, found CRMP2 anomalies in the postmortem brains of SCZ patients: the levels of active CRMP2 were abnormally high and correlated with relevant dendritic morphometric aberrations. Because CRMP2 is expressed also in lymphocytes, imbalances in the phosphorylation state of this intracerebral neural network modulator were reflected in the peripheral blood of SCZ patients, as confirmed by a blinded, prospective clinical study, making this finding pertinent to the search for a mechanistically based “bedside” diagnostic marker.     

Nitric oxide mediates local activity-dependent excitatory synapse development

Learning related paradigms play an important role in shaping the development and specificity of synaptic networks, notably by regulating mechanisms of spine growth and pruning. The molecular events underlying these synaptic rearrangements remain poorly understood. Here we identify NO signaling as a key mediator of activity-dependent excitatory synapse development. We find that chronic blockade of NO production in vitro and in vivo interferes with the development of hippocampal and cortical excitatory spine synapses. The effect results from a selective loss of activity-mediated spine growth mechanisms and is associated with morphological and functional alterations of remaining synapses. These effects of NO are mediated by a cGMP cascade and can be reproduced or prevented by postsynaptic expression of vasodilator-stimulated phosphoprotein phospho-mimetic or phospho-resistant mutants. In vivo analyses show that absence of NO prevents the increase in excitatory synapse density induced by environmental enrichment and interferes with the formation of local clusters of excitatory synapses. We conclude that NO plays an important role in regulating the development of excitatory synapses by promoting local activity-dependent spine-growth mechanisms.Neuronal activity and experience critically control the development and organization of synaptic networks by regulating the mechanisms of synapse formation and elimination. Sensory experience, motor training tasks, fear-conditioning, song-learning in birds, or exposure to novel environments in rodents are associated with major structural rearrangements of connectivity (1–8). An interesting aspect of these structural rearrangements is that they are spatially organized: the new spines formed as a result of activity tend to grow in the vicinity of activated synapses and repetitive learning promotes the formation of synaptic clusters (9–11). Despite the importance of these mechanisms for the development of brain circuits, the molecular events underlying these activity-mediated structural rearrangements of connectivity remain still essentially unknown.Here we tested whether the diffusible messenger nitric oxide (NO) could contribute to these mechanisms. NO is produced at excitatory synapses as a result of synaptic activation through the close association of its synthesizing enzyme, neuronal nitric oxide synthase (nNOS), with the postsynaptic density and NMDA receptors (12–14). NO has thus been implicated in several aspects of synaptic function and plasticity (15–18), notably as a retrograde messenger regulating presynaptic properties, such as synaptic vesicle recycling in terminals and growth and remodeling of presynaptic varicosities (19–22). At inhibitory synapses in the ventral tegmental area, NO has even heterosynaptic effects, mediating a form of GABA-mediated long-term potentiation triggered by excitatory synapse activation (23). Studies of nNOS-deficient mice further suggest an important role of NO in cognitive functions and social behavior (24–26) and recent genetic analyses have reported associations between genetic variants of nNOS and schizophrenia (27, 28), suggesting a possible developmental role of NO for brain circuit formation. Our study provides direct evidence for such a role by demonstrating that NO is required for activity-mediated synapse formation.     

The hypothalamic link between arousal and sleep homeostasis in mice

Sleep and wakefulness are not simple, homogenous all-or-none states but represent a spectrum of substates, distinguished by behavior, levels of arousal, and brain activity at the local and global levels. Until now, the role of the hypothalamic circuitry in sleep–wake control was studied primarily with respect to its contribution to rapid state transitions. In contrast, whether the hypothalamus modulates within-state dynamics (state “quality”) and the functional significance thereof remains unexplored. Here, we show that photoactivation of inhibitory neurons in the lateral preoptic area (LPO) of the hypothalamus of adult male and female laboratory mice does not merely trigger awakening from sleep, but the resulting awake state is also characterized by an activated electroencephalogram (EEG) pattern, suggesting increased levels of arousal. This was associated with a faster build-up of sleep pressure, as reflected in higher EEG slow-wave activity (SWA) during subsequent sleep. In contrast, photoinhibition of inhibitory LPO neurons did not result in changes in vigilance states but was associated with persistently increased EEG SWA during spontaneous sleep. These findings suggest a role of the LPO in regulating arousal levels, which we propose as a key variable shaping the daily architecture of sleep–wake states.

Interspecies variation in the daily amount of sleep is strongly influenced by genetic factors (1). However, individuals also possess a striking ability to adapt the timing and duration of wakefulness and sleep in response to a variety of intrinsic and extrinsic factors (2). Among the key regulators of “adaptive sleep architecture” are 1) homeostatic sleep need, 2) the endogenous circadian clock, and 3) the necessity to satisfy other physiological and behavioral needs such as feeding or the avoidance of danger (3–5). It is unknown how and in what form these numerous signals are integrated within the neural circuitry that generates the rapid and stable transitions between sleep and wake states.Brain state switching has been the main focus of circuit-oriented sleep research for decades. Early studies identified the preoptic hypothalamus as a primary candidate for a hypothesized “key sleep center” (6–8), and subsequent studies have confirmed the existence of sleep-active neurons in the ventrolateral and median preoptic areas (VLPO and MPO) of the hypothalamus (9–11). Combined with the findings that orexin/hypocretin neurons are necessary to maintain wakefulness (12, 13), a model was proposed in which the sleep/wake-promoting circuitries function as a flip-flop switch (14). This model was able to account for rapid and complete transitions between sleep and wakefulness and preventing state instability (15) or the occurrence of mixed hybrid states of vigilance (16). Over the last decade, our knowledge of subcortical brain nuclei that control sleep has expanded steadily, leading to the identification of functional specialization within the sleep–wake controlling network and, in parallel, highlighting a previously underappreciated complexity (17–35).A key question to emerge is how signals regulating sleep–wake architecture are represented and integrated in hypothalamic state-switching circuitries to ultimately maximize ecological fitness (36). Although sleep homeostasis has been considered an important factor influencing sleep/wake transitions (37–40), relatively few studies have addressed whether and how sleep–wake controlling brain areas overlap with those involved in homeostatic sleep regulation (26) or the underlying neurophysiological mechanisms (41–44). One recent study pointed to an important role of galanin neurons in the lateral preoptic hypothalamus, as was demonstrated through their selective ablation, which abolished the rebound of electroencephalogram (EEG) slow-wave activity (SWA; EEG power density between 0.5 to 4 Hz) after sleep deprivation (26). Other studies suggest that while homeostatic sleep pressure, reflected in SWA, builds up as a function of global wake duration, it is also locally regulated by specific activities during wakefulness (45, 46). The property of sleep and wake as brain states with flexible intensities on a global and local level suggests an additional complexity, which is difficult to reconcile with the existence of a single center solely responsible for complete sleep–wake switching (47). For example, there is evidence to suggest that wake “intensity” contributes to the build-up of global homeostatic sleep need (48–51), and the balance between intrinsic and extrinsic arousal-promoting and sleep-promoting signals ultimately determines the probability and degree of state switching (3, 52).Here, we investigate the role of the hypothalamus in the bidirectional interactions between sleep–wake switching, arousal, and sleep homeostasis. Firstly, we applied optogenetic stimulation of glutamate decarboxylase 2 (GAD2) neurons in the lateral preoptic area (LPO) of mice (17) and found that photoactivation of the LPO during sleep led to rapid wake induction, but this effect was also observed when structures surrounding the LPO were stimulated. Unexpectedly, GAD2^LPO neuronal stimulation did not merely trigger wakefulness, but the awake state produced by this stimulation was characterized by increased EEG theta activity—the established measure of arousal (53, 54). In turn, subsequent sleep was associated with increased levels of EEG SWA, indicative of higher homeostatic sleep pressure (45). In contrast, unilateral inhibition of GAD2^LPO neurons decreased the drive for arousal, as was reflected in a persistent increase in nonrapid eye movement (NREM) EEG SWA across the day. In summary, our experiments demonstrate an important role of GAD2^LPO neurons not only in the control of state transitions but also in linking arousal to sleep homeostasis. We find that the kinetics of the response to photoactivation and photoinhibition were different, and so they may arise from distinct mechanisms while converging on the dynamic modulation of arousal levels, ultimately shaping the daily architecture of sleep–wake states.     

Ena/VASP clustering at microspike tips involves lamellipodin but not I-BAR proteins,and absolutely requires unconventional myosin-X

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi is essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared with B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.

Thin, sheet-like membrane protrusions at the leading edge of cells, known as lamellipodia, drive cell migration under physiological and pathological conditions. The protrusion of the cell front is driven by actin polymerization with the expansion of the polymer directly pushing the membrane forward (1). Actin assembly in the leading edge is driven by the actin-related protein 2/3 (Arp2/3) complex-mediated formation of dendritic actin-filament networks downstream of WAVE regulatory complex (WRC) activation and Rac subfamily GTPase signaling (2, 3). The barbed ends of newly formed filament branches are eventually capped by heterodimeric capping protein (CP) (4). However, actin-filament elongation factors such as Ena/VASP or formin family proteins can protect filament ends from capping and also significantly accelerate filament elongation rate (5).Aside from the branched network, the lamellipodium harbors actin-filament bundles, called microspikes, that span the lamellipodium without protruding beyond the cell edge (6, 7). These structures can display various tilt angles relative to the moving network, the extent of which determines both rates of bundle polymerization and lateral motion (6, 7). Microspikes can convert into filopodia by protruding beyond the cell periphery, and thus separating their polymerization rates from the lamellipodium network (6, 8). However, despite being comprised of parallel actin-filament bundles sharing common constituents, microspikes and filopodia differ considerably. Unlike microspikes, filopodia can form independently of lamellipodial actin networks (9–11). Moreover, filopodia can be formed around the entire cell periphery or the dorsal surface (12–14), and kink, bend, or even contribute to the formation of contractile arrays (6). In line with this, ultrastructural analyses recently revealed microspike filaments to be less densely bundled and straight and more bent as compared with filopodial filaments (15).Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of filament-barbed ends (5, 16). Vertebrates express three paralogs: vasodilator-stimulated phosphoprotein (VASP), mammalian Ena (Mena), and Ena-VASP-like (Evl). All family members harbor an N-terminal Ena/VASP-homology 1 (EVH1) domain essential, but not sufficient, for subcellular localization, followed by a proline-rich region capable of recruiting profilin-actin complexes for actin assembly or interaction with Src-homology 3 (SH3) domain-containing proteins, and a C-terminal EVH2 domain encompassing G-actin and F-actin–binding sites and a short coiled-coil motif (Tet) mediating tetramerization (17).Compared with formins, Ena/VASP tetramers are weakly processive and poorly protect barbed ends from CP in solution (5, 16). However, when clustered at high density on beads, their mode of action is markedly changed allowing them to drive processive and long-lasting actin-filament elongation despite high CP concentrations (5, 18). Ena/VASP proteins were shown to localize to various sites of active actin assembly including the periphery of the leading edge (lamellipodium tip) and the distal tips of actin-bundles termed microspikes and filopodia (17). Consistently, Ena/VASP proteins were previously shown to be important for filopodium formation in Dictyostelium and mammalian cells (19, 20). Moreover, contrary to previous assumptions (21), but in agreement with their ability to enhance intracellular motility of Listeria (22) or motility of beads in reconstituted systems (23), Ena/VASP proteins have recently been shown to positively regulate cell migration in B16-F1 melanoma cells and fibroblasts (24). Of note, Ena/VASP-deficient B16-F1 cells displayed a severely perturbed lamellipodium architecture and were virtually devoid of microspikes, albeit still capable of forming filopodia (24).Since Ena/VASP clustering is presumably key for the formation of microspikes, it is instrumental to identify participating factors and determine their specific contributions. According to our current state of knowledge, both insulin receptor substrate of 53 kDa (IRSp53) and lamellipodin (Lpd, also known as Raph1) have been implicated in Ena/VASP clustering (16, 25–27). IRSp53 is a member of the inverse Bin-Amphiphysin-Rvs167 (I-BAR) protein family that senses and/or creates negative membrane curvature by its N-terminal I-BAR domain, which forms crescent-shaped homodimers (28) that can interact with acidic phospholipids such as PI(4, 5)P2 (29). The C-terminal SH3 domain of IRSp53 mediates interactions with proline-rich regions of other proteins such as Ena/VASP members (30) or WAVE2 (31). The remaining four I-BAR protein family members include IRTKS (Baiap2l1), ABBA (MTSS1L), MIM (MTSS1), and Pinkbar (Baiap2l2), albeit the latter appears to be specifically expressed in epithelial cells (32). Notably, IRSp53 was shown to promote recruitment of VASP into leading-edge foci in fibroblasts and mediate clustering on beads in vitro to initiate VASP-mediated actin assembly in the presence of CP (25). This is supported by very recent work using elaborate in vitro reconstitution systems, showing that IRSp53 can recruit and cluster VASP to assemble actin filaments locally on PI(4, 5)P2-containing membranes, leading to the generation of actin-filled membrane protrusions resembling filopodia (27).Lpd on the other hand is a vertebrate member of the Mig-10/RIAM/Lpd (MRL) family of adaptor proteins that localizes to the rim of the leading edge and the tips of microspikes and filopodia (2). The Lpd homodimer is though to be targeted through its Ras-Association and Pleckstrin Homology (RA-PH) domain to the membrane (33), but actin-filament binding through highly basic C-terminal sequences lacking the RA–PH domains was also implicated in Lpd recruitment to the leading edge (34). Lpd was assumed to be obligatory for Ena/VASP protein recruitment by its six EVH1-binding motifs to the leading edge to promote lamellipodia protrusion (33, 34), but its genetic elimination challenged this view (35). The actin filament-binding activity of Lpd, which can occur independently of Ena/VASP binding, was proposed to trigger Ena/VASP clustering and tether the complexes to growing barbed ends, thereby increasing their processive polymerase activity (34). More recent work proposed the initial formation of small leading-edge-Lpd clusters devoid of IRSp53 that expand, fuse, and subsequently recruit VASP to induce filopodia/microspike formation in B16-F1 cells (26).To resolve the controversy on which proteins are required for VASP clustering at the leading edge and for microspike formation, we employed CRISPR/Cas9-technology combined with proteomics to systematically analyze VASP clustering and microspike formation in genetic mutants derived from B16-F1 cells, which have emerged as excellent model system for the dissection of lamellipodium protrusion and microspike formation.     

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.Filopodia are actin-rich cell membrane protrusions, involved in processes as diverse as cell migration, wound closure, and cell invasion by pathogens (1–3). During cell migration, filopodia can exert forces on the substrate (4, 5) and act as precursors of focal adhesions (6–8). Filopodia initiate contacts during wound closure and contribute to dorsal closure of the fruit fly embryo in a zipper-like fashion (9–12). Viruses can hijack filopodia and filopodia-like cell–cell bridges to surf toward the cell body (13, 14). Filopodia from macrophages and epithelial cells actively pull pathogens bound to their tips (15–18). In all these examples filopodial retraction and retrograde force production are crucial. However, although filopodia formation and growth have been well studied (1–3), the mechanisms underlying their retraction are poorly understood.Filopodia show continuous rearward movement of their actin filaments in a process called “retrograde flow” (3, 19). In the lamellipodium, from which filopodia often emanate, the retrograde flow originates from actin treadmilling due to actin depolymerization at the rear and polymerization at the front of the lamellipodium. This retrograde flow is further amplified by the motor activity of myosins (20–23). In neurons, the filopodial shaft is deeply anchored in the growth cone and filopodial dynamics depends on the balance between actin polymerization at the filopodial tip and its retrograde flow (19). In other cell types actin depolymerization at the tip has been associated with retracting filopodia (24).Different contributions to filopodial force production during retraction can be considered. A connection between the filopodial tip and retracting actin filaments through transmembrane receptors such as integrins could transduce cortical forces applied on the actin shaft. In macrophages, force measurements on retracting filopodia suggested a major role for cortical myosins pulling on filopodial actin bundles (16). These measurements showed that retraction could be slowed down for forces below 20 pN. Applied forces higher than 20 pN inverted filopodial retraction of macrophages (25).Filopodial force production can also be due to membrane mechanics (26). Forces exerted by actin-free tubes extruded from the cell plasma membrane typically range between 5 pN and 30 pN (27). Membrane tension could drive filopodial retraction by exerting inward forces against the actin filaments. Moreover, filopodial actin filaments have been found disconnected from the membrane at the tip (28, 29), underlining the importance of membrane properties in filopodial mechanics. The contributions of membrane- and actin-based forces, as well as the mechanical links controlling force production during filopodial retraction, are still unclear.Here, we studied the retraction dynamics and the forces exerted by a single filopodium that is contacting an optically trapped bead at its tip. We found that filopodia retracted in association with a reduced actin polymerization at their tip at rates below those needed to compensate for the retrograde flow. The speed of filopodial retraction was only marginally affected by counteracting forces up to 50 pN, suggesting that the driving forces for retraction were not limiting within this range. We argue that actin treadmilling in the cell cortex, that functions far from its stall regime, transduces inward forces to the filopodial actin shaft at the base via high friction. In addition we found that filopodia can exert passive inward forces of 15 pN by using cell membrane-based forces. External counterforces that are only 5 pN higher than the membrane force can lead to rupture of connections between the actin shaft and the membrane at the filopodial tip. These weak contacts at the tip define the maximal pulling force of filopodia and allow cytoskeletal inward forces to operate only for short time intervals (<25 s). We found that the mechanical disconnection between membrane and actin filaments is only transient as actin dynamics at the tip are altered after disconnection. A continuous load-and-fail behavior allows thus tip-bound filopodia to probe the mechanics of their environment.     

GluN3A expression restricts spine maturation via inhibition of GIT1/Rac1 signaling

NMDA-type glutamate receptors (NMDARs) guide the activity-dependent remodeling of excitatory synapses and associated dendritic spines during critical periods of postnatal brain development. Whereas mature NMDARs composed of GluN1 and GluN2 subunits mediate synapse plasticity and promote spine growth and stabilization, juvenile NMDARs containing GluN3A subunits are thought to inhibit these processes via yet unknown mechanisms. Here, we report that GluN3A binds G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles actin regulatory complexes, including the Rac1 guanine nucleotide exchange factor βPIX, to promote Rac1 activation in spines. Binding to GluN3A limits the synaptic localization of GIT1 and its ability to complex βPIX, leading to decreased Rac1 activation and reduced spine density and size in primary cultured neurons. Conversely, knocking out GluN3A favors the formation of GIT1/βPIX complexes and increases the activation of Rac1 and its main effector p21-activated kinase. We further show that binding of GluN3A to GIT1 is regulated by synaptic activity, a response that might restrict the negative regulatory effects of GluN3A on actin signaling to inactive synapses. Our results identify inhibition of Rac1/p21-activated kinase actin signaling pathways as an activity-dependent mechanism mediating the inhibitory effects of GluN3A on spine morphogenesis.During the development of neural circuits, a phase of intense synaptogenesis is followed by a period of activity-dependent remodeling (or “synaptic refinement”) in which more than half of the initially formed synapses are eliminated, whereas other connections will mature and be kept (1, 2). The subunit composition of NMDA-type glutamate receptors (NMDARs) expressed by individual synapses during this critical period is a key factor influencing functional and structural synaptic plasticity and, in turn, synapse fate (3). Mature NMDARs composed of GluN1 and GluN2 subunits drive the maturation of active synapses by detecting coincident pre- and postsynaptic activity and coupling this activity to signaling pathways that trigger the enlargement and stabilization of synapses and associated dendritic spines (4–6). This structural plasticity is critical for coupling the wiring of neural circuits to experience and supporting the long-term maintenance of spines and memories. During the refinement stage, NMDARs additionally contain GluN3A subunits that serve as a brake on synapse maturation and stabilization, which might provide a counterbalance to limit synapse numbers. Supporting this idea, loss of GluN3A increases spine density and size (7) and accelerates the expression of markers of synaptic maturation (8), whereas overexpression reduces synapse and spine density and yields a higher proportion of smaller, immature spines (9). However, the downstream mechanisms by which GluN3A inhibits synapse and spine maturation remain unknown.Spines are actin-rich, and their structural remodeling relies on rearrangements of the actin cytoskeleton (10–13). Cytoskeletal rearrangements are regulated by the Rho family of small GTPases, and two members of this family, Rac1 and RhoA, are major regulators of spine remodeling (14, 15). Rho-GTPases act as molecular switches that cycle between an inactive GDP-bound conformation and an active GTP-bound conformation (16). Their activation state is controlled by guanine exchange factors (GEFs), which promote the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis. Several Rac1-specific GEFs, including Kalirin7, Tiam1, and βPIX, are targeted to synapses via interactions with scaffolding proteins, which allows local regulation of actin remodeling in spines and its coupling to synaptic activity (12, 17–22). Although many studies have shown that NMDAR activation induces cytoskeletal and spine remodeling by activating Rac1-GEFs (23–25), much less is known about pathways that restrict excitatory synapse maturation and/or promote elimination.Here, we identify a physical association between the intracellular C-terminal domain of GluN3A subunits and G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles a multiprotein signaling complex with Rac1 and the Rac1-GEF βPIX to regulate actin dynamics in spines (19, 26). GIT1 selectively bound juvenile NMDARs containing GluN3A but not mature NMDAR subtypes, and binding was regulated by activity because it could be enhanced or reduced, respectively, by brief episodes of synaptic inactivity or synaptic stimulation. A functional analysis demonstrates that binding to GluN3A interferes with the synaptic localization of GIT1 and its ability to recruit βPIX, leading to decreased Rac1 activation. We finally show that GluN3A-induced reductions in spine density and size critically require GIT1 binding. We propose that the coupling of GIT1/GluN3A binding to synapse use might provide an effective mechanism with which to restrict the maturation and growth of inactive synapses in a selective manner.     

Peripheral elevation of TNF-α leads to early synaptic abnormalities in the mouse somatosensory cortex in experimental autoimmune encephalomyelitis

Sensory abnormalities such as numbness and paresthesias are often the earliest symptoms in neuroinflammatory diseases including multiple sclerosis. The increased production of various cytokines occurs in the early stages of neuroinflammation and could have detrimental effects on the central nervous system, thereby contributing to sensory and cognitive deficits. However, it remains unknown whether and when elevation of cytokines causes changes in brain structure and function under inflammatory conditions. To address this question, we used a mouse model for experimental autoimmune encephalomyelitis (EAE) to examine the effect of inflammation and cytokine elevation on synaptic connections in the primary somatosensory cortex. Using in vivo two-photon microscopy, we found that the elimination and formation rates of dendritic spines and axonal boutons increased within 7 d of EAE induction—several days before the onset of paralysis—and continued to rise during the course of the disease. This synaptic instability occurred before T-cell infiltration and microglial activation in the central nervous system and was in conjunction with peripheral, but not central, production of TNF-α. Peripheral administration of a soluble TNF inhibitor prevented abnormal turnover of dendritic spines and axonal boutons in presymptomatic EAE mice. These findings indicate that peripheral production of TNF-α is a key mediator of synaptic instability in the primary somatosensory cortex and may contribute to sensory and cognitive deficits seen in autoimmune diseases.Sensory, motor, and cognitive dysfunctions are common at the early stages of autoimmune diseases, including in more than half of multiple sclerosis (MS) patients (1–5). The increased production of proinflammatory cytokines and infiltration of peripheral immune cells into the central nervous system (CNS) are closely associated with neuronal damage in the spinal cord and many brain regions (6–10). Elevation of several cytokines such as TNF-α and IFN-γ precedes infiltration of peripheral immune cells and could have a significant impact on neuronal function (11–19), potentially contributing to early sensory and cognitive impairments. However, the link between the cytokine elevation and CNS deficits in autoimmune diseases remains unclear.Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model to study the pathogenesis and treatment of MS (20). Previous studies have shown early behavioral changes, including decreased exploratory behavior and increased startle response before the onset of paralysis in EAE mice (21). The mechanisms underlying these early behavioral changes remain to be addressed. In EAE, activated antigen-specific T cells migrate through the blood brain barrier (BBB) and subsequently recruit additional myeloid and lymphoid cells to the spinal cord and susceptible brain regions (22–24). The resulting inflammatory cascade eventually leads to axonal loss and neuronal death. However, inflammatory infiltrates usually accompany the onset of clinical EAE and are therefore unlikely to contribute to sensory and behavioral deficits in presymptomatic mice. On the other hand, elevation of proinflammatory cytokines occurs during the early phase of EAE induction (25). Cytokines such as TNF-α are produced by activated cells within peripheral lymphoid tissues as well as by CNS-resident microglia in response to a number of inflammatory stimuli. Peripherally produced TNF-α can enter the circulation and cross the BBB through active transport (26–28) or passively after pathologic BBB disruption (27, 29). TNF-α has been shown to affect synaptic transmission and synaptic scaling (30–32), as well as regulate the production of other cytokines and chemokines to impact neuronal function. The induction and progression of EAE can be inhibited by either i.p. or intracranial injection of anti-TNF antibodies (33). Therefore, TNF-α elevation under pathological conditions could cause significant deficits in the CNS and contribute to behavioral abnormalities in EAE.TNF-α is known to affect neuronal functions by acting at two receptor types, TNF receptor (TNFR)1 and TNFR2, each having divergent effects in the CNS (16, 34). TNFR2 responds mainly to transmembrane TNF-α, which is thought to be a prosurvival signal. In contrast, TNFR1 responds mainly to circulating soluble TNF-α (solTNF) and plays a role in inflammation, proliferation, and neural migration. Activation of TNFR1 has been found to affect synaptic scaling by recruiting AMPA receptors to the postsynaptic cell membrane (30–32). TNFR1 activation also initiates the JNK pathway thought to be involved in dendritic cytoskeleton stability via regulation of microtubules (16). A dominant negative TNF inhibitor, XPro1595, blocks the effects of solTNF to selectively decrease the activation of TNFR1 (35, 36). Recently, decreased activation of TNFR1 via XPro1595 has been shown to promote axonal integrity and improve clinical outcome of mice with EAE (10).In this study, we examined the changes in synaptic connections in the primary somatosensory cortex in response to inflammation in an EAE mouse model. Using transcranial two-photon microscopy (37, 38), we followed postsynaptic dendritic spines and presynaptic axonal boutons over time in mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG_35–55). We found that, in conjunction with an elevation of peripheral TNF-α production, the turnover of dendritic spines and axonal boutons was significantly increased in the cortex within 7 d of MOG immunization, several days before the onset of paralysis. The turnover of dendritic spines and axonal boutons continued to increase as the disease progressed. This early instability of cortical synaptic connections in EAE mice is independent of T-cell infiltration and microglia activation, but depends on the elevation of peripheral TNF-α in EAE mice.